EP1194565A1

EP1194565A1 - Genes from corynebacterium glutamicum for the biosynthesis of folic acid and their use for the microbial production of folic acid

Info

Publication number: EP1194565A1
Application number: EP00945815A
Authority: EP
Inventors: Matthias Mack; Karin Herbster
Original assignee: Axaron Bioscience AG
Current assignee: Sygnis Pharma AG
Priority date: 1999-06-25
Filing date: 2000-06-23
Publication date: 2002-04-10
Also published as: CN1371418A; CA2377458A1; DE19929363A1; KR20020026469A; WO2001000845A1; ZA200200582B; AU5978200A

Abstract

The invention relates to nucleotide sequences of four genes (folE, folP, folB and folK) from Corynebacterium glutamicum for the biosynthesis of folic acid and their use for the microbial production of folic acid.

Description

Genes from Corynebacterium glutami cum for folic acid biosynthesis and their use in the microbial production of folic acid

description

The present invention relates to the production process for folic acid by fermentation using a genetically modified organism. This invention consists of the nucleotide sequences of four genes (folE, folP, folB and folK) from Corynebacterium glutamicum for folic acid biosynthesis and their use for the microbial production of folic acid. These four genes form an operon and are transcribed in the following order: folE, folP, folB, folK.

Folic acid is essential for animal organisms. Its derivative tetrahydrofolate is a very versatile carrier of activated single-carbon units in cells of the animal organism. Folic acid consists of three groups: a substituted pteridine ring, p-aminobenzoate and glutamate. Mammals cannot synthesize a pteridine ring. They take in folic acid from food and from microorganisms in their intestinal tract. Folic acid deficiency mainly leads to lesions in the mucous membranes.

The commercial importance of folic acid lies in the feed and food market. Folic acid is mainly used as a food additive.

Microorganisms can be used for the fermentative production of folic acid. They can be optimized in their folic acid biosynthesis performance by genetically modifying the biosynthetic pathway of folic acid. In this context, genetic engineering means increasing the number of copies and / or the rate of transcription of the genes of the biosynthetic pathway for folic acid. As a result, the proportion of gene product and thus also the intracellular enzyme activity increases. Increased enzyme activity leads to an increased rate of conversion of food (eg glucose) to folic acid and thus to an increased product concentration. For genetic modification, the nucleotide sequences of the genes of the folic acid biosynthetic pathway must be identified. This invention is concerned with four new gene sequences for the folic acid biosynthesis from Corynebacterium glutamicum and with their use for the microbial production of folic acid. Part of the invention is the folE gene product. SEQ ID NO. 2 describes a polypeptide sequence. The folE gene product encodes a polypeptide of 202 amino acids with a molecular weight of 22029 Da. The present invention is also concerned with functional derivatives of this polypeptide, which can be obtained if one in SEQ ID NO. 2 one or more amino acids, preferably up to 25% of the amino acids, preferably up to 15% of the amino acids, are replaced by deletion, insertion or substitution or by a combination of deletion, insertion and substitution. The term functional derivative means that the enzymatic activity of the derivative is still in the same order of magnitude as that of the polypeptide with the sequence SEQ ID NO. 2. Another part of the invention is the folP gene product. SEQ ID NO. 4 describes a polypeptide sequence. The folP gene product encodes a polypeptide of 285 amino acids with a molecular weight of 29520 Da. The present invention is also concerned with functional derivatives of this polypeptide, which can be obtained if one in SEQ ID NO. 4 one or more amino acids, preferably up to 40% of the amino acids, preferably up to 25% of the amino acids, are replaced by deletion, insertion or substitution or by a combination of deletion, insertion and substitution. The term functional derivative means that the enzymatic activity of the derivative is still of the same order of magnitude as that of the polypeptide with the sequence SEQ ID NO. 4th

Another part of the invention is the folB gene product. SEQ ID NO. 6 describes a polypeptide sequence. The folB gene product encodes a polypeptide of 131 amino acids with a molecular weight of 14020 Da. The present invention is also concerned with functional derivatives of this polypeptide, which can be obtained if one in SEQ ID NO. 6 one or more amino acids, preferably up to 30% of the amino acids, preferably up to 20% of the amino acids, are replaced by deletion, insertion or substitution or by a combination of deletion, insertion and substitution. The term functional derivative means that the enzymatic activity of the derivative is still of the same order of magnitude as that of the polypeptide with the sequence SEQ ID R. 6.

Another part of the invention is the folfC gene product. SEQ ID NO. 8 describes a polypeptide sequence. The folK gene product encodes a polypeptide of 160 amino acids with a molecular weight of 18043 Da. The present invention is also concerned with functional derivatives of this polypeptide, which can be obtained if one in SEQ ID NO. 8 by deletion, insertion or substitution or by a combination of deletion, Insertion and substitution of one or more amino acids, preferably replacing up to 40% of the amino acids, preferably up to 30% of the amino acids. By the term functional derivative it is meant that the enzymatic activity of the derivative is still in the same order of magnitude as that of the polypeptide with the sequence SEQ ID NO. 8th.

Another part of the invention consists in the polynucleotide sequences which encode the polypeptides described above. The polynucleotide sequences can be generated starting from sequences which are isolated from Corynebacterium glutamicum (ie SEQ ID NO. 1, 3, 5 and 7) by modifying these sequences by site-directed mutagenesis or after back-translating the corresponding polypeptide with genetic code carries out a total chemical synthesis.

These polynucleotide sequences can preferably be used for the transformation of host organisms, and preferably of microorganisms, in the form of gene constructs which contain at least one copy of one of these polynucleotides together with at least one regulatory sequence. Regulatory sequences include promoters, terminators, enhancers and ribosomal binding sites.

Preferred host organisms for the transformation with these gene constructs are Coryneibacterium and Bacillus species. Any eukaryotic microorganism can also be used, preferably yeast strains of the genus Ashbya, Candlda, Plchla, Saccharomyces and Hansenula.

Another part of the invention consists in the process for the preparation of folic acid by culturing a host organism which is transformed in the manner described above and in the subsequent isolation of the folic acid.

The trained personnel are familiar with the processes and procedures for cultivating microorganisms and isolating folic acid from a microbial production.

The invention is described in more detail in the following examples, as is its application for the genetic modification of microorganisms to increase the synthesis performance of folic acid. example 1

Representation of a genome library from Corynebacterium glutamlcum ATCC 13032

DNA from the genome of Corynebacterium glutamlcum ATCC 13032 can be obtained by standard methods which have already been described, e.g. B. by J. Altenbuchner and J. Cullum (1984, Mol. Gen. Genet. 195: 134-138). The genome library can be prepared according to standard regulations (e.g. Sambrook, J. et al. (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press) with any cloning vector, e.g. pBluescript II KS- (Stratagene) or ZAP Express ™ (Stratagene). Any fragment size can be used, preferably 5'au3AI fragments with a length of 2-9 kb, which can be integrated into cloning vectors with digested BamHI.

Example 2

Analysis of the nucleic acid sequence of the genome library

Individual E. coli clones can be selected from the genome library shown in Example 1. E. coli cells are cultivated according to standard ancestors in suitable media (e.g. LB supplemented with 100 mg / 1 ampicillin), after which the plasmid DNA can be isolated. If one clones genome fragments from the DNA of Corynebacterium glutamlcum in pBluescript II KS- (see Example 1), the DNA can be sequenced with the help of the oligonucleotides 5 '-AATTAACCCTCACTAAAGGG-3' and 5'-GTAATACGACTCACTATAGGGC-3 '.

Example 3

Computer analysis of the sequences of the isolated nucleic acids

The nucleotide sequences can e.g. using the BLASTX algorithm (Altschul et al. (1990) J. Mol. Biol. 215: 403-410). In this way, one can discover new sequences and elucidate the function of these new genes.

Example 4

Identification of an E. coii clone that contains a nucleotide sequence of the gene for GTP cyclohydrolase I (EC 3.5.4.16). When analyzing the E. coli clones as described in Example 2, to which the one described in Example 3 relates

Analysis of the sequences obtained in this way resulted in a sequence as described with SEQ ID NO. 1 is described. When using Using the BLASTX algorithm (see Example 3), this sequence showed similarity to GTP cyclohydrolases I (FolE; EC 3.5.4.16) from different organisms. The greatest similarity was with GTP cyclohydrolase-I (FolE) from Mycobacterlum tuberculoslε (NRDB 5 006273; 72% agreement at the amino acid level).

Example 5

Identification of an E. coli clone which contains a nucleotide sequence 0 of the gene for the dihydropteroate synthase (EC 2.5.1.15)

The analysis of the E. coli clones, as described in Example 2, which was followed by the analysis of the sequences obtained in Example 3, resulted in a sequence as described with SEQ ID NO. 3 is described. When using the BLASTX algorithm (see Example 3), this sequence showed similarity to dihydropteroate synthases (FolP; EC 2.5.1.15) from different organisms. The greatest similarity was with the dihydropteroate synthase (FolP) from Mycobacterlum tuberculosls (NRDB 006274; 53% agreement at the amino acid level).

Example 6

5 Identification of an E. coli clone that contains a nucleotide sequence of the gene for dihydroneopterin aldolase (EC 4.1.2.25)

When the E. coli clones were analyzed as described in Example 2, followed by the analysis of the sequences obtained in Example 3, a sequence was obtained as described with SEQ ID NO. 5 is described. When using the BLASTX algorithm (see Example 3), this sequence showed similarity to dihydroneopterin aldolases (FolB; EC 4.1.2.25) from different organisms. The greatest similarity 5 was with the dihydroneopterin aldolase (FolB) from Mycobacterlum tuberculosis (NRDB 006275; 61% agreement at the amino acid level).

Example 7 0

Identification of an E. coli clone which contains a nucleotide sequence of the gene for the 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (EC 2.7.6.3)

5 The analysis of the E. coli clones as described in Example 2, which was followed by the analysis of the sequences obtained in Example 3, resulted in one Sequence as shown with SEQ ID NO. 7 is described. When using the BLASTX algorithm (see Example 3), this sequence showed similarity with 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinases (FolK; EC 2.7.6.3) from different organisms. The greatest similarity was with that

2-Amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (FolK) from Nyco acteriurn leprae (EMBL AL023093; 43% agreement at the amino acid level).

Example 8

Use of the genes for GTP cyclohydrolase I, for dihydropteroate synthase, for dihydroneopterin aldolase and for 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase from Corynebacterium glutamicum for the production of folic acid

The genes for the GTP cyclohydrolase I, for the dihydropteroate synthase, for the dihydroneopterin aldolase and for the 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase from Corynebacterium glutamicum can be obtained with the aid of suitable cloning and expression systems introduce into Corynebacterium glutamicum or into any other microorganism. Genetically modified microorganisms can be produced which differ from the wild-type organism with regard to the activity or the number of gene copies. These new, genetically modified strains can be used to produce folic acid.

sequence Listing

(I) General information

[1) Applicant:

(A) Name: BASF-LYNX Bioscience AG

(B) Street: Im Neuenheimer Feld 515

(C) City: Heidelberg

(D) Country: Germany

(E) Postcode 69120

(F) Phone: 06221/4546

(G) Fax: 06221/454770

(2) Title: Genes from Corynebacterium glutamicum for the biosynthesis of folic acid and its use in the microbial production of folic acid (3) Number of sequences: 8

SEQ ID NO. 1: DNA (folE)

ATGAAGGAGACAACCGTGGATAACCACGCTGCAGTTCGCGAGTTCGATGAGGAGCGCGCAACAGC TGCGATTCGTGAGTTGCTCATCGCTGTGGGTGAGGATCCAGATCGCGAAGGCCTGTTGGAAACCC CAGCTCGAGTGGCTAGGGCGTACAAGGAAACTTTCGCGGGTCTGCATGAGGATCCCACCACTGTG CTGGAGAAGACGTTCTCTGAGGGCCATGAAGAGTTGGTTCTGGTTCGTGAGATCCCGATTTACTC CATGTGTGAGCACCACTTGGTGCCGTTCTTTGGCGTGGCGCACATTGGTTACATTCCGGGTAAGT CCGGCAAGGTGACTGGCCTGTCCAAGCTGGCGCGTTTAGCGGATATGTTTGCTAAGCGACCTCAG GTTCAGGAGCGCTTGACCTCCCAAATTGCGGATGCTCTCGTCGAAAAGCTTGATGCCCAGGCCGT GGCCGTGGTGATTGAAGCTGAGCACCTGTGCATGGCCATGCGCGGAATCCGTAAGCCTGGTGCTG TGACCACGACGTCTGCGGTGCGCGGCGGTTTTAAGAACAACGCTGCCTCCCGCGCTGAGGTGTTC TCCCTGATTCGGGGGCACTAA

SEQ ID NO. 2: amino acid (FolE)

MKETTVDNHAAVREFDEERATAAIRELLIAVGEDPDREGLLΞTPARVARAYKETFAGLHEDPTTV LEKTFSEGHEELVLVREIPIYSMCEHHLVPFFGVAHIGYIPGKSGKVTGLSKLARLADMFAKRPQ VQERLTSQIADALVKRGGHASKL

SEQ ID NO. 3: DNA (folP)

ATGAACGTATCCTCTTTGACCATCCCGGGACGCTGTTTGGTCATGGGAATTGTCAATGTCACTGA GGATTCCTTTTCGGACGGTGGCAAGTACATTGACGTTGATCAGGCGATCGCGCATGCCAAGGAAT TGGTGGCTGCTGGCGCCGACATGATTGATGTCGGCGGCGAGTCCACCCGGCCTGGGGCAGTGCGC GTCGACGCGTCCGTGGAACGGGACCGGGTTGTGCCGGTCATTAAGGCGCTTCACGACGCCGGCAT CCACACTTCCGTAGACACCATGCGGGCCTCCGTGGCGCAGGCTGCCGCGGGCGCTGGCGTCTCCA TGATCAACGACGTCTCTGGCGGTTTGGCTGATCCTGAGATGTTTTCTGTCATGGCGGAAGCGCAA ATTCCCGTGTGTTTGATGCACTGGCGCACCCTCCAATTCGGTGATGCCGCAGGTCAGGCAGATCA CGGTGGAGACGTTGTAGCCGATGTGCACGCAGTGCTTGATGATCTTGTCGCCCGCGCCACCGCTG CTGGTGTGGCCGAAAACCAGATCGTGCTTGATCCAGGTTTGGGTTTTGCCAAATCACGTGAAGAC AACTGGCGTTtGCTGCAAGCACTGCCCGAGTTTATTTCTGGACCTTTCCCCATCCTGGTGGGAGC ATCCCGGAAGCGATTCCTGGCTGGCGTGCGCAAAGACCGTGGCCTAGATGTCACCCCCATTGATG CCGACCCAGCAACCGCAGCGGTGACCGCAGTGTCTGCACATATGGGAGCATGGGGTGTGCGCGTG CACGATGTCCCAGTATCAAGGGACGCTGTTGATGTTGCCGCATTGTGGCGAAGTGGAGGAACTCA CCATGGCTGA

SEQ ID NO. 4: amino acid (FolP)

MNVSSLTIPGRCLVMGIVNVTEDSFSDGGKYIDVDQAIAHAKELVAAGADMIDVGGESTRPGAVR VDASVERDRWPVIKALHDAGIHTSVDTMRASVAQAAAGAGVSMINDVSGGLADPEMFSVMAEAQ IPVCLMHWRTLQFGDAAGQADHGGDWADVHAVLDDLVARATAAGVAENQIVLDPGLGFAKSRED ^{'NWRLLQALPEFISGPFPILVGASRKRFLAGVRKDRGLDVTPIDADPATAAVTAVSAHMGAWGVRV} HDVPVSRDAVDVAALWRSGGTHHG SEQ ID NO. 5: DNA (folB)

ATGGCTGATCGTATTGAACTTAAAGGCCTTGAATGCTTCGGACACCACGGTGTGTTCGACTTTGA AAAAGAGCAAGGCCAGCCCTTCATTGTGGATGTCACCTGCTGGATGGATTTCGATGCCGCAGGTG CCAGCGATGACCTTTCCGACACCGTAGATTACGGCGCGTTGGCATTGTTGGTTGCTGAAATCGTG GAAGGCCCATCCAGGGATTTGATCGAGACGGTGGCCACGGAATCTGCGGATGCTGTGATGGCTAA ATTTGATGCGCTTCATGCGGTGGAAGTAACCATCCATAAGCCCAAAGCACCGATCCCACGTACTT TTGCTGACGTCGCGGTGGTTGCCCGACGTTCCAGGAAATCCATGGCTGCTGGAAGGAGCAACGCC TAA

SEQ ID NO. 6: amino acid (FolB)

MADRIELKGLECFGHHGVFDFEKEQGQPFIVDVTCWMDFDAAGASDDLSDTVDYGALALLVAEIV EGPSRDLIETVATESADAVMAKFDALHAVEVTIHKPKAPIPRTFADVAWARRSRKSMAAGRSNA

SEQ ID NO. 7: DNA (folK)

ATGCATGCAGTTTTGTCCATCGGTTCCAACATGGATGATCGCTACGCGCTGCTCAACACAGTGAT CGAGGAATTCAAAGATGAGATCGTGGCGCAGTCTGCGATCTACTCAACCCCACCGTGGGGCATTG AGGATCAGGATGAATTCCTCAACGCAGTGCTCGTTGTTGAGGTTGAAGAAACCCCCATCGAGTTG CTGCGCCGTgGCCAAAAACTCGAAGAAGCCGCCGAGCGGGTCCGCGTCCGCAAATGGGGGCCACG CACCCTCGATGTGGATATCGTGCAGATCATTAAAGATGGGGAAGAGATCCTTTCTGAGGATCCCG AACTGACCTTGCCACACCCTTGGGCTTGGCAGCGTGCCTTCGTGTTGATCCCTTGGTTGGAAGCA GAACCTGATGCCGTCCTGCACGGCACGACCATTGCAGAACATGTGGATAATCTTGATCCCACAGA CATTGAAGGTGTCACCAAGATTTAA

SEQ ID NO. 8: amino acid (FolK)

MHAVLSIGSNMDDRYALLNTVIEEFKDEIVAQSAIYSTPPWGIEDQDEFLNAVLWEVEETPIEL LRRGQKLEEAAERVRVRKWGPRTLDVDIVQIIKDGEEILSEDPELTLPHPWAWQRAFVLIPWLEA EPDAVLVGTI

Claims

claims

1. A polypeptide with GTP cyclohydrolase I activity, which is selected from the following group:

(a) a polypeptide with the amino acid sequence shown in SEQ ID NO. 2 is described

(b) a polypeptide which is modified compared to that in (a) by deletion, insertion or substitution of one or more amino acids.

2. A polypeptide with dihydropteroate synthase activity, which is selected from the following group:

(a) a polypeptide with the amino acid sequence shown in SEQ ID NO. 4 is described;

3. A polypeptide with dihydroneopterin aldolase activity, which is selected from the following group:

(a) a polypeptide with the amino acid sequence shown in SEQ ID NO. 6 is described

(b) a polypeptide compared to that in (a) by

Deletion, insertion or substitution of one or more amino acids is changed.

4. A polypeptide with 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase activity, which results from the following

Group is selected:

(a) a polypeptide with the amino acid sequence shown in SEQ ID NO. 8 is described

a polypeptide which is modified compared to (a) by deletion, insertion or substitution of one or more amino acids.

5. A polynucleotide encoding a polypeptide corresponding to claim 1, 2, 3 or 4.

6. A gene construct with at least one copy of a polynucleotide corresponding to claim 5 together with at least one regulatory sequence.

7. A host organism which is transformed with a gene construct corresponding to claim 6.

Process for the preparation of folic acid by cultivating a host organism corresponding to claim 7 with subsequent isolation of the folic acid.