CN103820473A - ToyG expression-enhanced recombined streptomyces diastatochromogenes and construction method and uses thereof - Google Patents

Abstract

The invention discloses a toyG expression-enhanced recombined streptomyces diastatochromogenes and a construction method and uses thereof. The recombined streptomyces diastatochromogenes excessively expresses that the key enzyme of biosynthetic toyocamycin-an adenosine succinic acid synthetase encoding gene toyG has a toyocamycin synthesis capacity higher than streptomyces diastatochromogenes 1628. The construction process is as follows: (1) constructing an expression vector pIB139-toyG; (2) utilizing a conjugational transfer method to integrate the expression vector into a chromosome of streptomyces diastatochromogenes to obtain engineering bacteria. A promoter permE on the vector pIB139 is utilized to open the expression of the toyG gene, and the conjugational transfer method is utilized to integrate the specificity of the vector pIB139-toyG into the chromosome of streptomyces diastatochromogenes 1628 to obtain the genetic-stability engineering bacteria. Compared with parent strains, the enzyme activity of recombinant strain adenosine succinic acid synthetase is increased by at least 1.2 times, and the yield of toyocamycin is increased by at least 27.1 percent.

Description

Restructuring streptomyces diastatochromogenes and construction process and purposes that toyG expresses are strengthened

Technical field

The present invention relates to improve toyokamycin output by strengthening toyG gene in the expression of streptomyces diastatochromogenes, belong to gene engineering technology field.

Background technology

Toyokamycin is a kind of novel nucleoside microbiotic, and molecular formula is C ₁₂h ₁₃n ₅o ₄, ribose C ₁connect the deazapurine ring of similar guanine, core texture is pyrroles's pyrimidine nucleoside analoys.The mechanism of action is mainly the growth that affects thalline of transcribing by suppressing microorganism, and its bioactivity research report mainly concentrates on clinical medicine domain.Studies have found that in the recent period toyokamycin has good prevention effect to various plants epidemic disease, life-time service can not cause environmental pollution, and plant-growth is also had to certain regulating effect.Therefore the application potential that, toyokamycin has in agricultural plants disease control field.With respect to chemical synthesis, the synthetic toyokamycin of biological process is take renewable resources as raw material, has reaction conditions gentleness, pollutes less and the advantage such as with low cost, and therefore, biological synthesis process is the both economical effective means of current toyokamycin suitability for industrialized production.

Toyokamycin belongs to secondary metabolite, and route of synthesis complexity is subject to restriction and the regulation and control of many factors, causes the synthetic level of toyokamycin lower, is difficult to large-scale production.The existing way addressing this problem is all generally the production bacterial strain as Main Means screening high yield and high quality by traditional selection by mutation, but screen, the uncertain factor of high efficient strain is many, the cycle is long.

Output how to utilize the advanced persons' such as molecular biology technique means improvement microorganism further to improve toyokamycin becomes a feasible thinking.The people such as McCarty are take the synthetic toyokamycin bacterial strain Streptomyces rimosus (ATCC14673) of a strain as initial research object, clone biosynthesizing toyokamycin gene cluster, building-up process and the regulatory mechanism of toyokamycin are illustrated, research finds that toyokamycin is precursor by GTP, through the synthetic toyokamycin of polystep reaction, be wherein one of key enzyme of pathways metabolism by the adenosine succsinic acid synthetic enzyme of toyG genes encoding.Have no relevant report but utilize metabolic engineering technology further to improve toyokamycin output by increasing toyokamycin metabolic flux because relate to the challenge such as foundation of expression system.

Summary of the invention

In order to overcome the deficiencies in the prior art, the invention provides a kind of strengthened restructuring streptomyces diastatochromogenes and construction process and purposes that toyG expresses.

Streptomyces diastatochromogenes ( streptomyces diastatochromogenes) 1628 are strain Antagonistic Actinomycetes, its fermented liquid has stronger restraining effect to various plants pathogenic fungi, through separation and Extraction, determine that its main effective constituent is toyokamycin, there is not yet streptomyces diastatochromogenes metabolic engineering molecular modification aspect research report.

The present invention first from streptomyces diastatochromogenes ( streptomyces diastatochromogenes) clone the toyG gene of coding adenosine succsinic acid synthetic enzyme in 1628 (deposit number of bacterial strain is CGMCC NO. 2060), and this gene is connected with streptomycete integrative plasmid pIB139, successfully build and carried toythe recombinant vectors pIB139-of G gene toyg, and utilize conjugal transfer method by its specific being incorporated on streptomyces diastatochromogenes 1628 karyomit(e)s.

A toyG gene for streptomyces diastatochromogenes, his sequence is as shown in SEQ ID NO:1.

Strengthened the restructuring streptomyces diastatochromogenes that toyG expresses, it has expressed the key enzyme-adenosine succsinic acid synthetase-coding gene of biosynthesizing toyokamycin toyg, have than streptomyces diastatochromogenes ( streptomyces diastatochromogenes) 1628 higher toyokamycin abilities to express.

Described original bacterium be streptomyces diastatochromogenes ( streptomyces diastatochromogenes) 1628.

Described adenosine succsinic acid synthetase-coding gene toyG come from original bacterium streptomyces diastatochromogenes to be reorganized ( streptomyces diastatochromogenes).

Described adenosine succsinic acid synthetase-coding gene toyG is integrated into original bacterium streptomyces diastatochromogenes karyomit(e).

Described reinforcement the toyG construction process of restructuring streptomyces diastatochromogenes of expressing, process is as follows:

1) construction of expression vector pIB139-toyG;

2) utilize conjugal transfer method that described expression vector is integrated into streptomyces diastatochromogenes karyomit(e), obtain described engineering bacteria.

Utilize promotor permE* on pIB139 carrier to start the expression of toyG gene, utilize conjugal transfer method by specific carrier pIB139-toyG be integrated into streptomyces diastatochromogenes ( streptomyces diastatochromogenes) 1628 karyomit(e), obtain the engineering bacteria of inheritance stability.

Described reinforcement the purposes of restructuring streptomyces diastatochromogenes expressed of toyG, compared with original strain, recombinant bacterium adenosine succsinic acid synthetic enzyme enzyme is lived and has at least been improved 1.2 times, toyokamycin output has at least improved 27.1%.

Beneficial effect of the present invention:

The present invention strengthens toyokamycin biosynthetic pathway key gene toyG in streptomyces diastatochromogenes after overexpression, and the work of recombinant bacterium adenosine succsinic acid synthetic enzyme enzyme has improved 1.2 times, and the original bacterium of energy force rate of synthetic toyokamycin has improved 27.1%.For further improving toyokamycin output, realize early suitability for industrialized production and lay a good foundation.

Accompanying drawing explanation

Fig. 1 is recombinant plasmid pIB139 -the structure schematic diagram of toyG.

Fig. 2 is that the enzyme of recombinant plasmid pMD18-T-toyG is cut proof diagram.

1. toyG gene; 2. pMD18-T-toyG/ ndei+ noti; 3. DNA Marker DL2000.

Fig. 3 is that toyG gene exists e.colithe SDS-PAGE analysis chart of BL21.

1. pET28a- toyg/ e.colibL21 induction; 2. Protein Marker; 3. pET28a- toyG/ e.colibL21 does not induce.

Fig. 4 is the restriction enzyme digestion and electrophoresis proof diagram of restructuring shuttle expression plasmid pIB139-toyG.

1. toyG gene；2. pIB139-toyG/ Nde I+ Not I；3. pIB139/ Nde I+ Not I；4. λ DNA/ Hind III Marker。

Fig. 5 is the PCR electrophoresis proof diagram of recombinant bacterium 1628-TOYG.

1. original strain; 2-5. engineering strain; 6. DL2000 Marker.

specific implementation method

Below in conjunction with the drawings and specific embodiments, the present invention is described further.

embodiment 1:the structure of the amplification of goal gene and restructuring streptomyces diastatochromogenes

With s. diastatochromogenes1628 chromogene groups are template, with PtoyG F ndei and PtoyG R noti is primer, and pcr amplification obtains and contains ndei and notthe toyG gene of two restriction enzyme sites of I, is connected with pMD18-T Vector, builds cloning vector pMD18-T-toyG( ndei+ noti), by cloning vector pMD18-T-toyG( ndei+ noti) be converted in acceptor intestinal bacteria, coat on the LB agar plate containing ammonia benzyl resistance, after 37 ℃ of overnight incubation, random picking positive transformant enzyme is cut and is served that Hai Shenggong check order and carry out sequential analysis after evaluation, use ndei and noti double digestion is contained ndei and notthe toyG gene of two restriction enzyme sites of I, connects with the streptomycete integrative shuttle expression vector pIB139 of same double digestion, obtains recombinating shuttle expression carrier pIB139-toyG after enzyme is cut checking, is proceeded to e.colieT12567 (pUZ8002) screens positive transformant on the LB flat board of that resistance of card and apramycin resistance e.colieT12567 (pUZ8002, pIB139-toyG), with e.colieT12567 (pUZ8002, pIB139-toyG) is donor, and streptomyces diastatochromogenes is acceptor, utilizes conjugal transfer method by the specific pIB139-toyG streptomyces diastatochromogenes that is incorporated into s. diastatochromogeneson 1628 karyomit(e)s, on apramycin resistance MS flat board, screen positive transformant, streptomyces diastatochromogenes 1628-TOYG obtains recombinating.

With s. diastatochromogenes1628 karyomit(e)s are template, design two primers, pcr amplification toyG, and design of primers is as follows:

toyG F Nde I：5’-CGC CATATGGTGCCCGCACTTGTGCTG -3’

toyG R Not I：5’-CGC GCGGCCGCCTACAGGAAGGAGTTGATC-3’

Recombinant plasmid pMD18-T-toyG is with restriction enzyme ndei and noti double digestion verifies as shown in Figure 2, and recombinant plasmid pMD18-T-toyG double digestion obtains the DNA fragmentation of about 1.3kb and 2.7kb, and in the same size with toyG gene fragment and plasmid pMD18-T respectively, illustrates that recombinant cloning vector connects correct.To recombinant plasmid, pMD18-T-toyG checks order, sequential analysis shows that Insert Fragment is the sequence of 1284 bp, 427 amino acid of encoding, relative molecular weight size is about respectively 47 kDa, there is higher homology with the adenosine succsinic acid synthase gene of many streptomyces of reporting, be wherein up to 89%.ToyG nucleotide sequence is as shown in SEQ ID NO:1.ToyG gene is connected into pET28a carrier, build pET28a-toyG recombinant vectors, and recombinant vectors is proceeded to e. coli bl21, and picking positive transformant is in the LB of 10mL substratum, and 37 ℃ of shaking culture are spent the night, switching next day, IPTG abduction delivering, as shown in Figure 3, after induction, recombinant bacterium has obvious signature band to occur, recording adenosine succsinic acid synthetic enzyme enzyme activity is 23 U/mg total proteins, shows toyG gene successful expression in intestinal bacteria.

The enzyme of integrated restructuring shuttle expression plasmid pIB139-toyG is cut the result as shown in Figure 4, recombinant plasmid pIB139-toyG warp ndei and notit is in the same size that I double digestion discharges fragment and the toyG gene of 1.3 kb, illustrates that plasmid pIB139-toyG builds correct, with conjugal transfer method by its specific streptomyces diastatochromogenes that is incorporated into s. diastatochromogeneson 1628 karyomit(e)s, after the random some single bacterium colonies of picking are cultivated repeatedly in CP substratum in apramycin resistant panel, extract karyomit(e).PCR experiment all can amplify apramycin resistant gene apr(Fig. 5), prove that restructuring streptomyces diastatochromogenes 1628-TOYG successfully constructs, and inheritance stability.

embodiment 2:the leavening property checking of the original bacterium of streptomyces diastatochromogenes and recombinant bacterium

Compared with original strain, the adenosine succsinic acid synthetic enzyme enzyme work of recombinant bacterium has improved 1.2 times; To recombinant bacterium 1628-TOYG and original bacterium s. diastatochromogenes1628 carry out 250 mL shake flask fermentation experiments, and the enhancing of adenosine succsinic acid synthetic enzyme enzyme streptomyces diastatochromogenes being lived from fermentation angle is verified the raising effect of toyokamycin output.Reciprocating shaking speed is 200 r/min, 28 ℃, and fermentation 96h, control group is the streptomyces diastatochromogenes strain of setting out.As shown in table 1, the output of recombinant bacterium toyokamycin is higher than original bacterium, and recombinant bacterium toyokamycin ultimate capacity reaches 171.54 mg/L, and more original bacterium has improved approximately 27.1%, and repeatability is good.Illustrate at toyokamycin and produce bacterial strain s. diastatochromogenesthe expression that strengthens the key enzyme adenosine succsinic acid synthetic enzyme-ToyG of biosynthetic pathway in 1628 contributes to improve the output of toyokamycin in fermenting process.

The comparison of table 1 recombinant bacterium and the final toyokamycin output of original bacterium

Thalline	Toyokamycin output (mg/L)
		1628	134.97
1628-TOYG	171.54

Sequence table

SEQUENCE LISTING

The <110> China Measures Institute

<120> has strengthened restructuring streptomyces diastatochromogenes and construction process and purposes that toyG expresses

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1284

<212> DNA

<213> Streptomyces diastatochromogenes

<400> 1

gtgcccgcac ttgtgctgct cggtgctcag tggggtgatg agggcaaggg aaaggccacc 60

gatctgctcg gtgggtcagt ggactacgtc gtgcgttatc agggcggcaa caacgccggc 120

cacacggtcg tcgtgggcga ccagaagtac gcactgcacc tcctcccttc cggaatcctc 180

tcgccggggt gcaccccggt catcggcaac ggcgtcgtcg tcgacccggc ggtcctgctc 240

tccgagctga gcggactcaa cgagcgcggc gtcgacacgt ccaagctgct gttgagcggt 300

aacgcgcatc tgatcacgcc gtacaacatc accaccgaca aggtgacgga acggttcctc 360

ggcaagcgga agatcggcac caccggtcgg ggcatcggcc cgacctacgc cgacaagatc 420

aaccgcaccg gcatccgggt ccaggacctc tacgacgagt cgatcctgac ccagaaggtc 480

gaggcggccc tcgacgtcaa gaaccagctg ctcaccaagc tctgcaaccg ccgcgccatc 540

gaggccgggc aggtcgtcga ggagctgctg ggctacgccg accagatcaa gggctacgtc 600

gccgacacca ccctgatcct caacaaggcc ctggacgacg acaaggtcgt gctcttcgag 660

ggcgggcagg gcaccctgct cgacatcgac cacggcacgt accccttcgt gacctcctcg 720

aacccgaccg cgggcggtgc ctgcacgggt gcgggcgtcg gccccacgaa gatcagccgg 780

gtcatcggca tcctcaaggc gtacaccacc cgggtgggcg ccggaccgtt cccgacggag 840

ctgttcgacg aggacggcga ggcgctgcgc cggatcggcg gcgagcgtgg tgtcaccacc 900

gggcgcgacc gtcgctgcgg ctggttcgac gcggtcatcg cccgctacgc gacccgggtc 960

aacggcctga cggacttctt cctcgccaag ctcgacgtcc tcaccggctg ggagcagatc 1020

ccggtctgcg tggcctacga gatcgacggc aagcgcgtcg aggagctgcc ctacagccag 1080

accgacttcc accacgcgaa gccgatctac gagatgctgc cgggctggtc cgaggacatc 1140

accaaggcca agaccttcgc cgagctgccg aagaacgccc aggcgtacgt caaggcgctg 1200

gaggagatgt cgggcgcccc gatctccgcg atcggcgtcg gccccggccg gaccgagacg 1260

atcgagatca actccttcct gtag 1284

Claims (8)

1. a toyG gene for streptomyces diastatochromogenes, is characterized in that: his sequence is as shown in SEQ ID NO:1.

2. strengthened the restructuring streptomyces diastatochromogenes that toyG expresses, it is characterized in that: it has expressed the key enzyme-adenosine succsinic acid synthetase-coding gene of biosynthesizing toyokamycin toyg, have than streptomyces diastatochromogenes ( streptomyces diastatochromogenes) 1628 higher toyokamycin abilities to express.

3. as claimed in claim 2ly strengthened the restructuring streptomyces diastatochromogenes that toyG expresses, it is characterized in that: described original bacterium be streptomyces diastatochromogenes ( streptomyces diastatochromogenes) 1628.

4. as claimed in claim 2ly strengthened the restructuring streptomyces diastatochromogenes that toyG expresses, it is characterized in that: described adenosine succsinic acid synthetase-coding gene toyG come from original bacterium streptomyces diastatochromogenes to be reorganized ( streptomyces diastatochromogenes).

5. the restructuring streptomyces diastatochromogenes of having strengthened toyG expression as claimed in claim 2, is characterized in that: described adenosine succsinic acid synthetase-coding gene toyG is integrated into original bacterium streptomyces diastatochromogenes karyomit(e).

6. a construction process for the restructuring streptomyces diastatochromogenes of having strengthened toyG expression as claimed in claim 2, is characterized in that, process is as follows:

1) construction of expression vector pIB139-toyG;

2) utilize conjugal transfer method that described expression vector is integrated into streptomyces diastatochromogenes karyomit(e), obtain described engineering bacteria.

7. method as claimed in claim 6, is characterized in that, utilizes promotor permE* on pIB139 carrier to start the expression of toyG gene, utilize conjugal transfer method by specific carrier pIB139-toyG be integrated into streptomyces diastatochromogenes ( streptomyces diastatochromogenes) 1628 karyomit(e), obtain the engineering bacteria of inheritance stability.

8. one kind is utilized the purposes of the restructuring streptomyces diastatochromogenes of having strengthened toyG expression as claimed in claim 2, it is characterized in that, compared with original strain, recombinant bacterium adenosine succsinic acid synthetic enzyme enzyme is lived and has at least been improved 1.2 times, and toyokamycin output has at least improved 27.1%.

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