CN101302528B

CN101302528B - Polypeptides involved in the biosynthesis of spiramycins, nucleotide sequences coding these polypeptides and their uses

Info

Publication number: CN101302528B
Application number: CN2008100823963A
Authority: CN
Inventors: M-H·布隆德莱-鲁奥; H·多明格斯; E·达尔邦-龙热尔; C·热尔博; A·贡德朗; F·卡拉伊; P·拉克鲁瓦; N·厄斯特赖歇尔-梅尔梅-布维耶; J-L·佩尔诺代; K·蒂皮海尔
Original assignee: Centre National de la Recherche Scientifique CNRS; Aventis Pharma SA
Current assignee: Centre National de la Recherche Scientifique CNRS; Aventis Pharma SA
Priority date: 2002-10-08
Filing date: 2003-10-08
Publication date: 2012-05-30
Anticipated expiration: 2023-10-08
Also published as: FR2845394A1; CN101302528A; ZA200502139B; CN1711355A

Abstract

The invention concerns isolation and identification of novel genes of spiramycin biosynthesis process and novel polypeptides involved in said biosynthesis. The invention also concerns a method for producing sand polypeptides. The invention further concerns the use of said genes for increasing the rate of production and purity in the resulting spiramycin. The invention concerns in particular a micro-organism producing spiramycin I but not producing spiramycin II and III and the use of such a micro-organism. The invention also concerns the use of genes in spiramycin biosynthesis process for constructing mutants capable of leading to synthesis of novel antibiotics or derivative forms of spiramycin. Finally the invention concerns molecules produced through expression of said genes and phamacologically active compositions of one molecule produced through expression of such genes.

Description

The nucleotide sequence and the application thereof of the biosynthetic polypeptide of involved in spiramycin, these polypeptide of encoding

The application is dividing an application of one Chinese patent application 200380102966.6, and the applying date of original application is on October 8th, 2003, and denomination of invention is " nucleotide sequence and the application thereof of the biosynthetic polypeptide of involved in spiramycin, these polypeptide of encoding ".

The present invention relates to the separation and the evaluation of Spiramycin Base biosynthetic pathway sino-singaporean gene, and relate to this biosynthetic novel polypeptide of participation.The invention still further relates to these genes and be used to increase the output of the Spiramycin Base that is produced and the purposes of purity level.

The invention still further relates to these genes and be used to make up the purposes of the two mutants that can cause the synthetic or Spiramycin Base derivative form of new antibiotic.The invention still further relates to the molecule that these genes of overexpression are produced, and finally relate to the pharmaceutical active compsn of the molecule that this genoid of overexpression produced.

Streptomyces (Streptomyces) is the thread soil bacteria of gram-positive.Because they secrete multiple degrading enzyme, so in the decomposition of organic substance and mineralising, have important effect.Streptomyces shows morphologic differentiating phenomenon, and this is unique in prokaryotic organism, the differentiation in the metabolism that it is characteristic that the while streptomyces has with generation chemical structure and the remarkable different secondary metabolites of BA.In these metabolites, exist by bacterium and give birth to the natural synthetic Spiramycin Base of dyadic streptomycete (Streptomycesambofaciens).

Spiramycin Base is a macrolide antibiotics, at veterinary drug with humanly all have purposes aspect medical.Macrolide is characterised in that deposits the lactonic ring that is connected with one or more sugar above that.Give birth to the natural generation Spiramycin I of dyadic streptomycete, II and III (with reference to Fig. 1); Yet the antibiotic activity of Spiramycin I is apparently higher than the antibiotic activity (Liu etc., 1999) of spiramycin II and III.The Spiramycin I molecule is that forosamine, mycaminose and mycarose form (with reference to Fig. 1) by big ring (being called platenolide) and three sugar based on lactone.The antibiotic activity of Spiramycin Base is owing in prokaryotic organism, synthesize through relevant mechanism and the arrestin matter that this microbiotic is attached on the bacterial ribosome.

Some compound that belongs to the macrolide family member and also have a lactonic ring is used outside field of antibiotics more and more.Therefore, product FK506 has immunosuppressant activity and in filed of organ transplantation, rheumatoid arthritis field, more generally the pathologic conditions relevant with autoimmune response is had the treatment application prospect.Other macrolides, for example avermectin has desinsection and anthelmintic activity.

Up to the present many biosynthetic pathways are studied in streptomyces, wherein said biosynthetic pathway relates to different classes of microbiotic and the biosynthetic pathway of other secondary metabolites (summary is seen K, Chater, 1990).Yet up to the present, the understanding of Spiramycin Base biosynthetic pathway is still very limited.

The Spiramycin Base biosynthesizing be comprise many steps and relate to many enzymes complex process (Omura etc., 1979a, Omura etc., 1979b).Spiramycin Base belongs to one big type of polyketide (polyketide), and this type polyketide is included in complicated molecule abundant especially in the mikrobe of finding in the soil.It is not owing to similar that these molecules are classified as one type, but because certain some similarity in the step of their biosynthetic pathway.Particularly, polyketide is that the series reaction through complicacy produces, and wherein the common aspect is, in their biosynthetic pathway, it is to be called " polyketide synthase " (PKS) enzymatic by one or more that series reaction is arranged.In giving birth to the dyadic streptomycete, Spiramycin Base is (S.Kuhstoss, 1996, USP 5,945,320) of carrying out through a series of eight assemblies by five PKS genes encodings based on the biosynthesizing of the big ring (platenolide) of lactone.Spiramycin Base obtains from this lactonic ring.Yet, a plurality of steps of involved in sugar synthetic and enzyme, to such an extent as to and they be connected with lactonic ring and this ring modified and obtain Spiramycin Base and remain unknown at present.

USP 5,514,544 have described the clone who is called the sequence of srmR in the living dyadic streptomycete.In that patent, the protein regulation that has proposed the srmR coded by said gene is participated in the hypothesis of the biosynthetic gene transcription of macrolide.

1987, Richardson and colleague thereof (Richardson etc., 1987) research showed that the Spiramycin Base resistance of living dyadic streptomycete given by at least three genes; Said gene is called srmA, srmB and srmC.USP 4,886,757 have more specifically described the characteristics that living dyadic streptomycete contains the dna fragmentation of srmC gene.Yet the sequence of this gene is unexposed.Nineteen ninety, Richardson and colleague thereof (Richardson etc., 1990) have proposed near three biosynthetic hypothesis of gene involved in spiramycin the srmB.USP 5,098,837 have reported the clone of biosynthetic five genes of possibility involved in spiramycin.These unnamed genes are srmD, srmE, srmF, srmG and srmH.

One of main difficulty of the compound of generation such as Spiramycin Base is to produce the essential such fact of very large amount of fermentation of relatively small amount product.Therefore the efficient of expecting to increase this quasi-molecule generation is to reduce its production cost.

The Spiramycin Base biosynthetic pathway is that the association reaction that in this process, possibly exist is identified and removed in complicated process and expectation.The purpose of this kind operation is to obtain purer microbiotic and/or improve output.In this respect, give birth to the natural generation Spiramycin I of dyadic streptomycete, II and III (with reference to Fig. 1); Yet the antibiotic activity of Spiramycin I is apparently higher than spiramycin II and III (Liu etc., 1999).Therefore expectation can have the bacterial strain that only produces Spiramycin I.

Because the commercial value of macrolide antibiotic presses for the generation novel derivative, particularly has the Spiramycin Base analogue of advantageous feature.Expectation can be given birth to the biosynthesizing midbody of Spiramycin Base or Spiramycin Base verivate biosynthetic pathway with enough volume productions, particularly in order to produce Spiramycin Base deutero-hydridization microbiotic (hybrid antibiotics).

Summary of the invention

The present invention relates to clone to the biosynthetic gene of its product involved in spiramycin.At first, the present invention relates to the new gene and this biosynthetic novel polypeptide of participation of Spiramycin Base biosynthetic pathway.

The dna sequence dna of each gene has been cloned and confirmed to the gene of biosynthetic pathway and correlative coding sequence.Hereinafter the encoding sequence of being cloned is called orf1 ^*C, orf2 ^*C, orf3 ^*C, orf4 ^*C, orf5 ^*, orf6 ^*, orf7 ^*C, orf8 ^*, orf9 ^*, orf10 ^*, orf1, orf2, orf3, orf4, orf5, orf6, orf7, orf8; Orf9c, orf10, orf11c, orf12, orf13c, orf14, orf15c, orf16, orf17; Orf18, orf19, orf20, orf21c, orf22c, orf23c, orf24c, orf25c; Orf26, orf27, orf28c, orf29, orf30c, orf31, orf32c, orf33 and orf34c.The function of protein in the Spiramycin Base biosynthetic pathway by these sequences are coded discussed hereinafter, by Fig. 4,5,6 and 8 graphic extensions.

1) first theme of the present invention relates to the polynucleotide of the biosynthetic polypeptide of coding involved in spiramycin, and the sequence of wherein said polynucleotide is:

(a) sequence SEQ ID No.3,5,7,9,11,13,15,17,19,21,23,25,28,30,34,36; 40,43,45,47,49,53,60,62,64,66,68,70,72,74,76,78; One of 80,82,84,107,109,111,113,115,118,120,141,143,145,147 and 149

(b) one of sequence of forming by the variant of sequence (a),

(c) owing to the genetic code degeneracy and derived from one of sequence (a) and sequence (b).

2) theme of the present invention also relates under high stringent hybridization condition, and at least one according to above paragraph 1) the polynucleotide of multi-nucleotide hybrid.

3) the invention still further relates to and comprise above paragraph 1) polynucleotide at least 10,12,15,18,20 to 25,30,40,50,60,70,80; 90,100,150,200,250,300,350,400,450,500,550; 600,650,700,750,800,850,900,950,1000,1050; 1100,1150,1200,1250,1300,1350,1400,1450,1500,1550; 1600,1650,1700,1750, the polynucleotide of 1800,1850 or 1900 continuous nucleotides have at least 70%, 80%, and 85%, 90%, 95% or the polynucleotide of 98% Nucleotide identity.

4) the invention still further relates to according to above paragraph 2) or 3) polynucleotide, said polynucleotide separate from the bacterium of streptomyces.

5) the invention still further relates to according to above paragraph 2), 3) or 4) polynucleotide, said polynucleotide encoding is participated in the biosynthetic protein of macrolide.

6) the invention still further relates to according to above paragraph 2), 3) or 4) polynucleotide, the activity that the protein of said polynucleotide encoding has is similar to the coded protein of polynucleotide that presents identity with its hybridization or with it.

7) the invention still further relates to expression according to above paragraph 1), 2), 3), 4), 5) or 6) the resulting polypeptide of polynucleotide.

8) another aspect of the present invention relates to the biosynthetic polypeptide of involved in spiramycin, and the sequence of wherein said polypeptide is:

(a) sequence SEQ ID No.4,6,8,10,12,14,16,18,20,22,24,26,27,29,31,32; 33,35,37,38,39,41,42,44,46,48,50,51,52,54,55; 56,57,58,59,61,63,65,67,69,71,73,75,77,79,81; One of 83,85,108,110,112,114,116,117,119,121,142,144,146,148 and 150

(b), but in said full length sequence, one or more amino acid have been carried out replacement, insertion or deletion and have not been influenced its functional performance like one of (a) middle sequence that defines,

(c) one of sequence of forming by sequence (a) and variant (b).

9) another theme of the present invention relates to and comprises above paragraph 8) polypeptide at least 10,15,20,30 to 40,50,60,70,80,90,100; 120,140,160,180,200,220,240,260,280,300; 320,340,360,380,400,420,440,460,480,500; 520,540,560,580, the polypeptide of 600,620 or 640 continuous amino acids has at least 70%, 80%, and 85%, 90%, 95% or the polypeptide of 98% amino acid identity.

10) another aspect of the present invention also relates to according to above paragraph 9) polypeptide, said polypeptide separates from the bacterium of streptomyces and obtains.

11) another aspect of the present invention also relates to according to above paragraph 9) or 10) polypeptide, said peptide coding is participated in the biosynthetic protein of macrolide.

12) another aspect of the present invention also relates to according to above paragraph 9), 10) or 11) polypeptide, the activity that said polypeptide has is similar to the activity that polypeptide had that presents identity with it.

13) another aspect of the present invention also relates to recombinant DNA, and it contains at least one according to above paragraph 1), 2), 3), 4), 5) and 6) one of polynucleotide.

14) another aspect of the present invention relates to according to above paragraph 13) recombinant DNA, wherein said recombinant DNA is contained in the carrier.

15) another aspect of the present invention relates to according to above paragraph 14) recombinant DNA, wherein said carrier is selected from phage, plasmid, phagemid, integrative vector, fosmids, clay, shuttle vectors, BAC and PAC.

16) another aspect of the present invention relates to according to above paragraph 15) recombinant DNA, this recombinant DNA is selected from pOS49.1, pOS49.11, pOSC49.12, pOS49.14, pOS49.16, pOS49.28, pOS44.1, pOS44.2; POS44.4, pSPM5, pSPM7, pOS49.67, pOS49.88, pOS49.106, pOS49.120, pOS49.107, pOS49.32; POS49.43, pOS49.44, pOS49.50, pOS49.99, pSPM17, pSPM21, pSPM502, pSPM504, pSPM507; PSPM508, pSPM509, pSPM1, pBXL1111, pBXL1112, pBXL1113, pSPM520, pSPM521, pSPM522; PSPM523, pSPM524, pSPM525, pSPM527, pSPM528, pSPM34, pSPM35, pSPM36, pSPM37; PSPM38, pSPM39, pSPM40, pSPM41, pSPM42, pSPM43, pSPM44, pSPM45, pSPM47; PSPM48, pSPM50, pSPM51, pSPM52, pSPM53, pSPM55, pSPM56, pSPM58, pSPM72; PSPM73, pSPM515, pSPM519, pSPM74, pSPM75, pSPM79, pSPM83, pSPM107, pSPM543 and pSPM106.

17) another aspect of the present invention relates to expression vector, wherein contains at least one coding according to above paragraph 7), 8), 9), 10), 11) or 12) the nucleotide sequence of polypeptide.

18) the invention still further relates to expression system, it allows one or more according to above paragraph 7), 8), 9), 10), 11) or 12) expression of polypeptides, comprise suitable expression and host cell.

19) the invention still further relates to expression system according to above paragraph 18, this system is selected from prokaryotic expression system and eukaryotic expression system.

20) the invention still further relates to according to above paragraph 19) expression system, this system is selected from bacteria Escherichia coli (E.coli) system expressed, allows the baculovirus expression system in expressed in insect cells, the expression system that allows the expression system of in yeast cell, expressing and allow in mammalian cell, to express.

21) the invention still further relates to will be according to above paragraph 1), 2), 3), 4), 5), 6), 13), 14), 15), 16) and 17) one of the host cell that imported of at least one polypeptide and territory at least one recombinant DNA and/or at least one expression vector.

22) the invention still further relates to generation according to above paragraph 7), 8), 9), 10), 11) or 12) the method for polypeptide, wherein said method may further comprise the steps:

A) nucleic acid with at least one coding said polypeptide inserts appropriate carriers;

B) in suitable substratum, cultivate in advance with the carrier conversion of step a) or the host cell of transfection;

C) recovering condition substratum or cell extract;

D) from the said substratum that step c) obtains or in the cell extract, separate and the said polypeptide of purifying;

E) as required, characterize the recombinant polypeptide that is produced.

23) another aspect of the present invention relates to the mikrobe of the biosynthesizing step of having blocked at least a macrolide.

24) another aspect of the present invention relates to according to above paragraph 23) mikrobe, said mikrobe is to obtain through the biosynthetic proteinic function of at least a participation of inactivation this (these) macrolide in producing the mikrobe of this (these) macrolide.

25) another aspect of the present invention relates to according to above paragraph 24) mikrobe, wherein this (these) proteinic inactivation is to implement through the gene of mutagenesis code for said proteins or the overexpression one or more and the messenger RNA(mRNA) complementary sense-rna of code for said proteins.

26) another aspect of the present invention relates to according to above paragraph 25) mikrobe, wherein this (these) proteinic inactivation is implemented through radioinduction, chemical mutagen, site-directed mutagenesis or gene substitution.

27) another aspect of the present invention relates to according to above paragraph 25) or 26) mikrobe, one of them mutagenesis or a plurality of mutagenesis is in external or the inhibition through one or more bases in to specific gene in position, substitute, deletion and/or add or implement through gene inactivation.

28) another aspect of the present invention relates to according to above paragraph 23), 24), 25), 26) or 27) mikrobe, wherein said mikrobe is the bacterium of streptomyces.

29) another aspect of the present invention relates to according to above paragraph 23), 24), 25), 26), 27) or 28) mikrobe, wherein macrolide is a Spiramycin Base.

30) another aspect of the present invention relates to according to above paragraph 23), 24), 25), 26), 27), 28) or 29) mikrobe, wherein said mikrobe is to give birth to the dyadic streptomycete bacterial strain.

31) another aspect of the present invention relates to according to above paragraph 23), 24), 25), 26), 27), 28), 29) or 30) mikrobe, wherein mutagenesis is to contain with good grounds above paragraph 1 at least one), 2), 3), 4), 5) and 6) one of the gene of sequence in carry out.

32) another aspect of the present invention relates to according to above paragraph 25), 26), 27), 28), 29), 30) or 31) mikrobe, wherein mutagenesis is to contain corresponding to one or more sequence SEQ ID No.3,5,7,9,11,13,15,17,19 one or more; 21,23,25,28,30,34,36,40,43,45; 47,49,53,60,62,64,66,68,70; 72,74,76,78,80,82,84,107,109; Carry out in one of 111,113,115,118,120,141,143,145,147 and 149 the gene.

33) another aspect of the present invention relates to according to above paragraph 25), 26), 27), 28), 29), 30), 31) or 32) mikrobe, wherein mutagenesis is for carrying out gene inactivation to the gene that comprises corresponding to the sequence of sequence SEQ ID No.13.

34) another aspect of the present invention relates to living dyadic streptomycete bacterial strain; Said bacterial strain be selected from state-run microbial preservation center [National Collection of Cultures and Microorganisms] (CNCM) preservation, preservation day is that July 10, preserving number in 2002 are I-2909, I-2911, I-2912; I-2913; I-2914, I-2915, one of bacterial strain of I-2916 or I-2917.

35) another aspect of the present invention relates to the method for preparing macrolide biosynthesizing midbody, and this method may further comprise the steps:

A) in suitable substratum, cultivate according to above paragraph 23), 24), 25), 26), 27), 28), 29), 30), 31), 32), 33) or 34) one of mikrobe,

B) recovering condition substratum or cell extract,

C) separate and the said biosynthesizing midbody of purifying in said substratum that from step b), obtains or the cell extract.

36) another aspect of the present invention relates to the method for preparation derived from the molecule of macrolide, and wherein the biosynthesizing midbody is according to above paragraph 35) method preparation and the midbody that produces like this through modifying.

37) another aspect of the present invention relates to according to above paragraph 36) the preparation method, wherein said midbody is modified through chemistry, biological chemistry, zymetology and/or microbiology.

38) another aspect of the present invention relates to according to above paragraph 36) or 37) preparation method; Wherein one or more such genes are imported said mikrobe, the albumen mass-energy of wherein said genes encoding is modified said midbody as substrate with midbody.

39) another aspect of the present invention relates to according to above paragraph 36), 37) or 38) preparation method, wherein macrolide is a Spiramycin Base.

40) another aspect of the present invention relates to according to above paragraph 36), 37), 38) or 39) method of preparation, wherein employed mikrobe is to give birth to the dyadic streptomycete bacterial strain.

41) another aspect of the present invention relates to the mikrobe that produces Spiramycin I but do not produce spiramycin II and III.

42) another aspect of the present invention relates to according to above paragraph 41) mikrobe; This mikrobe contains the required full gene of biosynthesizing Spiramycin I, but wherein contains one of sequence SEQ ID No.13 or its variant, or because genetic code degeneracy and not expressed or by inactivation by the gene of the polypeptide of one of its deutero-sequence and one of encoding sequence SEQ ID No.14 or its variant.

43) another aspect of the present invention relates to according to above paragraph 42) mikrobe, wherein said inactivation is that the messenger RNA(mRNA) complementary sense-rna through mutagenesis or overexpression and code for said proteins in the gene of code for said proteins carries out.

44) another aspect of the present invention relates to according to above paragraph 43) mikrobe, wherein said mutagenesis is in the promotor of this gene, carries out in the encoding sequence or in the non-coding sequence, thereby makes encoded protein matter inactivation or stop by its expression or its translation.

45) another aspect of the present invention relates to according to above paragraph 43) or 44) mikrobe, wherein mutagenesis is to carry out through effect, site-directed mutagenesis or the gene substitution of radiation, chemical mutagen.

46) another aspect of the present invention relates to according to above paragraph 43), 44) or 45) mikrobe, wherein mutagenesis be in external or the inhibition through one or more bases in to specific gene in position, substitute, deletion and/or add or carry out through gene inactivation.

47) another aspect of the present invention relates to according to above paragraph 41) or 42) mikrobe; Wherein said mikrobe is that the gene in the overexpression Spiramycin Base biosynthetic pathway obtains, and wherein said gene does not comprise and containing corresponding to SEQ ID No.13 or its variant sequence or because genetic code degeneracy and by these genes of the gene of the polypeptide of one of its deutero-sequence and one of encoding sequence SEQ ID No.14 or its variant.

48) another aspect of the present invention relates to according to above paragraph 41), 42) 43), 44), 45), 46) or 47) mikrobe, wherein said mikrobe is the bacterium of streptomyces.

49) another aspect of the present invention relates to according to above paragraph 41), 42) 43), 44), 45), 46), 47) or 48) mikrobe, wherein said mikrobe is to be obtained by the initial strain that produces Spiramycin I, II and III.

50) another aspect of the present invention relates to according to above paragraph 41), 42) 43), 44), 45), 46), 47), 48) or 49) mikrobe, said mikrobe is through containing corresponding to SEQ ID No.13 or its variant sequence or because genetic code degeneracy and had by one of its deutero-sequence and encoding sequence SEQ ID No.14 or its and to carry out mutagenesis in gene of polypeptide of one of variant of identical function and obtain.

51) another aspect of the present invention relates to according to above paragraph 41), 42), 43), 44), 45), 46), 47), 48), 49) or 50) mikrobe; Wherein said mikrobe is to obtain from the livings dyadic streptomycete bacterial strain that produces Spiramycin I, II and III, has wherein carried out gene inactivation to containing corresponding to SEQ ID No.13 or owing to the genetic code degeneracy by the gene of one of its deutero-sequence.

52) another aspect of the present invention relates to living dyadic streptomycete bacterial strain, and said bacterial strain is the bacterial strain that is deposited in state-run microbial preservation center (CNCM) on July 10th, 2002, and preserving number is I-2910.

53) another aspect of the present invention relates to the method that produces Spiramycin I, and this method may further comprise the steps:

(a) in suitable substratum, cultivate according to above paragraph 41), 42), 43), 44), 45), 46), 47), 48), 49), 50), 51) and 52) one of mikrobe,

(b) recovering condition substratum or cell extract,

(c) separate and the purifying Spiramycin I said substratum that obtains from step b) or the cell extract.

54) another aspect of the present invention relates to according to above paragraph 1), 2), 3), 4), 5) and 6) one of the purposes of polynucleotide, be used to improve the macrolide turnout of mikrobe.

55) another aspect of the present invention relates to the mutant microbial that produces macrolide, and this mutant microbial contains corresponding to according to above paragraph 1 at least one), 2), 3), 4), 5) and 6) one of the gene of sequence in carried out genetic modification and overexpression at least one contained with good grounds above paragraph 1), 2), 3), 4), 5) and 6) one of the gene of sequence.

56) another aspect of the present invention relates to according to above paragraph 55) mutant microbial; Wherein genetic modification by the inhibition of one or more bases in specific gene, substitute, deletion and/or add and forms, purpose has more highly active protein or expresses higher levels of this or these protein for expression is one or more.

57) another aspect of the present invention relates to according to above paragraph 55) mutant microbial, wherein the overexpression of specific gene is to obtain through increasing gene copy number and/or importing the promotor stronger than wild-type promoter activity.

58) another aspect of the present invention relates to according to above paragraph 55) or 57) mutant microbial; Wherein the overexpression of specific gene is through using the recombinant DNA construction body according to

above paragraph

13,14 or 17 to transform the mikrobe that produces macrolide, allowing this gene overexpression to obtain.

59) another aspect of the present invention relates to according to above paragraph 55), 56), 57) or 58) mutant microbial, wherein genetic modification is to contain corresponding to one or more sequence SEQ IDNo.3,5,7,9,11,13,15,17,19 one or more; 21,23,25,28,30,34,36,40,43,45; 47,49,53,60,62,64,66,68,70; 72,74,76,78,80,82,84,107,109; One of 111,113,115,118,120,141,143,145,147 and 149 or one of its variant or because genetic code degeneracy and carry out in the gene by one of its deutero-sequence.

60) another aspect of the present invention relates to according to above paragraph 55), 56), 57), 58) or 59) mutant microbial, wherein the mikrobe overexpression is one or more contains corresponding to one or more sequence SEQ ID No.3,5,7,9,11,13,15,17,19; 21,23,25,28,30,34,36,40,43,45; 47,49,53,60,62,64,66,68,70; 72,74,76,78,80,82,84,107,109; One of 111,113,115,118,120,141,143,145,147 and 149 or one of its variant or because genetic code degeneracy and by the gene of one of its deutero-sequence.

61) another aspect of the present invention relates to according to above paragraph 55), 56), 57), 58), 59) or 60) mutant microbial, wherein said mikrobe is the bacterium of streptomyces.

62) another aspect of the present invention relates to according to above paragraph 55), 56), 57), 58), 59), 60) or 61) mutant microbial, wherein macrolide is a Spiramycin Base.

63) another aspect of the present invention relates to according to above paragraph 55), 56), 57), 58), 59), 60), 61) or 62) mutant microbial, wherein said mikrobe is to give birth to the dyadic streptomycete bacterial strain.

64) another aspect of the present invention relates to the method that produces macrolide, and this method may further comprise the steps:

(a) in suitable substratum, cultivate according to above paragraph 55), 56), 57), 58), 59), 60), 61), 62), 63) or 64) one of mikrobe,

(b) recovering condition substratum or cell extract,

(c) in said substratum that step b) obtains or cell extract, separate and macrolide that purifying produced.

65) another aspect of the present invention relates to according to above paragraph 1), 2), 3), 4), 5), 6), 7), 8), 9), 10), 11), 12), 13), 14), 15), 16) and 17) one of sequence and/or the purposes of recombinant DNA and/or carrier, be used to prepare the hydridization microbiotic.

66) another aspect of the present invention relates to according to above paragraph 1), 2), 3), 4), 5), 6), 7), 8), 9), 10), 11), 12), 13), 14), 15), 16), 17) and 21) one of at least one polynucleotide and/or the purposes of at least one recombinant DNA and/or at least one expression vector and/or at least one polypeptide and/or at least a host cell, be used to carry out one or more bio-transformations.

67) another aspect of the present invention relates to polynucleotide, said polynucleotide be with according to above paragraph 1), 2), 3), 4), 5) or 6) one of polynucleotide complementary polynucleotide.

68) another aspect of the present invention relates to the mikrobe that produces at least a Spiramycin Base, said mikrobe overexpression:

-use following aligning primer right through polymerase chain reaction (PCR):

5 ' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3 ' (SEQ IDNo.138) and 5 ' GGATCCCGCGACGGACACGACCGCCGCGCA 3 ' (SEQID No.139) and the gene that can obtain as template with clay pSPM36 or the total DNA of living dyadic streptomycete

-or owing to the genetic code degeneracy and by its deutero-gene.

69) another aspect of the present invention relates to the mikrobe according to paragraph 68 or 90, and this mikrobe is the bacterium of streptomyces.

70) another aspect of the present invention relates to the mikrobe according to paragraph 68,69 or 90, and this mikrobe is the bacterium that gives birth to the dyadic streptomyces strain.

71) another aspect of the present invention relates to the mikrobe according to

paragraph

68,69,70 or 90, and the overexpression of wherein said gene obtains through transform said mikrobe with expression vector.

72) another aspect of the present invention relates to living dyadic streptomycete bacterial strain; Said bacterial strain is bacterial strain OSC2/pSPM75 (1) or bacterial strain OSC2/pSPM75 (2), is deposited in state-run microbial preservation center (CNCM) [National Collection of Cultures and Microorganisms] Institute Pasteur, 25; Rue du Docteur Roux 75724 Paris Cedex 15; France, preservation day is on October 6th, 2003, preserving number is I-3101.

73) another aspect of the present invention relates to recombinant DNA, and it comprises:

-use following aligning primer right through polymerase chain reaction (PCR):

5 ' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3 ' (SEQ IDNo.138) and 5 ' GGATCCCGCGACGGACACGACCGCCGCGCA 3 ' (SEQID No.139) and the polynucleotide that can obtain as template with clay pSPM36 or the total DNA of living dyadic streptomycete

-or these polynucleotide at least 10,12,15,18,20 to 25,30,40,50,60,70,80; 90,100,150,200,250,300,350,400,450,500,550; 600,650,700,750,800,850,900,950,1000,1050,1100; 1150,1200,1250,1300,1350,1400,1450,1460, the fragment of 1470,1480,1490 or 1500 continuous nucleotides.

74) the present invention relates to the recombinant DNA according to paragraph 73 or 91 on the other hand, and said recombinant DNA is a carrier.

75) the present invention relates to the recombinant DNA according to paragraph 73,74 or 91 on the other hand, and said recombinant DNA is an expression vector.

76) the present invention relates to at least one recombinant DNA importing host cell wherein according to paragraph 73,74,75 or 91 on the other hand.

77) the present invention relates to the method that produces polypeptide on the other hand, and wherein said method may further comprise the steps:

A) with at least one expression vector transformed host cell according to paragraph 75;

B) in appropriate culture medium, cultivate said host cell;

C) recovering condition substratum or cell extract;

D) separate and the said polypeptide of purifying said substratum that obtains from step c) or the cell extract;

E) as required, characterize the recombinant polypeptide that is produced.

78) the present invention relates to the mikrobe according to paragraph 51 on the other hand, and wherein gene inactivation is to contain corresponding to the sequence of SEQ ID No.13 or owing to the genetic code degeneracy through homophase (in-phase) deletion to be undertaken by the gene or the Gene Partial of one of its deutero-sequence.

79) the present invention relates to according to paragraph 41 on the other hand; 42; 43; 44; 45; 46; 47; 48; 49; 50; One of 51 and 78 mikrobe; This mikrobe is overexpression also :-use following aligning primer right through polymerase chain reaction (PCR): 5 ' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3 ' (SEQ ID No.138) and 5 ' GGATCCCGCGACGGACACGACCGCCGCGCA 3 ' (SEQ ID No.139) and the gene that can obtain as template with clay pSPM36 or the total DNA of living dyadic streptomycete

-or owing to the genetic code degeneracy and by its deutero-gene.

80) the present invention relates to expression vector on the other hand; The polynucleotide of sequence SEQ ID No.47 or because genetic code degeneracy and by its deutero-polynucleotide wherein are to place to allow to give birth to the dyadic streptomycete by under the promotor control of the protein expression of said polynucleotide encoding.

81) the present invention relates to the expression vector according to paragraph 80 on the other hand, and this carrier is plasmid pSPM524 or pSPM525.

82) the present invention relates to the living dyadic streptomycete bacterial strain that the carrier used according to paragraph 80 or 81 has transformed on the other hand.

83) the present invention relates to the mikrobe according to one of

paragraph

41,42,43,44,45,46,47,48,49,50,51,78,79 and 92 on the other hand, said mikrobe also overexpression have encoding sequence SEQ ID No.47 or since the genetic code degeneracy by the gene of its deutero-encoding sequence.

84) the present invention relates to the mikrobe according to paragraph 83 on the other hand; This mikrobe is bacterial strain SPM502 pSPM525; Be deposited in state-run microbial preservation center (CNCM) Institute Pasteur, 25rue du Docteur Roux 75724 Paris Cedex 15, France; Preservation day is on February 26th, 2003, and preserving number is I-2977.

85) the present invention relates to the method that produces Spiramycin Base on the other hand, and this method may further comprise the steps:

(a) in appropriate culture medium, cultivate mikrobe according to one of

paragraph

68,69,70,71,72,78,79,82,83,84,90 and 92,

(b) recovering condition substratum or cell extract,

(c) separate and the purifying Spiramycin Base said substratum that obtains from step b) or the cell extract.

86) the present invention relates to polypeptide on the other hand, and its sequence comprises sequence SEQ ID No.112 or sequence SEQ ID No.142.

87) the present invention relates to polypeptide on the other hand, and its sequence is corresponding to the sequence from the translation of following encoding sequence:

-use following aligning primer right through polymerase chain reaction (PCR):

5 ' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3 ' (SEQ IDNo.138) and 5 ' GGATCCCGCGACGGACACGACCGCCGCGCA 3 ' (SEQID No.139) and with clay pSPM36 or give birth to the sequence of the gene that the total DNA of dyadic streptomycete can obtain as template

-or because genetic code degeneracy and by the sequence of its deutero-gene.

88) the present invention relates to the expression vector that permission is expressed in giving birth to the dyadic streptomycete according to the polypeptide of paragraph 86,87 or 93 on the other hand.

89) the present invention relates to the expression vector according to paragraph 88 on the other hand, and this expression vector is plasmid pSPM75.

90) the present invention relates to the mikrobe according to paragraph 68 on the other hand, wherein can be through the gene that the polymerase chain reaction obtains encoding sequence SEQ ID No.141 gene or since the genetic code degeneracy by its deutero-gene.

91) the present invention relates to the recombinant DNA according to paragraph 73 on the other hand, can be polynucleotide of sequence SEQ ID No.141 through the polynucleotide that the polymerase chain reaction obtains wherein.

92) the present invention relates to the mikrobe according to paragraph 79 on the other hand, wherein can be through the gene that the polymerase chain reaction obtains encoding sequence SEQ ID No.141 gene or since the genetic code degeneracy by its deutero-gene.

93) the present invention relates to polypeptide on the other hand, and its sequence is SEQ ID No.142.

Overall definition

For the purposes of the present invention, the biological substance (nucleic acid or protein) that shifted out of term " isolating " expression from its original environment (its naturally occurring environment).

For example, the polynucleotide that are present in plant or the animal with native state are unsegregated.The polynucleotide that separate from its natural adjacent nucleic acid that is inserted into plant or the animal gene group are considered to " isolating ".

This kind polynucleotide can be included in the carrier and/or this kind polynucleotide can be included in the compsn, yet still are separate stage because carrier or compsn do not constitute this kind of fact polynucleotide of its natural surroundings.

Term " purifying " does not require that material exists with the absolute pure form of getting rid of other compound and existing.It is a relative definition more.

The polynucleotide that after starting substance or crude substance purifying, are in " purifying " state improve an one magnitude at least, are preferably 2 or 3, and more preferably are 4 or 5 one magnitude.

For the object of the invention, term ORF (" ORFs ") has been used for representing particularly the encoding sequence of gene.

For the object of the invention, " nucleotide sequence " of statement can be used to represent polynucleotide or nucleic acid with being equal to." nucleotide sequence " of statement comprises genetic material itself but therefore is not limited to the information relevant with its sequence.

" nucleotide sequence " that term " nucleic acid ", " polynucleotide ", " oligonucleotide " or conduct are selected comprises the RNA/DNA heterozygosis sequence of RNA, DNA or cDNA sequence or Nucleotide, comprises single stranded form or double chain form equally.

Natural nucleotide (A promptly represented in term " Nucleotide "; T; G representes to comprise C) and also that the for example Nucleotide of (1) purine analogue, (2) pyrimidine analogue or (3) sugar analogue of at least a modification, the example of this type of modification are described in such as among the PCT application WO 95/04064.

For the object of the invention, when each base pairings of first polynucleotide in the complementary base of second polynucleotide, their direction is opposite, then thinks first polynucleotide and second polynucleotide " complementation ".Complementary base is A and T (or A and U), or C and G.

Term " gene of Spiramycin Base biosynthetic pathway " also comprises regulatory gene and the gene of giving producer's microbial resistance.

According to the present invention, term with reference to " fragment " of nucleic acid meaning be meant than with reference to the short nucleotide sequence of nucleic acid and identical part comprise with reference to the identical nucleotide sequence of nucleic acid.

According to the present invention, this kind nucleic acid " fragment " as required, can be used as its component and is included in the bigger polynucleotide.

This type of fragment comprise or we can say by according to the length range of nucleic acid of the present invention from 8,10,12,15,18,20 to 25,30,40,50,60,70,80,90; 100,150,200,250,300,350,400,450,500,550,600,650; 700,750,800,850,900,950,1000,1050,1100,1150,1200,1250; 1300,1350,1400,1450,1500,1550,1600,1650, the polynucleotide of 1700,1750,1800,1850 or 1900 continuous nucleotides are formed.

According to the present invention, " fragment " of term polypeptide meaning be meant than with reference to the short polypeptid acid sequence of polypeptide and with comprise identical aminoacid sequence with reference to all identical parts of polypeptide.

This type of fragment as required, can be used as its part and is included in the bigger polypeptide.

This type of fragment amino acid length according to polypeptide of the present invention can be 10,15,20,30 to 40,50,60,70,80,90; 100,120,140,160,180,200,220,240,260; 280,300,320,340,360,380,400,420,440; 460,480,500,520,540,560,580,600,620 or 640.

For the object of the invention, express " high stringency hybridization condition " meaning and be meant the hybridization conditions that is unfavorable for non-homogeneous nucleic acid chains hybridization.For example, high stringency hybridization condition can be described as the hybridization conditions of temperature between 55 ℃ and 65 ℃ in Church and the described damping fluid of Gilbert (Church & Gilbert, 1984); Being preferably hybridization temperature is 55 ℃; Even it is 60 ℃ more preferably for hybridization temperature; And being most preferably hybridization temperature is 65 ℃; In 2X SSC damping fluid (1X SSC damping fluid corresponding to 0.15M NaCl, the aqueous solution of 15mM Trisodium Citrate), carry out the one or many washing afterwards, temperature is between 55 ℃ and 65 ℃; Preferably this temperature is 55 ℃, even more preferably this temperature is 60 ℃, and to be most preferably this temperature be 65 ℃, in 0.5X SSC damping fluid, carries out the one or many washing afterwards, and temperature is between 55 ℃ and 65 ℃; Preferably this temperature is 55 ℃, even more preferably this temperature is 60 ℃, and to be most preferably this temperature be 65 ℃.

Much less, can above-mentioned hybridization conditions can be adjusted into the function of the length nucleic acid of the hybridization of being sought according to technology well known to those skilled in the art.For example, can be according to the suitable hybridization conditions of work adjustment of (2002) such as F.Ausubel.

" variant " of term nucleic acid meaning is meant and has the different nucleic acid of one or more bases with reference to polynucleotide according to the present invention.Variant nucleic acid can be natural origin, and for example the allele variant of natural discovery perhaps also can be the non-natural variant that obtains such as through induced-mutation technique.

Generally speaking, very little with reference to not being both between nucleic acid and the variant nucleic acid, to such an extent as to very close with reference to the nucleotide sequence of the nucleotide sequence of nucleic acid and variant nucleic acid, and be identical in some zones.The nucleotide modification that is present in the variant nucleic acid can be reticent, this means that they do not change the aminoacid sequence by said variant nucleic acid encoding.

Yet, Nucleotide in the variant nucleic acid change also can cause with by comparing with reference to the peptide of nucleic acid encoding by substituting, adding and/or deletion in the polypeptide of variant nucleic acid encoding.In addition, the nucleotide modification in the coding region can produce conservative or nonconservative substituting in aminoacid sequence.

Preferably, according to the present invention the variant nucleic acid encoding keep basically with reference to the polypeptide identical functions of nucleic acid and the polypeptide of BA, perhaps encoded polypeptide can be directed against the antibody recognition of initial nucleic acid encoded polypeptide.

So the polypeptide of some variant nucleic acid encoding mutant forms, the systematic study of this respect possibly derived in question proteinic structure-activity relation.

According to the present invention; " variant " meaning of term polypeptide mainly is meant its aminoacid sequence and compares with reference to amino acid sequence of polypeptide; Contain substituting, add or the polypeptide of deletion of one or more at least one amino-acid residue, be appreciated that amino acid replacement can be with being that guard or nonconservative.

Preferably, according to variant polypeptide of the present invention have basically with reference to polypeptide identical functions or activity, perhaps keep by ability to the antibody recognition of initial polypeptide.

For the object of the invention; Be meant to be suitable for measuring when measuring in the biological analysis with reference to the polypeptide BA to have and polypeptide close with reference to polypeptide active but must identical BA with the polypeptide meaning with reference to polypeptide " active similar ".

For the object of the invention, term " hydridization microbiotic " meaning is meant through using recombinant DNA technology to make up the compound that artificial bio-membrane's route of synthesis produces.

Detailed Description Of The Invention

Theme of the present invention more specifically be the Spiramycin Base biosynthetic pathway that appears like following detailed Description Of The Invention new gene with participate in this biosynthetic novel polypeptide.

Clone the gene of biosynthetic pathway and confirmed the dna sequence dna of these genes.The sequence of utilizing FramePlot program (J.Ishikawa and K.Hotta, 1999) analysis to be obtained.In these ORFs, identified that those present the ORFs that typical chain mould codon is selected.44 ORF that are positioned any side of 5 codases " polyketide synthase " gene (PKS) are contained in this zone of this analysis revealed, and have presented typical streptomycete codon selection.In any side of these 5 coding PKS, downstream and the upper reaches 10 and 34 ORF (through all defining upstream and downstream) (with reference to Fig. 3 and 37) have been identified respectively with the direction of localized 5 the PKS genes of equidirectional.Therefore; Such 34 ORFs; First regional SEQ ID No.1 that presents the 31kb that contains 25 ORF and the SEQ ID No.140 that presents about 12.1kb (are seen in the zone that occupies about 41.7kb; The overlapping above-mentioned sequence of 1.4kb (SEQ ID No.1) and wherein approximately 10.7kb is consistent with subsequently sequence wherein; The about back part of 10.7kb also Fig. 3 of face and 37 as follows of part (partial sequence that comprises an ORF) of containing 9 extra ORF) be accredited as the upper reaches of the gene of 5 coding PKS, and 10 ORF (SEQ ID No.2 and Fig. 3) that occupy about 11.1kb zone is accredited as the downstream of PKS gene.10 unnamed genes that are positioned 5 PKS gene downstream are orf1 ^*C, orf2 ^*C, orf3 ^*C, orf4 ^*C, orf5 ^*, orf6 ^*, orf7 ^*C, orf8 ^*, orf9 ^*And orf10 ^*(SEQ ID No.3,5,7,9,11,13,15,17,19 and 21).For the ORF that is discussing, " c " presentation code sequence that adds in the gene title be reverse (so coding strand be with SEQ ID No.2 in the sequence complementary chain of these genes of providing).Use identical nomenclature, 34 ORF called after orf1 of PKS upstream region of gene, orf2, orf3, orf4, orf5, orf6, orf7, orf8, orf9c, orf10, orf11c; Orf12, orf13c, orf14, orf15c, orf16, orf17, orf18, orf19, orf20, orf21c, orf22c, orf23c; Orf24c, orf25c, orf26, orf27, orf28c, orf29, orf30c, orf31, orf32c, orf33 and orf34c (SEQ ID Nos 23,25,28; 30,34,36,40,43,45,47,49,53,60,62; 64,66,68,70,72,74,76,78,80,82,84; 107,109,111,113,115,118,120,141,143,145,147 and 149) (with reference to Fig. 3 and 37).

Use the protein sequence that exists protein sequence that various programs will derive from these ORFs and the various DB to compare: BLAST (Altschul etc., 1990) (Altschul etc., 1997), CD-search, (these three programs especially can be from (the Bethesda of NCBI (NCBI) for COGs (Cluster of Orthologous Groups); Maryland USA) obtains), FASTA ((W.R.Pearson and D.J.Lipman, 1988) and (W.R.Pearson; 1990); BEAUTY (K.C.Worley etc., 1995)), (these two programs especially can be from INFOBIOGEN resource center; Evry, France obtains).These relatively make it possible to clearly describe the biosynthetic gene of those possibility involved in spiramycin of hypothesis and evaluation about the function of these gene products.

The gene in gene downstream of PKS is positioned to encode

In Fig. 3, provided the graphic extension of should the zone forming structure.To illustrate as follows, in 10 genes that identify in the downstream of the gene of coding PKS, 9 show involved in spiramycin biosynthesizing or Spiramycin Base resistance.They are following 9 gene: orf1 ^*C, orf2 ^*C, orf3 ^*C, orf4 ^*C, orf5 ^*, orf6 ^*, orf7 ^*C, orf8 ^*And orf9 ^*

Provided in the table 1 below 10 genes identifying in 5 PKS gene downstream with reference to dna sequence dna and aminoacid sequence.

Table 1

" c " presentation code sequence that adds in the gene title be reverse (so coding strand be with SEQID No.2 in the sequence complementary chain of these genes of providing).

The function of the polypeptide of identifying in order to measure; Three type of experiment have been carried out: the sequence of sequence of being identified and known function is compared; The gene inactivation experiment causes making up mutant strain and analyzes Spiramycin Base and the Spiramycin Base biosynthesizing midbody that is produced by these mutant strains.

At first use the protein sequence that exists protein sequence that distinct program will derive from these ORFs and the disparate databases to compare: BLAST (Altschul etc.; 1990) (Altschul etc.; 1997), CD-search, COGs (Cluster of Orthologous Groups), FASTA ((W.R.Pearson and D.J.Lipman, 1988) and (W.R.Pearson, 1990); BEAUTY (K.C.Worley etc., 1995)).These relatively make it possible to clearly to describe about the hypothesis of the function of the product of these genes with identify that those maybe the biosynthetic gene of involved in spiramycin.Table 2 has shown that the product with 10 genes that are positioned 5 PKS gene downstream presents the protein of strong similarity.

Table 2

^*Bigger sequence similarity relevant with higher BLAST score value (Altschul etc., 1990).

Carried out the gene inactivation experiment in order to prove conclusively these results.Employed method is to carry out gene substitution.The target gene that the copy replacement of this gene that the box of its microbiotic (for example apramycin, Geneticin or Totomycin) resistance interrupts will be interrupted is given in use.Employed box any side adjacent all read in frames translation stop codon and in streptomycete the active transcription terminator of tool.With box insert target gene can with or can be without the deletion in this target gene.The size of box two side areas can be individual to several thousand base pairs from hundreds of.Second type box can be used in gene inactivation: box is called " can excise box ".These boxes have after being imported into living dyadic streptomyces gene group through locus specificity and are binned in the streptomycete advantage of being excised.Purpose is some gene in the inactivation streptomycete bacterial strain and in final bacterial strain, do not stay the selected marker that do not belong to this bacterial strain or big dna sequence dna.After the excision, the short sequence of only about 30 base pairs (being called " scar " site) remains in the genome of bacterial strain (with reference to Figure 10).The purposes of this system is with excising the wild-type copy (owing to two homologous recombination incidents, with reference to Fig. 9) that box is inserted into the construct replacement target gene in this target gene at first.The insertion of this box is with the deletion in the target gene (with reference to Fig. 9).Next causes from the genome excision of bacterial strain and can excise box.Can excise box plays a role through site specific recombination system and has and can obtain the advantage of finally not carrying the streptomycete two mutants of resistant gene.Also avoided being positioned the issuable polarity effect of genetic expression (with reference to Figure 10) in inactivation gene downstream.The Spiramycin Base output that the bacterial strain that so makes up is used to test them.

Because the sequence comparative experiments makes it possible to confirm orf1 ^*C, orf2 ^*C, orf3 ^*C and orf4 ^*The c gene has high relatively similarity with the biosynthetic gene of the more proximate microbiotic of participation, so non-inactivation orf1 ^*C, orf2 ^*C, orf3 ^*C and orf4 ^*The c gene.Therefore, orf1 ^*The protein of c genes encoding and coded by said gene presents the protein of 66% identity (utilizing blast program to measure); TylMI genes encoding N-methyltransgerase wherein; 3-N-methylation (A.R.Gandecha etc., 1997 in the biosynthesizing of this enzyme participation tylosin and the mycaminose production process of catalysis streptomyces fradiae; GenBank accession number: CAA57473; BLAST score value: 287).The another kind of proteinic similarity of this and participation than the associated antibiotic biosynthetic pathway, and more specifically participate in the mycaminose biosynthesizing, hint out orf1 ^*The c genes encoding is responsible for the N-methyltransgerase (with reference to Fig. 5 and 6) of N-methylation in forosamine or the mycaminose biosynthesizing.By orf1 ^*Other protein with identity function presents this true this hypothesis (with reference to table 3) of supporting of high similarity in the protein of c genes encoding and other organism.

Table 3

^*Bigger sequence similarity and higher BLAST score value relevant (Altschul etc., 1990).

Orf2 ^*The protein of c genes encoding is participated in the biosynthetic NDP hexose 3 of tylosin with being coded in the streptomyces fradiae, and the protein of the tylMIII coded by said gene of 4-isomerase presents strong relatively similarity (35% identity) (A.R.Gandecha etc., 1997; GenBank accession number: CAA57471; BLAST score value: 130).Itself and the another kind of associated antibiotic biosynthetic pathway of participation more specifically are to participate in the biosynthetic proteinic similarity of mycaminose, strong hint orf2 ^*The c genes encoding possibly be a NDP hexose 3 of being responsible for isomerization in the mycaminose biosynthetic process at a kind of sugar of Spiramycin Base, 4-isomerase (with reference to Fig. 5 and 6).

Orf3 ^*The protein of c genes encoding presents strong relatively similarity (59% identity) (A.R.Gandecha etc., 1997 with the protein that is coded in the tylMII coded by said gene of the biosynthetic glycosyltransferase of participation tylosin in the streptomyces fradiae; GenBank accession number: CAA57472; BLAST score value: 448).Itself and the proteinic similarity strong hint of the another kind of associated antibiotic biosynthetic pathway of participation orf3 ^*C genes encoding glycosyltransferase.By ort3 ^*Other protein that have identity function in the protein of c coded by said gene and other organism present fact support this hypothesis (with reference to table 4) of strong similarity.

Table 4

Orf4 ^*C genes encoding and crotonoyl-CoA reductase enzyme presents the protein of strong relatively similarity.Particularly, orf4 ^*C encoded protein matter with from crotonoyl-CoA reductase enzyme (M.Redenbach etc., 1996 of streptomyces coelicolor; GenBank accession number: NP_630556; BLAST score value: 772) have quite high similarity.Itself and the proteinic similarity strong hint of the another kind of associated antibiotic biosynthetic pathway of participation orf4 ^*The c gene crotonoyl-CoA reductase enzyme of also encoding.By orf4 ^*Other protein that have identity function in the protein of c coded by said gene and other organism present fact support this hypothesis (with reference to table 5) of strong similarity.

Table 5

Orf6 ^*Gene and the mdmB gene (Hara and Hutchinson, 1992 that are present in generation macrolide antibiotic in the Streptomyces Macrofaciens; GenBank accession number: A42719; The BLAST score value:

489) present certain similarity.In this producer, said gene is participated in the acylation of lactonic ring.Therefore think orf6 ^*The genes encoding acyltransferase.By orf6 ^*Other protein that have identity function in the protein of coded by said gene and other organism present fact support this hypothesis (with reference to table 6) of strong similarity.

Table 6

Can produce orf6 through homophase deletion/inversion ^*The inactivation of gene and show that resulting bacterial strain no longer produces spiramycin II and III and only produces Spiramycin I (with reference to Fig. 1).This confirms orf6 ^*Certain involved in spiramycin II of gene and III's is synthetic.Be responsible for through forming spiramycin II and III on the carbon that ethanoyl or butyryl radicals is connected in the 3-position by the enzyme of this genes encoding.Owing to no longer express by orf6 ^*The proteinic bacterial strain of genes encoding no longer produces spiramycin II and III and only produces Spiramycin I, so they are particularly advantageous.As top pointed, the antibiotic activity of Spiramycin I is obviously greater than spiramycin II and III (Liu etc., 1999).

Orf5 ^*Genes encoding and several kinds of O-methyltransgerases present the protein of similarity relatively by force.Particularly, orf5 ^*Encoded protein matter with from O-methyltransgerase (EC 2.1.1.-) MdmC (Hara and Hutchinson, 1992 of Streptomyces Macrofaciens; GenBank accession number: B42719; BLAST score value: 355) have quite high similarity.Itself and the proteinic similarity strong hint orf5 that participates in another kind of microbiotic biosynthetic pathway ^*The gene O-methyltransgerase of also encoding.Think orf5 ^*The formation of the precursor of lactonic ring is integrated with in the gene participation.In fact, according to sequence relatively, orf5 ^*The product of gene also is similar to FkbG relatively, is responsible for methylation (Wu etc., 2000 of hydroxyl malonyl--ACP according to document FkbG; Hoffmeister etc., 2000; GenBank accession number: AAF86386; BLAST score value: 247) (with reference to Fig. 8).By orf5 ^*Other protein that have identity function in the protein of coded by said gene and other organism present fact support this hypothesis (with reference to table 7) of strong similarity.

Table 7

Because non-excision box inserts orf6 ^*The polar effect of gene can be confirmed orf5 ^*Gene is essential for the biosynthetic pathway of Spiramycin Base.More clearly say, can excise box and insert orf6 ^*The encoding part of gene causes the Spiramycin Base generation to stop fully.Yet, in case the box that is inserted is excised (and therefore orf6 only ^*During gene inactivation, with reference to embodiment 14 and 15), then observe Spiramycin I once more and produce.This shows orf5 ^*Gene is essential for the biosynthetic pathway of Spiramycin Base, so the inactivation of this gene causes the Spiramycin Base generation to stop fully.

Orf5 ^*Genes encoding and several kinds of O-methyltransgerases present the protein of similarity relatively by force.Think orf5 ^*Gene is to participate in the synthetic precursor that methylates (the methoxyl group malonyl-is with reference to Fig. 8) the synthetic O-methyltransgerase of perhaps participating in integrating with through PKS platenolide of platenolide directly.In order to prove this hypothesis, to genotype be: orf6 ^*:: the living dyadic streptomycete bacterial strain of att1 Ω hyg+ has carried out LC/SM and NMR analyzes experiment.In this bacterial strain, be inserted into orf6 owing to contain the box of transcription terminator ^*Polar effect in the gene, orf5 ^*Gene is not expressed (with reference to embodiment 27).Shown that this bacterial strain produces a kind of its UV spectrum and seems the molecule similar with the Spiramycin I UV spectrum, but mass spectrum shows molion at 829 places.The difference of comparing 14 quality of existence with the Spiramycin Base quality can be interpreted as the deletion (in Figure 39, having provided the structure of this compound) of methyl on the oxygen of No. 4 carbon loads of lactonic ring.Result and this hypothesis by NMR obtained are consistent.The compound that exists at 829 places makes confirms orf5 ^*Hypothesis in the effect of Spiramycin Base becomes possibility.In addition, when when testing, comparing, present very weak microbiological activity (weak 10 times) corresponding to the product of methylic Spiramycin Base not with the Spiramycin Base of unmodified at mikrobe micrococcus luteus (Micrococcus luteus).

Orf7 ^*The protein of the mdmA coded by said gene of c genes encoding and Streptomyces Macrofaciens presents the protein of strong relatively similarity, participates in protein (Hara etc., 1990 of mydecamycin resistance in latter's genes encoding producer's the enzyme; GenBank accession number: A60725; BLAST score value: 380).This and the proteinic similarity strong hint orf7 that participates in another kind of microbiotic biosynthetic pathway ^*Also the encode protein of involved in spiramycin resistance of c gene.More specifically, by orf7 ^*The enzyme of c coded by said gene has methyl transferase activity and involved in spiramycin resistance in giving birth to the dyadic streptomycete.Verified this gene is given MLS I type resistance, and known this resistance is because (Pernodet etc., 1996) that the monomethylation effect produces in the A2058 position of 23S ribosome-RNA(rRNA).By orf7 ^*Other protein that have identity function in the protein of coded by said gene and other organism present fact support this hypothesis (with reference to table 8) of strong similarity.

Table 8

Orf8 ^*Abc transport subtype protein presents protein (Campelo, 2002, the GenBank accession number: CAC22119 of strong relatively similarity in genes encoding and the streptomyces griseus; BLAST score value: 191).This and the proteinic similarity strong hint of abc transport subtype orf8 ^*Gene also encode maybe involved in spiramycin the abc transport subtype protein of resistance.By orf8 ^*Other protein that have identity function in the protein of coded by said gene and other organism present fact support this hypothesis (with reference to table 9) of strong similarity.

Table 9

Orf9 ^*Abc transport subtype protein presents protein (Campelo, 2002, the GenBank accession number: CAC22118 of strong relatively similarity in genes encoding and the streptomyces griseus; BLAST score value: 269).This and the proteinic similarity strong hint of abc transport subtype orf9 ^*Gene also encode maybe involved in spiramycin the abc transport subtype protein of resistance.By orf9 ^*Other protein that have identity function in the protein of coded by said gene and other organism present fact support this hypothesis (with reference to table 10) of strong similarity.

Table 10

Orf10 ^*Genes encoding and a kind of unknown function protein present the protein of strong relatively similarity.Yet, in the middle of several groups of genes of the biosynthetic gene of participation microbiotic, found and orf10 ^*Similar gene.Therefore, with orf10 ^*Proximate gene discovery is in streptomyces coelicolor (Redenbach etc., 1996, GenBank accession number: NP_627432, BLAST score value: 109).Approximate gene (CouY) also is found in (Wang etc., 2000, GenBank accession number: AAG29779, BLAST score value 97) in the sharp buttocks streptomycete (S.rishiriensis).

The gene of upstream region of gene of PKS is positioned to encode

(through all with the direction definition downstream and the upper reaches of localized 5 the PKS genes of equidirectional) (with reference to Fig. 3) identified 34 ORF (with reference to top) in the dna sequence dna of the upstream region of gene of the PKS that is positioned to encode.Therefore; The zone that 34 ORFs of the type have occupied about 41.7kb (presents the zone that 31kb first district of containing 25 ORF and SEQID No.140 present about 12.1kb with reference to SEQ ID No.1; Wherein 1.4kb and first presequence (SEQ ID No.1) overlapping and wherein approximately 10.7kb corresponding to subsequently sequence); About 10.7kb's contains 9 other ORF (comprising the ORF partial sequence) than aft section, also with reference to following Fig. 3 and 37).In Fig. 5 and 37, illustrate the composition in this zone.34 unnamed genes being identified are: orf1, orf2, orf3, orf4, orf5, orf6, orf7, orf8, orf9c; Orf10, orf11c, orf12, orf13c, orf14, orf15c, orf16, orf17; Orf18, orf19, orf20, orf21c, orf22c, orf23c, orf24c, orf25c; Orf26, orf27, orf28c, orf29, orf30c, orf31, orf32c, orf33 and orf34c.

Below provided the reference dna and the aminoacid sequence of 34 genes that identified at 5 PKS upstream region of gene in the table 11.

Table 11

¹" c " presentation code sequence that adds in the gene title is reverse (so the sequence of given these genes of coding strand and SEQ IDNo.1 or SEQ ID No.140 is complementary).

²When several protein sequences show that when being in same ORFs, corresponding proteins matter is from several possible translation initiation sites.

For the function of the polypeptide identified in the table 11 above confirming, three type of experiment have been carried out: the identity of the sequence of relatively being identified, gene inactivation experiment and analyze the generation of Spiramycin Bases through these mutant strains with known function sequence.

Utilize various programs: BLAST (Altschul etc.; 1990) (Altschul etc.; 1997), CD-search, COGs (orthogenesis homology crowd clustering cluster), FASTA ((W.R.Pearson and D.J.Lipman, 1988) and (W.R.Pearson, 1990), BEAUTY (K.C.Worley etc.; 1995)) (with reference to top), the sequence that the protein sequence that at first will derive from these ORFs and several data storehouse have existed compares.These relatively make it possible to clearly to describe about the hypothesis of these gene product functions and identify that those tend to the biosynthetic gene of involved in spiramycin.Table 12 has been listed the protein that presents strong similarity with 34 genes that are positioned at 5 PKF upstream region of gene.

Table 12

In order to confirm that these results have carried out the gene inactivation experiment.Method therefor is to carry out gene substitution.Use the copy of this gene that interrupts by the box of giving microbiotic (for example apramycin or Totomycin) resistance to replace the target gene that to interrupt.Employed box any side adjacent all read in frames translation stop codon and in streptomycete the active transcription terminator of tool.With box insert target gene maybe with or also maybe be without the deletion in this target gene.The size of box two side areas can be from hundreds of to several thousand base pairs.Second type the box that can be used for gene inactivation is: be called " can excise box " box of (with reference to top).The bacterial strain that so makes up is used to measure the generation of their Spiramycin Bases.

Orf1 genes encoding and several kinds of Cytochrome P450s present the protein of similarity relatively by force.Particularly, participate in protein (L.A.Merson-Davies etc., 1994 of the biosynthetic tylI coded by said gene of tylosin in orf1 dna encoding the protein and the streptomyces fradiae; GenBank accession number: S49051; BLAST score value: 530) have quite high similarity.This and the proteinic similarity strong hint orf1 gene of participating in another kind of associated antibiotic biosynthetic pathway also Codocyte cytochrome p 450.Intimate other protein present fact support this hypothesis (with reference to table 13) of strong similarity in the protein of orf1 coded by said gene and other organisms.

Table 13

The Orf2 genes encoding with have a liking for the dTDP-6-deoxidation-3 of the hot VitB1 genus bacillus of gas (Aneurinibacillusthermoaerophilus), 4-ketone tagatose isomerase (Pfoestl, A etc., 2003, GenBank accession number: AAO06351; BLAST score value: 118) present the protein of similarity relatively by force.This similarity strong hint Orf2 genes encoding isomerase, it is responsible for the necessary isomerization reaction of a kind of sugared biosynthesizing that exists in the Spiramycin Base molecule, and this sugar possibly be mycarose (with reference to Fig. 5).Carry out the orf2 gene inactivation.The inactivation experiment can show that resulting bacterial strain no longer produces Spiramycin Base.This confirms the certain involved in spiramycin biosynthesizing of orf2 gene.

Orf3 genes encoding and several kinds of transaminases present the protein of similarity relatively by force.Particularly, orf3 encoded protein matter and the biosynthetic antibiosis streptomycete transaminase of participation romicil (G.Draeger etc., 1999; GenBank accession number: AAF59939; BLAST score value: 431) have quite high similarity.The 3-transaminase (with reference to Fig. 5) that this necessary transamination of biosynthesizing of being responsible for a kind of aminosugar of Spiramycin Base with the proteinic similarity strong hint orf3 genes encoding of participating in another kind of associated antibiotic biosynthetic pathway is reacted.Intimate other protein present fact support this hypothesis (with reference to table 15) of strong similarity in the protein of Orf3 coded by said gene and other organisms.

Table 15

Inactivation orf3 gene.Therefore might show that resulting bacterial strain no longer produces Spiramycin Base.This confirms the certain involved in spiramycin biosynthesizing of orf3 gene.Therefore be responsible for the necessary biotransformation step of Spiramycin Base biosynthesizing clearly by the enzyme of this genes encoding.The TylB protein of expressing streptomyces fradiae can compensate the generation (with reference to embodiment 23) of Spiramycin Base.This shows that the orf3 genes encoding is responsible for the 3-transaminase (with reference to Fig. 5) of the necessary transamination of mycaminose biosynthesizing reaction.Because mycaminose is that first is connected to the sugar on the platenolide, so expection orf3 knock-out bacterial strain (OS49.67) can accumulate platenolide.

After deliberation the biosynthesizing midbody of orf3 gene knock-out bacterial strain (with reference to embodiment 20).These experiments make it possible to prove that this bacterial strain produces the platenolide:platenolide A and the platenolide B of two kinds of forms, have provided the structure of these two kinds of molecules of being derived among Figure 36.This bacterial strain also produces platenolide A+mycarose and platenolide B+mycarose (with reference to embodiment 20 and Figure 40).These compounds contain sugar, but do not contain any mycaminose.In addition, if they are compared with Spiramycin Base (with reference to Fig. 1), these compounds contain mycarose and do not contain mycaminose.The work that these results and orf3 gene product are participated in the mycaminose biosynthesizing and had 3 transaminases is consistent (with reference to Fig. 5) in order to be responsible for the necessary transamination reaction of mycaminose biosynthesizing.Can notice, be connected the position that normal circumstances should be taken by mycaminose owing to found its mycarose of some molecules, be not absolute (with reference to Figure 40) so demonstrate the specificity of glycosylation.

Orf4 genes encoding and several kinds of NDP-glucose synthetic enzyme present the protein of similarity relatively by force.Particularly, the alpha-D-glucose of orf4 encoded protein matter and streptomyces venezuelae-1-thymine phosphate transferase (Y.Xue etc., 1998; GenBank accession number: AAC68682; BLAST score value: 404) have quite high similarity.This and the proteinic similarity strong hint orf4 genes encoding NDP-glucose synthetic enzyme of participating in another kind of associated antibiotic biosynthetic pathway, this enzyme is responsible for synthetic three kinds of atypia sugar necessary NDP-glucose of biosynthesizing (with reference to Fig. 4,5 and 6) that mix the Spiramycin Base molecule.Intimate other protein present fact support this hypothesis (with reference to table 16) of strong similarity in the protein of orf4 coded by said gene and other organisms.

Table 16

Orf5 genes encoding and several kinds of gluconate dehydratase enzymes present the protein of similarity relatively by force.Particularly, the dTDP-glucose 4 of orf5 encoded protein matter and streptomyces tenebrarius, 6-dehydratase (T.B.Li etc., 2001; GenBank accession number: AAG18457, BLAST score value: 476) have quite high similarity.This and the proteinic similarity strong hint off5 genes encoding NDP-gluconate dehydratase enzyme of participating in another kind of associated antibiotic biosynthetic pathway, this enzyme is essential (with reference to Fig. 4,5 and 6) for the biosynthesizing of three kinds of atypia sugar that mix the Spiramycin Base molecule.Intimate other protein present fact support this hypothesis (with reference to table 17) of strong similarity in the protein of Orf5 coded by said gene and other organisms.

Table 17

Orf6 genes encoding and several kinds of thioesterases present the protein of similarity relatively by force.Particularly, orf6 encoded protein matter and deinsectization streptomycete thioesterase (S.Omura etc., 2001; GenBank accession number: BAB69315; BLAST score value: 234) have quite high similarity.This and the proteinic similarity strong hint orf6 genes encoding thioesterase of participating in another kind of close microbiotic biosynthetic pathway.Intimate other protein present fact support this hypothesis (with reference to table 18) of strong similarity in the protein of Orf6 coded by said gene and other organisms.

Table 18

Orf7 genes encoding and several kinds of hexose dehydratases present the protein of similarity relatively by force.Particularly, orf7 encoded protein matter and the dNTP-hexose 2 of participating in the biosynthetic streptomyces fradiae of tylosin, 3-dehydratase (by the TylCVI genes encoding) (L.A.Merson-Davies etc., 1994; GenBank accession number: AAF29379; BLAST score value: 461) have quite high similarity.This and the proteinic similarity strong hint orf7 gene of the participating in another kind of close microbiotic biosynthetic pathway hexose 2-3-dehydratase of also encoding, this enzyme is essential (with reference to Fig. 4 and 6) for the biosynthesizing of two kinds of atypia sugar that mix the Spiramycin Base molecule.Intimate other protein present fact support this hypothesis (with reference to table 19) of strong similarity in the protein of Orf7 coded by said gene and other organisms.

Table 19

Orf8 genes encoding and several kinds of transaminases present the protein of similarity relatively by force.Particularly, possibly participate in the biosynthetic transaminase of forosamine (C.Waldron etc., 2001 in orf8 encoded protein matter and the thorn saccharopolyspora strain; GenBank accession number: AAG23279; BLAST score value: 465) have quite high similarity.This and the proteinic similarity strong hint orf8 genes encoding 4-transaminase of participating in another kind of close microbiotic biosynthetic pathway, this enzyme is responsible for the necessary transamination reaction of forosamine biosynthesizing (with reference to Fig. 6).Intimate other protein present fact support this hypothesis (with reference to table 20) of strong similarity in the protein of Orf8 coded by said gene and other organisms.

Table 20

Inactivation orf8 gene.Therefore might show that resulting bacterial strain no longer produces Spiramycin Base.This confirms the certain involved in spiramycin biosynthesizing of orf8 gene.Therefore be responsible for the necessary biotransformation step of Spiramycin Base biosynthesizing clearly by the enzyme of this genes encoding.The orf8 gene inactivated mutants produces forocidin; Thereby this two mutants block in the forocidin stage and do not produce any new-Spiramycin Base, this fact has carried out confirming (with reference to Fig. 7 and embodiment 25) to the orf8 gene product has effect in the forosamine biosynthesizing hypothesis.It is consistent (with reference to Fig. 6) that these results participate in the forosamine biosynthesizing with the orf8 gene product.

Identified in the living dyadic streptomycete the orf9c gene and by (M.Geistlich etc., 1992) called after srmX such as Geistlich.By protein and several kinds of this similarity strong hint orf9c8 genes encoding methyltransgerases of participating in the methyltransgerase of other close microbiotic biosynthetic pathway of this coded by said gene, this enzyme is responsible for mycaminose or the necessary methylation reaction of forosamine biosynthesizing (with reference to Fig. 5 and 6).Intimate other protein present fact support this hypothesis (with reference to table 21) of strong similarity in the protein of orf9c coded by said gene and other organisms.

Table 21

Identified in the living dyadic streptomycete the orf10 gene and by (M.Geistlich etc., 1992) called after srmR such as Geistlich.Participate in giving birth to the regulation and control of Spiramycin Base biosynthetic pathway in the dyadic streptomycete by the protein of this coded by said gene.Inactivation orf10 gene.Therefore might show that resulting bacterial strain no longer produces Spiramycin Base.This confirms the certain involved in spiramycin biosynthesizing of orf10 gene.Therefore the albumen by this genes encoding obviously is essential for the Spiramycin Base biosynthesizing.

In addition, confirmed that the translation initiation site of orf10 and the overexpression that might show this gene cause the raising of Spiramycin Base output.Translation initiation site is equivalent to be positioned at the ATG at the ATG upper reaches that (M.Geistlich etc., 1992) such as Geistlich proposed.Because the courier of 5 '-brachymemma is a non-activity, proved that also this 5 ' terminal function for Orf10 is essential (with reference to embodiment 17).Therefore in order to obtain the desired result of Spiramycin Base output aspect, the courier that the effective orf10 of overexpression and noting does not express orf105 '-brachymemma is necessary.

Identified in the living dyadic streptomycete the orf11c gene and by (B.Schoner etc., 1992) called after srmB such as (M.Geistlich etc., 1992) such as Geistlich and Schone.By the protein of this coded by said gene involved in spiramycin resistance and be ABC-type transhipment in giving birth to the dyadic streptomycete.

Orf12 genes encoding and several kinds of hexose dehydratases present the protein of similarity relatively by force.Particularly, the NDP-hexose 3 of orf12 encoded protein matter and streptomyces fradiae UrdQ coded by said gene, 4-dehydratase have quite high similarity and participate in crow and reach mycin biosynthesizing (D.Hoffmeister etc., 2000; GenBank accession number: AAF72550; BLAST score value: 634).This and the proteinic similarity strong hint orf12 genes encoding 3 of participating in another kind of close microbiotic biosynthetic pathway, 4-dehydratase, this enzyme are responsible for the necessary dehydration reaction of forosamine biosynthesizing (with reference to Fig. 6).Intimate other protein present fact support this hypothesis (with reference to table 22) of strong similarity in the protein of Orf12 coded by said gene and other organisms.

Table 22

Inactivation orf12 gene.Might show that resulting bacterial strain no longer produces Spiramycin Base.This confirms the certain involved in spiramycin biosynthesizing of orf12 gene.Therefore obviously be responsible for the necessary biotransformation step of Spiramycin Base biosynthesizing by the enzyme of this genes encoding.The orf12 gene inactivated mutants no longer produces the fact of forosamine Orf12 is produced evidence in the hypothesis that the forosamine biosynthesizing has effect.Yet two mutants produces a small amount of forocidin.Therefore this two mutants the forocidin stage block and does not produce any newly-Spiramycin Base (with reference to Fig. 7 and embodiment 26).This two mutants also produces the compound with structure shown in Figure 38.Latter's compound contains two kinds of sugar, i.e. mycaminose and mycarose, but do not contain any forosamine.In addition, if compare (with reference to Fig. 1) with the structure of Spiramycin Base, this compound contains mycarose in the desired location of forosamine.It is consistent (with reference to Fig. 6) that these results participate in the forosamine biosynthesizing with the orf12 gene product.Can notice, be connected generally on the position that takies by forosamine, so the specificity of glycosylation is not absolute (seeing Figure 38) owing to observe mycarose in some molecules.

A kind of unknown function protein presents the protein of strong relatively similarity in orf13c genes encoding and the streptomyces coelicolor.This protein called after SC4H2.17 (GeneBank accession number: T35116; BLAST score value: 619).Protein by the orf13c coded by said gene also presents strong similarity (with reference to table 23) with other organic other protein.

Table 23

Still do not identify the proteinic exact function approaching with the orf13c coded protein.In order to study the function of this gene in giving birth to dyadic streptomycete Spiramycin Base biosynthetic pathway, the orf13c gene is carried out inactivation.Might show that resulting bacterial strain produces Spiramycin Base.This shows that the orf13c gene is optional for the Spiramycin Base biosynthesizing, and this gene also is nonessential for the survival of bacterium.Therefore by the necessary biotransformation step of the obvious not responsible Spiramycin Base biosynthesizing of the enzyme of this genes encoding.

The orf14 genes encoding presents protein (M.Redenbaeh etc., 1996 of similarity relatively by force with the reductase enzyme of inferring; Bentley etc., 2002; GenBank accession number: CAB90862; BLAST score value: 147).

Inactivation orf14 gene.Therefore might show that resulting bacterial strain no longer produces Spiramycin Base.This confirms the certain involved in spiramycin biosynthesizing of orf14 gene.Therefore obviously be responsible for the necessary biotransformation step of Spiramycin Base biosynthesizing by the enzyme of this genes encoding.Studied the biosynthesizing midbody (with reference to embodiment 20) of orf14 gene knock-out bacterial strain.These experiments make and might show that this bacterial strain produces platenolide A and do not produce platenolide B (with reference to Figure 36).

Orf15c genes encoding and several kinds of ketoreductases present the protein of similarity relatively by force.Particularly, orf15c encoded protein matter and antibiosis streptomycete 3-ketoreductase (GenBank accession number: T51102, BLAST score value: 285) have quite high similarity.This similarity strong hint orf15c genes encoding 3-ketoreductase, this enzyme is responsible for the necessary reduction reaction of forosamine biosynthesizing (with reference to Fig. 6).Other protein that has identity function in the protein of orf15c coded by said gene and other organism presents fact support this hypothesis (with reference to table 24) of strong similarity.

Table 24

Orf16 genes encoding and several kinds of isomerases present the protein of similarity relatively by force.Particularly, orf16 coded protein and streptomyces fradiae NDP-hexose 3,4-isomerase (Gandecha etc., 1997; GenBank accession number: CAA57471, BLAST score value: 209) have quite high similarity.The biosynthetic protein (with reference to Fig. 5 and 6) of a kind of sugar of this similarity strong hint orf16 genes encoding involved in spiramycin.Other protein that has identity function in the protein of orf16 coded by said gene and other organism presents fact support this hypothesis (with reference to table 25) of strong similarity.

Table 25

Orf17 genes encoding and several kinds of glycosyltransferases present the protein of similarity relatively by force.Particularly, orf17 coded protein and streptomyces venezuelae glycosyltransferase (Y.Xue etc., 1998; GenBank accession number: AAC68677; BLAST score value: 400) have quite high similarity.This gene of similarity strong hint of several kinds of glycosyltransferases of the protein of orf17 genes encoding and other close microbiotic biosynthetic pathway of participation is the encoding glycosyl transferring enzyme also.Other protein that has identity function in the protein of orf17 coded by said gene and other organism presents fact support this hypothesis (with reference to table 26) of some similarity.

Table 26

Orf18 genes encoding and several kinds of glycosyltransferases present the protein of similarity relatively by force.Particularly, orf18 coded protein and sharp buttocks streptomycete glycosyltransferase (Wang etc., 2000; GenBank accession number: AAG29785; BLAST score value: 185) have quite high similarity.This gene of similarity strong hint of several kinds of glycosyltransferases of the protein of orf18 genes encoding and other close microbiotic biosynthetic pathway of participation is the encoding glycosyl transferring enzyme also.Other protein that has identity function in the protein of orf18 coded by said gene and other organism presents fact support this hypothesis (with reference to table 27) of strong similarity.

Table 27

Orf19 genes encoding and several kinds of ketoreductases present the protein of similarity relatively by force.Particularly, orf19 encoded protein matter and streptomyces fradiae NDP-hexose 4-ketoreductase (TylCIV) (Bate etc., 2000; GenBank accession number: AAD41822; BLAST score value: 266) have quite high similarity.The protein of orf19 genes encoding and this gene of similarity strong hint of this ketoreductase of participating in other close microbiotic biosynthetic pathway 4-ketoreductase of also encoding, this enzyme is responsible for the necessary reduction reaction of mycarose biosynthesizing (with reference to Fig. 4).Other protein that has identity function in the protein of orf19 coded by said gene and other organism presents fact support this hypothesis (with reference to table 28) of strong similarity.

Table 28

Orf20 genes encoding and several kinds of hexose reductase enzymes present the protein of similarity relatively by force.Particularly, orf20 coded protein and red saccharopolyspora coding dTDP-4-ketone-L-6-deoxyhexamethylose 2, the EryBII of 3-reductase enzyme (R.G.Summers etc., 1997), GenBank accession number: AAB84068; BLAST score value: 491) have quite high similarity.This genes encoding 2 of similarity strong hint of several kinds of hexose reductase enzymes of the protein of orf20c genes encoding and other close microbiotic biosynthetic pathway of participation, 3-reductase enzyme, this enzyme are responsible for the necessary reduction reaction of mycarose biosynthesizing (with reference to Fig. 4).Other protein that has identity function in the protein of orf20c coded by said gene and other organism presents fact support this hypothesis (with reference to table 29) of strong similarity.

Table 29

Orf21c genes encoding and several kinds of hexose methyltransgerases present the protein of similarity relatively by force.Particularly, TylCIII gene (N.Bate etc., 2000 of orf21c encoded protein matter and streptomyces fradiae coding NDP-hexose 3-C-methyltransgerase; GenBank accession number: AAD41823; The BLAST score value: EryBII 669) has quite high similarity.This genes encoding hexose methyltransgerase of similarity strong hint of several kinds of hexose methyltransgerases of the protein of orf21c genes encoding and other close microbiotic biosynthetic pathway of participation, this enzyme is responsible for the necessary methylation reaction of mycarose biosynthesizing (with reference to Fig. 4).Other protein that has identity function in the protein of orf21c coded by said gene and other organism presents fact support this hypothesis (with reference to table 30) of strong similarity.

Table 30

Coding is participated in protein (K.Wu etc., 2000 of the fkbH coded by said gene of the biosynthetic enzyme of methoxy propyl diacyl in the protein of orf22c genes encoding and the streptomyces hygroscopicus WS7238A mutation (Streptomyceshygroscopicus var.ascomyceticus); GenBank accession number: AAF86387; BLAST score value: 463) present strong relatively similarity.The protein of orf22c coded by said gene and proteinic this gene of similarity strong hint of participating in other relevant macrolide biosynthetic pathway are also encoded and are participated in the biosynthetic enzyme of methoxy propyl diacyl (with reference to Fig. 8) in the living dyadic streptomycete.

Coding is participated in protein (K.Wu etc., 2000 of the fkbI coded by said gene of the biosynthetic fatty acyl-CoA dehydrogenase of methoxy propyl diacyl in the protein of orf23c genes encoding and the mutation of streptomyces hygroscopicus WS7238A; GenBank accession number: AAF86388; BLAST score value: 387) present strong relatively similarity.The protein of orf23c genes encoding is participated in the biosynthetic fatty acyl-CoA dehydrogenase of methoxy propyl diacyl (with reference to Fig. 8) with this genes encoding of similarity strong hint of several kinds of fatty acyl-CoA dehydrogenases of participating in other close microbiotic biosynthetic pathway.Other protein that has identity function in the protein of orf23c coded by said gene and other organism presents fact support this hypothesis (with reference to table 31) of strong similarity.

Table 31

Be considered to coding in the protein of orf24c genes encoding and the mutation of streptomyces hygroscopicus WS7238A and participate in protein (K.Wu etc., 2000 of the fkbJ coded by said gene of the biosynthetic acyl carrier protein of methoxy propyl diacyl (ACP); GenBank accession number: AAF86389; BLAST score value: 87) present strong relatively similarity.The protein of orf24c genes encoding participates in giving birth to the biosynthetic protein of methoxy propyl diacyl (with reference to Fig. 8) in the dyadic streptomycete with this genes encoding of the proteinic similarity strong hint of participating in another kind of close macrolide biosynthetic pathway.

Coding is participated in protein (K.Wu etc., 2000 of the fkbK coded by said gene of the biosynthetic fatty acyl-CoA dehydrogenase of methoxy propyl diacyl in the protein of orf25c genes encoding and the mutation of streptomyces hygroscopicus WS7238A; GenBank accession number: AAF86390; BLAST score value: 268) present strong relatively similarity.The protein of orf25c genes encoding is participated in the biosynthetic fatty acyl-CoA dehydrogenase of methoxy propyl diacyl (with reference to Fig. 8) with this genes encoding of similarity strong hint of several kinds of fatty acyl-CoA dehydrogenases of participating in other close microbiotic biosynthetic pathway.Other protein that has identity function in the protein of orf25c coded by said gene and other organism presents fact support this hypothesis (with reference to table 32) of strong similarity.

Table 32

Coding is participated in protein (N.Bate etc., 2000 of the tylCV coded by said gene of the biosynthetic mycarose glycosyltransferase of tylosin in the protein of orf26 coded by said gene and the streptomyces fradiae; GenBank accession number: AAD41824, BLAST score value: 471) present 65% identity (using blast program to confirm).More specifically, TylCV is the glycosyltransferase that in the tylosin building-up process, combines the mycarose molecule.This protein with another close relatively microbiotic biosynthetic pathway of participation more specifically is that the similarity hint orf26 gene of mycarose transhipment is a glycosyltransferase.Other protein that has identity function in the protein of orf26 coded by said gene and other organism presents fact support this hypothesis (with reference to table 33) of strong similarity.

Table 33

Coding is participated in the biosynthetic NDP-hexose 3 of tylosin, protein (N.Bate etc., 2000 of the tylCVII coded by said gene of 5-(or 5-) epimerase in the protein of orf27 coded by said gene and the streptomyces fradiae; GenBank accession number: AAD41825, BLAST score value: 243) present 70% identity (using blast program to confirm).More specifically, TylCVII participates in the biosynthetic hexose 3 of mycarose, 5-(or 5-) epimerase.This more specifically is that the biosynthetic proteinic similarity hint orf27 gene of mycarose is an epimerase with participating in another close relatively microbiotic biosynthetic pathway.Other protein that has identity function in the protein of orf27 coded by said gene and other organism presents fact support this hypothesis (with reference to table 34) of strong similarity.The analysis strong hint orf27 genes encoding 5-epimerase that utilizes blast program that the close sequence that is obtained is carried out, this enzyme are responsible for the necessary epimerization reaction of mycarose biosynthesizing (with reference to Fig. 4).

Table 34

Initial only part has been measured the sequence of orf28c, and the sequence of about 450 base zones is being confirmed (in incomplete sequence SEQ ID No.106 should zone with " N " expression) again after the sequential analysis.Partial sequence (SEQ ID No.111) that even so will this ORF is used for using multiple as stated computer program analysis.Therefore can be through determined sequence (SEQ ID No.112; This sequence is the proteinic partial sequence of Orf28c); Confirm that coding in protein and the heat-resisting streptomycete of orf28c genes encoding participates in protein (A.Arisawa etc., 1993 of the proteinic acyB2 coded by said gene of the biosynthetic adjusting of Magnamycin A; GenBank accession number: JC2032, BLAST score value: 329) present 64% identity (using blast program to confirm).This proteinic similarity with another close relatively microbiotic biosynthetic pathway of participation hints the biosynthetic adjusting protein of orf28c genes encoding involved in spiramycin.The protein of orf28c coded by said gene also presents this hypothesis of fact support of strong similarity with TylR protein, TylR protein is to participate in the biosynthetic adjusting protein of tylosin (N.Bate etc., 1999 in the streptomyces fradiae; GenBank accession number: AAF29380, BLAST score value: 167).

Utilization is positioned not confirm the oligonucleotide amplification orf28c gene of sequence both sides and its subclone is gone into expression vector is possible.Therefore might prove that the orf28c gene overexpression significantly improves the output of Spiramycin Base in the OSC2 bacterial strain (with reference to embodiment 24).This shows that overexpression orf28c causes Spiramycin Base output to increase and has confirmed that it is as the sub effect of the conciliation of Spiramycin Base biosynthetic pathway.

Accomplish the partial sequence of orf28c subsequently and confirmed the zone of ignorance (with reference to SEQ ID No.140 and SEQ ID No.141) of about 450 base pairs.Use above-mentioned multiple computer program that the complete sequence (SEQ ID No.141) of this ORF is analyzed.Therefore protein (Arisawa, A. etc., 1993 of the proteinic acyB2 coded by said gene of the coding participation biosynthetic adjusting of Magnamycin A in the protein that might confirm the orf28c coded by said gene and the heat-resisting streptomycete; GenBank accession number: JC2032, the BLAST score value: 451) (SEQ ID No.142, this sequence is the proteinic complete sequence of Orf28c) presents 69% identity (utilizing blast program to confirm) on determined sequence.The biosynthetic adjusting protein of the biosynthetic proteinic similarity hint orf28c genes encoding involved in spiramycin of close relatively microbiotic is regulated in this and participation.The protein of orf28c coded by said gene also presents this hypothesis of fact support of strong similarity with TylR protein, TylR protein is to participate in regulating the biosynthetic adjusting protein of tylosin (N.Bate etc., 1999 in the streptomyces fradiae; GenBank accession number: AAF29380, BLAST score value: 224).The result of this gene of overexpression has confirmed the effect (with reference to embodiment 24) of this gene as the regulon of Spiramycin Base biosynthetic pathway.

Inactivation orf28c gene.Might show that so resulting bacterial strain no longer produces Spiramycin Base.This confirms the obvious involved in spiramycin biosynthesizing of orf28c gene and biosynthesizing is essential for Spiramycin Base.Combine the result (with reference to embodiment 24) of this gene overexpression to show that Orf28c is as the necessary effect that activates son of Spiramycin Base biosynthesizing these results.

Be positioned to participate in possible glycosyl hydrolase (J.Ligon etc., 2002 in the biosynthetic gene group of soraphen A (a kind of polyketide is antifungal agents based) in the protein of orf29 coded by said gene and the Mierocrystalline cellulose capsule bacterium; GenBank accession number: AAK19890, BLAST score value: 139) present 31% identity (using blast program to confirm).This have the active protein of glycosyl hydrolase with the proteinic similarity hint orf29 genes encoding of participating in close relatively molecular biosciences route of synthesis.Other protein that has identity function in the protein of orf29 coded by said gene and other organism presents fact support this hypothesis (with reference to table 35) of strong similarity.Utilize CD-search utility (with reference to top) that the coded proteinic sequence of orf29 is analyzed and also show orf29 genes encoding glycosyl hydrolase.

Table 35

Use SignalP program (http://www.cbs.dtu.dk/services/SignalP/) (Nielsen; H. etc.; 1997), analyze and to show by the protein sequence that orf29 derived that this protein has and between 30 and 31 (QSA/QA), contain the C-terminus signal sequence of predicting cleavage site.Can predict that this protein is extracellular protein.As glycosyl hydrolase, this protein maybe be carrying out glycosylation through glycosyltransferase GimA and/or GimB has effect (Gourmelen etc., 1998) in the reactivate process of the Spiramycin Base of inactivation.

Nucleosides in the protein of Orf30c coded by said gene and the Corynebacterium glutamicum-di-phosphate sugar epimerase (GenBank accession number: NP_600590, BLAST score value: 89) present 31% identity (using blast program to confirm).This similarity hint orf30c genes encoding epimerase.The fact of utilizing CD-search utility (with reference to top) that this sequence is analyzed is supported this hypothesis, also shows orf30c genes encoding epimerase.

Orf30c presents two possible initiator codons (with reference to SEQ ID No.115), and this makes and can form two kinds of possible 345 and 282 amino acid whose protein (SEQ ID No.116 and 117) that are respectively.Yet the codon of typical chain mould is selected only since second ATG; In addition, can not compare with the close sequence of being identified from the protein sequence that the sequence between first ATG and second is derived, otherwise the shortest protein sequence is (initial from second ATG: as SEQ ID No.144) correctly to compare with these protein.Therefore can reach a conclusion thus: second ATG is correct initiator codon, and the sequence of this orf is shown in SEQ ID No.143, in case translate, its corresponding proteins matter sequence is SEQ ID No.144.

The protein of Orf31 coded by said gene and the oxydo-reductase in the streptomyces coelicolor (GenBank accession number: NP_631148, BLAST score value: 261) present 52% identity (using blast program to confirm).This similarity hint orf31 genes encoding reductase enzyme.The fact of utilizing CD-search utility (with reference to top) that this sequence is analyzed is supported this hypothesis, also shows orf31 genes encoding reductase enzyme.The fact that is presented strong similarity by other protein that has identity function in the protein of orf31 coded by said gene and other organism is also supported this hypothesis (with reference to table 36).

Table 36

Inactivation orf31 gene.Might show that so resulting bacterial strain no longer produces Spiramycin Base.This confirms the certain involved in spiramycin biosynthesizing of orf31 gene.Therefore obviously be responsible for the necessary biotransformation step of Spiramycin Base biosynthesizing by the enzyme of this coded by said gene.

At first part has been confirmed the sequence (with reference to embodiment 19) of orf32c, and the encoding sequence of 5 ' position is just confirmed in second step.Yet the partial sequence of this orf (SEQ ID No.120) is analyzed with above-mentioned multiple computer program.Therefore might confirm protein (SEQ ID No.121 in the sequence scope of having measured of orf32c genes encoding; This sequence is the proteinic partial sequence of Orf32c); With the adjusting protein of GntR family in the streptomyces coelicolor (GenBank accession number: NP_625576, BLAST score value: 229) present 47% identity (utilizing blast program to confirm).This similarity hints transcriptional regulatory of orf32c genes encoding GntR family.Other protein that has identity function in the protein of orf32c genes encoding and other organism presents this hypothesis of fact support of strong similarity.

Measure again subsequently and accomplished the partial sequence of orf32c and measured unknown zone (with reference to SEQID No.140 and SEQ ID No.145).The adjusting protein of GntR family in this orf full sequence coding and the deinsectization streptomycete (GenBank accession number: NP_824604, BLAST score value: 282) present 44% identity (utilizing blast program to confirm).This similarity hints transcriptional regulatory of orf32c genes encoding GntR family.Other protein that has identity function in the protein of orf32c genes encoding and other organism presents fact support this hypothesis (with reference to table 37) of strong similarity.

Table 37

In order to study the function of this gene in the Spiramycin Base biosynthetic pathway of giving birth to the dyadic streptomycete, the orf32c gene is carried out inactivation.Can show that resulting bacterial strain produces Spiramycin Base.This shows the orf32c gene, and optional and this gene neither be essential for the existence of bacterium for the Spiramycin Base biosynthesizing.

The protein of orf33 coded by said gene and xanthomonas campestris infer protein (GenBank accession number: NP_635564, BLAST score value: 54) present 49% identity (utilizing blast program to confirm).

The sequence of orf34c is a partial sequence.In fact, that between the product of this orf and DB, carries out comparison shows that this proteinic C-terminal portions is not the product of being derived by nucleotide sequence, so this orf should be longer and contain the part that checks order beyond the zone.Yet, the partial sequence of this ORF is analyzed with above-mentioned multiple computer program.Might confirm the protein of orf34c coded by said gene, in whole fixed sequence scopes (SEQ ID No.150, this sequence is the proteinic partial sequence of Orf34c), with arabinofuranosidase (Bentley etc., 2002 from streptomyces coelicolor; GenBank accession number: NP_630049, BLAST score value: 654) present 91% identity (utilizing blast program to confirm).In streptomyces coelicolor, the gene of this arabinofuranosidase of encoding shows does not participate in the biosynthesizing of secondary metabolites.So not involved in spiramycin biosynthesizing of this gene in giving birth to the dyadic streptomycete.

Theme of the present invention also is such polynucleotide, under the height stringent condition, these polynucleotide at least with sequence SEQ ID Nos 3,5,7,9,11,13,15,17; 19,21,23,25,28,30,34,36,40,43; 45,47,49,53,60,62,64,66,68,70; 72,74,76,78,80,82,84,107,109; One of one of polynucleotide in 111,113,115,118,120,141,143,145,147 and 149 or its variant or since the genetic code degeneracy hybridize from one of its deutero-sequence.Preferably; These said polynucleotide obtain from the separation of streptomyces bacterium; More preferably these polynucleotide encodings are participated in the biosynthetic protein of macrolides, and even more preferably the activity that has of the coded protein of these polynucleotide be similar to the protein coded with the polynucleotide of its hybridization.High stringent hybridization condition can be defined as the hybridization conditions that is unfavorable for that non-homogeneous nucleic acid chains is hybridized.For example, high stringent hybridization condition can be described as such hybridization conditions: temperature is between 55 ℃ and 65 ℃ in Church and the described damping fluid of Gilbert (Church and Gilbert, 1984); Hybridization temperature is preferably 55 ℃, and hybridization temperature more preferably is 60 ℃, and hybridization temperature is most preferably 65 ℃, next carries out the one or many washing with the temperature of 2X SSC damping fluid between 55 ℃ and 65 ℃; This temperature is preferably 55 ℃, and this temperature more preferably is that 60 ℃ and this temperature are most preferably 65 ℃, next carries out the one or many washing with the temperature of 0.5X SSC damping fluid between 55 ℃ and 65 ℃; This temperature is preferably 55 ℃, and this temperature more preferably is that 60 ℃ and this temperature are most preferably 65 ℃.Above-mentioned hybridization conditions can be according to the length of prehybridization nucleic acid, or adjust according to technology well known to those skilled in the art according to selected labeling pattern.For example, suitable hybridization conditions can be according to F.Ausubel etc., and 2002 work is adjusted.

The invention still further relates to and comprise and be selected from nucleotide sequence SEQ ID Nos 3,5,7,9,11,13,15,17,19,21,23,25,28; 30,34,36,40,43,45,47,49,53,60,62,64,66; 68,70,72,74,76,78,80,82,84,107,109,111; 113,115,118,120,141,143,145,147 and 149 or one of its variant or because genetic code degeneracy and by at least 10,12,15,18,20 to 25 in the polynucleotide of the polynucleotide of one of its deutero-sequence or complementary sequence; 30,40,50,60,70,80,90,100,150,200,250,300; 350,400,450,500,550,600,650,700,750,800,850,900; 950,1000,1050,1100,1150,1200,1250,1300,1350,1400,1450,1500; The polynucleotide of 1550,1600,1650,1700,1750,1800,1850 or 1900 continuous nucleotides have at least 70%, more preferably are 80%, more preferably are 85%, even more preferably are 90%, even more preferably are 95%, and are most preferably the polynucleotide of 98% Nucleotide identity.Preferably; These said polynucleotide obtain from the separation of streptomyces bacterium; More preferably be that these polynucleotide encodings are participated in the biosynthetic protein of macrolide, and even more preferably be that the activity that the coded protein of these polynucleotide has is similar to the coded protein of polynucleotide that presents identity with it.Most preferably be that polynucleotide according to the present invention are selected from nucleotide sequence SEQ ID Nos 3,5,7,9,11,13,15,17,19,21,23; 25,28,30,34,36,40,43,45,47,49,53,60; 62,64,66,68,70,72,74,76,78,80,82,84; 107,109,111,113,115,118,120,141,143,145,147 and 149, the perhaps polynucleotide of complementary sequence.

Utilize known algorithm; The for example algorithm of FASTA software package (W.R.Pearson and D.J.Lipman, 1988) and (W.R.Pearson, 1990); Particularly can obtain, can carry out the best comparison to the sequence that is used for comparison on computers by the INFOBIOGEN resource center of French Evry.Explanation utilizes LFASTA (K.-M.Chao etc., 1992) or LALIGN (X.Huang and W.Miller, 1991) software can confirm sequence identity percentage by way of example.LFASTA and LALIGN program are the parts of FASTA software package.LALIGN provides best local comparison, and this program is more accurate than LFASTA, but speed is slower.

Another aspect of the present invention relates to overexpression such as top defined nucleotide sequence and the polypeptide that obtains.Preferably, be selected from SEQ ID No4,6,8,10,12,14,16,18,20,22,24,26 according to these polypeptide of the present invention with comprising; 27,29,31,32,33,35,37,38,39,41,42,44,46; 48,50,51,52,54,55,56,57,58,59,61,63,65; 67,69,71,73,75,77,79,81,83,85,108,110,112; 114,116,117,119,121,142,144,146,148 and 150 or in said sequence, carried out the replacement of one or more amino acid, insert or deletion and do not influence at least 10,15,20,30 to 40 in one of one of these sequences of its functional performance or variant of these sequences; 50,60,70,80,90,100,120,140,160,180,200,220; 240,260,280,300,320,340,360,380,400,420,440,460; The polypeptide of 480,500,520,540,560,580,600,620 or 640 continuous amino acids presents at least 70%, more preferably is 80%, more preferably is 85%, even more preferably is 90%, even more preferably is 95% and is most preferably 98% amino acid identity.Preferably; These polypeptide according to the present invention are expressed with native state by the streptomyces bacterium; More preferably be the biosynthesizing that these polypeptide are participated in macrolide, and even more preferably be that the activity that these polypeptide have is similar to the polypeptide that has identity with it.Preferably, polypeptide according to the present invention is selected from peptide sequence SEQ ID No4,6,8,10,12,14,16,18,20,22,24,26; 27,29,31,32,33,35,37,38,39,41,42,44; 46,48,50,51,52,54,55,56,57,58,59,61; 63,65,67,69,71,73,75,77,79,81,83,85; 108,110,112,114,116,117,119,121,142,144,146,148 and 150 or in said sequence, carried out the replacement of one or more amino acid, insert or deletion and do not influence one of one of these sequences of its functional performance or variant of these sequences.More preferably, polypeptide according to the present invention is selected from peptide sequence SEQ ID No4,6,8,10,12,14,16,18,20,22,24,26,27,29,31; 32,33,35,37,38,39,41,42,44,46,48,50,51,52,54; 55,56,57,58,59,61,63,65,67,69,71,73,75,77,79; 81,83,85,108,110,112,114,116,117,119,121,142,144,146,148 and 150.

Utilize known algorithm; The for example algorithm of FASTA software package (W.R.Pearson and D.J.Lipman, 1988) and (W.R.Pearson, 1990); Particularly can obtain, can carry out the best comparison to the sequence that is used for comparison on computers by the INFOBIOGEN resource center of French Evry.Explanation by way of example utilizes LFASTA (K.-M.Chao etc., 1992) or LALIGN (X.Huang and W.Miller, 1991) software to use INFOBIOGEN resource center, Evry, and French defined default parameter can be confirmed sequence identity percentage.LFASTA and LALIGN program are the parts of FASTA software package.LALIGN provides best local comparison; This program is more accurate than LFASTA, but speed is slower.

Another aspect of the present invention relates to the recombinant DNA that contains at least one above-mentioned polynucleotide.Preferably, this recombinant DNA is a carrier.Even more preferably be that this carrier is selected from phage, plasmid, phagemid, integrative vector, fosmids, clay, shuttle vectors, BAC (bacterial artificial chromosome) and PAC (artificial chromosome in P1-source).Explanation by way of example, lambda particles phage and M13 phage can be described as bacteriophage.About plasmid; What can mention is the plasmid that in intestinal bacteria (E.coli), duplicates; PBR322 and verivate thereof, pUC18 and verivate thereof, pUC19 and verivate thereof, pGB2 and verivate thereof (G.Churchward etc., 1984), pACYC177 (GenBank accession number: X06402) and verivate and pACYC184 (GenBank accession number: X06403) and verivate for example.What can mention is the plasmid that in streptomycete, duplicates, for example pIJ101 and verivate thereof, pSG5 and verivate thereof, SLP1 and verivate thereof and SCP2 ^*And verivate (Kieser etc., 2000).About phagemid; Explanation by way of example, what can mention is that pBluescript II and verivate thereof are (particularly by Stratagene (LaJolla, California; USA) company's sale), pGEM-T and verivate thereof are (by Promega (Madison; Wisconsin, USA) company sells), [lacuna] IS117 integration system (Kieser etc., 2000).About fosmids, explanation by way of example, what can mention is fosmid pFOS1 (by New England Bioloabs ltd, Beverly, Massachussetts, the sale of USA company) and verivate thereof.About clay, explanation by way of example, what can mention is that clay SuperCos and verivate thereof are (particularly by Stratagene (USA) company sells for LaJolla, California) and clay pWED15 (Wahl etc., 1987) and verivate thereof.

About shuttle vectors, explanation by way of example, what can mention is intestinal bacteria/streptomycete shuttle plasmid; For example; PIJ903 and verivate thereof, plasmid pUWL, pCAO106, pWHM3 and pOJ446 series and verivate thereof (Kieser etc., 2000), and intestinal bacteria/streptomycete BAC that shuttles back and forth; For example, at the BAC that shuttles back and forth described in the patented claim WO 01/40497.About BAC (bacterial artificial chromosome), explanation by way of example, what can mention is BAC pBeloBAC11 (GenBank accession number: U51113).About PAC (artificial chromosome in P1-source), explanation by way of example, what can mention is carrier pCYPAC6 (GenBank accession number: AF133437).Most preferably be, support according to the present invention is selected from pOS49.1, pOS49.11, pOSC49.12, pOS49.14, pOS49.16, pOS49.28, pOS44.1, pOS44.2, pOS44.4, pSPM5; PSPM7, pOS49.67, pOS49.88, pOS49.106, pOS49.120, pOS49.107, pOS49.32, pOS49.43, pOS49.44, pOS49.50, pOS49.99; PSPM17, pSPM21, pSPM502, pSPM504, pSPM507, pSPM508, pSPM509, pSPM1, pBXL1111, pBXL1112; PBXL1113, pSPM520, pSPM521, pSPM522, pSPM523, pSPM524, pSPM525, pSPM527, pSPM528, pSPM34; PSPM35, pSPM36, pSPM37, pSPM38, pSPM39, pSPM40, pSPM41, pSPM42, pSPM43, pSPM44; PSPM45, pSPM47, pSPM48, pSPM50, pSPM51, pSPM52, pSPM53, pSPM55, pSPM56, pSPM58; PSPM72, pSPM73, pSPM515, pSPM519, pSPM74, pSPM75, pSPM79, pSPM83, pSPM107, pSPM543 and pSPM106.

Another aspect of the present invention relates to the expression system that contains suitable expression vector and host cell that allows one or more aforementioned polypeptides in biosystem, to express.Expression vector according to the present invention comprises the nucleotide sequence of one or more aforementioned polypeptides of encoding, and expression vector can on purpose be expressed multiple polypeptides in multiple host cell well known to those skilled in the art.By way of example; What can mention is prokaryotic expression system (the for example expression system in bacteria Escherichia coli) and eukaryotic expression system; The expression system that for example allows at the baculovirus expression system of expressed in insect cells and allow in yeast cell, to express, or allow at mammalian cell the expression system of especially expressing among the human cell.The expression vector that can be used in this type systematic is well-known to those skilled in the art, about prokaryotic cell prokaryocyte, and explanation by way of example; What can mention is the expression vector in the intestinal bacteria, for example Stratagene (LaJolla, California; USA) pET, the Invitrogen (Carlsbad of company's sale; California, USA) GATEWAY serial carrier, Invitrogen (Carlsbad, the California of company's sale; USA) pBAD serial carrier, the New England Bioloabs (Beverly of ltd of company's sale; Massachussetts, USA) the pMAL serial carrier sold of company, and at B.Wilms etc. (2001) but publication in rhamnosyl-inducible expression carrier and the derivative vector thereof mentioned; Mentioned content also comprises the expression vector in the streptomycete, carrier pIJ4123 for example, and pIJ6021, pPM927, pANT849, pANT 850, and pANT 851, pANT1200, pANT1201 and pANT1202 and verivate thereof (Kieser etc., 2000).About yeast cell, explanation by way of example, the content of being referred to comprises Stratagene (LaJolla, California, USA) the carrier pESC of company's sale.About allowing baculovirus expression system, explain that by way of example the content of being referred to comprises BD Biosciences Clontech, (Palo Alto, California, USA) the carrier B acPAK6 of company's sale in expressed in insect cells.About mammalian cell; Explanation by way of example, the content of being referred to comprises that the carrier that contains CMV (cytomegalovirus (Cytomegalovirus)) immediately-early genes promotor is (for example by Stratagene (LaJolla, California; USA) the carrier pCMV and the verivate thereof of company's sale); Perhaps the SV40 early promoter of simian virus 40 (vacuolating simian virus) is (for example by Stratagene (LaJolla, California, USA) the carrier pSG5 of company's sale).

The invention still further relates to and produce the method for polypeptide as stated, said method comprising the steps of:

A) nucleic acid with coding said polypeptide inserts appropriate carriers;

B) in suitable substratum, cultivate in advance host cell with conversion of the carrier in the step a) or transfection;

C) recovering condition substratum or cell extract are for example through ultrasonic method or through the osmotic shock method;

D) separate and the said polypeptide of purifying from the said substratum that step c), obtains or in the cell extract;

E) as required, identify the characteristic of the polypeptide that is produced.

Know and such as F.Ausubel etc., the method described in (2002) can purifying recombinant polypeptide of the present invention through passing a series of suitable chromatography columns according to those skilled in the art.Explanation by way of example, the content of being referred to comprises " Histidine-label " technology, this technology is short poly Histidine sequence is added on the polypeptide that is produced, and makes that this polypeptide might be by purifying on the nickel post.Also can prepare according to polypeptide of the present invention through external synthetic technology.To illustrating of this type of technology, utilize by Roche Diagnostics France S.A., Meylan, " translation system (RTS) fast " that France company sells also can prepare polypeptide of the present invention.

Another aspect of the present invention relates to at least one polynucleotide of the present invention and/or at least one recombinant DNA and/or at least one expression vector importing host cell wherein.

Another aspect of the present invention relates to the mikrobe of the step of having blocked at least a macrolide biosynthetic pathway.Advantage at first be to study mutein function and, next is to produce the mikrobe that can produce the biosynthesizing midbody.Through in producing substratum, adding special composition; Perhaps through other gene that will suddenly change (these genes encodings through utilize midbody as substrate and can be to its protein of modifying) import mikrobe; Can modify these midbodys, randomly modify at after separating.Therefore can carry out chemistry, biological chemistry, zymetology and/or microbiology to these midbodys modifies.One or more participate in this kind or the biosynthetic proteinic function of these macrolides through inactivation in the mikrobe that produces this kind or these macrolides, can obtain the mikrobe of the step blocking-up of macrolide biosynthetic pathway.According to the protein of institute's inactivation, thereby can obtain the mikrobe of this kind or these macrolide biosynthetic pathway different steps blocking-up.Any means of knowing by one of skill in the art can be carried out this kind or these proteinic inactivations, for example through in the gene of code for said proteins, carrying out mutagenesis or the inactivation through messenger RNA(mRNA) complementary one or more sense-rna of expression and code for said proteins.For example, through radiation, through the effect of mutagenesis chemical reagent, through site-directed mutagenesis, through gene substitution or other method arbitrarily well known to those skilled in the art, can carry out mutagenesis.For example, the instruction in working according to (2002) such as (2000) such as T.Kieser and Ausubel can be adjusted the condition that is suitable for this kind mutagenesis.Through in the gene of considering, suppressing, replace, delete and/or adding one or more bases,, can carry out mutagenesis in external or original position perhaps through gene inactivation.In containing the gene of above-mentioned sequence, can carry out this kind mutagenesis.Preferably, the mikrobe of macrolide biosynthetic pathway step blocking-up is the bacterium of streptomyces.More preferably be biosynthetic one or more the proteinic functions of macrolide of participating in being discussed to be carried out inactivation through mutagenesis.Even more preferably be that the macrolide of being discussed is that Spiramycin Base and the mikrobe that carries out mutagenesis or repeatedly mutagenesis therein are to give birth to the dyadic streptomycete bacterial strain.More preferably be to contain the No3 corresponding to SEQ ID, 5,7,9,11,13,15,17,19,21,23; 25,28,30,34,36,40,43,45,47,49,53,60; 62,64,66,68,70,72,74,76,78,80,82,84; 107,109,111,113,115,118,120,141,143,145,147 and one or more genes of one of the sequence of 149 one or more sequences in carry out mutagenesis.Preferably, carry out mutagenesis or repeatedly mutagenesis through gene inactivation.Most preferably be that mutagenesis is by carrying out gene inactivation and form containing gene corresponding to the sequence of sequence SEQ ID No.13.

Explanation by way of example; Following mikrobe can be used as the example of this quasi-microorganism and is mentioned: OS49.16 (orf3::.hyg is with reference to embodiment 2), OS49.67 (deletion of orf3 homophase is with reference to embodiment 6), OS49.107 (orf8::.hyg; With reference to embodiment 7), OS49.50 (orf10::.hyg; With reference to embodiment 8), SPM21 (orf2::att3 Ω aac-is with reference to embodiment 10), SPM22 (deletion of orf2::att3 homophase is with reference to embodiment 10), SPM501 (orf6 ^*:: att1 hyg+, with reference to embodiment 14), SPM502 (orf6 ^*:: att1 homophase deletion, with reference to embodiment 14), SPM507 (orf12::att3 Ω aac-is with reference to implementing 11), SPM508 (orf13c::att3 Ω aac-; With reference to embodiment 12), and SPM509 (orf14::att3 Ω aac-is with reference to implementing 13); SPM107 (orf28c::att3aac+; With reference to embodiment 29), SPM543 (orf31::att3aac+, with reference to implement 30), SPM106 (orf32c::att3aac+ is with reference to embodiment 31).

Another aspect of the present invention relates to the method for preparing macrolide biosynthesizing midbody, and this method is used the mikrobe of the step blocking-up of above-mentioned macrolide biosynthetic pathway.This method is included in the mikrobe of the step blocking-up of cultivating above-mentioned macrolide biosynthetic pathway in the suitable substratum; Recovering condition substratum or cell extract; For example pass through ultrasonic method or osmotic shock method, and separate and the said biosynthesizing midbody of purifying in said substratum that from previous step, obtains or the cell extract.Can confirm to cultivate the condition of this quasi-microorganism according to technology well known to those skilled in the art.For example, substratum can be to be used for streptomycete, in particular for giving birth to MP5 substratum or the SL11 substratum (Pernodet etc., 1993) of dyadic streptomycete.About the cultivation of streptomycete, those skilled in the art especially can be with reference to Kieser etc., the work of (2000).Can both reclaim the midbody that is produced through any technology well known to those skilled in the art.For example, those skilled in the art can be with reference to USP 3,000, the technology of being instructed in 785, and more specifically be the method for the extraction Spiramycin Base described in this patent.

Another theme of the present invention relates to the method that preparation is derived from the molecule of macrolide, and this method is used the mikrobe of the step blocking-up of above-mentioned this macrolide biosynthetic pathway.This method comprises the midbody that obtains biosynthesizing midbody and modification generation like this according to top method, randomly after from substratum, separating, modifies.Can confirm to cultivate the condition of this quasi-microorganism according to technology well known to those skilled in the art.For example, substratum can be to be used for streptomycete, in particular for giving birth to MP5 substratum or the SL11 substratum (Pernodet etc., 1993) of dyadic streptomycete.About the cultivation of streptomycete, technology well known in the art particularly can be with reference to Kieser etc., the work of (2000).Through in producing substratum, add proper composition or through with other gene (these genes encodings through utilize midbody as substrate and can be to its protein of modifying) the importing mikrobe; Can modify the midbody that produces, randomly modify at after separating.Therefore can carry out chemistry, biological chemistry, zymetology and/or microbiology to these midbodys modifies.More preferably be that the macrolide of being discussed is that Spiramycin Base and the mikrobe that carries out one or many mutagenesis therein are to give birth to the dyadic streptomycete bacterial strain.

The invention still further relates to the mikrobe that produces Spiramycin I but do not produce spiramycin II and III.This mikrobe contains the biosynthesizing Spiramycin I but does not produce spiramycin II and necessary all genes of III, this be since contain corresponding to one of the sequence of SEQ ID No.13 or its variant or since the genetic code degeneracy and by the gene of one of the polypeptide of one of its deutero-sequence and encoding sequence SEQ ID No.14 or its variant be do not express or by inactivation.Can carry out this proteinic inactivation through any arbitrary method well known to those skilled in the art; For example through in the gene of code for said proteins, carrying out mutagenesis; The perhaps messenger RNA(mRNA) complementary sense-rna of overexpression and code for said proteins, this be appreciated that into: if by this operating influence orf5 ^*Expression, modify and to make orf5 thereby must carry out another kind ^*Gene is correctly expressed.In order to make coded protein inactivation or to stop its expression or, can carry out mutagenesis in encoding sequence or in the non-coding sequence by its translation.Through site-directed mutagenesis, gene substitution or any other method well known to those skilled in the art, can carry out mutagenesis.For example, the content of being taught in the work according to (2000) such as T.Kieser and Ausubel etc. 2002 can be adjusted the condition that is suitable for this kind mutagenesis.Inhibition through in the gene of being considered, carrying out one or more bases, replacement, deletion and/or add or, can carry out mutagenesis in external or original position through gene inactivation.The gene of overexpression Spiramycin Base biosynthetic pathway; Do not contain one of sequence SEQ ID No.13 or its variant or because genetic code degeneracy and, yet can obtain this mikrobe and do not express by the gene of one of the polypeptide of one of deutero-sequence and encoding sequence SEQ ID No.14 or its variant.Preferably, mikrobe is the bacterium of streptomyces.More preferably be that this kind only produces Spiramycin I and the mikrobe that do not produce spiramycin II and III is to obtain from the initial mikrobe that produces Spiramycin I, II and III.Even more preferably be; Through in the sequence that contains corresponding to SEQ ID No.13; One of or its variant; Or because genetic code degeneracy and by one of its deutero-sequence, and the polypeptide of encoding sequence SEQ ID No.14 or its have one of variant of identical function, gene in carry out mutagenic obtained this mikrobe.Even more preferably be to carry out this mutagenesis through gene inactivation.Preferably; Obtain said mikrobe from the livings dyadic streptomycete bacterial strain that produces Spiramycin I, II and III, wherein carried out gene inactivation by the gene of one of its deutero-sequence containing corresponding to the sequence of SEQ ID No.13 or owing to the genetic code degeneracy.Most preferably, contain corresponding to the sequence of SEQ ID No.13 or because genetic code degeneracy and, carry out gene inactivation through homophase deletion by the part of the gene or the gene of one of its deutero-sequence.Explanation by way of example, bacterial strain SPM502 (orf6 ^*:: att1, with reference to embodiment 14) can be described as producing Spiramycin I and the mikrobe that do not produce spiramycin II and III.

The invention still further relates to above-mentioned generation Spiramycin I and do not produce the mikrobe of spiramycin II and III, this mikrobe is overexpression also:

-utilize the primer of following sequence right: 5 ' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3 ' (SEQ ID No.138) and 5 ' GGATCCCGCGACGGACACGACCGCCGCGCA 3 ' (SEQ IDNo.139); And with clay pSPM36 or give birth to the total DNA of dyadic streptomycete as template; The gene that can obtain through the polymerase chain reaction; It more preferably is the gene of encoding sequence SEQ ID No.141

-or because the degeneracy of genetic code and by its deutero-gene.

In SEQ ID No.111 (DNA), provided the example of the sequence of this kind gene; Yet, because this sequence does not contain 3 ' part of corresponding encoded sequence, so this sequence is a partial sequence.SEQ ID No.112 has provided the protein that this encoding sequence part is translated into.Particularly utilize the content of being taught among the embodiment 24, those skilled in the art can easily accomplish this work.In second step, confirm undetermined sequence among the SEQ ID No.111, and in SEQ ID No.141, provided the complete sequence of this orf (orf28c).SEQ ID No.142 has provided the protein that this encoding sequence is translated into.Embodiment 24 has provided the method and the method that produces the expression vector that allows the orf28c expression of clone orf28c gene.This embodiment is further illustrated among the bacterial strain OSC2 overexpression orf28c gene and causes that Spiramycin Base output increases in this bacterial strain.Copy number through increasing this gene and/or through importing the promotor stronger than wild-type promoter activity can obtain the orf28c gene overexpression.Preferably, import the overexpression that mikrobe obtains said gene through the recombinant DNA construction body that will allow this gene overexpression.Preferably, this recombinant DNA construction body increases the copy number of said gene and makes it possible to obtain the overexpression of said gene.In this recombinant DNA construction body, can the encoding sequence of this gene be placed under the control of the promotor stronger than wild-type promoter activity.Explanation by way of example, the content of being referred to is included in has active ptrc promotor (E.Amann etc., 1988) and ermE in the living dyadic streptomycete ^*Promotor.Therefore, preferably, the one or more orf28c gene copies that imported place ermE ^*Under the control of promotor, like the situation in construct pSPM75 (with reference to embodiment 24).

The invention still further relates to the mikrobe that produces Spiramycin I as stated and do not produce spiramycin II and III, this mikrobe also overexpression has encoding sequence SEQ ID No.47 or has because genetic code degeneracy and by the gene of its deutero-encoding sequence.Preferably; This mikrobe is bacterial strain SPM502 pSPM525, and it is preserved in (CNCM) Institute Pasteur of state-run microbial preservation center [National Collection ofCultures and Microorganisms], 25; Rue duDocteur Roux 75724 Paris Cedex 15; France, preservation day is on February 26th, 2003, preserving number is I-2977.

The invention still further relates to the method that produces Spiramycin I; This method is included in the suitable substratum; Cultivate Spiramycin I as stated and do not produce the mikrobe of spiramycin II and III; Recovering condition substratum or cell extract, and separate and the purifying Spiramycin I in said substratum that from previous step, obtains or the cell extract.Can confirm to cultivate the condition of this kind mikrobe according to technology well known to those skilled in the art.For example, substratum can be to be used for streptomycete, in particular for giving birth to MP5 substratum or the SL11 substratum (Pernodet etc., 1993) of dyadic streptomycete.About the cultivation of streptomycete, those skilled in the art particularly can be with reference to Kieser etc., 2000 work.Can both reclaim the Spiramycin I that is produced through any technology well known to those skilled in the art.For example, those skilled in the art can be with reference to USP 3,000, the technology of being taught in 785, and more specifically be the method for the extraction Spiramycin Base described in this patent.

Another aspect of the present invention relates to the purposes that nucleotide sequence according to the present invention is used to increase mikrobe macrolide output.Therefore, the present invention relates to produce the mutant microbial of macrolide, this mikrobe has carried out genetic modification at least one contains the gene of above-mentioned sequence, and/or this mikrobe overexpression at least one contain the gene of above-mentioned sequence.In order to express one or more more highly active protein or express higher levels of this protein or these protein of having, genetic modification can be included in inhibition, replacement, deletion and/or the interpolation of carrying out one or more bases in the gene of being discussed.Copy number through increasing this gene and/or through importing the promotor stronger than wild-type promoter activity, the overexpression of the gene that can obtain to be discussed.Explanation by way of example, the content of being referred to is included in has active ptrc promotor (E.Amann etc., 1988) and ermE in the living dyadic streptomycete ^*Promotor (Bibb etc., 1985), (Bibb etc., 1994).Therefore, through importing the mikrobe of the generation macrolide of being considered according to the recombinant DNA construction body of this gene overexpression of permission of the present invention, the overexpression of the gene that can obtain to be considered.Particularly; Biosynthetic some step of macrolide be conditioning step and; Have more high reactivity or the more protein of high expression level if expressed one or more of participating in these conditioning steps than wild-type protein, might improve the output of related macrolide.For example, through the gene of the restricted methyltransgerase of replica code (this enzyme changes into tylosin with Tylosin C), the tylosin productive rate (R.Baltz, 1997) that in streptomyces fradiae, has obtained to increase.Especially can obtain active higher protein expression through mutagenesis; For example, those skilled in the art can be with reference to F.Ausubel etc., the related work of (2002).Preferably, these mutant microbials of being improved of macrolide output are streptomyces bacteriums.More preferably be that the macrolide of being discussed is that Spiramycin Base and the mikrobe that has carried out one or many mutagenesis therein are to give birth to the dyadic streptomycete bacterial strain.More preferably be to contain corresponding to one or more sequence SEQ ID No3,5,7,9,11,13,15,17,19,21,23 one or more; 25,28,30,34,36,40,43,45,47,49,53,60; 62,64,66,68,70,72,74,76,78,80,82,84; 107,109,111,113,115,118,120,141, one of one of sequence of 143,145,147 and 149 or its variant or because genetic code degeneracy and by having carried out genetic modification in the gene of one of its deutero-sequence.Preferably, this mikrobe overexpression is one or more contains corresponding to one or more sequence SEQ ID No3,5,7,9,11,13,15,17,19,21,23; 25,28,30,34,36,40,43,45,47,49,53,60; 62,64,66,68,70,72,74,76,78,80,82,84; 107,109,111,113,115,118,120,141, one of one of sequence of 143,145,147 and 149 or its variant or because genetic code degeneracy and by the gene of one of its deutero-sequence.Preferably, this mikrobe overexpression contains corresponding to one of the sequence of SEQ ID No.111 or 141 or its variant or because genetic code degeneracy and by the gene of one of its deutero-sequence.The sequence that provides among the SEQ ID No.111 is a partial sequence; Yet technology given among those skilled in the art embodiment 24 especially capable of using can easily be accomplished this sequence.In second step, confirm undetermined sequence among the SEQ IDNo.111, and in SEQ ID No.141, provided the complete sequence of this orf (orf28c).SEQ ID No.142 has provided the protein that this encoding sequence is translated into.Embodiment 24 has provided the method and the method that produces the expression vector that allows the orf28c expression of clone orf28c gene.This embodiment is further illustrated among the bacterial strain OSC2 overexpression orf28c gene and causes that Spiramycin Base output increases in this bacterial strain.Copy number through increasing this gene and/or through importing the promotor stronger than wild-type promoter activity can obtain the orf28c gene overexpression.Preferably, import the overexpression that mikrobe obtains said gene through the recombinant DNA construction body that will allow this gene overexpression.Preferably, this recombinant DNA construction body increases the copy number of said gene and makes it possible to obtain the overexpression of said gene.In this recombinant DNA construction body, can the encoding sequence of this gene be placed under the control of the promotor stronger than wild-type promoter activity.Explanation by way of example, the content of being referred to is included in has active ptrc promotor (E.Amann etc., 1988) and ermE in the living dyadic streptomycete ^*Promotor.Therefore, preferably, the one or more orf28c gene copies that imported place ermE ^*Under the control of promotor, like the situation in construct pSPM75 (with reference to embodiment 24).

Another aspect of the present invention relates to the method for the microorganisms macrolide that utilizes described in the first previous paragraphs.This method is included in and cultivates defined mikrobe in the first previous paragraphs in the suitable substratum, recovering condition substratum or cell extract, and separate the macrolide that also purifying produced in said substratum that from previous step, obtains or the cell extract.Can confirm to cultivate the condition of this quasi-microorganism according to technology well known to those skilled in the art.For example, substratum can be to be used for streptomycete, in particular for giving birth to MP5 substratum or the SL11 substratum (Pernodet etc., 1993) of dyadic streptomycete.About the cultivation of streptomycete, those skilled in the art especially can be with reference to Kieser etc., 2000 work.Can reclaim the macrolide that is produced through any technology well known to those skilled in the art.For example, those skilled in the art can be with reference to USP 3,000, the technology of being taught in 785, and more specifically be the method for the extraction Spiramycin Base described in this patent.Preferably, employed mikrobe is the bacterium of streptomyces in this kind method.More preferably be, the macrolide of being discussed is a Spiramycin Base, and the mutant microbial that its Spiramycin Base output is improved is to give birth to the dyadic streptomycete bacterial strain.

Another aspect of the present invention relates to according to sequence of the present invention and/or carrier and is used to prepare the antibiotic purposes of hydridization.Particularly; Can be used to obtain mikrobe according to polynucleotide of the present invention; The one or more muteins of modifying that aspect substrate specificity, produce of these microbial expressions perhaps can be expressed these polynucleotide in order to produce the hydridization microbiotic in the antibiotic mikrobe of many generations.Therefore, through producing the transgenosis between the mikrobe, can be so that can produce hydridization microbiotic (Hopwood etc., 1985a, Hopwood etc., 1985b, Hutchinson etc., 1989) with favourable pharmacological characteristics according to polynucleotide of the present invention.Hopwood (Hopwood 1981) at first proposes to utilize this principle, genetically engineered to cause and produces the hydridization microbiotic.Therefore proposing to participate in the biosynthetic enzyme of microbiotic usually accepts on the structure relevant but be different from the substrate of its natural substrate.The enzyme that generally believes the coded by said gene of microbiotic biosynthetic pathway has more undemanding substrate specificity (Hopwood 1981, and Hutchinson 1988, and Robinson 1988) than the enzyme of analytic metabolism.Therefore having shown might be through producing a large amount of non-natural substrates of enzymatic conversion (Hutchinson 1988) of antibiotic microorganism, its two mutants or purified these microbiotic biosynthetic pathways.Utilizing should instruction, those skilled in the art can construction expression one or more on substrate specificity, produces the mikrobe of the mutein of modifying, with generation hydridization microbiotic.

The invention still further relates to the purposes that is used to carry out one or more bio-transformations according at least one polynucleotide of the present invention and/or at least one recombinant DNA and/or at least one expression vector and/or at least one polypeptide and/or at least a host cell.Therefore, the invention enables to make up such bacterium or fungal bacterial strain, one or more protein wherein according to the present invention are expressed under the control of suitable expression signal.This type of bacterial strain can be used in and carries out one or more bio-transformations then.Use full cell perhaps to use the cell-free extract of said cell, can carry out these bio-transformations.These bio-transformations make and might utilize the enzyme of biosynthetic pathway that molecule is changed into derivative form.For example, Carreras etc. (Carreras etc., 2002) have described the purposes that red saccharopolyspora and streptomyces coelicolor bacterial strain are used to produce new erythromycin derivatives.Walczak etc. (Walczak etc., 2001) have described streptomycete P450 monooxygenase and have been used for the purposes with the anthracycline of desacetyladriamycin (the similar thing of anthracycline) bio-transformation Cheng Xin.The bacterial strain that Olonao etc. (Olonao etc., 1999) have described shallow Streptomyces glaucoviolaceus (Streptomyces lividans) modification is used for ε-rhodomycinon bio-transformation is become the purposes of rhodomycetin D.Those skilled in the art can be applied to any biosynthesizing midbody with these principles.

The invention still further relates to such recombinant DNA, it comprises:

-utilize primer right with following sequence: 5 ' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3 ' (SEQ ID No.138) and 5 ' GGATCCCGCGACGGACACGACCGCCGCGCA 3 ' (SEQ ID No.139); And be template with clay pSPM36 or total DNA of giving birth to the dyadic streptomycete; The polynucleotide that can obtain through the polymerase chain reaction; More preferably these polynucleotide are polynucleotide of sequence SEQ ID No.141

-or have 10,12,15,18 in the said polynucleotide, 20 to 25,30,40,50,60,70,80 at least; 90,100,150,200,250,300,350,400,450,500,550; 600,650,700,750,800,850,900,950,1000,1050,1100; 1150,1200,1250,1300,1350,1400,1450,1460, the fragment of 1470,1480,1490 or 1500 continuous nucleotides.Preferably, this recombinant DNA is a carrier.Even more preferably be that this carrier is selected from phage, plasmid, phagemid, integrative vector, fosmids, clay, shuttle vectors, BAC (bacterial artificial chromosome) and PAC (artificial chromosome in P1-source).Explanation by way of example, lambda particles phage and M13 phage can be described as bacteriophage.As plasmid; What can mention is the plasmid that in intestinal bacteria, duplicates; PBR322 and verivate thereof, pUC18 and verivate thereof, pUC19 and verivate thereof, pGB2 and verivate thereof (G.Churchward etc., 1984), pACYC177 (GenBank accession number: X06402) and verivate and pACYC184 (GenBank accession number: X06403) and verivate for example.What also can mention is the plasmid that in streptomycete, duplicates, for example pIJ101 and verivate thereof, pSG5 and verivate thereof, SLP1 and verivate thereof and SCP2 ^*And verivate (Kieser etc., 2000).About phagemid, explanation by way of example, what can mention is that pBluescript II and verivate thereof are (especially by Stratagene (LaJolla; California; USA) company sells), pGEM-T and verivate thereof be (by Promega (USA) company sells for Madison, Wisconsin) and λ ZAPII and verivate thereof (especially by Stratagene (LaJolla; California, USA) company sells).About integrative vector, explanation by way of example, what can mention is the carrier of in streptomycete, integrating; For example derive from the carrier (Kieser etc., 2000) of SLP1, the carrier (Kieser etc., 2000) that derives from pSAM2, use PhiC31 phage integration system (Kieser etc.; 2000) carrier of (for example pSET152 (Bierman etc., 1992)) or VWB integration system (L.van Mellaert etc., 1998); And comprise the carrier that uses IS117 integration system (Kieser etc., 2000).About fosmids, explanation by way of example, what can mention is fosmid pFOS1 (by New England Bioloabs ltd, Beverly, Massachussetts, the sale of USA company) and verivate thereof.About clay, explanation by way of example, what can mention is that clay SuperCos and verivate thereof are (especially by Stratagene (USA) company sells for LaJolla, California) and clay pWED15 (Wahl etc., 1987) and verivate thereof.About shuttle vectors, explanation by way of example, what can mention is intestinal bacteria/streptomycete shuttle plasmid; For example; PIJ903 and verivate thereof, plasmid pUWL, pCAO106, pWHM3 and pOJ446 series and verivate thereof (Kieser etc., 2000), and intestinal bacteria/streptomycete BAC that shuttles back and forth; For example, at the BAC that shuttles back and forth described in the patented claim WO 01/40497.About BAC (bacterial artificial chromosome), explanation by way of example, what can mention is BAC pBeloBAC11 (GenBank accession number: U51113).As PAC (artificial chromosome in P1-source), explanation by way of example, what can mention is carrier pCYPAC6 (GenBank accession number: AF133437).More preferably be that this recombinant DNA is an expression vector.The expression vector that can be used in this type systematic is well-known to those skilled in the art; About prokaryotic cell prokaryocyte, explanation by way of example, what can mention is the expression vector in the intestinal bacteria; Stratagene (LaJolla, California, USA) pET, the Invitrogen (Carlsbad that sell of company for example; California, USA) GATEWAY serial carrier, Invitrogen (Carlsbad, the California of company's sale; USA) pBAD serial carrier, the New EnglandBioloabs (Beverly of ltd of company's sale; Massachussetts, USA) the pMAL serial carrier sold of company, and at B.Wilms etc. (2001) but publication in rhamnosyl-inducible expression carrier and the derivative vector thereof mentioned; Mentioned content also comprises the expression vector in the streptomycete, carrier pIJ4123 for example, and pIJ6021, pPM927, pANT849, pANT 850, and pANT 851, pANT1200, pANT1201 and pANT1202 and verivate thereof (Kieser etc., 2000).About yeast cell, explanation by way of example, the content of being referred to comprises Stratagene (LaJolla, California, USA) the carrier pESC of company's sale.About allowing baculovirus expression system, explain that by way of example the content of being referred to comprises BD Biosciences Clontech, (Palo Alto, California, USA) the carrier B acPAK6 of company's sale in expressed in insect cells.About mammalian cell; Explanation by way of example, the content of being referred to comprises that the carrier that contains CMV (cytomegalovirus (Cytomegalovirus)) immediately-early genes promotor is (for example by Stratagene (LaJolla, California; USA) the carrier pCMV and the verivate thereof of company's sale); Perhaps the SV40 early promoter of simian virus 40 is (for example by Stratagene (LaJolla, California, USA) the carrier pSG5 of company's sale).Another aspect of the present invention relates to the importing of the recombinant DNA described at least one this paragraph host cell wherein.

The invention still further relates to the method that produces polypeptide, wherein said method may further comprise the steps:

A) with the expression vector transformed host cell described in the paragraph above at least one;

B) in suitable substratum, cultivate said host cell;

C) recovering condition substratum or cell extract;

D) separate and the said polypeptide of purifying in the said substratum that from step c), obtains or in the cell extract;

E) as required, identify the characteristic of the recombinant polypeptide that is produced.

Know and such as F.Ausubel etc. according to those skilled in the art, the method described in (2002), through passing a series of suitable chromatography columns, recombinant polypeptide that can purifying produced.Explanation by way of example, the content of being referred to comprises " Histidine-label " technology, this technology is short poly Histidine sequence is added on the polypeptide that is produced, and makes that this polypeptide might be by purifying on the nickel post.Also can prepare polypeptide through external synthetic technology.Through illustrating of this type of technology, utilize " translation system (RTS) fast ", especially by Roche Diagnostics France S.A., Meylan, France company sells, and also can prepare polypeptide.

Another aspect of the present invention relates to the mikrobe that produces at least a Spiramycin Base, this mikrobe overexpression:

-utilize the primer of following sequence right: 5 ' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3 ' (SEQ ID No.138) and 5 ' GGATCCCGCGACGGACACGACCGCCGCGCA 3 ' (SEQ ID No.139); And with clay pSPM36 or give birth to the total DNA of dyadic streptomycete as template; The gene that can obtain through the polymerase chain reaction; It more preferably is the gene of encoding sequence SEQ ID No.141

-or owing to the genetic code degeneracy and by its deutero-gene.In SEQ ID No.111 (DNA), provided the example of the sequence of this kind gene; Yet, because this sequence does not contain 3 ' part of corresponding encoded sequence, so this sequence is a partial sequence.SEQ ID No.112 has provided the protein that this encoding sequence part is translated into.Especially utilize the content of being taught among the embodiment 24, those skilled in the art can easily accomplish this sequence.Therefore this embodiment has provided the method and the method that is used to produce the expression vector that allows the orf28c expression that is used to clone the orf28c gene.This embodiment is further illustrated among the bacterial strain OSC2 overexpression orf28c gene and causes that Spiramycin Base output increases in this bacterial strain.Preferably, the following gene of overexpression

-utilize the primer of following sequence right: 5 ' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3 ' (SEQ ID No.138) and 5 ' GGATCCCGCGACGGACACGACCGCCGCGCA 3 ' (SEQ ID No.139); And with clay pSPM36 or give birth to the total DNA of dyadic streptomycete as template; The gene that can obtain through the polymerase chain reaction; It more preferably is the gene of the preparatory sequence SEQ ID No.141 of coding

-or because genetic code degeneracy and by its deutero-gene, mikrobe be the streptomyces bacterium; Even more preferably be that this mikrobe is the bacterium that gives birth to dyadic streptomycete species.Preferably, through transform the overexpression that said mikrobe obtains said gene with expression vector; Most preferably; Microorganism strains is bacterial strain OSC2/pSPM75 (1) or bacterial strain OSC2/pSPM75 (2), is preserved in state-run microbial preservation center (CNCM) [NationalCollection of Cultures and Microorganisms] Institute Pasteur, 25; Rue duDocteur Roux 75724 Paris Cedex 15; France, preservation day is on October 6th, 2003, preserving number is I-3101.

Another aspect of the present invention relates to the method that produces Spiramycin Base, and this method is used the mikrobe described in the first previous paragraphs.This method is included in and cultivates defined mikrobe in the first previous paragraphs in the suitable substratum, recovering condition substratum or cell extract, and from the cell extract that said substratum perhaps obtains from previous step, separate and the purifying Spiramycin Base.Can confirm to cultivate the condition of this quasi-microorganism according to technology well known to those skilled in the art.For example, substratum can be to be used for streptomycete, in particular for giving birth to MP5 substratum or the SL11 substratum (Pernodet etc., 1993) of dyadic streptomycete.About the cultivation of streptomycete, those skilled in the art especially can be with reference to Kieser etc., the work of (2000).Can both reclaim the Spiramycin Base that is produced through any technology well known to those skilled in the art.For example, those skilled in the art can be with reference to USP 3,000, the technology of being taught in 785, and more specifically be the method for the extraction Spiramycin Base described in this patent.Preferably, the mikrobe that is used for this kind method is the streptomyces bacterium.More preferably be that this mikrobe is to give birth to the dyadic streptomycete bacterial strain.

Another aspect of the present invention relates to expression vector, wherein the polynucleotide of sequence SEQ ID No.47 or because genetic code degeneracy and placed by its deutero-polynucleotide and to allow the coded protein of said polynucleotide under the control of giving birth to dyadic streptomycete expression promoter.Provided the example of the expression vector that can be used in streptomycete above.Preferably, this kind expression vector is plasmid pSPM524 or pSPM525.

Another aspect of the present invention relates to the living dyadic streptomycete bacterial strain that has transformed with defined carrier in the first previous paragraphs.

Another aspect of the present invention relates to the polypeptide that its sequence comprises sequence SEQ ID No.112.The invention still further relates to its sequence corresponding to the polypeptide of sequence of translating from following encoding sequence:

-utilize following aligning primer right: 5 ' AAGCTTGTGTGCCCGGTGTACCTGGGGAGC 3 ' (SEQ ID No.138) and 5 ' GGATCCCGCGACGGACACGACCGCCGCGCA 3 ' (SEQ ID No.139); And with clay pSPM36 or give birth to the total DNA of dyadic streptomycete as template; The polypeptide of the gene that can obtain through the polymerase chain reaction; It more preferably is the gene of encoding sequence SEQ ID No.141

-or owing to the genetic code degeneracy and by its deutero-gene.Preferably, express these polypeptide with native state, more preferably be these polypeptide involved in spiramycin biosynthesizing by the streptomyces bacterium.

Another aspect of the present invention relates to the expression vector that defined polypeptide is expressed in the first previous paragraphs of permission in giving birth to the dyadic streptomycete.Provided the example of the expression vector that can be used in streptomycete above.Preferably, the expression vector of being discussed is plasmid pSPM75.

The accompanying drawing summary:

Fig. 1: the chemical structure of Spiramycin I, II and III.

Fig. 2: the clay that is used for the zone is carried out sequential analysis.

Fig. 3: the composition diagram of the gene group of involved in spiramycin biosynthetic pathway.

Fig. 4: the mycarose biosynthetic pathway that is proposed.

Fig. 5: the mycaminose biosynthetic pathway that is proposed.

Fig. 6: the forosamine biosynthetic pathway that is proposed.

Fig. 7: add sugar on Spiramycin Base molecule and the midbody preferred sequence.

Fig. 8: the biosynthetic pathway of methoxyl group malonyl-in the living dyadic streptomycete that is proposed.Through with the mutation of streptomyces hygroscopicus WS7238A in biosynthesizing (K.Wu etc., the 2000) analogy of methoxyl group malonyl-this approach is proposed.

Fig. 9: the step that causes gene inactivation:

A) target gene is cloned in the carrier that in intestinal bacteria, duplicates and in streptomycete, do not duplicate;

B) the resistance box is inserted target gene (through clone or the reorganization between the short identical sequence);

C) plasmid is imported the clone who gives birth to dyadic streptomycete (through transforming or engaging with intestinal bacteria) and select to have the integration box, thereby the clone that carrier part has gene substitution is lost in screening then;

D) chromosomal region of mutants which had, wherein target gene is through the gene substitution inactivation.

Figure 10: carry out gene inactivation optional step afterwards according to method described in Fig. 9, be used to interrupt target gene, can implement these steps if can excise box;

E) will carry the xis of pSAM2 and the plasmid pOSV508 of int gene and import mutants which had, its product effectively excises allowing through the attL and the reorganization of the fixed point between the attR sequence of box avris;

F) generation has been lost and can have been excised box and to being directed against box the clone of resistance sensitive antibiotics is provided;

G) growth and forming after the gemma on not containing antibiotic solid medium, plasmid pOSV508 is lost with high frequency.Therefore might obtain the responsive clone of thiostrepton, wherein target gene contains the homophase deletion.Can control the sequence of deletion target gene through the sequential analysis of pcr amplification and PCR product.

Figure 11: amplification can be excised box to use it for the homologous recombination experiment.Chaveroche etc., 2000 have described the technology of carrying out homologous recombination through short homologous sequence.Be positioned these oligonucleotide 5 ' 39 or 40 terminal deoxynucleotides and comprise sequence, and be positioned most of 3 ' terminal 20 deoxynucleotides corresponding to excising one of the terminal sequence of box corresponding to gene order that will inactivation.

Figure 12: utilize Chaveroche etc., 2000 described technology produce the construct that is used for the inactivation target gene.

Figure 13: the collection of illustrative plates of plasmid pWHM3.Strepto ori: streptomycete replication orgin.

Figure 14: the collection of illustrative plates of plasmid pOSV508.

Figure 15: can excise the box example of structure.It comprises that avris is the Ω hyg box (Blondelet-Rouault etc., 1997) of attR and attL site (Raynal etc., 1998), because xis and int expression of gene, the recombination event between attR and attL site will allow the cutting of box.

Figure 16: the collection of illustrative plates of plasmid pBXL1111.

Figure 17: the collection of illustrative plates of plasmid pBXL1112.

Figure 18: microbiological assay Spiramycin Base output, based on the susceptibility of micrococcus luteus bacterial strain to Spiramycin Base.Used micrococcus luteus bacterial strain is natural to the Spiramycin Base sensitivity and to killing the bacterial strain of the plain tool resistance of firm trypanosome.Many streptomyces strains of being tested are incubated in the 500ml Erlenmeyer flask that contains 70ml MP5 substratum, with 2.5 * 10 ⁶The initial concentration of gemma/ml is inoculated and is vibrated 27 ℃ of growths with 250 rev/mins of orbit determination.After cultivating 48,72 and 96 hours, take out fermented liquid sample and centrifugal.To be used for test with these supernatants of 10 times of dilutions of aseptic culture medium.It is anti-that to kill firm trypanosome plain but the responsive micrococcus luteus indicator (Gourmelen etc., 1998) of Spiramycin Base is incubated in the plate that area is 12 * 12cm.Every kind of supernatant with 10 times of dilutions of 70 μ l soaks Whatman AA filter paper disk and is positioned over the plate surface.To use the Spiramycin Base solution wetted dish dilution of different concns (2-4-8 μ g/m1 in the MP5 substratum) to be standard range.Ware was hatched 24-48 hour at 37 ℃.If filter paper disk contains Spiramycin Base, it will diffuse into agar and suppress the growth of micrococcus luteus indicator.This filter paper disk that is suppressed at produces " swooning " on every side, swoons and has reflected the zone that the micrococcus luteus bacterial strain is not grown.Therefore this dizzy existence is to have the indication of Spiramycin Base and whether the livings dyadic streptomycete bacterial strain that makes it possible to confirm discussed is or be not Spiramycin Base production bacterium.The inhibition diameter that is obtained and standard range are compared the indication that makes it possible to obtain Spiramycin Base amount that this bacterial strain is produced.

Figure 19: the HPLC color atlas of bacterial strain OSC2 through the filtration culture supernatant liquid.

Figure 20: the HPLC color atlas of bacterial strain SPM501 through the filtration culture supernatant liquid.

Figure 21: the HPLC color atlas of bacterial strain SPM502 through the filtration culture supernatant liquid.

Figure 22: the HPLC color atlas of bacterial strain SPM507 through the filtration culture supernatant liquid.

Figure 23: the HPLC color atlas of bacterial strain SPM508 through the filtration culture supernatant liquid.

Figure 24: the HPLC color atlas of bacterial strain SPM509 through the filtration culture supernatant liquid.

Figure 25: use of the comparison of FASTA program to TylB protein (SEQ IDNo.87) with the Orf3 protein (SEQ ID No.29) of the streptomyces fradiae that produced.

Figure 26: use the comparison of SrmD protein (SEQ ID No.16) with the MdmA protein (SEQ ID No.88) of Streptomyces Macrofaciens of the generation of FASTA program.

Figure 27: the example that can excise the sequence in remaining site, box excision back.That represent with runic is Raynal etc., and in 1998 defined minimum att26 site.Provide the sequence of 33 Nucleotide of phase place 1 (att1) among the SEQ ID No.104, provided the sequence of 34 Nucleotide of phase place 2 (att2) among the SEQ ID No.105, and provided the sequence of 35 Nucleotide of phase place 3 (att3) among the SEQ ID No.95.

Figure 28: the location of orf10 gene, used PCR primer and by the graphic extension of the construct that every pair of primer obtained.

Figure 29: the structure of pac-oritT box.

Figure 30: the collection of illustrative plates of clay pWED2.

Figure 31: the location (with reference to embodiment 19) of the gene group of graphic extension involved in spiramycin biosynthetic pathway and three probes of the clay of the genome dna library that is used for the living dyadic streptomycete of separating Escherichia coli OSC2 bacterial strain.

Figure 32: the insertion site (with reference to embodiment 19) of from intestinal bacteria, giving birth to the isolating clay of genome dna library of dyadic streptomycete OSC2 bacterial strain.

Figure 33: the segmental subclone of clay pSPM36 PstI-PstI (plasmid pSPM58 insertion), the segmental subclone of clay pSPM36 StuI-StuI (plasmid pSPM72 insertion) and the segmental subclone of EcoRI-StuI (plasmid pSPM73 insertion).

Figure 34: the location of the ORFs of being identified during the PstI-PstI insertion of plasmid pSPM58 and the EcoRI-StuI of plasmid pSPM73 insert.

Figure 35: the 238 bacterial strain OS49.67 that produce with 280nm through the HPLC color atlas that filters culture supernatant liquid stacked (above) during with 33.4 minutes and 44.8 minutes the UV of the molecule of wash-out compose (below).

Figure 36: the molecular structure of platenolide A and platenolide B molecule.

Figure 37: the composition diagram of the gene group of involved in spiramycin biosynthetic pathway.

Figure 38: the molecular structure of the biosynthesizing midbody that bacterial strain SPM507 is produced.

Figure 39: the genotype from excessive generation Spiramycin Base bacterial strain is orf6 ^*:: the structure of the biosynthesizing midbody that the living dyadic streptomycete bacterial strain of att1 Ω hyg+ is produced.Box att1 Ω hyg+ is inserted orf6 ^*Produce and stop orf5 ^*The polar effect of expressing.

Figure 40: the platenolide A+mycarose that is produced by bacterial strain OS49.67 and the molecular structure of platenolideB+mycarose molecule.

Figure 41: the segmental subclone of clay pSPM36 PstI-PstI (plasmid (pSPM79) insertion), the location of the location of the ORFs that the PstI-PstI of plasmid pSPM79 is identified in inserting and sequence SEQ IDN ° 140.

The present invention will be described through following examples, and these embodiment can be considered non-limiting and illustrate.

In a word, the gene of Spiramycin Base biosynthetic pathway is from the genome dna library of giving birth to the dyadic streptomycete, to separate to obtain.Obtained this library through using the BamHI Restriction Enzyme that living dyadic streptomyces gene group DNA is carried out part digestion.With big dna fragmentation, average 35-45kb is cloned into by among clay pWED15 (Wahl etc., 1987) the deutero-clay pWED1 (Gourmelen etc., 1998).Use phage particle that these clays are imported intestinal bacteria.With the library that so obtains use corresponding to streptomyces fradiae tylB gene (Merson-Davies and Cundliffe, 1994, the GenBank accession number: U08223) probe (sequence SEQ ID No.86) of part is hybridized.After the hybridization, with 4 clays of probe hybridization in more specifically select a clay.Then with this clay of SacI digestion called after pOS49.1, and will comprise with the 3.3kb fragment subclone in this probe hybridization zone and go into carrier pUC19 and order-checking.Identify 4 ORFs and one of them coding and streptomyces fradiae TylB protein (SEQ ID No.87) and presented the protein (SEQ ID No.29) (with reference to Figure 25) of strong similarity.This unnamed gene be orf3 (SEQ ID No.28) and in giving birth to the dyadic streptomycete this gene of inactivation.The clone that might prove wherein orf3 gene inactivation no longer produces Spiramycin Base.The gene involved in spiramycin biosynthesizing that this shows the orf3 gene or is positioned downstream.

In case above-mentioned aspect has obtained affirmation, with the big zone of clay pOS49.1, the segmental any side of promptly studying in the past of SacI checks order.Therefore, might obtain to comprise the sequence in 7 complete ORFs and other 2 that are positioned these 7 any sides of complete ORFs imperfect ORFs zones from clay pOS49.1.Through in DB, searching for, can show that an imperfect ORFs is corresponding to srmG locus (coding is called the zone of " polyketide synthase " enzyme (PKS)).Utilize S.Burgett etc., the method for 1996 (USP 5,945,320) is carried out subclone with corresponding gene.And, in DB, do not find other ORFs, called after: orf1, orf2, orf3; Orf4, orf5, orf6 and orf7 (SEQ ID No.23,25,28; 30,34,36,40) starting point of 7 complete ORF and the 8th ORF of called after orf8 (sequence SEQ ID No.43).

Subsequently, in this same area, contain segmental other clay of living dyadic streptomyces gene group in order to clone biosynthetic other gene of involved in spiramycin, to have separated.For this reason, use 3 probes on bacterium colony, to carry out other a series of hybridization.The 1st probe correspondence is from the orf1 that comprises of pOS49.1 subclone, the 3.7kb dna fragmentation of orf2 and orf3 starting point.The 2nd probe correspondence comprises the orf7 part 2kb dna fragmentation a part of with orf8 from the pOS49.1 subclone.Also used the 3rd probe.Back one probe correspondence contains the 1.8kb dna fragmentation of srmD gene.The srmD gene is to separate the gene that can give the Spiramycin Base resistance that obtains from giving birth to the dyadic streptomycete.Particularly; Previous research has made it possible to clone several RDs of living dyadic streptomycete; Said RD is given grey pale red yellow streptomycete bacterial strain (Spiramycin Base-sensitive strain) Spiramycin Base resistance (Pernodet etc., 1993) (Pernodet etc., 1999).For the separation resistance gene, in clay pKC505, produced the cosmid library (Richardson MA etc., 1987) of living dyadic streptomycete bacterial strain genomic dna.This clay storehouse is imported the natural grey pale red yellow streptomycete responsive to Spiramycin Base.5 clays that can give grey pale red yellow streptomycete apramycin resistance and Spiramycin Base resistance have so been obtained.In these 5 clays; The clay of called after pOS44.1; In its insertion fragment, contain the srmD gene; The protein of this genes encoding and Streptomyces Macrofaciens mdmA coded by said gene and protein that in this producer, participate in the mydecamycin resistance (Hara etc., 1990, the GenBank accession number: A60725) (Figure 26) presents certain similarity.The 3rd probe that is used for setting screw mycin biosynthesis gene is the segmental insertion of about 1.8kb that comprises the srmD gene.

These three probes are used to hybridize above-mentioned genome dna library and might select two clays (pSPM7 and pSPM5), and they tend to contain the longest insertion fragment and do not have the common band.Clay pSMP5 and first and second probe hybridizations, but not with the 3rd probe hybridization, and clay pSPM7 only with the 3rd probe hybridization.Use " shotgun sequential analysis " technology that these two clays are checked order fully.The sequence that these two clay: pSPM7 and pSPM5 insert can make up, although they are not overlapping, each inserts fragment and all comprises known sequences at an one of which end.Particularly, each insertion fragment all comprises the sequence fragment that a coding is called " polyketide synthase " gene (PKS).S.Burgett etc., this five genes (with reference to Fig. 2) have been cloned in 1996 (USP 5,945,320).Therefore, might confirm single living dyadic streptomyces gene group dna sequence dna.Provided in 5 ' position the initial sequence (with reference to Fig. 2 and 3) that is positioned first PKS gene and extends to 30 943 Nucleotide in BamHI site, 3 ' position among the SEQ ID No.1 from the EcoRI site.Provided in 5 ' position initial and extend to BstEII site, 3 ' position, be positioned second zone of 11 171 Nucleotide of the 5th PKS gene among the SEQ ID No.2 from the PstI site.This zone is PKS gene downstream area (downstream and the upper reaches are by the direction definition of 5 PKS genes or with the equidirectional orientation) (with reference to Fig. 2 and 3).

Then, in this same area, contain segmental other clay of living dyadic streptomyces gene group (with reference to embodiment 18 and 19) in order to clone biosynthetic other gene of involved in spiramycin, to have separated.

Embodiment 1: the structure of giving birth to the genome dna library of dyadic streptomycete bacterial strain ATCC23877 in the intestinal bacteria

1.1. give birth to the extraction of dyadic streptomycete bacterial strain ATCC23877 genomic dna.

Giving birth to dyadic streptomycete bacterial strain ATCC23877 (especially can be by American type culture collection (ATCC) (Manassas; Virginia USA) obtains, and preserving number is 23877) be incubated at YEME (yeast extract-malt extract) (Kieser; T etc.; 2000), and according to cracking and sedimentary standard technique (Kieser T etc., 2000) extract the also genomic dna of this bacterial strain of purifying.

1.2. the structure of genome dna library

Partly digest as above isolating living dyadic streptomycete bacterial strain ATCC23877 genomic dna with the BamHI Restriction Enzyme, thus obtain size about 35 and 45kb between dna fragmentation.These fragment clonings are gone in advance the clay pWED1 (Gourmelen etc., 1998) that has digested with BamHI.Contain the 4.1kbHpaI-HpaI fragment (Gourmelen etc., 1998) of the active expression module of tool in Mammals through deletion, obtain clay pWED1 from clay pWE15 (Wahl etc., 1987).Then; " Packagene

the λ DNA packaging system " of utilizing Promega company to sell; According to the step of manufacturer recommendation, be packaged in the lambda particles phage particle at the external mixture that will connect.Resulting phage particle is used to infect (the LaJolla of Stratagene company; California, intestinal bacteria SURE

bacterial strain of USA) selling.Because clay pWED1 given amicillin resistance, thus on the flat board of LB substratum+penbritin (50 μ g/ml) screening and cloning.

Embodiment 2: the separation and the sign of giving birth to the biosynthetic gene of involved in spiramycin in the dyadic streptomycete

2.1 give birth to the colony hybridization of dyadic streptomycete ATCC23877 genomic library escherichia coli cloning

About 2000 escherichia coli clonings in the top library that obtains are transferred on the filter membrane hybridize.The probe that is used to hybridize comprises the NaeI-NaeI dna fragmentation (SEQ ID No.86) of streptomyces fradiae part tylB gene.This fragment is corresponding to dna fragmentation (L.A.Merson-Davies and the E.Cundliffe of L.A.Merson-Davies and the described Nucleotide 2663-3702 of E.Cundliffe; 1994; The GenBank accession number: U08223), wherein the encoding sequence of tylB gene is corresponding to Nucleotide 2677-3843.

Use random primer technology (test kit that Roche company sells) to use ³²The NaeI-NaeI dna fragmentation (SEQ ID No.86) of P mark streptomyces fradiae tylB Gene Partial, and with its 2000 clones as the probe hybridization library, be transferred on the filter membrane.Used film is Amersham company (Amersham Biosciences, Orsay, the Hybond N nylon membrane of France) selling and in Church and the described damping fluid of Gilbert (Church and Gilbert, 1984), hybridizing at 55 ℃.55 ℃ were washed 15 minutes in 2X SSC, in 0.5X SSC, washed twice continuously for 55 ℃ then, each 15 minutes.Under these hybridization and wash conditions, in 2000 hybridization clones, there are 4 clones to present strong hybridization signal.These 4 clones are incubated in LB substratum+penbritin (50 μ g/ml), and extract corresponding 4 clays through standard. alkaline type cracking (Sambrook etc., 1989).Proved that then hybridization is owing to be present in the dna fragmentation that inserts in these 4 clays really.For this reason, use several kinds of enzymes (BamHI, PstI and SacI) digestion clay respectively.The separating digesting product is transferred on the nylon membrane on sepharose, and under above-mentioned identical condition, hybridizes with the NaeI-NaeI dna fragmentation that comprises streptomyces fradiae tylB Gene Partial (above the reference).This might verify these 4 clays and more specifically be and the called after pOS49.1 that selects in these clays.

2.2 checking institute identifies regional participation and to the sequential analysis of clay pOS49.1 insertion portion

Several fragments of subclone clay pOS49.1 insertion portion are also confirmed their sequence.With SacI enzymic digestion clay pOS49.1, and show that through the Southern trace that carries out under these conditions the 3.3kb fragment comprises the zone with the tylB probe hybridization.Separate this 3.3kb fragment through electroelution from 0.8% sepharose, then it is cloned into carrier pUC19 (GenBank accession number: M77789) also order-checking.The plasmid called after pOS49.11 that so obtains.Use FramePlot program (J.Ishikawa and K.Hotta, 1999), in this fragment, can identify 4 and present the ORFs that typical streptomycete codon selects (two complete with ORFs two brachymemmas).Utilize the FASTA program (referring to (W.R.Pearson & D.J Lipman; 1988) and (W.R.Pearson; 1990), especially can obtain from the INFOBIOGEN of French Evry resource center) sequence of carrying out relatively makes it possible to show protein and streptomyces fradiae TylB protein (the SEQ ID No.87 that is derived by one of these 4 ORFs; GenBank accession number: U08223) present strong sequence similarity (with reference to Figure 25).This protein called after Orf3 (SEQ ID No.29).

Whether participate in giving birth to the Spiramycin Base biosynthesizing in the dyadic streptomycete in order to test corresponding gene (orf3 gene (SEQ ID No.28)), with Ω hyg box (M-H.Blondelet-Rouault etc., 1997, GenBank accession number: X99315) interrupt this gene.For this reason; With XhoI enzymic digestion plasmid pOS49.11; And will comprise these 4 ORFs (two complete and ORFs two brachymemmas; Comprise total length orf3) the fragment subclone go into Stratagene company (LaJolla, California, the XhoI site of the carrier pBC SK+ that USA) sells.The plasmid called after pOS49.12 that so obtains.For inactivation orf3, Ω hyg box is cloned into back one plasmid to replace the PmlI-BstEII fragment in the orf3 through the blunt ends clone.For this reason, with PmlI and BstEII enzymic digestion plasmid pOS49.12, a PmlI and BstEII site are only arranged in the orf3 gene coded sequence.Through handle the segmental terminal blunt ends that produces of respective carrier with Klenow enzyme (the big fragment of dna polymerase i).Through with BamHI enzymic digestion plasmid pHP45 Ω hyg (Blondelet-Rouault etc., 1997, GenBank accession number: X99315) obtain Ω hyg box.On sepharose, reclaim the fragment of corresponding Ω hyg box, and make its end become blunt ends through handling with the Klenow enzyme.Two the blunt ends fragments (Ω hyg box and plasmid pOS49.12) that so obtain are connected and will connect product be used for the transformed into escherichia coli bacterium.The plasmid called after pOS49.14 that so obtains and this plasmid comprise the orf3 gene that is interrupted with Ω hyg box.

To exist and its two ends handle to form the terminal plasmid pOS49.14 of non-sticky through the Klenow enzyme and insert fragment cloning and go into the EcoRV site of plasmid pOJ260 (plasmid pOJ260 can duplicate in intestinal bacteria and the conjugative plasmid (M.Bierman etc., 1992) that can not in giving birth to the dyadic streptomycete, duplicate with the XhoI-XhoI pieces.This plasmid is given the apramycin resistance in intestinal bacteria and streptomycete).Resulting plasmid (the insertion fragment cloning of plasmid pOS49.14 is gone into plasmid pOJ260) called after pOS49.16.Use the coli strain S17-1 that engages, of (Mazodier et al., 1989) such as Mazodier, through engaging back one plasmid is transferred to living dyadic streptomycete bacterial strain ATCC23877.Coli strain S17-1 derives from coli strain 294 (Simon etc., 1983) (Simon etc., 1986).Might obtain trans joint clone, these clones have the entrained hygromycin resistance mark of Ω hyg box and have lost the entrained apramycin resistance marker of carrier pOJ260.For this reason, after the joint, select to have the clone of hygromycin resistance.Then Totomycin-resistance clone is gone down to posterity respectively to be incubated at and have Totomycin (microbiotic B) and have on the substratum of apramycin (microbiotic A) (with reference to Fig. 9).By and large, Totomycin (HygR) is had resistance and be those clones of dual recombination event wherein having taken place and therefore having had the orf3 gene that is interrupted by Ω hyg box the responsive clone of apramycin (ApraS).Replace by the copy that is interrupted through two successive hybridization proof wild-type orf3 copies.Therefore, have box in order to prove in gained cloned genes group DNA, the total DNA with plurality of enzymes digestion institute DCRP separates on sepharose, it is transferred on the film and with the probe of corresponding Ω hyg box (with reference to top) hybridize.Use contains the XhoI-XhoI fragment of plasmid pOS49.11 of four ORFs (two complete and two brachymemmas, comprise whole orf3) as probe, carries out the hybridization second time.Any means of knowing by one of skill in the art, and the PCR through utilizing suitable oligonucleotide and to the sequential analysis of PCR product especially also can be carried out genotypic checking.Selected an orf3::. Ω hyg who so obtains to clone and, this cloned genes type is verified with its called after OS49.16.

Gained OS49.16 like this clone's the Spiramycin Base output (with reference to embodiment 15) of having used following turnout test determines.Therefore might prove that this bacterial strain no longer produces Spiramycin Base, confirm orf3 and/or be positioned the for example orf4 involved in spiramycin biosynthesizing of gene in downstream.

In case obtained this confirmation, just to the big zone of clay pOS49.1, any side of the SacI fragment of former research has been carried out sequential analysis.Therefore, might obtain this regional sequence from clay pOS49.1, this zone comprises seven complete ORFs and other two the imperfect ORFs that are positioned these seven any sides of ORFs.Through in DB, searching for, might show that one of imperfect ORFs is corresponding to srmG locus (coding is called the zone of " polyketide " enzyme (PKS)).S.Burgett etc., corresponding gene has been cloned in 1996 (USP 5,945,320).And, in DB, do not find other ORFs: seven complete ORF called afters: orf1, orf2, orf3; Orf4, orf5, orf6 and orf7 (SEQ ID No23; 25,28,30; 34,36 and 40) and the starting point of the 8th ORF of called after orf8 (having provided the full sequence of this orf among the SEQ ID No.43).

Embodiment 3: the separation and the sign of participating in giving birth to biosynthetic other gene of Spiramycin Base in the dyadic streptomycete

Secondly, in this same area, contain segmental other clay of living dyadic streptomyces gene group in order to clone biosynthetic other gene of involved in spiramycin, to have separated.For this reason, use following three probes to carry out other a series of colony hybridization:

BamHI-PstIDNA fragment (the S.Burgett etc. that contain the PKS gene fragment of the corresponding 3.7kb of-employed article one probe; 1996 (USPs 5; 945,320) cloned corresponding PKS gene), from orf1, orf2 and the orf3 starting point of pOS49.1 subclone; The BamHI site of containing 1 300 base pairs in the upper reaches, EcoRI site that are positioned to be defined as among the SEQ ID No.1 position 1 is to the PstI site that is positioned 2472 positions (SEQ ID No.1).This BamHI-PstI fragment is gone into plasmid pBC SK+ from the pOS49.1 subclone, and this makes might obtain plasmid pOS49.28.The corresponding approximately 2kb's of-employed second probe contains orf7 and orf 8 segmental PstI-BamHIDNA fragments; This fragment obtains from the pOS49.1 subclone, contains from the PstI site of 6693 positions that are positioned SEQ ID No.1 to the BamHI site of 8714 positions that are positioned SEQ ID No.1.This PstI-BamHI fragment is gone into plasmid pBC SK+ from the pOS49.1 subclone, and this makes might obtain plasmid pOS49.76.

-also used the 3rd probe.The EcoRI-HindIII dna fragmentation that comprises the srmD gene of the corresponding 1.8kb of this probe.The srmD gene is to separate the gene that can give the Spiramycin Base resistance that obtains from giving birth to the dyadic streptomycete.Particularly; Previous research has made it possible to clone several RDs of living dyadic streptomycete; Said RD is given grey pale red yellow streptomycete (S.griseofuscus) bacterial strain (Spiramycin Base sensitive strain) Spiramycin Base resistance (Pernodet etc.; 1993) (Pernodet etc., 1999).For the separation resistance gene, in clay pKC505, prepared the cosmid library (M.A.Richardson etc., 1987) of living dyadic streptomycete bacterial strain ATCC23877 genomic dna.Therefore, for obtain size about 30 and 40kb between fragment, carried out partly digestion to giving birth to dyadic streptomycete bacterial strain ATCC23877 genomic dna with Sau3AI.The genomic dna that will so digest (3 μ g) be connected (Pernodet etc., 1999) with 1 μ g pKC505 of BamHI enzymic digestion in advance.To connect then that mixture is external to be packaged in the phage particle.The phage particle that is obtained has been used for ehec infection bacterial strain HB101 (especially can be from American type culture collection (ATCC) (Manassas, Virginia USA) obtain, and preserving number is 33694).Collect about 20000 apramycin resistance escherichia coli clonings and extracted these clones' clay.This clay storehouse is imported grey pale red yellow streptomycete strain DSM 10191 (K.L.Cox and R.H.Baltz, 1984) through protoplast transformation, this bacterial strain is natural in the responsive (R.N.Rao etc. of Spiramycin Base; 1987; This bacterial strain especially from German mikrobe and cell culture preservation center (Deutsche Sammlung von Mikro-organismen und ZellkulturenGmbH, DSMZ), (Braunschweig; Germany) obtain, be numbered DSM 10191).Screened transformant containing on the substratum of apramycin.1300 are transferred on the substratum that contains 5 μ g/ml Spiramycin Bases containing the clone who grows on the substratum of apramycin.There are several apramycin resistance clones to grow on the substratum that contains Spiramycin Base, and extracted the clay of these bacterium colonies and used it for transformed into escherichia coli and grey pale red yellow streptomycete (Pernodet etc., 1999).Therefore 5 clays that can give intestinal bacteria apramycin resistance and give grey pale red yellow streptomycete apramycin and the common resistance of Spiramycin Base have been obtained.In these 5 clays; Confirmed the clay of called after pOS44.1; Insert in the fragment at it, contain gene (SEQ ID No.15), the protein of this coded by said gene (SEQ ID No.16) presents certain similarity with the protein (SEQ ID No.88) of Streptomyces Macrofaciens mdmA coded by said gene; This unnamed gene is srmD (comparison of carrying out with reference to the use FASTA program shown in Figure 26 (with reference to (W.R.Pearson and D.J.Lipman, 1988) and (W.R.Pearson, 1990), especially can available from the INFOBIOGEN resource center of French Evry)).

In order to separate the RD that is contained among the plasmid pOS44.1; Use the Sau3AI Restriction Enzyme partly to digest this plasmid and be the fragment of about 1.5-3kb to obtain size; And these fragments are connected into among the linearizing carrier pIJ486 of BamHI enzyme (Ward etc., 1986).Select plasmid (R.N.Rao etc., 1987) according to its ability of in natural grey pale red yellow streptomycete strain DSM 10191 (above the reference), giving the Spiramycin Base resistance to the Spiramycin Base sensitivity.For this reason, through protoplast transformation, the segmental plasmid of the pOS44.1 Sau3AI storehouse (with reference to top) that has connected into accordingly among the carrier pIJ486 is imported strain DSM 10191, and screen its thiostrepton resistance (because tsr gene that pIJ486 carries).Be transferred to and contain on the Spiramycin Base substratum growing in clone on the sulfur-bearing Streptothrix peptide substratum.Several thiostrepton resistance clones also grow on the substratum that contains Spiramycin Base and extract the plasmid of these bacterium colonies.Selection can be given resistance and contain about 1.8kb and inserted segmental plasmid and called after pOS44.2.Because the insertion portion both sides exist HindIII site and EcoRI site in the carrier, insert fragment so can easily excise this 1.8kb.This 1.8kb HindIII-EcoRI is inserted that fragment is carried out sequential analysis and with its resistant gene called after srmD that contains.Therefore can easily this fragment subclone that contains the srmD gene be gone into carrier pUC19 (the GenBank accession number: M77789) and with gained plasmid called after pOS44.4 of cutting with EcoRI-HindIII.The 1.8kb HindIII-EcoRI that contains the srmD gene in this plasmid inserts fragment and is used as probe with setting screw mycin biosynthesis gene (with reference to following).

The sample that contains the e.colistraindh5 of plasmid pOS44.4 is preserved in (CNCM) Institute Pasteur of state-run microbial preservation center [National Collection of Cultures and Microorganisms]; 25; Rue du Docteur Roux 75724Paris Cedex 15; France, preservation day is on July 10th, 2002, preserving number is I-2918.

According to routine techniques (Sambrook etc., 1989), about 2000 clones in the library that top (with reference to embodiment 1) obtained are transferred on the filter membrane to carry out colony hybridization.

Use random primer technology (test kit that Roche sells) to use ³²Above-mentioned three probes of P mark also are used to hybridize 2000 clones in library, are transferred on the filter membrane then.In Church and the described damping fluid of Gilbert (Church and Gilbert, 1984), hybridize at 65 ℃.With 65 ℃ of 2XSSC washing 15 minutes and use 65 ℃ of continuous washing twice of 0.5X SSC then, washed 15 minutes at every turn.Under these hybridization and wash conditions, 16 clones and at least one probe among 2000 clones that hybridized present strong hybridization signal.Yet, do not have the clay of all hybridizing with three probes.Extracting these 16 clays also digests with the BamHI Restriction Enzyme.The restriction map of these different clays is compared feasible two clays of selecting each other, and they tend to contain maximum insertion district and do not have identical band.Therefore, select this two clays, a called after pSPM5 and another called after pSPM7.Clay pSPM5 and probe orf1-orf4 and probe orf8 hybridization, but do not hybridize with probe srmD.PSPM7 only with probe srmD hybridization and not with other two probe hybridizations.

Use " air gun sequential analysis " technology that these 2 clays are carried out the sufficient sequence analysis.Although this 2 clay: pSPM7 and pSPM5, the sequence of insertion portion is not overlapping, all comprises known array at an end of each insertion portion, so can the sequence of these 2 clay insertion portions be carried out assembled arrangement.Particularly, each insertion portion all comprises the sequence fragment that a coding is called the gene of " polyketide synthase " enzyme (PKS).S.Burgett etc., this 5 genes (with reference to Fig. 2) have been cloned in 1996 (USP 5,945,320).Therefore, might confirm single living dyadic streptomyces gene group dna sequence dna.Provided the sequence of 30 943 Nucleotide among the SEQ ID N ° 1, this sequence is initial and extend to the BamHI site of 3 ' position in the EcoRI site of 5 ' position from be positioned first PKS gene.The zone (with reference to Fig. 2 and 3) of the corresponding PKS upstream region of gene of this sequence.Provided second sequence area of 11 171 Nucleotide among the SEQ ID No. 2, this zone is initial and extend to 3 ' position and be positioned the 5th the NcoI site the PKS gene from PstI site, 5 ' position.The zone that this second sequence area is PKS gene downstream (by all with the direction of localized 5 the PKS genes of the equidirectional definition downstream and the upper reaches) (with reference to Fig. 2 and 3).

Embodiment 4: the definite and sign of the nucleotide sequence analysis of the biosynthetic gene of involved in spiramycin, ORFs

Use FramePlot programanalysis institute's calling sequence (J.Ishikawa and K.Hotta 1999).This might identify from ORFs and present the ORFs that typical streptomycete codon is selected.This analysis might confirm that this zone comprises 35 ORF that are positioned coding " polyketide synthase " 5 any sides of gene (PKS).Identify 10 and 25 ORF (by all with the direction definition downstream and the upper reaches of localized 5 the PKS genes of equidirectional) (with reference to Fig. 3) respectively at the downstream of these genes and the upper reaches.Therefore, identify 25 ORFs of the type (SEQ ID No.1 and Fig. 3) of occupying about 31kb zone and identify 10 ORFs of the type (SEQ ID No.2 and Fig. 3) that occupy about 11.1kb zone in PKS gene downstream at 5 PKS upstream region of gene.The unnamed gene of upstream region is orf1, orf2, orf3, orf4, orf5, orf6, orf7, orf8, orf9c, orf10, orf11c, orf12; Orf13c, orf14, orf15c, orf16, orf17, orf18, orf19, orf20, orf21c, orf22c, orf23c, orf24c and orf25c (SEQ ID No23; 25,28,30,34,36,40,43,45,47,49,53,60; 62,64,66,68,70,72,74,76,78,80,82 and 84).The unnamed gene of downstream area is orf1 ^*C, orf2 ^*C, orf3 ^*C, orf4 ^*C, orf5 ^*, orf6 ^*, orf7 ^*C, orf8 ^*, orf9 ^*And orf10 ^*(SEQ ID No3,5,7,9,11,13,15,17,19 and 21).For the ORF that is discussed, be added to " c " meaning in the gene title be meant encoding sequence be reciprocal (so coding strand be with SEQ ID No.1 or SEQ ID No.2 in the sequence complementary chain of given these genes) (with reference to Fig. 3).

Use that existing those sequences compare in protein sequence that various programs will be derived by these ORFs and the several data storehouse: BLAST (Altschul etc., 1990) (Altschul etc., 1997), CD-search, (these three programs especially can be from (the Bethesda of NCBI (NCBI) for COGs (Cluster of Orthologous Groups); Maryland USA) obtains), FASTA ((W.R.Pearson and D.J.Lipman, 1988) and (W.R.Pearson; 1990); BEAUTY (K.C.Worley etc., 1995)), (these two programs especially can be from INFOBIOGEN resource center; Evry, France obtains).These relatively make it possible to clearly describe the biosynthetic gene of those possibility involved in spiramycin of function hypothesis and evaluation about these gene products.

Embodiment 5: gene inactivation: make up the principle that knocks out living dyadic streptomycete bacterial strain

Method therefor comprises and carries out gene substitution.The target gene that the copy replacement of the target gene that interrupts with the box that can give microbiotic (for example apramycin or Totomycin) resistance as shown in Figure 9, will interrupt.Employed box any side adjacent all read in frames translation stop codon and in streptomycete the active transcription terminator of tool.

With box insert target gene can with or can be without the deletion in this target gene.The size of box two side areas can be individual to several thousand base pairs from hundreds of.

Obtain in intestinal bacteria with the required construct of box inactivation gene, intestinal bacteria be obtain the recombinant DNA construction body with reference to organism.Obtain the gene that interrupted in the plasmid that can not in streptomycete, duplicate can in intestinal bacteria, duplicating.

Then the construct subclone is gone into carrier to allow to transform and give birth to the inactivation of goal gene in the dyadic streptomycete.For this reason, used two kinds of plasmids:

-pOJ260 (M.Bierman etc., 1992) (with reference to embodiment 2), this plasmid give the apramycin resistance and when target gene interrupts with the box that can give hygromycin resistance, use this plasmid in intestinal bacteria and streptomycete.

-pOSK1205(4726bp)。This plasmid derives from plasmid pBK-CMV (by (LaJolla of Stratagene company; California; USA) sell), the AvrII fragment that wherein contains the sequence of coding Xin Meisu/kalamycin resistance is replaced by the sequence of coding hygromycin resistance, and has kept the PSV40 promotor simultaneously.For this reason; With NotI and PflmI enzymic digestion plasmid pHP45-Ω hyg (Blondelet-Rouault etc.; 1997), and when handle with the Klenow enzyme make all ends become blunt ends after, the fragment subclone of giving hygromycin resistance is gone into the AvrII site of carrier pBK-CMV.In pOSK1205, the pSV40 promotor is in the box front of giving hygromycin resistance.This plasmid is given hygromycin resistance and when the box that target gene is endowed the apramycin resistance interrupts, is used in intestinal bacteria and streptomycete.

Restriction enzyme site through existing in this target gene is cloned, and perhaps through the reorganization between the identical sequence of above-mentioned weak point, for example through the method for (M.K.Chaveroche et al., 2000) such as Chaveroche, box is imported in the target gene.

Then, for example through the conjugation (P.Mazodier etc., 1989) between intestinal bacteria and the streptomycete, can the plasmid that carry the gene that is interrupted by box be imported and give birth to the dyadic streptomycete.When underlying carrier is carrier pOJ260, using should technology.In order to increase frequency, also can use second technology: through the technology of the protoplast transformation behind the alkaline purification denatured DNA (T.Kieser etc., 2000) like Oh and the said reorganization of Chater (Oh & Chater, 1997).When underlying carrier is pOJ260 or pOSK1205, using should technology (below the reference).Use microbiotic (with reference to Fig. 9, microbiotic B) to screen these transformant then corresponding to existing box in the target gene.So select clone's mixture, wherein integrate through recombination event once or twice.Next, found out the clone responsive to the microbiotic (with reference to Fig. 9, microbiotic A) of existing resistant gene in the carrier (reorganization box outside).Therefore might filter out some such clones in principle, wherein carry out twice recombination event, cause wild type gene to be replaced by the copy that is interrupted by box.These step diagrams are shown in Fig. 9.

Several boxes can be used in and interrupt target gene.For example can use Ω hyg box (Blondelet-Rouault etc., 1997, the GenBank accession number: X99315) of giving hygromycin resistance.

Embodiment 6: the structure that in the orf3 gene, has the living dyadic streptomycete bacterial strain that homophase knocks out

Interrupt orf 3 genes (with reference to embodiment 2.2) with Ω hyg box, and might prove that orf3::. Ω hyg bacterial strain no longer produces Spiramycin Base, this confirms one or more gene involved in spiramycin biosynthesizing (with reference to embodiment 2.2) in the zone of cloning.Consider their direction, but corotation record ORF1-7 (with reference to Fig. 3), and viewed phenotype (non-helical mycin is produced bacterium) possibly be because the inactivation of the gene of one or more and the record of orf3 corotation.In order to confirm the biosynthesizing of orf3 involved in spiramycin, carried out another time inactivation of orf3 gene, back one inactivation is that homophase carries out.For this reason, deleted the DraIII fragment of 504 base pairs in the orf3.To be cloned into plasmid pOJ260 (M.Bierman etc. by the dna fragmentation that pOS49.1 obtains; 1992); This dna fragmentation is contained from the EcoRI site that is positioned at position 1 (SEQ ID No.1) to the SacI site that is positioned at position 5274 (SEQ ID No.1), and between two DraIII sites of position 2563 and 3067, comprises deletion (having removed 504 Nucleotide).The plasmid called after pOS49.67 that so obtains.

So the insertion portion of pOS49.67 comprises the following dna fragmentation of living dyadic streptomycete, this fragment comprises orf1 gene, orf2 gene, has the part of orf3 gene, orf4 gene and the orf5 of homophase deletion.The carrier that wherein this insertion portion is carried out subclone is pOJ260, so plasmid pOS49.67 can give the apramycin resistance and through protoplast transformation it imported bacterial strain OS49.16 (with reference to embodiment 2).Because bacterial strain OS49.16 is a hygromycin resistance, has obtained hygR and apraR transformant.This type of is cloned in goes down to posterity on the non-selection substratum after twice, just found apramycin and the responsive clone of Totomycin (apraS and hygS).In some clones, the recombination event between the homologous sequence expects that in fact causing replacing the orf3 that interrupts with Ω hyg box with the orf3 copy with homophase deletion that exists on the carrier copies (being contained in the bacterial strain OS49.16 genome).Expect that the clone who is obtained by this reorganization is apraS and hygS after removing the carrier sequence.Can obtain the genotype (only there is the orf3 copy of a homophase deletion in proof in gained cloned genes group) of this bacterial strain through hybridization or through PCR and the sequential analysis of PCR product.Therefore obtained only to have the homophase deletion the orf3 copy the clone and confirmed their genotype.Also screen more in particular to appear and expect the clone and the called after OS49.67 of characteristic.

The sample of bacterial strain OS49.67 is preserved in state-run microbial preservation center (CNCM) Institute Pasteur, and 25, rue du Docteur Roux 75724 Paris Cedex 15, France, preservation day is on July 10th, 2002, preserving number is I-2916.

Embodiment 7: the structure with the living dyadic streptomycete bacterial strain that knocks out in the orf8 gene

In order to carry out the orf8 gene inactivation, obtained wherein Ω hyg box to be imported the construct of orf8 encoding sequence.For this reason, at first made up plasmid pOS49.88.3.7kb fragment through will containing orf7 end, orf8 and orf9 initiating terminal (the PstI-EcoRI fragment that obtains from clay pSPM5) is cloned into the PstI-EcoRI site of pUC19, from pUC19 (GenBank accession number: M77789) obtain plasmid pOS49.88.After making all terminal blunt endsization, Ω hyg box (with through handle the segmental form of BamHI of blunt endsization with the Klenow enzyme) is cloned into unique SalI site of the pOS49.88 that is positioned orf8 with the processing of Klenow enzyme.

Because the clone is a blunt ends, the direction that depends on the box insertion has obtained two types plasmid: pOS49.106, and wherein hyg and orf8 gene are in the same way, and pOS49.120, and wherein hyg and orf8 gene are reverse.Then the insertion portion subclone of plasmid pOS49.106 is gone into plasmid pOJ260, to produce pOS49.107.For this reason, make terminal blunt endsization with Asp718I enzymic digestion plasmid pOS49.106 and through handling with the Klenow enzyme; Fragment cloning with the PstI enzyme digests this digestion product once more and will contain the orf8 gene that has wherein inserted Ω hyg box is gone into carrier pOJ260 (with reference to top).For this reason, with EcoRV with PstI enzymic digestion carrier pOJ260 and be used to be connected.Because it is blunt ends and opposite side is a PstI enzyme simple stage property end that each fragment in these two fragments is a side, so this operation makes it possible to obtain directed the connection.Gained plasmid called after pOS49.107.

Through protoplast transformation plasmid pOS49.107 is imported living dyadic streptomycete bacterial strain ATCC23877 (T.Kieser etc., 2000).After the protoplast transformation, screening has the clone of hygromycin resistance.Upload to be commissioned to train at substratum that contains Totomycin (microbiotic B) and the substratum that contains apramycin (microbiotic A) respectively then and support these Totomycin-resistance clones (with reference to Fig. 9).Basically, Totomycin (HygR) is had resistance and be those clones of the double exchange incident wherein having taken place and having contained the orf8 gene that interrupts by Ω hyg box the responsive clone of apramycin (ApraS).Verified that through the Southern blotting orf8 copy that is interrupted by Ω hyg box has replaced orf8 wild-type copy.Therefore, have box in order to verify among the gained cloned genes group DNA, the total DNA with several kinds of enzymic digestion institute DCRPs separates on sepharose, transfers on the film and with the probe of corresponding Ω hyg box to hybridize.The PstI-EcoRI insertion portion that uses plasmid pOS49.88 is as probe, and this part size is about 3.7kb, comprises orf7 end, orf8 and orf9 start-up portion, carries out the hybridization second time.Any means of knowing by one of skill in the art, and, can carry out genotypic checking especially through utilizing PCR that suitable oligonucleotide carries out and the PCR product being carried out sequential analysis.

Filter out orf8:: Ω hyg clone and called after OS49.107.The sample of bacterial strain OS49.107 is preserved in state-run microbial preservation center (CNCM) Institute Pasteur, and 25, rue du DocteurRoux 75724Paris Cedex 15, France, preservation day is on July 10th, 2002, preserving number is I-2917.

Embodiment 8: have the living dyadic streptomycete bacterial strain that knocks out in the structure orf10 gene

Through being template, using following primer to carry out PCR and obtained 1.5kb dna fragmentation in the orf10 gene to give birth to dyadic streptomyces gene group DNA:

SRMR1：5’CTGCCAGTCCTCTCCCAGCAGTACG?3’(SEQ?ID?No.89)

SRMR2：5’TGAAGCTGGACGTCTCCTACGTCGG?3’(SEQ?ID?No.90)

To be cloned into Invitrogen company (Carlsbad, California, the carrier pCR2.1 that USA) is sold from the dna fragmentation of PCR.The plasmid called after pOS49.32 that so obtains.Through handling after all terminal blunt endsization, Ω hyg box (with the BamHI pieces, with reference to top) is cloned into BstEII site unique in the orf10 gene fragment with the Klenow enzyme.Because the clone is a blunt ends, the direction of inserting according to box has obtained two types plasmid: pOS49.43, and wherein hyg and orf10 gene are in the same way, and pOS49.44, and wherein hyg and orf10 gene are reverse.The insertion portion of plasmid pOS49.43 (with the Asp718I-XbaI pieces, through handled its latter end blunt ends change with the Klenow enzyme) is transferred to the EcoRV site of plasmid pOJ260, and this makes might obtain plasmid pOS49.50.The plasmid pOS49.50 that will contain the orf10 gene fragment that is interrupted by Ω hyg box imports and gives birth to dyadic streptomycete bacterial strain ATCC23877.After the conversion, screening has the clone of hygromycin resistance.Upload to be commissioned to train at substratum that contains Totomycin (microbiotic B) and the substratum that contains apramycin (microbiotic A) respectively then and support these Totomycin-resistance clones (with reference to Fig. 9).In principle, Totomycin (HygR) is had resistance and be those clones of the double exchange incident wherein having taken place and having contained the orf10 gene that interrupts by Ω hyg box the responsive clone of apramycin (ApraS).The clone who has so obtained to contain the hygromycin resistance mark that box carries and lost the apramycin resistance marker that carrier pOJ260 carries.Verified by orf10: through the Southern blotting: the orf10 copy that Ω hyg box interrupts replaces the incident of orf8 wild-type copy.Therefore, have box in order to verify among the gained cloned genes group DNA, the total DNA with several kinds of enzymic digestion institute DCRPs separates on sepharose, transfers on the film and with the probe of corresponding Ω hyg box to hybridize.Carry out the hybridization second time (with reference to top) with 1.5kb PCR product in the orf10 gene as probe.

Screened more in particular and demonstrated expection characteristic (orf10:: clone Ω hyg) and called after OS49.50.Hybridize through two-wheeled; In fact might verify that Ω hyg box is present in this cloned genes group really; And verified after dual group of incident, in this cloned genes group, used under the situation of the copy replacement wild type gene that interrupts by Ω hyg box, obtained the digestion spectrum of expection really.Any means of knowing by one of skill in the art, and particularly be through utilizing suitable oligonucleotide PCR and to the sequential analysis of PCR product, also can carry out genotypic checking.

Embodiment 9: gene inactivation: the principle (with reference to Fig. 9 and 10) of the living dyadic streptomycete bacterial strain that knocks out according to " can excise box " technique construction

Can be used in second type box of gene inactivation: the box that is called " can excise box ".These boxes have such advantage: after being imported into living dyadic streptomyces gene group, and can be through the site-specific recombination event with its excision in streptomycete.Purpose is some gene in the inactivation streptomycete bacterial strain and in final bacterial strain, do not lose selective marker or have the big dna sequence dna do not belong to this bacterial strain.After the excision, the short sequence of only about 30 left and right sides base pairs (being called " scar " site) is retained in (with reference to Figure 10) in the strain gene group.

Implementing this system at first comprises the wildtype target gene copy alternative with wherein having inserted the construct (through two homologous recombination incidents, with reference to Fig. 9) that can excise box.The insertion of this box is attended by the deletion (with reference to Fig. 9) in the target gene simultaneously.Secondly, carry out to excise box from the excision of strain gene group.Can excise box through the site specific recombination system functionating, and have the advantage that acquisition is not finally carried the streptomycete two mutants of resistant gene.Also avoided the polar effect (with reference to Figure 10) of expression and localization in the gene in inactivation gene downstream.

Describe the application that to excise box and can excise box and can be used for multiple organism, comprised (Bayley etc., 1992 in mammalian cell and yeast cell and the intestinal bacteria; Brunelli and Pall, 1993; Camilli etc., 1994; Dale and Ow, 1991; Russell etc., 1992; Lakso etc., 1992).These can excise box and all use lox site acting Cre site-specific recombinase.This recombination system comes from the P1 bacteriophage.

In order in streptomycete, to make up " can excise box " type system, developed the site specific recombination system that is used for giving birth to dyadic streptomycete mobile genetic element pSAM2 (Boccard etc., 1989a and b).The foundation of this system comprises that at first structure contains the recombinant vectors that is interrupted gene, has wherein inserted and can excise box.Can excise box insertion target gene and be attended by the deletion in the target gene.Clone through existing restriction enzyme site in the use target gene, perhaps, can carry out this insertion through the reorganization between (ChaverocheMK etc., 2000) described short identical sequences such as for example Chaveroche.For example, use Ω hyg box to make up and to excise box (Blondelet Rouault etc., 1997).This box edge is attR and attL sequence, is positioned at the flank (with reference to Figure 15) of the pSAM2 copy of integration under these two sequence normal circumstancess.AttR and attL sequence comprise permission excision pSAM2 or the required whole sites (Sezonov etc., 1997, Raynal etc., 1998) of the segmental site-specific reorganization of any DNA between these two zones.Clearly, the structure of this kind box is not limited to and uses Ω hyg box, and other resistance box also can be as basic box to make up this kind box (for example Ω aac or Ω vph box (BlondeletRouault etc., 1997)).

After having obtained this construct, with this recombinant plasmid transformed streptomycete bacterial strain.Use antibiotic-screening transformant (with reference to Fig. 9, microbiotic B derives from Ω hyg box if for example can excise box, and this relates to the use hygromycin selection) then to existing box in the target gene.Therefore screened wherein clone's mixture of integrating through single or twice recombination event.Subsequently, found the responsive clone of microbiotic (with reference to Fig. 9, microbiotic A) to the resistant gene that exists in the carrier (reorganization box outside).Therefore by screening some clones like this, wherein carried out twice recombination event basically, the gene copy that causes wild type gene to be interrupted by box is replaced.Illustrate these steps among Fig. 9; Verified gained cloned genes type like this and filtered out the bacterial strain that presents expection characteristic (having replaced wild type gene) through the Southern blotting by excising the copy that box interrupts.

Secondly, with the bacterial strain that is screened above the plasmid conversion that allows xis and int genetic expression, reorganization all is essential for the site-specific between attR and the attL site for xis and int gene.This reorganization causes excising box and leaves strain gene group (with reference to Figure 10) (Raynal etc., 1998) through recombination event.(for example derive from the carrier of streptomycete carrier pWHM3 from metastable carrier streptomycete; (Vara etc.; 1989) carrier of selecting to carry xis and int gene) is favourable; This makes might obtain such bacterial strain, and after carrying out several gemma formation circulations under the shortage selective pressure condition, this bacterial strain has been lost a back carrier.

In order to excise box, for example might use plasmid pOSV508 (with reference to Figure 14), through protoplast transformation this plasmid is imported and contain the living dyadic streptomycete bacterial strain that can excise the gene that box interrupts.Plasmid pOSV508 derives from plasmid pWHM3 (J.Vara etc., 1989) (with reference to Figure 13), and (F.Boccard etc. 1989b), and place under the control of ptrc promotor (E.Amann etc., 1988) in this plasmid, to have added xis and the int gene of pSAM2.Go into plasmid pWHM3 (Raynal etc., 1998) (with reference to Figure 14) with placing xis and int gene under the control of ptrc promotor from plasmid pOSint3 subclone.The plasmid pOSV508 that carries pSAM2xis and int gene is imported mutants which had, will allow to recombinate through site-specific, effective excision (A.Raynal etc., 1998) of between the attL of box both sides and attR site, carrying out excising box (Figure 10).In these transformant, select thiostrepton is produced resistance and there is the transformant of the antibiotic sensitive of the resistance that produces in box, wherein the thiostrepton resistance produces (with reference to Figure 10) by the tsr gene that pOSV508 carries.Excision is that transformant effective and that observe more than 90% belongs to this type.Lack on the solid medium of thiostrepton through one take turns above growth and gemma form circulate after, obtained to lose the clone of plasmid pOSV508.Can detect these clones through them to the susceptibility of thiostrepton.Can verify the sequence of the target gene of being deleted through PCR with to the sequential analysis of PCR product.

At last, resulting bacterial strain in the target gene of inactivation (for example inner deletion), carries " scar " att site (Raynal etc., 1998) in the minimum attB of a correspondence site, and this site derives from the reorganization between attR and the attL site.The minimum attB site of these remnants with give birth to dyadic streptomycete, beginning and revolve naturally occurring this site similar (Sezonov etc., 1997) in streptomycete (Streptomyces pristinaespiralis) and the shallow Streptomyces glaucoviolaceus.

Expect its inactivation gene can with other gene corotation record that is positioned downstream.The inactivation that has one of the gene of polar effect for fear of the expression to the operon downstream gene obtains the homophase deletion after importantly excising box.The above-mentioned box system of excising makes and might satisfy this demand.In fact, those skilled in the art can make up three different excised boxes at an easy rate, the sequence of said box remaining 33,34 or 35 Nucleotide of difference after excision, and in any reading frame, do not contain terminator codon.If the size of the sequence of having known target gene and the deletion relevant with the box insertion portion selects excision to produce the homophase deletion to such an extent as to then might can excise at these three between the box.In 33,34 or 35 Nucleotide that added, 26 Nucleotide correspondences are arranged in minimum attB sequence (with reference to Figure 27).

About the application, used two can excise box.These two boxes are following: att1 Ω hyg+ (SEQID No.91) and att3 Ω aac-(SEQ ID No.92); These boxes of excision back are remaining 33 and 35 Nucleotide respectively.They comprise Ω hyg box or Ω aac box respectively ,+with the direction of the corresponding resistance box of-symbol.Make up these two boxes and it is cloned into the EcoRV site of carrier pBC SK+, its HindIII site is by deletion in advance.The gained plasmid is called after patt1 Ω hyg+ and patt3 Ω aac-respectively.Through using the EcoRV digested plasmid, can obtain at an easy rate to excise box.

Embodiment 10: have the living dyadic streptomycete bacterial strain that knocks out in the structure orf2 gene

Use can be excised the inactivation (with reference to top) that the box technology is carried out the orf2 gene.Used initial strain is the living dyadic streptomycete bacterial strain OSC2 that derives from strains A TCC23877.Yet bacterial strain OSC2 is different from strains A TCC23877, because bacterial strain OSC2 has lost movably gene pSAM2 (Boccard etc., 1989a and b).During protoplasma turned usefulness (the mycelial effect of N,O-Diacetylmuramidase bacterial digestion wall and fragmentation (Kieser et al., 2000)) and strains A TCC23877 protoplast regeneration into, possible spontaneity was lost this removable factor.In order to select to have lost the clone of pSAM2 element, transcribe the restraining effect of inhibition based on KorSA to the pra gene, set up a kind of screening (Sezonov etc., 1995) (G.Sezonov etc., 2000).For this reason; The dna fragmentation that will contain the pra gene promoter that places aph gene (give kalamycin resistance and lack its oneself the promotor) upper reaches is cloned into unstable carrier pWHM3Hyg; The latter derives from plasmid pWHM3 (Vara etc.; 1989), wherein the tsr gene is replaced by hyg gene (giving hygromycin resistance).The plasmid called after pOSV510 that so obtains.After strains A TCC23877 is carried out the protoplast formation effect, transform bacterial strain ATCC23877 with plasmid pOSV510.Promotor Pra is the promotor that a kind of KorSA of receiving repressor checks, and the coding latter's gene is placed in (SezonovG. etc., 2000) in the pSAM2 displaceable element.After transforming with plasmid pOSV510, the bacterium after the conversion is screened because it has kalamycin resistance (coming from the aph gene that is carried by pOSV510).Have the clone that the pSAM2 integrated element loses and lost the KorSA repressor, and the Pra promotor is no longer checked and allowed to express the aph kalamycin resistance gene.Might filter out the clone that those have been lost pSAM2 integrated element (and therefore losing KorSA) and have contained plasmid pOSV510 with making with the kantlex screening again after the plasmid pOSV510 conversion.Because plasmid pOSV510 is unstable, under not containing the microbiotic condition, takes turns after gemma forms through several, containing on the substratum of kantlex, containing on the substratum of Totomycin and do not contain antibiotic substratum and upload the foster clone who is separated to that is commissioned to train.Kantlex and the responsive clone of Totomycin have been lost pOSV510.Verified the deletion of pSAM2 element through hybridization and PCR.Choose to appear and expect the clone and the called after OSC2 of characteristic.

The sample of bacterial strain OSC2 is preserved in state-run microbial preservation center (CNCM) Institute Pasteur, and 25, rue du Docteur Roux 75724 Paris Cedex 15, France, preservation day is on July 10th, 2002, preserving number is I-2908.

Use can be excised the inactivation (with reference to top and Figure 10) that the box technology is carried out the orf2 gene.For this reason; With the 4.5kb insertion portion; Its sequence originates in the EcoRI site that is positioned position 1, ends at the BamHI site that is positioned position 4521 (SEQ ID No.1), and subclone is gone into plasmid pUC19 (the GenBank accession number: EcoRI M77789) and BamHI site from clay pSPM5.Gained plasmid called after pOS49.99 like this.

This plasmid is imported coli strain KS272, and this bacterial strain has contained plasmid pKOBEG (Chaveroche etc., 2000) (with reference to Figure 12).

Parallel therewith; (plasmid pOSK1102 is the plasmid (Chaveroche etc. that derive from carrier pGP704Not to utilize plasmid pOSK1102; 2000) (V.L.Miller and J.J.Mekalanos, 1988), wherein att3 Ω aac-box is gone into EcoRV site unique among the pGP704Not as the EcoRV fragment cloning) as template and utilize following primer; Can excise box (SEQ ID No.92 is with reference to top) through pcr amplification att3 Ω aac-:

ORF2A

(SEQ?ID?No.93)

ORF2B

(SEQ?ID?No.94)

Be positioned these oligonucleotide 5 ' 40 terminal deoxynucleotides and comprise sequence, and be positioned the sequence (with reference to Figure 11) that the corresponding att3 Ω aac-of 20 deoxynucleotides (shown in the top runic) of 3 ' position can excise an end of box corresponding to sequence in the target gene (being orf2 in this example).

The PCR product that so obtains is used to transform the coli strain (Chaveroche etc., 2000) (with reference to Figure 12) that contains above-mentioned plasmid pKOBEG and pOS49.99.Therefore, through the electroporation transform bacteria and screen its apramycin resistance.The plasmid that extracts institute's DCRP is also with several kinds of Restriction Enzymes digestion; Purpose is to have inserted target gene (orf2) in order to verify like compartmentalized box for holding assorted fruits and candies (att3 Ω aac-); If homologous recombination (Chaveroche etc. have promptly taken place between the end of PCR product and target gene really; 2000), then resulting digestion spectrum is consistent with expection digestion spectrum.Known by one of skill in the art method, especially PCR and the sequential analysis of PCR product through utilizing suitable oligonucleotide also can be carried out the checking of construct.Choose that plasmid wherein has the clone of expection digestion spectrum and with corresponding plasmid called after pSPM17.This plasmid derives from pOS49.99, and wherein orf2 interrupts (with reference to Figure 12) by the apramycin box.The insertion of box is accompanied by the deletion between the Nucleotide 211 and 492 of encoding part among the orf2.With EcoRI enzymic digestion plasmid pSPM17 and through handling terminal blunt endsization with the Klenow enzyme; The insertion portion that also will contain through deletion orf2 gene with this product of this digestion of XbaI enzymic digestion then is cloned into carrier pOSK1205 (above the reference).For this reason, with BamHI enzymic digestion carrier pOSK1205 and through handling terminal blunt endsization with the Klenow enzyme; Then should this product of digestion with the XbaI enzymic digestion and with its with as above be connected from pSPM17 acquisition insertion portion.Since these two segmental each be that blunt ends and opposite side are that XbaI is terminal in a side, therefore this operation makes and might obtain the orientation connection.The plasmid called after pSPM21 that so obtains; It carry hygromycin gene (carrier part) and wherein tool through the orf2 gene of deletion by the insertion portion of att3 Ω aac-box replacement.

Through protoplast transformation (T.Kieser etc., 2000) carrier pSPM21 is imported living dyadic streptomycete bacterial strain OSC2 (with reference to top).After the conversion, through these clones of its apramycin resistance screening.On the substratum that contains apramycin (microbiotic B), upload foster these the apramycin-resistance clones (with reference to Fig. 9) of being commissioned to train respectively then with the substratum that contains Totomycin (microbiotic A).In principle, apramycin is had resistance (ApraR) and be that those double exchange incident have wherein taken place and have contained the clone who is interrupted the orf2 gene by att3 Ω aac-box the clone of Totomycin responsive (HygS).Choose these clones and verified that box interrupts the replacement of copy to orf2 wild-type copy.Verified the existence of att3 Ω aac-box through bacterium colony PCR.Also hybridize.For this reason, the total DNA with several kinds of enzymic digestion institute DCRPs separates on sepharose, transfers on the film and with the segmental probe of corresponding plasmid pOS49.99 insertion portion EcoRI-BamHI to hybridize (with reference to top).Known by one of skill in the art any means, especially PCR and the sequential analysis of PCR product through utilizing suitable oligonucleotide also can be carried out genotypic checking.

Selection appears expects the clone and the called after SPM21 of characteristic.Utilize PCR and hybridization, might prove to have att3 Ω aac-box in this clone gene group really and obtained really in this clone gene group owing to interrupt with att3 Ω aac-box after dual group of incident and expect that the digestion that obtains composes behind the copy replacement wild type gene.Therefore this clone has genotype: orf2::att3 Ω aac-.

The sample of bacterial strain SPM21 is preserved in state-run microbial preservation center (CNCM) Institute Pasteur, and 25, rue du Docteur Roux 75724 Paris Cedex 15, France, preservation day is on July 10th, 2002, preserving number is I-2914.

In order to produce the excision of box,, transform bacterial strain SPM21 (with reference to Figure 14) with carrier pOSV508 through the protoplast transformation method.Plasmid pOSV508 derives from plasmid pWHM3 (J.Vara etc., 1989) (with reference to Figure 13), and (F.Boccard etc., xis 1989b) and int gene also place under the control of ptrc promotor (E.Amann etc., 1988) (with reference to Figure 14) wherein to have added pSAM2.Through the site-specific reorganization, (Figure 10) with the excised box (A.Raynal etc., 1998) between plasmid pOSV508 importing bacterial strain SPM21 permission this box both sides attL of effective excision that carries pSAM2 xis and int gene and the attR site.In these transformant; Selection produces resistance and the transformant responsive to apramycin to thiostrepton; Wherein the thiostrepton resistance is produced by the tsr gene that pOSV508 carries, and the apramycin resistance is given by the entrained resistant gene of att3 Ω aac-box; In fact excision causes the deletion (with reference to Figure 10) of apramycin resistant gene.Plasmid pOSV508 be unsettled and, on not containing antibiotic substratum, carry out after the two-wheeled continuous passage, on the substratum of sulfur-bearing Streptothrix peptide and the substratum of sulfur-bearing Streptothrix peptide is not uploaded to be commissioned to train and is supported isolating clone.The responsive clone of thiostrepton has lost pOSV508.Through PCR and the sequential analysis of PCR product, verified that excising this box causes the homophase deletion in the orf2 gene really; The excision box has in fact stayed characteristic " scar " att3 sequence (after recombinating between attL and the attR site, this sequence is similar with the initiation site of attB):

5’ATCGCGCGCGCTTCGTTCGGGACGAAGAGGTAGAT?3’(SEQ?ID?No.95)。The bacterial strain called after SPM22 that so obtains with expection genotype (orf2::att3).

The sample of bacterial strain SPM22 is preserved in state-run microbial preservation center (CNCM) Institute Pasteur, and 25, rue du Docteur Roux 75724 Paris Cedex 15, France, preservation day is on July 10th, 2002, preserving number is I-2915.

Embodiment 11: have the living dyadic streptomycete bacterial strain that knocks out in the structure orf12 gene

For inactivation orf12, orf13c and orf14, identical initial plasmid (pSPM504) is used for importing in different positions the box of " can excise box " type.This plasmid comprises corresponding 15.1kb insertion portion regional from orf7 to orf17.In order to make up this plasmid, will go into plasmid pMBL18 (Nakano etc., 1995) from the 15.1kb BglII fragment cloning that digestion clay pSPM7 (above the reference) obtains with BamHI digestion.Because BamHI and BglII end are compatible, obtain plasmid pSPM502 after the connection.Then whole insertion portions (with the HindIII/NheI pieces) subclone of pSPM502 is gone into plasmid pOSK1205 (digesting with HindIII/NheI), this makes might obtain plasmid pSPM504.

This plasmid is imported the coli strain KS272 (Chaveroche etc., 2000) (with reference to Figure 12) that has contained plasmid pKOBEG.

Parallel therewith; (plasmid pOSK1102 is the plasmid (Chaveroche etc. that derive from carrier pGP704Not to utilize plasmid pOSK1102; 2000) (V.L.Miller and J.J.Mekalanos; 1988), wherein att3 Ω aac-box is gone into EcoRV site unique among the pGP704Not as the EcoRV fragment cloning) as template and utilize following primer, can excise box through pcr amplification att3 Ω aac-:

EDR8：

(SEQ?ID?No.96)

EDR9：

(SEQ?ID?No.97)

Be positioned these oligonucleotide 5 ' 40 terminal (for only 39 of EDR8) deoxynucleotides and comprise sequence, and be positioned 20 deoxynucleotides (shown in the top runic) of 3 ' position can excise an end of box corresponding to att3 Ω aac-sequence (with reference to Figure 11) corresponding to sequence in the target gene (being orf12 in this example).

The PCR product that so obtains is used for transforming (Chaveroche etc. such as containing Chaveroche; 2000) the coli strain KS272 of said plasmid pKOBEG and pSPM504 is (with reference to Figure 12; In principle; Plasmid pOS49.99 will be replaced by plasmid pSPM504, and the gained plasmid no longer is pSPM17 but pSPM507).Therefore, through the electroporation transform bacteria and screen its apramycin resistance.The plasmid that extracts institute's DCRP is also with several kinds of Restriction Enzymes digestion; Purpose is to have inserted target gene (orf12) in order to verify like compartmentalized box for holding assorted fruits and candies (att3 Ω aac-); If homologous recombination (Chaveroche etc. have promptly taken place between the end of PCR product and target gene really; 2000), then resulting digestion spectrum is consistent with expection digestion spectrum.Known by one of skill in the art method, especially PCR and the sequential analysis of PCR product through utilizing suitable oligonucleotide also can be carried out the checking of construct.Choose that plasmid wherein has the clone of expection digestion spectrum and with corresponding plasmid called after pSPM507.This plasmid derives from pSPM504, and wherein orf12 interrupts (with reference to Figure 12) by att3 Ω aac-box.The insertion of box is accompanied by the deletion among the orf12, interrupts the 30 codon that starts from orf12.Box has been preserved last 46 codons of orf12 at the back.

Through protoplast transformation (T.Kieser etc., 2000) carrier pSPM507 is imported living dyadic streptomycete bacterial strain OSC2 (with reference to top).After the conversion, through these clones of its apramycin resistance screening.On the substratum that contains apramycin (microbiotic B), upload foster these the apramycin-resistance clones (with reference to Fig. 9) of being commissioned to train respectively then with the substratum that contains Totomycin (microbiotic A).In principle, apramycin is had resistance (ApraR) and be those clones of the double exchange incident wherein having taken place and having contained the orf12 gene that interrupts by att3 Ω aac-box the clone of Totomycin responsive (HygS).More specifically choose these clones and verified that through hybridization box interrupts the replacement of copy to orf12 wild-type copy.Therefore, have box in order to prove among the gained cloned genes group DNA, the total DNA with several kinds of enzymic digestion institute DCRPs separates on sepharose, transfers on the film and with the probe of corresponding att3 Ω aac-box and hybridizes.With obtain through PCR and the very most dna fragmentation of corresponding orf12 gene coded sequence as probe, carried out hybridization for the second time.Known by one of skill in the art any means, especially PCR and the sequential analysis of PCR product through utilizing suitable oligonucleotide also can be carried out genotypic checking.

More specifically screen to appear and expect the clone (orf12::att3 Ω aac-) and the called after SPM507 of characteristic.Through two-wheeled hybridization, in fact might prove to have att3 Ω aac-box in this clone gene group really and obtained really in this clone gene group owing to expect behind the copy replacement wild type gene that interrupts with att3 Ω aac-box after dual group of incident that the digestion that obtains composes.Therefore this clone has genotype: orf12::att3 Ω aac-and called after SPM507.Provided the direction (with reference to Fig. 3) of these genes, need not excise the effect of this box with research orf12 inactivation.Particularly, the localized in the opposite direction fact of orf13c and orf12 shows that these genes are not the corotation records.On the other hand, use can be excised box makes the possibility of removing selection markers at any time become possibility, especially through transforming plasmid pOSV508.The sample of bacterial strain SPM507 is preserved in state-run microbial preservation center (CNCM) Institute Pasteur, and 25, rue du Docteur Roux 75724 Paris Cedex 15, France, preservation day is on July 10th, 2002, preserving number is I-2911.

Embodiment 12: have the living dyadic streptomycete bacterial strain that knocks out in the structure orf13c gene

Utilize plasmid pOSK1102 (with reference to top) as template, and use following primer, can excise box through pcr amplification att3 Ω aac-:

EDR3：

(SEQ?ID?No.98)

EDR4：

(SEQ?ID?No.99)

Be positioned these oligonucleotide 5 ' 40 terminal deoxynucleotides and comprise the sequence of sequence in the corresponding target gene (being orf13c in this example), and be positioned the sequence (with reference to Figure 11) that the corresponding att3 Ω aac-of 20 deoxynucleotides (shown in the top runic) of 3 ' position can excise an end of box.

The PCR product that so obtains is used for transforming (Chaveroche etc. such as containing Chaveroche; 2000) the coli strain KS272 of said plasmid pKOBEG and pSPM504 is (with reference to Figure 12; In principle; Plasmid pOS49.99 will be replaced by plasmid pSPM504, and the gained plasmid no longer is pSPM17 but pSPM508).Therefore, use this PCR product transform bacteria and screen the clone of apramycin resistance through electroporation.The plasmid that extracts institute's DCRP is also with several kinds of Restriction Enzymes digestion; Purpose is to have inserted target gene (orf13c) in order to verify like compartmentalized box for holding assorted fruits and candies (att3 Ω aac-); If homologous recombination (Chaveroche etc. have promptly taken place between the end of PCR product and target gene really; 2000), then resulting digestion spectrum is consistent with expection digestion spectrum.Known by one of skill in the art method, especially PCR and the sequential analysis of PCR product through utilizing suitable oligonucleotide also can be carried out the checking of construct.Choose that plasmid wherein has the clone of expection digestion spectrum and with corresponding plasmid called after pSPM508.This plasmid derives from pSPM504, and wherein orf13c interrupts (with reference to Figure 12) by the apramycin box.The insertion of box is accompanied by the deletion in the orf13c gene, interrupts the 6th codon that starts from orf13c.Box has kept last 3 codons of orf13c at the back.

Through protoplast transformation (T.Kieser etc., 2000) carrier pSPM508 is imported living dyadic streptomycete bacterial strain OSC2 (with reference to top).After the conversion, through these clones of its apramycin resistance screening.On the substratum that contains apramycin (microbiotic B), upload foster these the apramycin-resistance clones (with reference to Fig. 9) of being commissioned to train respectively then with the substratum that contains Totomycin (microbiotic A).In principle, apramycin is had resistance (ApraR) and be those clones of the double exchange incident wherein having taken place and having contained the orf13c gene that interrupts by att3 Ω aac-box the clone of Totomycin responsive (HygS).More specifically be to choose these clones and verified that through hybridization box interrupts the replacement of copy to orf13c wild-type copy.Therefore, have box in order to prove among the gained cloned genes group DNA, the total DNA with several kinds of enzymic digestion institute DCRPs separates on sepharose, transfers on the film and with the probe of corresponding att3 Ω aac-box and hybridizes.The PCR product of containing about 100 base-pair sequences of orf13c encoding sequence upstream and downstream with correspondence has carried out hybridization for the second time as probe.Known by one of skill in the art any means, especially PCR and the sequential analysis of PCT product through utilizing suitable oligonucleotide also can be carried out genotypic checking.

More specifically screen to appear and expect the clone (orf13c::att3 Ω aac-) and the called after SPM508 of characteristic.Through two-wheeled hybridization, in fact might prove to have att3 Ω aac-box in this clone gene group really and obtained really in this clone gene group owing to interrupt with att3 Ω aac-box after dual group of incident and expect that the digestion that obtains composes behind the copy replacement wild type gene.Therefore this clone has genotype: orf13c::att3 Ω aac-and called after SPM508.Provided the direction (with reference to Fig. 3) of these genes, need not excise the effect of this box with research orf13c inactivation.The localized in the opposite direction fact of orf14 and orf13c shows that these genes are not the corotation records.On the other hand, use can be excised the feasible possibility of removing selection markers at any time of box becomes possibility.The sample of bacterial strain SPM508 is preserved in state-run microbial preservation center (CNCM) Institute Pasteur, and 25, rue du DocteurRoux 75724Paris Cedex 15, France, preservation day is on July 10th, 2002, preserving number is I-2912.

Embodiment 13: have the living dyadic streptomycete bacterial strain that knocks out in the structure orf14 gene

EDR5：

(SEQ?ID?No.100)

EDR6：

(SEQ?ID?No.101)

Be positioned these oligonucleotide 5 ' 40 terminal deoxynucleotides and comprise the sequence of sequence in the corresponding target gene (being orf14 in this example), and be positioned the sequence (with reference to Figure 11) that the corresponding att3 Ω aac-of 20 deoxynucleotides (shown in the top runic) of 3 ' position can excise an end of box.

The PCR product that so obtains is used for transforming (Chaveroche etc. such as containing Chaveroche; 2000) the coli strain KS272 of said plasmid pKOBEG and pSPM504 is (with reference to Figure 12; In principle; Plasmid pOS49.99 will be replaced by plasmid pSPM504, and the gained plasmid no longer is pSPM17 but pSPM509).Therefore, use this PCR product transform bacteria and screen the clone of apramycin resistance through electroporation.The plasmid that extracts institute's DCRP is also with several kinds of Restriction Enzymes digestion; Purpose is to have inserted target gene (orf14) in order to verify like compartmentalized box for holding assorted fruits and candies (att3 Ω aac-); If homologous recombination (Chaveroche etc. have promptly taken place between the end of PCR product and target gene really; 2000), then resulting digestion spectrum is consistent with expection digestion spectrum.Known by one of skill in the art method, and especially PCR and the sequential analysis of PCR product through utilizing suitable oligonucleotide also can be carried out the checking of construct.Choose that plasmid wherein has the clone of expection digestion spectrum and with corresponding plasmid called after pSPM509.This plasmid derives from pSPM504, and wherein orf14 interrupts (with reference to Figure 12) by the apramycin box.The insertion of box is accompanied by the deletion in the orf14 gene, interrupts the 4th codon that starts from orf14.Box has kept the last codon of orf13c at the back.

Through protoplast transformation (T.Kieser etc., 2000) carrier pSPM509 is imported living dyadic streptomycete bacterial strain OSC2 (with reference to top).After the conversion, through these clones of its apramycin resistance screening.On the substratum that contains apramycin (microbiotic B), upload foster these the apramycin-resistance clones (with reference to Fig. 9) of being commissioned to train respectively then with the substratum that contains Totomycin (microbiotic A).In principle, apramycin is had resistance (ApraR) and be those clones of the double exchange incident wherein having taken place and having contained the orf14 gene that interrupts by att3 Ω aac-box the clone of Totomycin responsive (HygS).More specifically be to choose these clones and verified that through hybridization box interrupts the replacement of copy to orf14 wild-type copy.Therefore, have box in order to prove among the gained cloned genes group DNA, the total DNA with several kinds of enzymic digestion institute DCRPs separates on sepharose, transfers on the film and with the probe of corresponding att3 Ω aac-box and hybridizes.The PCR product of containing about 100 base-pair sequences of orf14 encoding sequence upstream and downstream with correspondence has carried out hybridization for the second time as probe.Known by one of skill in the art any means, especially PCR and the sequential analysis of PCT product through utilizing suitable oligonucleotide also can be carried out genotypic checking.

More specifically screen to appear and expect the clone (orf14::att3 Ω aac-) and the called after SPM509 of characteristic.Through two-wheeled hybridization, in fact might prove to have att3 Ω aac-box in this clone gene group really and obtained really in this clone gene group owing to interrupt with att3 Ω aac-box after dual group of incident and expect that the digestion that obtains composes behind the copy replacement wild type gene.Therefore this clone has genotype: orf14::att3 Ω aac-and called after SPM509.Provided the direction (with reference to Fig. 3) of these genes, need not excise the effect of this box with research orf14 inactivation.The localized in the opposite direction fact of orf15c and orf14 shows that these genes are not the corotation records.On the other hand, use can be excised the feasible possibility of removing selection markers at any time of box becomes possibility.The sample of bacterial strain SPM509 is preserved in state-run microbial preservation center (CNCM) Institute Pasteur, and 25, rue du Docteur Roux75724 Paris Cedex 15, France, preservation day is on July 10th, 2002, preserving number is I-2913.

Embodiment 14: make up orf6 ^*Has the living dyadic streptomycete bacterial strain that knocks out in the gene

Use can be excised the box technology and carried out orf6 ^*The inactivation of gene (with reference to top and Figure 10).For this reason, use clay pSPM7 as template and the following oligonucleotide amplification of use orf6 ^*The fragment of gene:

C9583:

(oligonucleotide intentionally)

(SEQ?ID?No.102)

C9584:

(antisense oligonucleotide)

(SEQ?ID?No.103)

Be positioned these oligonucleotide 3 ' 20 terminal deoxynucleotide correspondences and be positioned orf6 ^*The sequence (SEQ ID No.13) of genes encoding part and be positioned the sequence that 6 deoxynucleotide correspondences of 5 ' position are beneficial to clone's PstI site.The dna fragmentation size of amplification is about 1.11kb.The PCR product cloning is gone into carrier pGEM-T Easy (by Promega company (Madison, Wisconsin USA) sell), this feasible plasmid (with reference to Figure 16) that might obtain called after pBXL1111.

Then att1 Ω hyg+ can be excised box and import orf6 ^*Gene coded sequence.For this reason, also handle digestion product with SmaI and Asp718I Restriction Enzyme digested plasmid pBXL1111 with the Klenow enzyme.This operation makes might be at orf6 ^*Produce the inside deletion (with reference to Figure 15) of 120bp in the gene coded sequence.In addition, in the both sides of restriction enzyme site, kept the orf6 of 511bp and 485bp respectively ^*Sequence, this carries out homologous recombination with inactivation orf6 with allowing ^*Gene.Through with this plasmid of EcoRV digestion, prepare att1 Ω hyg+ from plasmid patt1 Ω hyg+ (above the reference) and can excise box.Then latter's subclone is gone into as stated the carrier pBXL1111 of preparation in advance (SmaI and Asp718I digest and handle with the Klenow enzyme then).Gained plasmid called after pBXL1112 (with reference to Figure 17).In this construct, orf6 ^*Gene comprises the deletion of 120bp and is interrupted by att1 Ω hyg+ box.

Use PstI enzyme (this site at the box avris, thereby be present in the PCR oligonucleotide) digested plasmid pBXL1112 then, and will contain the orf6 that has interrupted by att1 Ω hyg+ box then ^*The 3.7kbPstI of part inserts fragment cloning and goes into the PstI site of plasmid pOJ260 (with reference to top).The plasmid called after pBXL1113 that so obtains.

Through protoplast transformation (T.Kieser etc., 2000) carrier pBXL1113 is imported living dyadic streptomycete bacterial strain OSC2 (with reference to top).After the conversion, through these clones of its hygromycin resistance screening.On the substratum that contains Totomycin (microbiotic B), upload foster these the Totomycin-resistance clones (with reference to Fig. 9) of being commissioned to train respectively then with the substratum that contains apramycin (microbiotic A).In principle, Totomycin is had resistance (HygR) and be that those double exchange incident have wherein taken place and have contained the orf6 that is interrupted by att1 Ω hyg+ box the clone of apramycin responsive (ApraS) ^*The clone of gene.More specifically be to choose these clones and verified that through the Southern engram technology box interrupts copy to orf6 ^*The replacement of wild-type copy.Therefore, have box in order to prove among the gained cloned genes group DNA, the total DNA with several kinds of enzymic digestion institute DCRPs separates on sepharose, transfers on the film and with the probe (obtaining through PCR) of corresponding hyg gene to hybridize.Utilization is by the orf6 that contains of plasmid pBXL1111 acquisition ^*The PstI-PstI of gene inserts fragment (size is about 1.1kb) as probe, has carried out hybridization for the second time (with reference to top and Figure 16).Known by one of skill in the art any means, especially PCR and the sequential analysis of PCR product through utilizing suitable oligonucleotide also can be carried out genotypic checking.

More specifically screen and present expection characteristic (orf6 ^*:: clone att1 Ω hyg+) and called after SPM501.Through two-wheeled hybridization, in fact might prove to have att1 Ω hyg+ box in this clone gene group really and obtained really in this clone gene group owing to interrupt with att1 Ω hyg+ box after dual group of incident and expect that the digestion that obtains composes behind the copy replacement wild type gene.Therefore this clone has genotype: orf6 ^*:: att1 Ω hyg+ and called after SPM501.The sample of bacterial strain SPM501 is preserved in state-run microbial preservation center (CNCM) Institute Pasteur, and 25, rue du DocteurRoux 75724 Paris Cedex 15, France, preservation day is on July 10th, 2002, preserving number is I-2909.

In order to excise box,, transform bacterial strain SPM501 (with reference to Figure 14) with carrier pOSV508 through protoplast transformation.Plasmid pOSV508 derives from plasmid pWHM3 (J.Vara etc., 1989) (with reference to Figure 13), and (F.Boccard etc. 1989b), and place under the control of ptrc promotor (E.Amann etc., 1988) in this plasmid, to have added xis and the int gene of pSAM2.The plasmid pOSV508 that carries pSAM2 xis and int gene is imported bacterial strain SPM501, will allow to recombinate through site-specific, effective excision (A.Raynal etc., 1998) of between the attL of box both sides and attR site, carrying out excising box (Figure 10).In these transformant, select thiostrepton is produced resistance and the transformant responsive to Totomycin, wherein the thiostrepton resistance is produced by the tsr gene that pOSV508 carries, and hygromycin resistance is given by the entrained resistant gene of att1 Ω hyg+ box; In fact this excision causes the deletion (with reference to Figure 10) of resistant gene.Plasmid pOSV508 be unsettled and, after not containing on the antibiotic substratum through the two-wheeled continuous passage, with the substratum of sulfur-bearing Streptothrix peptide on the substratum that is cloned in sulfur-bearing Streptothrix peptide that is separated to and not upload be commissioned to train foster.The responsive clone of thiostrepton has lost pOSV508.Through PCR and the sequential analysis of PCR product, verified at orf6 ^*The excision of box has taken place in certain homophase in the gene.Interrupt and start from the 158th codon, deleted 40 codons (120bp), the excision of box has kept characteristic " scar " the att1 sequence of 33bp:

5’ATCGCGCGCTTCGTTCGGGACGAAGAGGTAGAT?3’(SEQ?ID?No.104)。

That so obtain and have an expection genotype (orf6 ^*:: bacterial strain called after SPM502 att1).

The sample of bacterial strain SPM502 is preserved in state-run microbial preservation center (CNCM) Institute Pasteur, and 25, rue du Docteur Roux 75724 Paris Cedex 15, France, preservation day is on July 10th, 2002, preserving number is I-2910.

Embodiment 15: at orf2, orf3, orf8, orf10, orf12, orf13c, orf14 or orf6 ^*The analysis that has the living dyadic streptomycete bacterial strain that knocks out in the gene

The Spiramycin Base output of the multi-strain bacteria strain that obtains in order to test has been researched and developed the microbiological assay (with reference to (A.Gourmelen etc., 1998)) based on micrococcus luteus bacterial strain susceptibility.(this bacterial strain especially can be available from German mikrobe and cell culture preservation center (Deutsche Sammlung vonMikro-organismen und Zellkulturen GmbH to the responsive strain DSM 1790 of Spiramycin Base derived from natural for used micrococcus luteus bacterial strain; DSMZ); (Braunschweig; Germany), preserving number is DSM1790); Because bacterial strain uses therefor has resistance to killing firm trypanosome element in this test, so be different from strain DSM 1790.This bacterial strain is through killing and select the spontaneous mutation strain that obtains on the plain substratum of firm trypanosome containing cumulative dosage.Produce Spiramycin Base and kill plain the two the fact of firm trypanosome in view of giving birth to the dyadic streptomycete, screened this bacterial strain.Because purpose is in order to use based on the microbiological assay of micrococcus luteus bacterial strain susceptibility the Spiramycin Base output of the multi-strain bacteria strain that obtained to be measured, so be necessary to obtain killing firm trypanosome element-resistant strain.

With the many streptomyces strains that will test be incubated in the tool plate washer 500ml Erlenmeyer flask (Erlenmeyer flask of band plate washer) that contains 70ml MP5 substratum (Pernodet etc., 1993).Many strains are given birth to the dyadic streptomycete bacterial strain with 2.5 * 10 ⁶The initial concentration of gemma/ml is inoculated in the Erlenmeyer flask of being with plate washer and at 27 ℃ of 250 rev/mins of orbit determination oscillating growths.Cultivate and take out 2ml suspension-s sample and centrifugal after 48,72 and 96 hours.Then that various supernatants are frozen in-20 ℃.To be used for test (with reference to Figure 18) with these supernatants of 10 times of dilutions of aseptic culture medium.

Will be to killing that firm trypanosome element has resistance and the responsive micrococcus luteus indicator of Spiramycin Base is killed in the plain 2TY substratum (Sambrook etc., 1989) of firm trypanosome 37 ℃ and cultivated 48 hours containing 5 μ g/ml.Measure the optical density(OD) (OD) of culture and dilute this culture so that optical density(OD) is adjusted to 4.This preparatory culture of 0.4ml is diluted in the 40ml DAM5 substratum (Difco microbiotic substratum 5 is sold by Difco company), makes temperature reach about 45 ℃ in advance.Pour into this substratum in 12 * 12cm plate then and be placed on room temperature.

In case substratum cooling is also solidified, be that the Whatman AA filter paper disk (with reference to A.Gourmelen etc., 1998) of 12mm soaks with every kind of supernatant of 10 times of dilutions of 70 μ l with diameter, and be positioned over the plate surface.To use different concns (2-4-8 μ g/ml in the MP5 substratum) Spiramycin Base solution wetted filter paper disk as the standard filter paper dish.In order to allow microbiotic to be dispensed into agar, with these plates place 4 ℃ 2 hours and hatched 24-48 hour at 37 ℃ then.

If this filter paper disk contains Spiramycin Base, Spiramycin Base diffuses into agar and suppresses the growth of micrococcus luteus indicator.This restraining effect produces " swooning " around filter paper disk, this is swooned and has reflected the zone that the micrococcus luteus bacterial strain is not grown.Therefore should dizzy existence be the indication that Spiramycin Base exists, and make the livings dyadic streptomycete bacterial strain that to confirm the corresponding filter paper disk of being discussed whether be or be not Spiramycin Base production bacterium.The inhibition diameter that is obtained compared with the inhibition diameter of standard filter paper dish make the indication of amount of the Spiramycin Base might obtain to produce by this bacterial strain.

In order to detect its Spiramycin Base output, used the multi-strain bacteria strain described in the previous embodiment in this test.The gained result is summarized in table 38.

Table 38

The feasible conclusion that might draw the some amount relevant of these results with the function of the biosynthetic range gene of involved in spiramycin.Therefore, biosynthesizing is essential to the orf3 gene for Spiramycin Base.Particularly, this intragenic homophase inactivation causes forming the bacterial strain (OS49.67, (with reference to embodiment 6)) that no longer produces Spiramycin Base.The homophase inactivation makes the box that might abandon importing might influence this hypothesis of genetic expression that is positioned the orf3 downstream.

Likewise, because bacterial strain OS49.107 and OS49.50 have nonproductive bacterium phenotype, so orf8 and the necessary protein of orf10 genes encoding Spiramycin Base biosynthesizing.In addition, in these two bacterial strains, owing to consider the direction (with reference to Fig. 3) of different orf, clearly being responsible for the phenotypic corresponding gene of this nonproductive bacterium is inactivation, and the construct that is imported can not have polar effect.

Might draw the some amount conclusion relevant to the research with the bacterial strain that can excise box is feasible with the function that is interrupted gene.Bacterial strain SPM507 has genotype: orf12::att3 Ω aac-.Consider the direction (with reference to Fig. 3) of gene, think to there is no need to excise the effect of box with research orf12 inactivation.The localized in the opposite direction fact of orf13c and orf12 shows that these genes are not the corotation records.On the other hand, can excise the feasible possibility that might obtain to remove at any time selection markers of use of box.The phenotype of bacterial strain SPM507 is nonproductive bacterium; Therefore can infer that the orf12 gene is to give birth to the necessary gene of Spiramycin Base biosynthesizing in the dyadic streptomycete by it.

Bacterial strain SPM508 has genotype: orf13c::att3 Ω aac-.Consider the direction (with reference to Fig. 3) of gene, think to there is no need to excise the effect of box with research orf13c inactivation.The localized in the opposite direction fact of orf14 and orf13c shows that these genes are not the corotation records.On the other hand, can excise the feasible possibility that might obtain to remove at any time selection markers of use of box.The phenotype of bacterial strain SPM508 is to produce bacterium; Therefore can infer that the orf13c gene is not to give birth to the necessary gene of Spiramycin Base biosynthesizing in the dyadic streptomycete by it.

Bacterial strain SPM509 has genotype: orf14::att3 Ω aac-.Consider the direction (with reference to Fig. 3) of gene, think to there is no need to excise the effect of box with research orf14 inactivation.The localized in the opposite direction fact of orf15c and orf14 shows that these genes are not the corotation records.On the other hand, can excise the feasible possibility that might obtain to remove at any time selection markers of use of box.The phenotype of bacterial strain SPM509 is nonproductive bacterium; Therefore can infer that the orf14 gene is to give birth to the necessary gene of Spiramycin Base biosynthesizing in the dyadic streptomycete by it.

Bacterial strain SPM21 has genotype: orf2::att3 Ω aac-.This bacterial strain has the nonproductive bacterium phenotype of Spiramycin Base.Yet gene orf1 is corotation records to these genes of direction hint of orf8.Therefore, viewed phenotype possibly be owing to import the box of orf2 the expression of gene that is positioned these operon downstream to be had polar effect.Bacterial strain SPM22 has genotype orf2::att3, and is that the box homophase excision back that is imported obtains.Characteristic " scar " sequence (with reference to embodiment 10) that the excision of box is only residual.Because bacterial strain SPM22 also has nonproductive bacterium phenotype, can be to give birth to the necessary gene of Spiramycin Base biosynthesizing in the dyadic streptomycete by its orf2 gene of reaching a conclusion.Here only observed the effect that causes by the orf2 inactivation.

Bacterial strain SPM501 has genotype: orf6 ^*:: att1 Ω hyg+.This bacterial strain has the nonproductive bacterium phenotype of Spiramycin Base.Yet, because orf5 ^*And orf6 ^*Gene (with reference to Fig. 3) has identical direction, and viewed phenotype possibly be owing to import orf6 ^*Box to orf5 ^*Expression have polar effect.They possibly be the corotation record for the arrangement hints of these genes.In order to answer this problem, obtained bacterial strain SPM502 behind the box that the homophase excision is imported.In this bacterial strain, only observed by orf6 ^*The effect that inactivation causes.This bacterial strain has genotype orf6 ^*:: att1 (with reference to embodiment 14).Homophase " scar " sequence (with reference to embodiment 14) that the excision of box is only residual.Bacterial strain SPM502 has the bacterium of production phenotype, and (yet this bacterial strain only produces Spiramycin I (reference ^*Embodiment 16)).Therefore can be by its orf5 that reaches a conclusion ^*Gene is to give birth to the necessary gene of Spiramycin Base biosynthesizing in the dyadic streptomycete, owing in bacterial strain SPM501, its indirect inactivation is caused nonproductive bacterium phenotype.On the other hand, orf6 ^*Gene is not to give birth to the necessary gene of Spiramycin I biosynthesizing in the dyadic streptomycete (on the other hand, it is to produce spiramycin II and III necessary (with reference to embodiment 16)).

Embodiment 16: the mensuration of Spiramycin I, II and III output in the mutants which had that is obtained

With the multi-strain bacteria strain that will test be incubated at separately in 7 500ml tool plate washer Erlenmeyer flasks that contain 70ml MP5 substratum (Pernodet etc., 1993).Many strains are given birth to the dyadic streptomycete bacterial strain with 2.5 * 10 ⁶Gemma/ml is inoculated in the Erlenmeyer flask and 27 ℃ of 250 rev/mins of orbit determination oscillating growths 72 hours.Collect corresponding identical clone's culture, randomly filter, and 7000 rev/mins centrifugal 15 minutes through pleating filter.Then various supernatants are stored in-30 ℃.

(HPLC) measures through the ion pairing HPLC.The HPLC of substratum analyzes the feasible concentration that might accurately measure three kinds of form of screw mycins.Fill used pillar (Macherey-Nagel) mutually with Nucleosil octyl group grafted silicon-dioxide.Particle size be 5 μ m and hole size be 100

.The internal diameter of pillar is that 4.6mm and length are 25cm.Moving phase is H ₃PO ₄Damping fluid (pH2.2) and contain 6.25g/L NaClO ₄The acetonitrile of perchlorate is with 70/30 (v/v) blended mixture.Flow velocity with fixed 1ml/ minute is analyzed in permanent solvent systems.With the pillar temperature control in 23 ℃.Pass through UV spectrophotometer measuring at 238nm.Also pass through the area estimation quantitative (through external calibration) at peak at+10 ℃ of refrigeration samples.In these cases, the RT of Spiramycin I, II and III is respectively about 17; 21 and 30 minutes, the commercialization sample that this use contains three kinds of form of screw mycins can prove.

Bacterial strain OSC2 has Spiramycin Base and produces the bacterium phenotype.Use it to have the bacterial strain (with reference to embodiment 15) that can excise box with acquisition as parent strain.Therefore this bacterial strain is as the positive control that produces three kinds of form of screw mycins.Clearly this bacterial strain produces the Spiramycin Base of three kinds of forms, has proved this point (with reference to Figure 19) through HPLC.

Research has the feasible conclusion that might draw the function of the relevant gene that interrupts of some amount of the bacterial strain that can excise box.Bacterial strain SPM507 has genotype: orf12::att3 Ω aac-.The phenotype of bacterial strain SPM507 is nonproductive bacterium (with reference to embodiment 15); Therefore can infer thus that the orf12 gene is to give birth to the necessary gene of Spiramycin Base biosynthesizing in the dyadic streptomycete.This bacterial strain no longer produces Spiramycin Base, and this can prove (with reference to Figure 22) through HPLC.This result confirms the necessity of orf12 gene in the Spiramycin Base biosynthesizing.

Bacterial strain SPM508 has genotype: orf13c::att3 Ω aac-.The phenotype of bacterial strain SPM508 is that Spiramycin Base is produced bacterium phenotype (with reference to embodiment 15); Therefore can infer thus that the orf13c gene is not to give birth to the necessary gene of Spiramycin Base biosynthesizing in the dyadic streptomycete.This bacterial strain produces Spiramycin I, II and III, and this can prove (with reference to Figure 23) through HPLC.This result confirms that the orf13c gene is not to give birth to Spiramycin I, II and the necessary gene of III biosynthesizing in the dyadic streptomycete.

Bacterial strain SPM509 has genotype: orf14::att3 Ω aac-.The phenotype of bacterial strain SPM509 is nonproductive bacterium; Therefore can infer thus that the orf14 gene is to give birth to the necessary gene of Spiramycin Base biosynthesizing in the dyadic streptomycete.This bacterial strain no longer produces Spiramycin Base, and this can prove (with reference to Figure 24) through HPLC.This result confirms the necessity of orf14 gene in the Spiramycin Base biosynthesizing.

Bacterial strain SPM501 has genotype: orf6 ^*:: att1 Ω hyg+.This bacterial strain has the nonproductive bacterium phenotype of Spiramycin Base.This bacterial strain no longer produces Spiramycin Base, and this can prove (with reference to Figure 20) through HPLC.Yet, because orf5 ^*And orf6 ^*Gene (with reference to Fig. 3) has identical direction, and viewed phenotype possibly be owing to import orf6 ^*Box to orf5 in the operon ^*The polar effect of expressing.These these genes of hint are corotation records.In order to answer this problem, the box through excision imports makes at or6 ^*Produce the homophase deletion in the gene and recover orf5 ^*Expression, obtained bacterial strain SPM502.This bacterial strain has genotype orf6 ^*:: att1 (with reference to embodiment 14 and 15).Homophase " scar " att sequence (with reference to embodiment 14) that the excision of box is only residual.Bacterial strain SPM502 has Spiramycin Base and produces the bacterium phenotype.Yet, such as through HPLC proof, this bacterial strain only produces Spiramycin I and does not produce spiramycin II and III (with reference to Figure 21).Therefore can reason out orf5 by these results ^*Gene is to give birth to the necessary gene of Spiramycin Base biosynthesizing in the dyadic streptomycete, owing in bacterial strain SPM501, its indirect inactivation is caused the nonproductive bacterium phenotype of Spiramycin Base (with reference to Figure 20).On the other hand, orf6 ^*Gene is not to give birth to the necessary gene of Spiramycin I biosynthesizing in the dyadic streptomycete, because this gene of inactivation causes Spiramycin I to produce bacterium phenotype (with reference to Figure 21).Yet, orf6 ^*Be to produce spiramycin II and III necessary (with reference to embodiment 16).Therefore orf6 clearly ^*Genes encoding is responsible for the acyltransferase (with reference to Fig. 1) of the 3 modification platenolide in the position.

Embodiment 17: the translation initiation site and raising Spiramycin Base output of confirming orf10

17.1 the structure of plasmid pSPM523, pSPM524 and pSPM525:

In giving birth to the dyadic streptomycete, identified the orf10 gene and by (M.Geistlich etc., 1992) called after srmR such as Geistlich.Carried out the inactivation (with reference to embodiment 8) of orf10 gene.Therefore might show that obtained strains no longer produces Spiramycin Base (with reference to embodiment 15).This confirms the obvious involved in spiramycin biosynthesizing of orf10 gene.Therefore obviously biosynthesizing is essential for Spiramycin Base by the protein of this coded by said gene.Yet sequential analysis shows that 2 ATG codons that are positioned in the identical reading frame can be used for the translation (with reference to Figure 28) of orf10.One of 2 possible codons (codons at the upper reaches) originate in 10656 positions of given sequence among the SEQ ID No.1, and another possible codon is positioned than downstream, originates in 10809 positions (with reference to SEQ ID No.1).To before possibly the acting on of Spiramycin Base output, is at first to confirm translation initiation site at test overexpression srmR importantly.

In order to confirm translation initiation site, three constructs that contain three kinds of form orf10 have been produced.The PCR that contains the HindIII restriction enzyme site or contain the oligonucleotide of BamHI restriction enzyme site through utilization has obtained this several kinds of forms.

The first pair of following oligonucleotide of primer correspondence that is used to increase:

EDR39：

(SEQ ID No.122) (runic is represented the HindIII site)

EDR42：

(SEQ ID No.123) (runic is represented the BamHI site)

This allows amplification orf10 fragment to primer EDR39-EDR42, and it contains and is positioned the ATG (10809 positions of given sequence among the SEQ ID No.1) (with reference to Figure 28) of 3 ' position.The gained clip size is about 2kb and was called as " short orf10 " afterwards; This fragment does not contain the orf10 promotor.This 2kb fragment cloning is gone into carrier pGEM-T easy, to produce plasmid pSPM520.

The second pair of following oligonucleotide of primer correspondence that is used to increase:

EDR40：

(SEQ ID No.124) (runic is represented the HindIII site)

EDR42：

(SEQ ID No.123) (runic is represented the BamHI site)

This allows amplification orf10 fragment to primer EDR40-EDR42, and it contains and is positioned the ATG (10656 positions of given sequence among the SEQ ID No.1) (with reference to Figure 28) of 5 ' position.With this 2.2kb fragment, be called as " long orf10 " afterwards and be cloned into carrier pGEM-T easy, to produce plasmid pSPM521; This plasmid does not contain the orf10 promotor.

The 3rd pair of following oligonucleotide of primer correspondence that is used to increase:

EDR41：

(SEQ ID No.125) (runic is represented the HindIII site)

EDR42：

(SEQ ID No.123) (runic is represented the BamHI site)

This allows amplification to have the orf10 gene of two ATG to primer EDR41-EDR42, and contains its oneself promotor (with reference to Figure 28).With this 2.8kb fragment, be hereinafter referred to as " former orf10 " and be cloned into carrier pGEM-T easy, to produce plasmid pSPM522.

Utilize bacterial strain OSC2 chromosomal DNA to obtain " former orf10 " fragment as template.Utilizing in advance, " former orf10 " sheet segment DNA of purifying has obtained " short orf10 " and " long orf10 " fragment self as template.

Then the HindIII-BamHI of plasmid pSPM520, pSPM521 and pSPM522 being inserted the fragment subclone goes into the carrier pUWL201 that has digested in advance with the HindIII-BamHI enzyme and (derives from plasmid pUWL199 (U.F.Wehmeier; 1995) plasmid; The KpnI-BamHI fragment that has wherein imported the ermE promoter region is (with reference to Bibb etc.; 1985, particularly be Fig. 2), this fragment carries the sudden change (ermE that increases promotor intensity ^*Promotor) (Bibb etc., 1994) (with reference to Doumith etc., 2000)).Therefore; Obtained three plasmids: pSPM523 (derives from pUWL201; The insertion portion of form that it has " short orf10 "), pSPM524 (derives from pUWL201; It has the insertion portion of " long orf10 " form) and pSPM525 (derive from pUWL201, it has the insertion portion of " former orf10 " form) is (Figure 28).

17.2 transform bacterial strain OS49.50 with construct pSPM523, pSPM524 and pSPM525:

Through protoplast transformation effect (T.Kieser etc., 2000), transform bacterial strain OS49.50 (in the orf10 gene, having the bacterial strain that knocks out) with plasmid pSPM523, pSPM524 and pSPM525 respectively with reference to embodiment 8.Also prepared negative control through transforming bacterial strain OS49.50 with the plasmid pUWL201 that does not contain insertion portion.After the protoplast transformation, through its thiostrepton resistance screening these clones.Verified the clone who uses each plasmid to transform through extracting these plasmids.Therefore, obtained the new bacterial strain of 4 strains: bacterial strain OSC49.50 pUWL201 derives from the conversion that plasmid pUWL201 that use do not contain insertion portion carries out; Bacterial strain OSC49.50 pSPM523 derives from the conversion of using plasmid pSPM523 to carry out; Bacterial strain OSC49.50 pSPM524 derives from the conversion of using plasmid pSPM524 to carry out; With last bacterial strain OS49.50 pSPM525, derive from the conversion of using plasmid pSPM525 to carry out.

Spiramycin Base output through each strain in this four strains bacterial strain of HPLC test.For this reason, with the many streptomyces strains that will test be incubated in the 500ml Erlenmeyer flask (Erlenmeyer flask of band plate washer) of the tool plate washer that contains 70ml MP5 substratum (Pernodet etc., 1993).When bacterial strain contains one of plasmid pUWL201 or derivatives thereof, add 5 μ g/ml thiostreptons.With various living dyadic streptomycete bacterial strains with 2.5 * 10 ⁶The initial concentration of gemma/ml is inoculated in the Erlenmeyer flask of band plate washer, and hatches these cultures 96 hours at 27 ℃ with 250 rev/mins of orbit determination vibrations.Then through centrifugal from substratum isolated cell and through HPLC (with reference to embodiment 16) analytically clear liquid with the amount of the Spiramycin Base that produced of mensuration.According to standard sample and the area of measuring the peak might be measured the amount of the every kind of Spiramycin Base that is produced by these bacterial strains.Provided the result of this analysis in the table 39, data are expressed as mg and whenever go up clear liquid.The ultimate production of the corresponding Spiramycin Base of result (the output addition through with Spiramycin I, II and III obtains).

Table 39. derives from the Spiramycin Base output of the bacterial strain of OS49.50, (result is expressed as mg/l).

Shown in the result who provides in the table 39, negative control (with the bacterial strain OS49.50 of plasmid pUWL201 conversion) does not produce Spiramycin Base.When plasmid pSPM523 (it contains " short orf10 " form) when importing bacterial strain OS49.50, is observed no Spiramycin Base and produces.On the other hand, the generation that exists plasmid pSPM524 (it contains " long orf10 " form) and plasmid pSPM525 (it contains " former orf10 " form) can recover Spiramycin Base among the host strain OS49.50.Therefore, the orf10 fragment that only contains upper reaches ATG makes might recover the Spiramycin Base Synthesis.

In order to confirm these results, with XhoI Restriction Enzyme (this enzyme has a unique site in this plasmid, be positioned between two ATG (with reference to Figure 28)) digested plasmid pSPM521 (the plasmid pGEM-T easy that contains " long orf10 " form).Handle through the Klenow enzyme then and make the terminal blunt endsization of XhoI.Effect through the T4DNA ligase enzyme makes the plasmid closed in itself then, to form plasmid pSPM527.If the ATG (sequence location 10656 that provides among the SEQ ID No.1) at the upper reaches is really as translation initiation site, this processing will cause reading in the frame in the displacement in XhoI site and will have to produce to appear does not have the active proteinic effect of activation.On the other hand; If translation initiation betides the ATG (sequence location 10809 that provides among the SEQ ID No.1) in downstream; This processing will have very little or not effect to the expression of Orf10 (having provided transcription initiation site), and the also not effect of protein to being produced.

The BamHI-HindIII of pSPM527 insertion fragment subclone is gone into carrier pUWL201 then, to produce plasmid pSPM528.This plasmid is imported bacterial strain OS49.50 and more specifically is to have screened the clone with expection plasmid.Test the Spiramycin Base output (with reference to embodiment 16 and top) of obtained strains then through HPLC.Be different from and use the viewed result of plasmid pSPM524 (with reference to table 39), exist plasmid pSPM528 not recover Spiramycin Base output among the bacterial strain OS49.50.This confirms that the translation initiation site of orf10 gene is the ATG (ATG1 is with reference to Figure 28) that is positioned downstream position.

17.3 improve the Spiramycin Base output of giving birth among the dyadic streptomycete bacterial strain OSC2:

In order to test the influence of overexpression orf10 gene pairs Spiramycin Base output, plasmid pSPM523, pSPM524, pSPM525 and pSPM528 are imported bacterial strain OSC2.For this reason, transform the protoplastis (T.Kieser etc., 2000) of bacterial strain OSC2 respectively independently with plasmid pSPM523, pSPM524, pSPM525 and pSPM528.Also prepared negative control through transforming bacterial strain OSC2 with the plasmid pUWL201 that does not contain insertion portion.After the protoplast transformation, through its thiostrepton resistance screening these clones.Verified the clone who uses each plasmid to transform through extracting these plasmids.Like this, obtained five new bacterial strains: bacterial strain OSC2 pUWL201 derives from the conversion that plasmid pUWL201 that use do not contain insertion portion carries out; Bacterial strain OSC2pSPM523 derives from the conversion of using plasmid pSPM523 to carry out; Bacterial strain OSC2 pSPM524 derives from the conversion of using plasmid pSPM524 to carry out; Bacterial strain OSC2 pSPM525 derives from the conversion of using plasmid pSPM525 to carry out; With, last, bacterial strain OSC2 pSPM528 derives from the conversion of using plasmid pSPM528 to carry out.Analyzed the Spiramycin Base output of these bacterial strains then through HPLC (with the mode identical) with embodiment 17.2.For the parallel analysis of more also carrying out bacterial strain OSC2 Spiramycin Base output.Provided the result of this analysis in the table 40, data are expressed as mg and whenever go up clear liquid.The ultimate production of the corresponding Spiramycin Base of result (the turnout addition through with Spiramycin I, II and III obtains).

Table 40. derives from the Spiramycin Base output of the bacterial strain of OSC2, (result is expressed as mg/l).

Therefore, the existence of observing plasmid pSPM524 or plasmid pSPM525 significantly increases the Spiramycin Base output of bacterial strain OSC2.This proves that clearly overexpression Orf10 has positive-effect to Spiramycin Base output.On the other hand, plasmid pSPM528 is to the not effect of Spiramycin Base output.

In an identical manner, plasmid pSPM525 and pUWL201 are imported bacterial strain SPM502 (with reference to embodiment 14).Like this, obtained two new bacterial strains: bacterial strain SPM502 pUWL201 derives from the conversion that plasmid pUWL201 that use do not contain insertion portion carries out; With bacterial strain SPM502pSPM525, derive from the conversion of using plasmid pSPM525 to carry out.

(this bacterial strain contains plasmid pSPM525 to bacterial strain SPM502 pSPM525; With reference to top) sample be preserved in state-run microbial preservation center (CNCM) Institute Pasteur; 25, rue du DocteurRoux 75724 Paris Cedex 15, France; Preservation day is on February 26th, 2003, and preserving number is I-2977.

Analyzed the Spiramycin Base output of these bacterial strains SPM502UWL201 and SPM502 pSPM525 through HPLC (with the mode identical) with embodiment 17.2.For the parallel analysis of more also carrying out bacterial strain SPM502 Spiramycin Base output.Provided the result of this analysis in the table 41, data are expressed as mg and whenever go up clear liquid.The turnout of the corresponding Spiramycin I of result.In fact, do not produce the bacterial strain of spiramycin II and III in these bacterial strains.

Table 41. derives from the Spiramycin I output (result is expressed as mg/l) of the bacterial strain of SPM502.

Therefore, might observe the output that in bacterial strain SPM502 overexpression orf10 gene significantly increases Spiramycin I.

Embodiment 18: the genome dna library that in intestinal bacteria, makes up the living dyadic streptomycete bacterial strain OSC2 that is arranged in clay pWED2.

18.1 the structure of clay pWED2:

In order to promote the inactivation of gene in the streptomycete, made up carry plasmid RK2 oriT sequence (this sequence allows to import streptomycete through engaging from suitable coli strain) but and be carried at the clay that is directed against antibiotic resistant gene of giving the detected representation type the streptomycete.This kind contains bigger living dyadic streptomyces gene group DNA and inserts segmental clay and can be used in gene inactivation experiment.

In order to make up this carrier, pac-oriT box (EcoRV fragment) is imported the clay pWED1 (Gourmelen etc., 1998) that derives from clay pWED15 (Wahl etc., 1987) in unique HpaI site.Obtained the pac-oriT box through PCR.For this reason, through PCR from plasmid pVF 10.4 (Vara etc., 1985; Lacalle etc.; 1989) the pac gene that increased; (sequence is

(SEQ ID No.126) to have used primer A as first primer; The EcoRV restriction enzyme site is rule and the upstream region of the corresponding pac gene promoters of 20 nucleotide sequences of runic below); (sequence is

(SEQ IDNo.127) with primer B as second primer; At its 5 ' end; 12 nucleotide sequences (double underline) and terminal 20 nucleotide sequences (runic) (with reference to Figure 29, first round PCR) of corresponding pac gene that comprise corresponding oriT sequence initiating terminal.

About the oriT gene; Through PCR from plasmid pPM803 (P.Mazodier etc.; 1989) this gene that increased; Used primer C (sequence is

(SEQ ID No.128)) as first primer; This primer comprises 12 nucleotide sequences (runic) of corresponding pac gene coded sequence downstream sequence and 20 nucleotide sequences of corresponding oriT sequence initiating terminal at its 5 ' end; With primer D (sequence is

(SEQ IDNo.129)) as second primer; This primer comprises EcoRV restriction enzyme site (single underscore) and terminal 20 nucleotide sequences (double underline) (with reference to Figure 29, second takes turns PCR) of corresponding oriT sequence.

Use primer A and B amplified production that obtains and the amplified production that uses primer C and D to obtain,, contain the common sequences of 24 Nucleotide at their end.These two amplified productions through mixing previous acquisition with use primer A and D as primer, carried out third round PCR (with reference to Figure 29, third round PCR).This makes and might obtain the amplified production that corresponding pac+oriT makes up.(by Promega company (Madison, Wisconsin USA) sell), this makes might obtain plasmid pGEM-T-pac-oriT then the pac-oriT fragment cloning to be gone into carrier pGEM-T Easy.Then the insertion portion subclone of this plasmid is gone into clay pWED1.For this reason, also will contain the EcoRV insertion portion that makes up pac+oriT with EcoRV enzymic digestion plasmid pGEM-pac-oriT and insert the clay pWED1 that cuts with the HpaI enzyme in advance.Gained clay called after pWED2 (with reference to Figure 30) like this.

This clay makes it can be beneficial to the inactivation of gene in the streptomycete.Particularly, this clay carries the oriT sequence, and this sequence not only allows to import streptomycete through engaging from suitable coli strain, but also is carried at the antibiotic resistant gene that is directed against of giving the detected representation type in the streptomycete.This kind contains bigger living dyadic streptomyces gene group DNA and inserts segmental clay and can be used in gene inactivation experiment.

Therefore, according to Chaveroche etc., 2000 described technology for example can import the coli strain KS272 that contains plasmid pKOBEG (with reference to Chaveroche etc., 2000) and box will import in the target gene with the clay derived from pWED2 that contains target gene.Can the clay (the wherein clay of target gene inactivation) that obtain through this technology be imported such as the coli strain of S17.1 bacterial strain or makes and might be transferred to any other bacterial strain of streptomycete through engaging the plasmid that will contain the oriT sequence then.

After engaging between intestinal bacteria and the streptomycete, described in embodiment 12, can obtain the streptomycete clone, wherein the wild-type of target gene copy is replaced by the copy that interrupts.

The resistant gene of in streptomycete, expressing that is present on this new clay is pac gene (J.Vara etc., 1985 of white black streptomycete (Streptomyces alboniger); Lacalle etc., 1989), this genes encoding tetracycline N-acetyltransferase is also given the tetracycline resistance.In the gene inactivation experiment, found the clone that dual group of incident wherein taken place.Therefore these clones will remove clay pWED2 and will become and responsive to tetracycline.

18.2 in intestinal bacteria, make up the genomic DNA of the living dyadic streptomycete bacterial strain OSC2 library that is arranged in clay pWED2

For obtain size about 35 and 45kb between dna fragmentation, use the BamHI Restriction Enzyme partly to digest the genomic dna of living dyadic streptomycete bacterial strain OSC2.Then these fragment clonings are gone into clay pWED2, the latter has digested with BamHI in advance, and uses alkaline phosphatase treatment.Utilize (the Madison of Promega company then; Wisconsin; " Packagene

λ DNA packs system " of USA) selling; According to the recommendation of manufacturers, will connect that mixture is external to be packaged in the lambda particles phage particle.The phage particle that is obtained is used to infect (the LaJolla of Stratagene company; California, the coli strain SURE

that USA) sells.These clones of screening on LB substratum+penbritin (50 μ g/ml) flat board, clay pWED2 give amicillin resistance.

Embodiment 19: separate segmental subclone and sequential analysis in the clay comprise the new library that covers Spiramycin Base biosynthetic pathway zone, these clays.

19.1 the hybridization that the bacterium colony of giving birth to dyadic streptomycete OSC2 genomic library is carried out:

The clay (with reference to embodiment 18) that has separated the new library of living dyadic streptomycete OSC2, this library covers orf1 ^*To orf10 ^*Or cover orf1 to orf25c partly or entirely, or covering orf25c upstream region more.For this reason, use following 3 probes that the colibacillary bacterium colony that is obtained according to embodiment 18 has been carried out hybridization (with reference to Figure 31) successively:

-about 0.8kb dna fragmentation that used article one probe correspondence arrives through pcr amplification, wherein said PCR uses clay pSPM5 to be template, and has used following primer:

ORF23c:5 '-ACGTGCGCGGTGAGTTCGCCGTTGC-3 ' (SEQ ID No.130) and

ORF25c：5’-CTGAACGACGCCATCGCGGTGGTGC-3’(SEQ?ID?No.131)。

The PCR product that so obtains comprises and contains the orf23c initiating terminal, orf24c is whole and the fragment (with reference to Figure 31, probe I) of orf25c terminal portions.

-about 0.7kb dna fragmentation that used second probe correspondence arrives through pcr amplification, wherein said PCR use gives birth to the total DNA of dyadic streptomycete bacterial strain ATCC23877 and is template, and has used following primer:

ORF1 ^*C:5 '-GACCACCTCGAACCGTCCGGCGTCA-3 ' (SEQ ID No.132) and

ORF2 ^*c：5’-GGCCCGGTCCAGCGTGCCGAAGC-3’(SEQ?ID?No.133)。

The PCR product that so obtains comprises and contains orf1 ^*C end and orf2 ^*The fragment (with reference to Figure 31, probe I I) of c initiating terminal part.

The EcoRI-BamHI fragment (with reference to Figure 31, probe I II) of the about 3kb that contains orf1, orf2 and orf3 that-used the 3rd probe correspondence obtained through digested plasmid pOS49.99.

According to routine techniques (Sambrook etc., 1989), about 2000 clones in the library that obtains among the embodiment 18.2 are transferred to be used for colony hybridization on the filter membrane.

After transferring on the filter membrane, also this probe is used for 2000 clones in library are hybridized with 32P mark article one probe (with reference to Figure 31, probe I) through random primer technology (test kit that Roche company sells).Hybridizing 65 ℃ the time in Church and the described damping fluid of Gilbert (Church and Gilbert, 1984).In containing 2 * SSC of 0.1%SDS, carry out twice washing 65 ℃ the time, washed 10 minutes for the first time and 20 minutes for the second time, the washing for the third time that in containing the 0.2X SSC of 0.1%SDS, continues 30 minutes 65 ℃ the time then.Under these hybridization and wash conditions, in 2000 clones that hybridized, there are 20 clones and article one probe to present strong hybridization signal.These 20 clones are incubated in LB+ penbritin (the 50 μ g/ml) substratum and extract corresponding 20 clays and digest with the BamHI Restriction Enzyme through alkaline lysis (Sambrook etc., 1989).Then digestion product is separated on sepharose, be transferred on the nylon membrane and under above-mentioned the same terms with article one probe hybridize (with reference to above: PCR product ORF23c-ORF25c, probe I).12 clays comprise and the strong BamHI fragment of hybridizing of used probe.These 12 clay called afters pSPM34, pSPM35, pSPM36, pSPM37, pSPM38, pSPM39, pSPM40, pSPM41, pSPM42, pSPM43, pSPM44 and pSPM45.With after the BamHI digestion, that the digestion spectrum of these 12 clays is compared to each other and compare with the digestion spectrum of clay pSPM5.The feasible position (with reference to Figure 32) that might locate the insertion portion of these clays that are relative to each other and also might confirm these insertion portions in the known orf1-orf25c zone of pcr amplification experiment of in addition, using the corresponding heterogeneic different primers of in the orf1-orf25c zone, having identified out to carry out.More specifically be to have chosen clay pSPM36, this clay tends to contain bigger orf25c upstream region (with reference to Figure 31 and 32).

Secondly, use the condition identical, with corresponding PCR product: ORF1 with above-mentioned condition ^*C-ORF2 ^*The second probe of c (with reference to Figure 31, probe I I) is hybridized with 2000 clones that give birth to dyadic streptomycete OSC2 library.This hybridization makes that might be separated to its insertion portion is positioned from orf1 ^*C is to orf10 ^*Clay in the c zone.Under used hybridization conditions, in 2000 clones that hybridized, there are 16 clones and second probe to present strong hybridization signal.These 16 clones are incubated in LB+ penbritin (the 50 μ g/ml) substratum and extract corresponding 16 clays and digest with the BamHI Restriction Enzyme through alkaline lysis (Sambrook etc., 1989).The digestion of these 16 clays spectrum (after BamHI digestion) is compared to each other and compare with the digestion spectrum of clay pSPM7.Use primer ORF1 ^*C and ORF2 ^*The pcr amplification experiment that c carries out makes that might choose two obviously contains orf1 ^*C and orf2 ^*Existing identical band is composed in the clay of c gene and their digestion also has different stripes.In addition, use correspondence at orf1 ^*C is to orf10 ^*The heterogeneic different primers of having identified out in the c zone carry out pcr amplification experiment to make and might locate the insertion portion of these clays that are relative to each other and also might confirm known orf1 ^*C is to orf10 ^*The position (with reference to Figure 32) of these insertion portions in the zone.This that more specifically screens 2 clay called afters pSPM47 and pSPM48 (with reference to Figure 32).

Use condition same as described above, hybridize with the 3rd probe (with reference to Figure 31, probe I II) of corresponding plasmid pOS49.99EcoRI-BamHI dna fragmentation and 2000 clones in living dyadic streptomycete OSC2 library.This hybridization makes and might be separated to the clay that contains from orf1 to orf3 zone, and these clays tend to contain the major part of PKS gene or contain the major part of Spiramycin Base biosynthetic pathway gene orf1 to orf25c.Under these hybridization conditions, in 2000 clones that hybridized, there are 35 clones and the 3rd probe to present strong hybridization signal.These 35 clones are incubated in LB+ penbritin (the 50 μ g/m) substratum and extract corresponding 35 clays and digest with the BamHI Restriction Enzyme through alkaline lysis (Sambrook etc., 1989).With after the BamHI digestion, that the digestion spectrum of these 35 clays is compared to each other and compare with the digestion spectrum of clay pSPM5.In addition; The pcr amplification experiment of using the corresponding heterogeneic different primers of identifying out in orf1 institute in the orf25c zone to carry out makes that might prove conclusively these clays has comprised the insertion fragment to orf25c from regional orf1, and feasible mutual the reaching with respect to the position (with reference to Figure 32) of known region orf1 to orf25c of insertion fragment that might locate these clays.5 clays have more specifically been screened, their called after pSPM50, pSPM51, pSPM53, pSPM55 and pSPM56 (with reference to Figure 32).

19.2 part clay pSPM36 inserts segmental subclone and sequential analysis

Use the probe (the top and Figure 31 of reference, probe I) of about 0.8kb that primer ORF23c and ORF25c obtain through PCR also be used for to the PstI enzymic digestion the total DNA of living dyadic streptomycete OSC2 carry out the Southern Blot experiment.Under above-mentioned hybridization conditions, when to the PstI enzymic digestion the total DNA of living dyadic streptomycete OSC2 when hybridizing, this probe shows the PstI fragment of a single about 6kb.The PstI site is present in (with reference to SEQ ID No.80) in the orf23e zone, but does not have other PstI site (with reference to SEQID No.1) up to the end (BamHI site) of known array.This PstI-BamHI clip size is about 1.4kb.Therefore should with the PstI enzymic digestion the total DNA of the living dyadic streptomycete OSC2 6kb PstI fragment of hybridizing to contain the about 4.6kb that is positioned the orf25c upper reaches regional.Other gene of its product involved in spiramycin biosynthetic pathway is tended to contain in this zone.Verified that through digestion clay pSPM36 contains this 6kbPstI fragment really.In order to confirm the more sequence at the upper reaches of orf25c, separate obtaining this fragment from pSPM36.For this reason, with PstI Restriction Enzyme digestion clay pSPM36.Obtain size through electroelution from the separation of 0.8% sepharose and be the PstI-PstI fragment of about 6kb, and then it is cloned into carrier pBK-CMV (4512bp) (by Stratagene company (La Jolla, California USA) sell).The plasmid called after pSPM58 (with reference to Figure 33) that so obtains has also confirmed the sequence of its insertion portion.Provided the sequence of this insertion portion among the SEQ ID No.134.Yet, be not that all sequences has all been confirmed, also have the breach of about 450 Nucleotide not confirm that the undetermined part of sequence is represented with successive " N " in corresponding sequence.

19.3 the sign of the definite and biosynthetic gene of involved in spiramycin of the analysis of new nucleotide sequence, ORFs

Use FramePlot program (J.Ishikawa and K.Hotta 1999) to analyze the sequence of the insertion portion of gained clay pSPM58.This makes might identify the ORFs that presents the selection of typical chain mould codon in ORFs.This analysis makes might confirm that this insertion portion comprises 4 new ORF (with reference to Figure 34) at the orf25c upper reaches.These unnamed genes are orf26 (SEQ ID No.107), orf27 (SEQ ID No.109), orf28c (SEQ ID No.111; The sequence of this orf is not confirmed fully and in the sequential analysis of pSPM58 insertion portion, is also had the breach of about 450 Nucleotide not check order; These 450 Nucleotide come across sequence SEQ ID No.111 with the form of a series of " N ") and orf29 (to the insertion portion of this orf, sequence is incomplete).Add " c " meaning in the gene title and be meant that for the ORF that is discussed, this encoding sequence is reciprocal (with reference to Figure 34).

Use that existing those sequences compare in protein sequence that various programs will be derived by these ORFs and the several data storehouse: BLAST (Altschul etc.; 1990) (Altschul etc.; 1997), (these two programs especially can be from (the Bethesda of NCBI (NCBI) in the CD-search; Maryland USA) obtains), FASTA ((W.R.Pearson and D.J.Lipman; 1988) and (W.R.Pearson, 1990) (this program especially can obtain from the INFOBIOGEN resource center of French Evry).These relatively make it possible to clearly describe the biosynthetic gene of those possibility involved in spiramycin of hypothesis and evaluation about the function of these gene products.

19.4 another part clay pSPM36 inserts segmental subclone and sequential analysis

Use the probe (with reference to top and Figure 31, probe I) of about 0.8kb that primer ORF23c and ORF25c obtain through PCR also to be used for and with the StuI enzymic digestion the total DNA of bacterial strain OSC2 carry out the Southern Blot experiment.Under the above-mentioned hybridization conditions that is used for this probe, when to the StuI enzymic digestion the total DNA of bacterial strain OSC2 when hybridizing, this probe shows the StuI fragment of a single about 10kb.Owing to exist the StuI site (with reference to SEQ ID No.80) and the location in this site relevant among the orf23c, might obtain the zone (with reference to Figure 33) that about 4kb studies not yet so this 10kb fragment comprises all PstI fragments (pSPM58 insertion portion) of previous research and makes with the PstI site.Verified that through digestion clay pSPM36 contains the StuI fragment of this 10kb really.In order to confirm the more sequence of other gene of the upper reaches of the terminal sequence of orf29 and orf29, obtain this fragment from the pSPM36 separation.For this reason, with StuI Restriction Enzyme digestion clay pSPM36.Obtain size through electroelution from the separation of 0.8% sepharose and be the StuI-StuI fragment of about 10kb, and then it is cloned into carrier pBK-CMV (4512bp) (by Stratagene company (La Jolla, California USA) sell).The plasmid called after pSPM72 (with reference to Figure 33) that so obtains.Use EeoRI (the EcoRI site in the pSPM58 insertion portion) and HindIII (the HindIII site in the carrier MCS is and then after the StuI site of insertion portion end) digestion back one plasmid (with reference to Figure 33) then.The fragment (with reference to Figure 33) of the corresponding plasmid pSPM72 of the EcoRI-HindIII dna fragmentation that so obtains insertion portion is also gone in advance the carrier pBC-SK+ that digested with EcoRI and HindIII (by (the La Jolla of Stratagene company with its subclone; California USA) sells).Like this gained plasmid called after pSPM73 and confirmed the sequence of its insertion portion.Provided the sequence of this insertion portion among the SEQ ID No.135.

Provided the assembled arrangement of the sequence of pSPM58 and pSPM73 insertion portion among the SEQ ID No.106.This sequence originates in the PstI site (with reference to SEQ ID No.80) among the orf23c and extends to the StuI site (with reference to Figure 34) among the orf32c.Because the sequence (SEQ ID No.111) of orf28c is incomplete (with reference to embodiment 19.3), sequential analysis is not carried out in the zone of wherein about 450 Nucleotide, and these 450 Nucleotide occur with the form of a series of " N " in sequence SEQ ID No.106.

19.5 the sign of the definite and biosynthetic gene of involved in spiramycin of the analysis of new nucleotide sequence, ORFs

Use FramePlot program (J.Ishikawa and K.Hotta 1999) to analyze the partial sequence of the insertion portion of gained clay pSPM73.This makes might identify the ORFs that presents the selection of typical chain mould codon in ORFs.Feasible this insertion portion (with reference to Figure 34) that might confirm to comprise 4 ORF (incomplete and three complete ORF) of this analysis.3 ' part of the corresponding orf29 encoding sequence of incomplete ORF, this makes and might accomplish the sequence of this gene according to the partial sequence (with reference to embodiment 19.2 and 19.3) of the same orf that in the sequential analysis of plasmid pSPM58 insertion portion, obtains; So feasible complete sequence that might obtain orf29 of the combination of two sequences.Therefore these 4 unnamed genes are orf29 (SEQ ID No.113), orf30c (SEQID No.115), orf31 (SEQ ID No.118) and orf32c (SEQ ID No.120).Add " c " meaning in the gene title and be meant that for the ORF that is discussed, this encoding sequence is reciprocal (with reference to Figure 34).

Protein sequence (the SEQ ID No.114 of corresponding orf29 that uses various programs to derive by these ORFs; The SEQ ID No.116 and 117 of corresponding orf30e; The SEQ ID No.119 of corresponding orf31 and the SEQ ID No.121 of corresponding orf32c) with various DBs in existing those sequences compare: BLAST (Altschul etc.; 1990) (these two programs especially can be from NCBI (NCBI) (Bethesda, Maryland in (Altschul etc., 1997), CD-search; USA) obtain); FASTA ((W.R.Pearson and D.J.Lipman, 1988) and (W.R.Pearson, 1990) (this program especially can obtain from the INFOBIOGEN resource center of French Evry).These relatively make it possible to clearly describe the biosynthetic gene of those possibility involved in spiramycin of hypothesis and evaluation about the function of these gene products.

19.6 clay pSPM36 inserts the subclone and the sequential analysis of fragment third part.

Obtained the probe (0.8kb dna fragmentation) of corresponding orf32c internal sequence through PCR, the PCR reaction is used and given birth to the total DNA of dyadic streptomycete bacterial strain is template and the following primer of use:

KF36:5 '-TTGCCGTAGCCGAGGACCAGCG-3 ' (SEQ ID No.151) and

KF37：5′-CACATGGCCCTGGAGGACCCTG-3′(SEQ?ID?No.152)。

The PCR product that so obtains is represented the orf32c internal sequence.This probe be used for with the PstI enzymic digestion bacterial strain OSC2 chromosomal DNA and clay pSPM36 DNA carry out the Southern Blot experiment.Use the hybridization conditions (with reference to embodiment 19.1) identical with above-mentioned condition, when to the PstI enzymic digestion the total DNA of bacterial strain OSC2 and clay pSPM36DNA when hybridizing, this probe shows two PstI fragments that are approximately 3.4kb and 2.5kb.Owing to have the PstI site in the used probe, can explain these results.First dna fragmentation, size is about 3.4kb, is the complete known fragment of its sequence.2.5kb fragments sequence only part is known, regional known to 700bp.For the sequence of definite orf32c end and the sequence of other gene of the latter upper reaches, obtain this fragment from clay pSPM36 separation.For this reason, with PstI Restriction Enzyme digestion clay pSPM36.Obtain size through purifying from the separation of 0.8% sepharose and be the PstI-PstI fragment of about 2.5kb, and then it is cloned into carrier pBK-CMV (4518bp) (by Stratagene company (LaJolla, California USA) sell).The plasmid called after pSPM79 (with reference to Figure 41) that so obtains has also confirmed the sequence of its insertion portion.

The sequence of orf28e (SEQ ID No.111) is incomplete (with reference to embodiment 19.3).Particularly, can not confirm the zone of about 450 Nucleotide, these 450 Nucleotide occur with the form of a series of " N " in sequence SEQ ID No.106.Through sequential analysis is carried out in this zone once more, confirmed the sequence of these whole zone of ignorances.Therefore, all confirmed the sequence of pSPM58 and pSPM73 insertion portion.Provided among the SEQ ID No.141 among complete sequence and the SEQ ID No.142 of orf28c encoding part and provided the protein of deriving by this sequence.Provided the sequence of plasmid pSPM79 insertion portion among the SEQ ID No.161.

Provided pSPM58 among the SEQ ID No.140, the combination (with reference to Figure 41) of the sequence of pSPM73 and pSPM79 insertion portion.This sequence originates in the PstI site (with reference to SEQ ID No.80) among the orf23c and extends to the PstI site (with reference to Figure 41) among the orf34c.

19.7 the sign of the definite and biosynthetic gene of involved in spiramycin of the analysis of new nucleotide sequence, ORFs

Use FramePlot program (J.Ishikawa and K.Hotta 1999) to analyze the sequence of the insertion portion of gained plasmid pSPM79.This makes might identify the ORFs that presents the selection of typical chain mould codon in ORFs.Feasible this insertion portion that might confirm to comprise 3 ORF of this analysis, two incomplete ORF (orf32c and orf34c) and a complete ORF (orf33) (with reference to Figure 41).

5 ' part of the corresponding orf32c encoding sequence of first incomplete ORF.This makes might be according to the partial sequence (with reference to embodiment 19.4 and 19.5) of the same orf that in the sequential analysis of plasmid pSPM73 insertion portion, obtains; Accomplish the sequence of this gene, therefore the feasible complete sequence (SEQ ID No.145) that might obtain orf32c of the combination of two sequences.Add " c " meaning in the gene title and be meant that for the ORF that is discussed, this encoding sequence is reciprocal (with reference to Figure 41).Complete orf is called orf33 (SEQ ID No.147).The 3rd ORF is called orf34c (SEQ ID No.149).Add " c " meaning in the gene title and be meant that for the ORF that is discussed, this encoding sequence is reciprocal (with reference to Figure 37).That between the product of this orf and DB, carries out comparison shows that this protein C-terminal portions not in the product of being derived by nucleotide sequence, and therefore shows that also this orf is longer and continue to after the zone of carrying out sequential analysis.

Use that existing those sequences compare in protein sequence that multiple program will be derived by these ORFs and the several data storehouse: BLAST (Altschul etc.; 1990) (Altschul etc.; 1997), (these two programs especially can be from (the Bethesda of NCBI (NCBI) in the CD-search; Maryland USA) obtains), FASTA ((W.R.Pearson and D.J.Lipman; 1988) and (W.R.Pearson, 1990) (this program especially can obtain from the INFOBIOGEN resource center of French Evry).These relatively make it possible to clearly describe the biosynthetic gene of those possibility involved in spiramycin of hypothesis and evaluation about the function of these gene products.

Embodiment 20: analyze the Spiramycin Base biosynthesizing midbody that produces:

20.1 specimen preparation:

With the multi-strain bacteria strain that will test be incubated at separately in 7 500ml tool plate washer Erlenmeyer flasks that contain 70ml MP5 substratum (Pernodet etc., 1993).Many strains are given birth to the dyadic streptomycete bacterial strain with 2.5 * 10 ⁶Gemma/ml is inoculated in the Erlenmeyer flask and 27 ℃ of 250 rev/mins of orbit determination oscillating growths 96 hours.Collect corresponding identical clone's culture, randomly filter, and 7000 rev/mins centrifugal 15 minutes through pleating filter.

With sodium hydroxide the pH value of supernatant is adjusted to 9 and with methyl isobutyryl ketone (MIBK) extracting supernatant then.Reclaim organic phase (MIBK) and evaporation drying then.Then dry extract is dissolved in the 1ml acetonitrile, before being used for liquid chromatography/mass spectrometry measurement (LC/MS) analysis, is diluted to 1/10 (water is diluted to 1ml with 100 μ l) then.

20.2 pass through the LC/MS analyzing samples:

In order to measure quality, through the LC/MS analyzing samples by the multiple product of being tested of bacterial strain synthetic.

Used HPLC post is Kromasil C8 150*4.6mmm; 5mmm, 100 pillar.The gradient solution that moving phase is made up of the mixture of acetonitrile and 0.05% trifluoroacetic acid aqueous solution, flow velocity were fixed as 1ml/ minute.The temperature maintenance of post thermostatted is at 30 ℃.Under two different wavelengths: 238nm and 280nm, carry out UV at the post spout and detect.Being connected to mass spectrograph on the chromatographic column is the Single Quadripole equipment that Agilent company sells, and cone voltage is 30 and 70V.

20.3 analyze the biosynthesizing midbody that produces by bacterial strain OS49.67:

Wherein the orf3 gene is not produced Spiramycin Base (with reference to embodiment 6 and 15) through the homophase deletion by the bacterial strain OS49.67 of inactivation.(with reference to paragraph 20.1) preparation sample is also analyzed (with reference to paragraph 20.2) as above-mentioned through LC/MS according to the method described above.More specifically, analyze through chromatography in mutually at gradient solvent, like moving phase: from the time be T=0 to 5 minute be 20% acetonitrile, linearity increases to 30% in the time of T=35 minute then, is maintained until T=50 minute subsequently.

Under these conditions, observe two products more significantly: the absorption (RT is 44.8 minutes) (with reference to Figure 35) at absorption at 238nm place (RT is 33.4 minutes) and 280nm place.Figure 35 has shown 238 with superimposed (top) of the HPLC color atlas of 280nm place generation and the UV spectrum (bottom) of the molecule that wash-out goes out when having shown 33.4 minutes and 44.8 minutes.

The mass spectrometry analysis condition of coupling is following: scan with scan mode, contain mass range 100 and 1000Da between.The gain of electron-multiplier is 1V.About the electronic spraying source, the pressure of atomizing gas is 35psig, and the flow velocity of dry gas is 12.0l/ minute, and the temperature of dry gas is 350 ℃, and capillary voltage be+/-3000V.These experiments make the quality of two kinds of products might confirming to be separated to.Its quality is respectively first the eluted product ([M-H ₂O] ⁺=353 primary products) 370g/mol and second product the 368g/mol ([M-H ₂O] ⁺=351 primary products).

In order to obtain their structure, separate above-mentioned product and under following condition, carry out purifying: moving phase is 70/30 (v/v) mixture of 0.05% trifluoroacetic acid aqueous solution and acetonitrile.In permanent solvent systems to carry out stratographic analysis in fixed flow rate 1ml/ minute.Under these conditions, the RT of 2 products was respectively 8 and 13.3 minutes.In addition, in this case, be injected into before the 10 μ l samples the not prepared sample (with reference to paragraph 20.1) of dilute with water.

Reclaim these 2 products and under following condition, separate at the delivery port of chromatographic column: use 1ml acetonitrile, 1ml water/acetonitrile (20v/80v) and 1ml 80/20 water/acetonitrile balance Oasis HLB 1cc30mg post (Waters) successively.Add sample then and use 1ml water/acetonitrile (95/5) and 1ml water/deuterate acetonitrile (95/5) washing column successively, use 1ml water/deuterate acetonitrile wash-out of 600 μ l 40/60 then.The solution that reclaims through the NMR direct analysis then.

For these two compounds, the NMR spectrum that is obtained is following:

-the first eluted product: Platenolide A: (spectrum 9646V)

1H spectrum (chemical transport ppm) among the CD3CN: 0.90 (3H, t, J=6Hz), 0.93 (3H, d, J=5Hz), 1.27 (3H, d, J=5Hz), between 1.27 and 1.40 (3H, m); 1.51 (1H, m), 1.95 (1H, m), 2.12 (1H, m), 2.30 (1H, d, J=12Hz), 2.50 (1H, d; J=11Hz), 2.58 (1H, dd, J=9 and 12Hz), 2.96 (1H, d, J=7Hz), 3.43 (3H, s), 3.70 (1H, d; J=9Hz), 3.86 (1H, d, J=7Hz), 4.10 (1H, m), 5.08 (1H, m), 5.58 (1H, dt; J=3 and 12Hz), 5.70 (1H, dd, J=8 and 12Hz), 6.05 (1H, dd, J=9 and 12Hz), 6.24 (1H, dd, J=9 and 12Hz).

-the second eluted product: Platenolide B: (spectrum 9647V)

1H spectrum (chemical transport ppm) among the CD3CN: 0.81 (3H, t, J=6Hz), 0.89 (1H, m), 1.17 (3H, d, J=5Hz), 1.30 (4H, m), 1.47 (2H; M), 1.61 (1H, t, J=10Hz), 2.20 (1H, m), 2.38 (1H, d, J=13Hz), 2.52 (1H, m); 2.58 (1H, m), 2.68 (1H, dd, J=8 and 13Hz), 3.10 (1H, d, J=7Hz), 3.50 (3H, s); 3.61 (1H, d, J=8Hz), 3.82 (1H, d, J=7Hz), 5.09 (1H, m), 6,20 (1H; M), 6.25 (1H, dd, J=9 and 12Hz), 6.58 (1H, d, J=12Hz), 7.19 (1H, dd, J=9 and 12Hz).

The feasible structure that might confirm these two compounds of these experiments.First eluted product is that the platenolide A and second eluted product are platenolide B; Provided the structure of these two molecules of being derived among Figure 36.

Use the LC/MS technology that combines NMR; Under the condition that is different from above-mentioned those conditions slightly; But the establishment of these conditions is well-known to those skilled in the art, might confirm that also except platenolide A and B, bacterial strain OS49.67 also produces the verivate of these two kinds of compounds.They are platenolide A+mycarose and platenolide B+mycarose (having provided the structure of these two kinds of compounds among Figure 40).Provided the result that the supernatant of bacterial strain OS49.67 is analyzed in the table 42.

The LC/MS analytical results of the supernatant of table 42. bacterial strain OS49.67

20.4 analyze the biosynthesizing midbody that is produced by bacterial strain SPM509:

Wherein the orf14 gene the bacterial strain SPM509 of inactivation (orf14::att3 Ω aac-) do not produce Spiramycin Base (with reference to

embodiment

13,15 and 16).(with reference to paragraph 20.1) preparation sample is also analyzed (with reference to paragraph 20.2 and 20.3) as above-mentioned through LC/MS according to the method described above.This bacterial strain of analysis revealed of the biosynthesizing midbody that exists in the culture supernatant liquid to the bacterial strain SPM509 that in the MP5 substratum, cultivates only produces the platenolide (" platenolide B " of form B; With reference to Figure 36) and do not produce the platenolide (" platenolide A " is with reference to Figure 36) of form A.

Embodiment 21: be interrupted the orf14 gene in the bacterial strain that in having the orf3 gene, knocks out (OS49.67)

The product of orf14 gene is Spiramycin Base biosynthesizing necessary (with reference to embodiment 13,15 and 16: wherein the bacterial strain SPM509 that interrupted of this gene no longer produces Spiramycin Base).This bacterial strain of analysis revealed of the biosynthesizing midbody that exists in the culture supernatant liquid to the bacterial strain SPM509 that in the MP5 substratum, cultivates only produces the platenolide of form B and does not produce the platenolide (with reference to embodiment 20) of form A.One of the hypothesis that can explain this observations is that the product of orf14 participates in through the enzyme reduction step form B platenolide being converted into form A platenolide.In order to verify this hypothesis, but the orf14 gene is not being produced Spiramycin Base producing inactivation in the two mutants of platenolide of form A and B.The situation (with reference to embodiment 6 and 20) of Here it is bacterial strain OS49.67, in this bacterial strain through homophase deletion inactivation orf3 gene (Δ orf3).For the orf14 gene in this bacterial strain of inactivation, plasmid pSPM509 is imported bacterial strain OS49.67 (T.Kieser etc., 2000) through protoplast transformation.The inactivation of orf14 gene has been described in the orf14 gene that (with reference to embodiment 13) in the example of bacterial strain OSC2 and same steps as also are used for deactivated strain OS49.67.After plasmid pSPM509 conversion, these clones are screened through its apramycin resistance.Then, with the clone of apramycin resistance respectively contain on the substratum of apramycin with the substratum that contains Totomycin upload be commissioned to train foster.In principle, apramycin is had resistance (ApraR) and be that double exchange incident and those clones of being replaced of the orf14 copy that interrupted by att3 Ω aac-box of orf14 gene have wherein wherein been taken place for those the clone of Totomycin responsive (HygS).More specifically be to have screened these clones and verified that through hybridization box interrupts the replacement of copy to wild-type orf14 copy.Therefore, in order to verify certain gene replacement that taken place, the total DNA with several kinds of enzymic digestion institute DCRPs separates on sepharose, is transferred on the film also to hybridize with the probe of corresponding att3 Ω aac-box.Any means of knowing by one of skill in the art, and the PCR through using suitable oligonucleotide and the PCR product carried out sequential analysis especially also can be carried out genotypic checking.

More specifically be to have screened the clone who presents expection characteristic (Δ orf3, orf14::att3 Ω aac-) to be named as SPM510.In fact might verify that through two-wheeled hybridization att3 Ω aac-box is present in this cloned genes group really; And might verify certain desired digestion spectrum that obtained; Promptly in this cloned genes group; Through after the two-wheeled recombination event, the copy that is interrupted by att3 Ω aac-box has been replaced the digestion spectrum behind the wild-type orf14 gene.

Embodiment 22: interrupt the functional compensation of orf14 gene

22.1 make up plasmid pSPM519:

Use following oligonucleotide right: EDR31:

(SEQ ID No.136) and EDR37:

(SEQ ID No.137); With bacterial strain OSC2 chromosomal DNA is template, through pcr amplification orf14 gene.Oligonucleotide EDR31 and EDR37 carry HindIII and XbaI restriction enzyme site (runic sequence) respectively.The 1.2kb fragment cloning that so obtains is gone into carrier pGEM-T easy (by Promega company (Madison, Wisconsin USA) sell), to form plasmid pSPM515.Then with HindIII and this plasmid of XbaI Restriction Enzyme digestion.With the 1.2kb HindIII/XbaI insertion portion that is obtained be cloned into use identical enzymic digestion in advance carrier pUWL201 (with reference to embodiment 17.1).Gained plasmid called after pSPM519 like this.

22.2 transform bacterial strain SPM509 and SPM510 with plasmid pSPM519:

Through protoplast transformation (T.Kieser etc., 2000) plasmid pSPM519 is imported bacterial strain SPM509 (with reference to embodiment 13) and SPM510 (with reference to embodiment 17).After the conversion, through these clones of its thiostrepton resistance screening.Then with these be cloned in the substratum that contains thiostrepton upload be commissioned to train foster.

Bacterial strain SPM509 be the nonproductive bacteria strain of Spiramycin Base (with reference to

embodiment

13,15 and 16 and Figure 24).Through in having the MP5 substratum of thiostrepton, cultivating this bacterial strain, analyzed the Spiramycin Base output of the bacterial strain SPM509 (bacterial strain called after SPM509 pSPM519) that transforms with carrier pSPM519.Analyze culture supernatant liquid (with reference to embodiment 16 and 17) through HPLC then.Provided the result of this analysis in the table 43, data are expressed as mg and whenever go up clear liquid.The corresponding total Spiramycin Base output of result (through with Spiramycin I, the output addition of II and III acquisition).Observe the carrier pSPM519 that exists among the bacterial strain SPM509 and can recover Spiramycin Base output (with reference to table 43).

Table 43. is with the Spiramycin Base output (result is expressed as the mg/l supernatant) of the bacterial strain SPM509 of carrier pSPM519 conversion.

Bacterial strain SPM510 called after SPM510 pSPM509 with plasmid pSPM519 conversion.

Embodiment 23: the interrupting of the tylB gene function property compensation orf3 gene through streptomyces fradiae

23.1 make up plasmid pOS49.52:

The corresponding permission of plasmid pOS49.52 expressed the proteinic plasmid of TylB in living dyadic streptomycete.In order to make up this plasmid, with encoding sequence (Merson-Davies and Cundliffe, 1994 of streptomyces fradiae tylB gene; GenBank accession number: U08223 (sequence that this is regional), SFU08223 (dna sequence dna) and AAA21342 (protein sequence)) importing plasmid pKC1218 (Bierman etc., 1992; Kieser etc.; 2000, the coli strain that contains this plasmid especially can be from ARS (NRRL) Agricultural Research Service Culture Collection) (Peoria, Illinois; USA) obtain, preserving number is B-14790).In addition, this encoding sequence places ermE ^*Under the promotor control (above the reference, particularly being embodiment 17.1, Bibb etc., 1985, Bibb etc., 1994)

23.1 transform bacterial strain OS49.67 with plasmid pOS49.52:

Wherein the orf3 gene is not produced Spiramycin Base (with reference to embodiment 6 and 15) through the homophase deletion by the bacterial strain OS49.67 of inactivation.Through protoplast transformation (T.Kieser etc., 2000) plasmid pOS49.52 is imported bacterial strain OS49.67.After the conversion, through these clones of its apramycin resistance screening.Then with these be cloned in the substratum that contains apramycin upload be commissioned to train foster.Screen this clone and called after OS49.67 pOS49.52 more specifically.

As above illustrate, bacterial strain OS49.67 does not produce Spiramycin Base (with reference to

embodiment

6 and 15).Through the Spiramycin Base output of the technical Analysis described in the embodiment 15 with the bacterial strain OS49.67 of carrier pOS49.52 conversion.Therefore might prove that this bacterial strain has Spiramycin Base and produces the bacterium phenotype.Therefore, TylB protein allows interrupting of functional compensation orf3 gene.

Embodiment 24: improve Spiramycin Base output through overexpression orf28c gene

24.1 make up plasmid pSPM75:

Use contains a pair of oligonucleotide of HindIII restriction enzyme site or BamHI restriction enzyme site, through pcr amplification orf28c gene.These primers have following sequence:

KF30:

(SEQ ID No.138) has HindIII restriction enzyme site (shown in the runic)

KF31:

(SEQ ID No.139) has BamHI restriction enzyme site (shown in the runic).

Primer KF30 and KF31 carry HindIII and BamHI restriction enzyme site (runic sequence) respectively.KF30 and KF31 primer contain orf28c gene size and be the dna fragmentation of about 1.5kb making to increase, and utilize clay pSPM36 as template (reference is top).The 1.5kb fragment cloning that so obtains is gone into carrier pGEM-T easy (by Promega company (Madison, Wisconsin USA) sell) to produce plasmid pSPM74.Go in advance the carrier pUWL201 (with reference to embodiment 17.1) that has digested with same enzyme then with HindIII and BamHI Restriction Enzyme digested plasmid pSPM74 and with the about 1.5kb HindIII/BamHI insertion portion subclone that is obtained.Gained plasmid called after pSPM75 like this; This plasmid comprises and places ermE ^*The whole encoding sequences of orf28c under the promotor control.

24.2 transform bacterial strain OSC2 with plasmid pSPM75:

Through protoplast transformation (T.Kieser etc., 2000) plasmid pSPM75 is imported bacterial strain OSC2.After the protoplast transformation, through these clones of its thiostrepton resistance screening.Then these being cloned in the substratum that contains thiostrepton uploads to be commissioned to train to support and extract through plasmid and has verified the conversion of carrying out with plasmid.Screen two clones and called after OSC2/pSPM75 (1) and OSC2/pSPM75 (2) more significantly.

The sample of bacterial strain OSC2/pSPM75 (2) is preserved in state-run microbial preservation center (CNCM) [National Collection of Cultures and Microorganisms] Institute Pasteur; 25; Rue du Docteur Roux 75724Paris Cedex 15; France, preservation day is on October 6th, 2003, preserving number is I-3101.

In order to test the influence of overexpression orf28c gene pairs Spiramycin Base output, through technical testing OSC2/pSPM75 (1) described in the embodiment 15 and OSC2/pSPM75 (2) clone's Spiramycin Base output.The parallel therewith Spiramycin Base output of also having analyzed bacterial strain OSC2 is to compare.Therefore the existence that might observe plasmid pSPM75 significantly increases the Spiramycin Base output of bacterial strain OSC2.This proof orf28c overexpression causes Spiramycin Base output to increase and confirms the effect of this gene as regulon.

Also through HPLC analyzed OSC2/pSPM75 (1) and OSC2/pSPM75 (2) clone Spiramycin Base output (with embodiment 17.2 in identical mode).The parallel therewith Spiramycin Base output of also having analyzed bacterial strain OSC2 is to compare.Provided the result of this analysis in the table 44, data are expressed as mg and whenever go up clear liquid.The corresponding Spiramycin Base ultimate production of result (through with Spiramycin I, the turnout addition of II and III acquisition).

Table 43. derives from the Spiramycin Base output (result is expressed as mg/l) of the bacterial strain that transforms with carrier pSPM75 of OSC2.

Therefore, the existence of observing plasmid pSPM75 significantly increases the Spiramycin Base output of bacterial strain OSC2.This proves that clearly the orf28c overexpression has positive-effect to Spiramycin Base output.

Embodiment 25: through its orf8 gene the bacterial strain of inactivation analyze the output of Spiramycin Base biosynthesizing midbody:

Bacterial strain OS49.107, wherein inactivation does not produce Spiramycin Base (with reference to embodiment 7 and 15) to the orf8 gene through inserting Ω hyg box.Orf8 genes encoding and several kinds of transaminases present the protein of strong relatively similarity and the 4-transaminase (with reference to Fig. 6) that strong hint orf8 genes encoding is responsible for the necessary transamination reaction of forosamine biosynthesizing.Therefore expect that the Spiramycin Base biosynthesizing will block in forocidin stage (with reference to Fig. 7).Therefore the nonproductive bacteria strain OS49.107 of Spiramycin Base will produce forocidin.

Prepare the supernatant samples (, not carrying out the MIBK extracting) of bacterial strain OS49.107 according to the method described above and analyze (with reference to paragraph 20.2 and 20.3) through LC/MS as above-mentioned with reference to embodiment 16.Under the SIM pattern, selected the quality 558 of corresponding forocidin molion and detected several peaks.The existence of quality 558 compounds and orf8 have the hypothesis of effect in forosamine is synthetic consistent.

Embodiment 26: through its orf12 gene the bacterial strain of inactivation analyze the output of Spiramycin Base biosynthesizing midbody:

Bacterial strain SPM507, wherein orf12 gene inactivation does not produce Spiramycin Base (with reference to

embodiment

11 and 15).Think that the orf12 genes encoding is responsible for 3 of the necessary dehydration reaction of forosamine biosynthesizing, 4-dehydratase (with reference to Fig. 6).Therefore expect that the Spiramycin Base biosynthesizing will block in forocidin stage (with reference to Fig. 7).Therefore the nonproductive bacteria strain SPM507 of Spiramycin Base will produce forocidin.

Prepare the supernatant samples (, not carrying out the MIBK extracting) of bacterial strain SPM507 according to the method described above and analyze (with reference to paragraph 20.2 and 20.3) through LC/MS as above-mentioned with reference to embodiment 16.Under these conditions, the forocidin RT is about 12.9 minutes.Under the SIM pattern, selected corresponding forocidin molion [M+H] ⁺Quality 558 and detect several peaks.The existence of 558 place's compounds makes might verify that the product of orf12 has the hypothesis of effect in the Spiramycin Base biosynthesizing.

Yet, forocidin exist with low relatively amount and, under these conditions, observed the product (RT is 17.1 minutes) that absorption is arranged at the 238nm place more significantly.LC/MS analyzes the feasible quality that might confirm this compound, is 744.3g/mol ([M+H] ⁺=744.3, primary product).

In order to obtain structure, separate above-mentioned product and purifying (with reference to paragraph 20.1) under these conditions.Reclaim organic phase (MIBK) and evaporation drying then.The exsiccant extract is water-soluble and carry out extracting with heptane.Then through being attached to Oasis HLB 1g post (Waters SAS, St-Quentin en-Yvelines, France) this aqueous solution of extracting.Through reclaiming this compound with 30/70 water/acetonitrile mixture wash-out.Then this injection of solution (100 μ l) is gone into analytical column and gone up recovery level branch at Oasis HLB 1cc 30mg post (Waters).Before using, with Oasis HLB 1cc 30mg post (Waters) successively with acetonitrile, be that water/acetonitrile mixture (20v/80v) and 80/20 water/acetonitrile mixture are regulated then.

Use 1ml water/acetonitrile (95/5) and 1ml water/deuterate acetonitrile (95/5) washing OasisHLB 1 cc 30mg post (Waters) then successively, use 600 μ l, 40/60 water/deuterate acetonitrile wash-out then.Then the solution that reclaims is directly analyzed through NMR.

The NMR spectrum of this compound of gained is (19312V NMR spectrum) as follows: the 1H spectrum (chemical transport ppm) among the CD3CN/D2O: 0.92 (3H, d, J=6Hz), not 1.10 (1H does not differentiate the peak), 1.14 (3H, s), 1.17 (3H, d, J=6Hz); 1.22 (3H, d, J=6Hz), 1.25 (3H, d, J=6Hz), 1.40 (1H does not differentiate the peak), 1.75 (1H, dd; J=12 and 2Hz), not 1.81 (1H does not differentiate the peak), 1.90 (1H, d, J=12Hz), not 2.05 (1H does not differentiate the peak), 2.12 (3H, s); (2.15 1H does not differentiate the peak), 2.35 (2H does not differentiate the peak), 2.45 (6H, wide s), 2.53 (1H does not differentiate the peak), 2.64 (1H, dd; J=12 and 9Hz), 2.80 (1H, dd, J=9 and 16Hz), 2.95 (1H, d, J=8Hz), 3.23 (2H does not differentiate the peak), 3.34 (1H; D, J=7Hz), 3.45 (3H, s), 3.49 (1H does not differentiate the peak), 3.93 (1H, dd, J=7 and 3Hz), 4.08 (1H; Do not differentiate the peak), 4.37 (1H, d, J=6Hz), 4.88 (1H does not differentiate the peak), 5.05 (2H does not differentiate the peak), 5.65 (2H; Do not differentiate the peak), 6.08 (1H, dd, J=8 and 12Hz), 6.40 (1H, dd, J=12 and 9Hz), 9.60 (1H, s).

These experiments make and the structure that might confirm this compound have provided this structure among Figure 38.

Embodiment 27: through its orf5 ^*Gene is the generation of the bacterial strain analysis Spiramycin Base biosynthesizing midbody of inactivation:

Bacterial strain SPM501 has genotype orf6 ^*:: att1 Ω hyg+.Be inserted into orf6 according to att1 Ω hyg+ box ^*The polar effect of gene might be confirmed orf5 ^*Gene is that the Spiramycin Base biosynthetic pathway is necessary.Particularly, according to orf5 ^*The polar effect of genetic expression can be excised box and is inserted into orf6 ^*Gene coded sequence causes Spiramycin Base production to be blocked fully.Yet, in case excised the box that inserts (and therefore inactivation orf6 only ^*During gene, with reference to embodiment 14 and 15), the generation of Spiramycin I recovers.This proves orf5 ^*Gene is that the Spiramycin Base biosynthesizing is necessary, because the inactivation of this gene causes Spiramycin Base to produce blocking-up fully.

Orf5 ^*Genes encoding and several kinds of O-methyltransgerases present the protein of similarity relatively by force.Think orf5 ^*Gene is to participate in the biosynthetic O-methyltransgerase of platenolide.In order to verify this hypothesis, the bacterial strain from excessive production Spiramycin Base is obtained, genotype is orf6 ^*:: the living dyadic streptomycete bacterial strain of att1 Ω hyg+ has carried out LC/MS and NMR analyzes experiment.

Prepare the sample (, not carrying out the MIBK extracting) of bacterial strain supernatant according to the method described above and analyze (with reference to paragraph 20.2 and 20.3) through above-mentioned LC/MS with reference to embodiment 16.Yet used pillar is X-Terra post (Waters SAS, St-Quentin en-Yvelines, a France), and the cone voltage of spectrometer (cone voltage) is set to 380V to obtain the fermentation of institute's analysis of compounds.Under these conditions, observing RT is about 13.1 minutes product.The mass spectrum of this compound and the mass spectrum of Spiramycin I have apparent similarity, but molion is 829.The difference of comparing 14 quality with the quality of Spiramycin Base can be interpreted as: on the oxygen that No. 4 carbon of lactonic ring are loaded, lack methyl (having provided the structure of this compound among Figure 39).Exist compound to make at 829 places and might confirm orf5 ^*The hypothesis that in the Spiramycin Base biosynthesizing, has effect.Utilize the sensitive strain micrococcus luteus to carry out microbiological assay, proved bacterial strain orf6 ^*:: the midbody molecule that Ω atthyg+ is produced (not methylic Spiramycin Base has provided this structure among Figure 39) has (weak 10 times) a little less than the Spiramycin Base activity of methyl than 4 places, position.

Embodiment 28: make up new " can excise box ":

Made up new excised box.The excised box of having described among these boxes and the embodiment 9 is closely similar.The former box is in the latter, to lack corresponding Ω to insert the terminal sequence of son (interposon) with main difference between the new box, and this sequence comprises the transcription terminator that derives from the T4 phage.

In the box that does not contain terminator, the both sides of giving the gene of antibiotics resistance are attR and the attL sequences that allows excision.Resistant gene is aac (3) the IV gene that coding is given the acetyltransferase of apramycin resistance.This gene is present in Ω aac box (GenBank accession number: X99313; Blondelet-Rouault; M.H. etc.; 1997) and through pcr amplification obtain, the PCR reaction with plasmid pOSK1102 (with reference to top) as template, to contain HindIII restriction enzyme site (runic) oligonucleotide KF42 and KF43 (AAGCTT) respectively in 5 ' position as primer:

KF42:

(SEQ IDNo.153) and

KF43：

(SEQ?IDNo.154)。

The PCR product cloning of the about 1kb that is obtained is gone into escherichia coli vector pGEMT Easy, to produce plasmid pSPM83.

With HindIII Restriction Enzyme digested vector pSPM83.Separate the HindIII-HindIII fragment through purifying from 0.8% sepharose; Then it is cloned into and is positioned multiple attL and the HindIII site between the attR sequence (with reference to embodiment 9 and Figure 27) that carries the plasmid that difference can excise box, so that the HindIII fragment of replacing corresponding Ω acc with the HindIII fragment of corresponding aac gene only.This makes might obtain att1aac, att2aac and att3aac box (according to purpose, with reference to embodiment 9).Different according to the relative attL of aac gene with the direction of attR sequence, att1aac+, att1aac-, att2aac+, att2aac-, att3aac+ is distinguishing (according to the identical convention that in embodiment 9, is adopted) with att3aac-.

Embodiment 29: have the living dyadic streptomycete bacterial strain that knocks out in the structure orf28c gene:

Utilization can be excised box technology inactivation orf28c gene.Can excise box att3aac+ (with reference to embodiment 28) through pcr amplification; (plasmid pSPM101 is the plasmid (Chaveroche etc. that derive from carrier pGP704Not with plasmid pSPM101 in the PCR reaction; 2000) (Miller VL and Mekalanos JJ; 1988), wherein the att3aac+ box has been cloned into EcoRV site unique among the pGP704Not as the EcoRV fragment) be template, and use following primer:

KF32：

(SEQ?ID?No.155)

And KF33:

(SEQ?ID?No.156)

Being positioned these oligonucleotide sequences 5 ' 39 terminal Nucleotide comprises the sequence of sequence in the corresponding orf28c gene and is positioned 26 Nucleotide (top underlining with runic represented) correspondence of 3 ' position and can excise the sequence of an end of box att3aac+.Gained PCR product like this is used to transform overweight group of coli strain DY330 (Yu etc. that contain clay pSPM36; 2000) (this bacterial strain contains the exo of lambda particles phage; Bet and gam gene are integrated into its karyomit(e), and these genes are expressed in the time of 42 ℃; This bacterial strain is used to replace coli strain KS272 (Chaveroche etc., 2000)).Therefore, also pass through these clones of its apramycin resistance screening through the fax hole with this PCR product transform bacteria.The clay that extracts institute's DCRP also digests with the BamHI Restriction Enzyme; Purpose is to have inserted the orf28c gene in order to verify like compartmentalized box for holding assorted fruits and candies (att3aac+); If homologous recombination (Chaveroche etc. have promptly taken place between the end of PCR product and target gene really; 2000), then resulting digestion spectrum is consistent with expection digestion spectrum.Known by one of skill in the art method, especially PCR and the sequential analysis of PCR product through utilizing suitable oligonucleotide also can be verified this construct.Choose that clay wherein has the clone of expection digestion spectrum and with corresponding clay called after pSPM107.This clay is the verivate of pSPM36, and wherein orf28c is interrupted by the att3aac+ box.The insertion of box is accompanied by the deletion in the orf28c gene, interrupts the 28th codon that originates in orf28c.Box has kept last 137 codons of orf28c at the back.

In the first step, clay pSPM107 is imported coli strain DH5 α, and import living dyadic streptomycete bacterial strain OSC2 through protoplast transformation then.After the conversion, through these clones of its apramycin resistance screening.Then apramycin-resistance clone is uploaded be commissioned to train foster (with reference to Fig. 9) with the substratum that contains tetracycline (microbiotic A) respectively on the substratum that contains apramycin (microbiotic B).In principle, apramycin being had resistance (ApraR) and tetracycline responsive (PuroS) is cloned is that those double exchange incident have wherein taken place and have contained the clone who is interrupted the orf28c gene by the att3aac+ box.More specifically choose these clones and verified that through hybridization box interrupts the replacement of copy to wild-type orf28c copy.Therefore, have box in order to prove the expection locus in gained cloned genes group DNA, the total DNA with several kinds of enzymic digestion institute DCRPs separates on sepharose, transfers on the film and with the probe of corresponding att3aac+ box and hybridizes.Known by one of skill in the art any means, especially PCR and the sequential analysis of PCR product through utilizing suitable oligonucleotide also can be carried out genotypic checking.

More specifically screen the clone and the called after SPM107 that present expection characteristic (orf28c::att3aac+).Therefore this clone has genotype: orf28c::att3aac+ and called after SPM107.Consider the direction (with reference to Fig. 3) of gene, there is no need to excise the effect of box with research orf28c inactivation.The localized in the opposite direction fact of orf29 and orf28c shows that these genes are not the corotation records.On the other hand, can excise the use of box, especially through transform the feasible possibility that might obtain to remove at any time selection markers with plasmid pOSV508.

In order to test the influence of closing orf28c gene pairs Spiramycin Base output, through the Spiramycin Base output of the technical testing bacterial strain SPM107 described in the embodiment 15.Therefore might prove that this bacterial strain has the nonproductive bacterium phenotype of Spiramycin Base.This proof orf28c gene is to give birth to the necessary gene of Spiramycin Base biosynthesizing in the dyadic streptomycete.

Embodiment 30: be structured in and have the living dyadic streptomycete bacterial strain that knocks out in the orf31 gene:

Utilization can be excised box technology inactivation orf31 gene.Can excise box att3aac+ through pcr amplification, PCR reaction is template with plasmid pSPM101 and is primer with oligonucleotide EDR71 and EDR72.

EDR71：

(SEQ?ID?No.157)

EDR72：

(SEQ?ID?No.158)

Being positioned these oligonucleotide sequences 5 ' 39 terminal Nucleotide comprises the sequence of sequence in the corresponding orf31 gene and is positioned 26 Nucleotide (top underlining with runic represented) correspondence of 3 ' position and can excise the sequence of an end of box att3aac+.

Like (Chaveroche et al. such as Chaveroche; 2000) said; Gained PCR product like this is used to transform the coli strain KS272 that contains plasmid pKOBEG and clay pSPM36 (with reference to the principle of Figure 12, plasmid pOS49.99 will no longer be pSPM17 but pSPM543 by clay pSPM36 replacement and gained plasmid).Therefore, also pass through these clones of its apramycin resistance screening through the fax hole with this PCR product transform bacteria.The clay that extracts institute's DCRP is also with several kinds of Restriction Enzymes digestion; Purpose is to have inserted the orf31 gene in order to verify like compartmentalized box for holding assorted fruits and candies (att3aac+); If homologous recombination has promptly taken place between the end of PCR product and target gene really, then resulting digestion spectrum is consistent with expection digestion spectrum.Known by one of skill in the art method particularly is PCR and the sequential analysis of PCR product through utilizing suitable oligonucleotide, also can verify this construct.Choose that clay wherein has the clone of expection digestion spectrum and with corresponding clay called after pSPM543.This clay is the verivate of pSPM36, and wherein orf31 interrupts (with reference to Figure 12) by the att3aac+ box.The insertion of box is accompanied by the deletion in the orf31 gene, and this interrupts and originates in the 36th codon of orf31.Box has kept last 33 codons of orf31 at the back.

Through protoplast transformation (Kieser, T etc., 2000) pSPM543 is imported living dyadic streptomycete bacterial strain OSC2 (with reference to top).After the conversion, through these clones of its apramycin resistance screening.Then apramycin-resistance clone is uploaded be commissioned to train foster (with reference to Fig. 9) with the substratum that contains tetracycline (microbiotic A) respectively on the substratum that contains apramycin (microbiotic B).In principle, apramycin is had resistance (ApraR) and be that those double exchange incident have wherein taken place and have contained the clone who is interrupted the orf31 gene by the att3aac+ box the clone of tetracycline responsive (PuroS).More specifically choose these clones and verified that through hybridization box interrupts the replacement of copy to wild-type orf31 copy.Therefore, have box in order to prove the expection locus in gained cloned genes group DNA, the total DNA with several kinds of enzymic digestion institute DCRPs separates on sepharose, transfers on the film and with the probe of corresponding att3aac+ box and hybridizes.To pass through PCR acquisition and the very most dna fragmentation of corresponding orf31 gene coded sequence, carried out second and taken turns hybridization as probe.

Known by one of skill in the art any means particularly is PCR and the sequential analysis of PCR product through utilizing suitable oligonucleotide, also can carry out genotypic checking.

More specifically screen the clone and the called after SPM543 that present expection characteristic (orf31c::att3aac+).Hybridize through two-wheeled; In fact might verify that the att3aac+ box is present in this cloned genes group really and checking after dual group of incident; In this cloned genes group, use under the situation of the copy replacement wild type gene that interrupts by the att3aac+ box, obtained the digestion spectrum of expection really.Therefore this clone has genotype: orf31::att3aac+ and called after SPM543.Consider the direction (with reference to Fig. 3) of gene, there is no need to excise the effect of box with research orf31 inactivation.The localized in the opposite direction fact of orf32c and orf31 shows that these genes are not the corotation records.On the other hand, can excise the use of box, especially through transform the feasible possibility that might obtain to remove at any time selection markers with plasmid pOSV508.

In order to test the influence of closing orf31 gene pairs Spiramycin Base output, through the technical testing described in the embodiment 15 the Spiramycin Base output of bacterial strain SPM543.Therefore might prove that this bacterial strain has the nonproductive bacterium phenotype of Spiramycin Base.This proof orf31 gene is to give birth to the necessary gene of Spiramycin Base biosynthesizing in the dyadic streptomycete.

Embodiment 31: be structured in and have the living dyadic streptomycete bacterial strain that knocks out in the orf32c gene:

Utilization can be excised box technology inactivation orf32c gene.Can excise box att3aac+ through pcr amplification, the PCR reaction is template with plasmid pSPM101 and uses following primer:

KF52：

(SEQ?ID?No.159)。

And KF53:

(SEQ?ID?No.160)。

Being positioned these oligonucleotide sequences 5 ' 40 terminal Nucleotide comprises the sequence of sequence in the corresponding orf32c gene and is positioned 26 Nucleotide (top underlining with runic represented) correspondence of 3 ' position and can excise the sequence of an end of box att3aac+.

Gained PCR product like this is used to transform the overweight group of coli strain DY330 (Yu etc., 2000) that contains clay pSPM36.Therefore, also pass through these clones of its apramycin resistance screening through the fax hole with this PCR product transform bacteria.The clay that extracts institute's DCRP also digests with the BamHI Restriction Enzyme; Purpose is to have inserted the orf32c gene in order to verify like compartmentalized box for holding assorted fruits and candies (att3aac+); If homologous recombination has promptly taken place between the end of PCR product and target gene really, then resulting digestion spectrum is consistent with expection digestion spectrum.Known by one of skill in the art method particularly is PCR and the sequential analysis of PCR product through utilizing suitable oligonucleotide, also can verify this construct.Choose that clay wherein has the clone of expection digestion spectrum and with corresponding clay called after pSPM106.This clay is the verivate of pSPM36, and wherein orf32c is interrupted by the att3aac+ box.The insertion of box is accompanied by the deletion in the orf32c gene, and this interrupts and originates in the 112nd codon of orf32c.Box has kept last 91 codons of orf32c at the back.

In the first step, clay pSPM106 is imported coli strain DH5 α, and through transforming it is imported living dyadic streptomycete bacterial strain OSC2 then.After the conversion, through these clones of its apramycin resistance screening.Then apramycin-resistance clone is uploaded be commissioned to train foster (with reference to Fig. 9) with the substratum that contains tetracycline (microbiotic A) respectively on the substratum that contains apramycin (microbiotic B).In principle, apramycin is had resistance (ApraR) and be that those double exchange incident have wherein taken place and have contained the clone who is interrupted the orf32c gene by the att3aac+ box the clone of tetracycline responsive (PuroS).More specifically be to choose these clones and verified that through hybridization box interrupts the replacement of copy to wild-type orf32c copy.Therefore, have box in order to prove in gained cloned genes group DNA, the total DNA with several kinds of enzymic digestion institute DCRPs separates on sepharose, transfers on the film and with the probe of corresponding att3aac+ box and hybridizes.Known by one of skill in the art any means particularly is PCR and the sequential analysis of PCR product through utilizing suitable oligonucleotide, also can carry out genotypic checking.

More specifically screen the clone who presents expection characteristic (orf32c::att3aac+).Therefore this clone has genotype: orf32c::att3aac+ and called after SPM106.Consider the direction (with reference to Fig. 3) of gene, there is no need to excise the effect of box with research orf32c inactivation.The localized in the opposite direction fact of orf33 and orf32c shows that these genes are not the corotation records.On the other hand, can excise the use of box, especially through transform the feasible possibility that might obtain to remove at any time selection markers with plasmid pOSV508.

In order to test the influence of closing orf32c gene pairs Spiramycin Base output, through the technical testing described in the embodiment 15 the Spiramycin Base output of bacterial strain SPM106.Therefore might prove that this bacterial strain has Spiramycin Base and produces the bacterium phenotype.This proof orf32c gene is not to give birth to the necessary gene of Spiramycin Base biosynthesizing in the dyadic streptomycete.

The tabulation of the construct described in the application

Shortenings tabulation: Am: penbritin; Hyg: Totomycin; Sp: Spiramycin Base; Ts: thiostrepton; Cm: paraxin; Kn: kantlex; Apra: apramycin.

The preservation of biomaterial

Following organism is deposited in state-run microbial preservation center [National Collection of Cultures and Microorganisms] (CNCM) according to budapest treaty on July 10th, 2002; 25rue du Docteur Roux; 75724 Paris Cedex 15, France.

-bacterial strain OSC2, preserving number are I-2908.

-bacterial strain SPM501, preserving number are I-2909.

-bacterial strain SPM502, preserving number are I-2910.

-bacterial strain SPM507, preserving number are I-2911.

-bacterial strain SPM508, preserving number are I-2912.

-bacterial strain SPM509, preserving number are I-2913.

-bacterial strain SPM21, preserving number are I-2914.

-bacterial strain SPM22, preserving number are I-2915.

-bacterial strain OS49.67, preserving number are I-2916.

-bacterial strain OS49.107, preserving number are I-2917.

-containing the coli strain DH5 α of plasmid pOS44.4, preserving number is I-2918.

Following organism is deposited in state-run microbial preservation center (CNCM), 25rue du Docteur Roux, 75724 Paris Cedex 15, France according to budapest treaty on February 26th, 2003.

-bacterial strain SPM502 pSPM525, preserving number are I-2977.

Following organism is deposited in state-run microbial preservation center (CNCM), 25rue du Docteur Roux, 75724Paris Cedex 15, France according to budapest treaty on October 6th, 2003.

-bacterial strain OSC2/pSPM75 (2), preserving number are I-3101.

All publications and patent are quoted as a reference in this application.

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Sequence table

Claims

1. the polynucleotide of coding involved in spiramycin biosynthetic polypeptide, the sequence of wherein said polynucleotide is:

(a) be selected from one of the sequence of SEQ ID No.111 and 141,

(b) because the degeneracy of genetic code and derived from one of sequence of sequence (a).

2. polynucleotide, its coding is compared with the described polynucleotide encoded polypeptide of claim 1, has the conservative alternate polypeptide of one or more amino-acid residues.

3. polynucleotide as claimed in claim 2, it separates the bacterium from streptomyces (Streptomyces).

4. polynucleotide as claimed in claim 2, the biosynthetic protein of its coding involved in spiramycin.

5. the polypeptide that produces by the expression of claim 1,2,3 or 4 said polynucleotide.

6. the biosynthetic polypeptide of involved in spiramycin, the sequence of wherein said polypeptide is one of sequence that is selected from SEQ ID No.112 and 142.

7. recombinant DNA, it comprises at least one like each described polynucleotide in the claim 1,2,3 or 4.

8. recombinant DNA as claimed in claim 7, wherein said recombinant DNA is included in the carrier.

9. recombinant DNA as claimed in claim 8, wherein said carrier is selected from phage, plasmid, phagemid, integrating vector, fosmids, clay, shuttle vectors, BAC and PAC.

10. expression vector, it comprises the nucleotide sequence of polypeptide described at least a coding such as claim 5 or 6.

11. expression system, it comprises the expression vector and the host cell of claim 10.

12. the expression system described in claim 11, it is selected from prokaryotic expression system and eukaryotic expression system.

13. expression system as claimed in claim 12, it is selected from the system in bacteria Escherichia coli, expressed, allows the baculovirus expression system in expressed in insect cells, the expression system that allows the expression system of in yeast cell, expressing and allow in mammalian cell, to express.

14. host cell has wherein imported at least a like any described recombinant DNA in any described polynucleotide and/or at least a claim 7,8 and 9 in the claim 1,2,3 and 4 and/or the described expression vector of at least a claim 10.

15. produce the method like claim 5 or 6 said polypeptide, wherein said method may further comprise the steps:

A) nucleic acid with at least a coding said polypeptide inserts appropriate carriers;

C) recovering condition substratum or cell extract;

D) the said substratum or the cell extract that obtain from step c) separate and the said polypeptide of purifying;

E) as required, characterize the recombinant polypeptide that is produced.

16. the purposes like any described polynucleotide in the claim 1,2,3 and 4 is used to improve the Spiramycin Base output of mikrobe, wherein said mikrobe is the streptomyces bacterium.

17. produce the mutant microbial of Spiramycin Base; At least one comprises the gene like any said polynucleotide in the claim 1,2,3 and 4 its overexpression; Wherein said mikrobe is the streptomyces bacterium, the overexpression of wherein said gene be through the copy number that increases this gene and/or through import than the higher promotor of wild-type promoter activity and/or through with as claim 7, the recombinant DNA described in 8 or 9 transform product Spiramycin Base mikrobe and obtain.

18. the mutant microbial described in claim 17, the overexpression of wherein said gene are to obtain through the copy number that increases this gene and/or through importing the promotor higher than wild-type promoter activity.

19. the mutant microbial described in claim 17 or 18, the overexpression of wherein said gene are through obtaining with transform product Spiramycin Base mikrobe like claim 7, the recombinant DNA described in 8 or 9.

20. the mutant microbial described in claim 17 or 18; One or more genes of mikrobe overexpression wherein, said one or more genes comprise corresponding to one of sequence one or more in sequence SEQ ID No.111 and 141 or because genetic code degeneracy and by one of one of said sequence deutero-sequence.

21. the mutant microbial described in claim 17 or 18, wherein said mikrobe are to give birth to the dyadic streptomycete bacterial strain.

22. produce the method for Spiramycin Base, it may further comprise the steps:

(a) in suitable substratum, cultivate like any described mikrobe in the claim 17,18,19,20 and 21,

(b) recovering condition substratum or cell extract,

(c) separate said substratum that obtains from step (b) or the cell extract and said Spiramycin Base that purifying produces.

23., be used to prepare the hydridization microbiotic like any described polynucleotide in the claim 1,2,3 and 4 and/or like any described polypeptide in claim 5 and 6 and/or like the purposes of any described recombinant DNA and/or carrier as claimed in claim 10 in the claim 7,8 and 9.

24. at least a like any described polynucleotide in the claim 1,2,3 and 4 and/or at least a like each described recombinant DNA in the claim 7,8 and 9 and/or at least a expression vector as claimed in claim 10 and/or at least a purposes like any described polypeptide and/or at least a host cell as claimed in claim 14 in claim 5 and 6, be used to carry out the required bio-transformation of one or more Spiramycin Base biosynthesizing.

25. polynucleotide, its be with like one of claim 1,2, the polynucleotide described in 3 or 4 complementary polynucleotide.