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EP1846564A2 - Procede destine a la production enzymatique d'alpha-cetobutyrate - Google Patents

Procede destine a la production enzymatique d'alpha-cetobutyrate

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Publication number
EP1846564A2
EP1846564A2 EP06708069A EP06708069A EP1846564A2 EP 1846564 A2 EP1846564 A2 EP 1846564A2 EP 06708069 A EP06708069 A EP 06708069A EP 06708069 A EP06708069 A EP 06708069A EP 1846564 A2 EP1846564 A2 EP 1846564A2
Authority
EP
European Patent Office
Prior art keywords
homoserine
synthase
cystathionine
ketobutyrate
phosphohomoserine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06708069A
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German (de)
English (en)
Inventor
Rainer Figge
Fabien Lux
Céline RAYNAUD
Philippe Soucaille
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Metabolic Explorer SA
Original Assignee
Metabolic Explorer SA
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Filing date
Publication date
Application filed by Metabolic Explorer SA filed Critical Metabolic Explorer SA
Publication of EP1846564A2 publication Critical patent/EP1846564A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids

Definitions

  • the present invention relates to a method for the enzymatic production of ⁇ - ketobutyrate, wherein ⁇ -ketobutyrate is obtained by ⁇ -elimination of an activated homoserine with an appropriate enzyme in an appropriate medium, in particular where the appropriate enzymes are modified enzymes, preferably enzymes that when expressed from the plasmid pMElOl-thrA* in strains MG1655 ⁇ metBJ rnetAU AiIvA ⁇ fcfcABCDEFG the ratio of isoleucine to methionine produced at least equal to two.
  • L- amino acids and their derivatives are used in animal nutrition, human medicine and the pharmaceutical industry.
  • Isoleucine which is an essential amino acid, is principally used as a starting material for drug synthesis and in nutritional supplements.
  • Isoleucine that includes two asymmetric carbon atoms is difficult to synthesize chemically as the pure L-stereoisomer and therefore fermentative processes for the production of isoleucine have been developed (EP0745679B1, US5474918A1, EP0685555A1). These processes rely on the natural biosynthetic pathway in which the aspartate-derived homoserine is transformed into threonine that in turn can be converted by a set of five reactions to isoleucine (see Fig. 1). Conversion of aspartate to homoserine requires the enzyme aspartate semialdehyde dehydrogenase and aspartokinase/ homoserine dehydrogenase that as a iusion protein harbors both activities.
  • ThrrA Two Aspartokinases/homoserine dehydrogenases are present in E. coli, one is encoded by the gene thrA that is part of the threonine operon (encoding also homoserine kinase (thrB) and threonine synthase (thrC), which catalyze the biosynthesis of threonine) and the other is encoded by the gene metL that is part of the methionine regulon.
  • the threonine operon is regulated by attenuation via threonine and isoleucine.
  • the enzyme ThrA is feedback controlled by threonine, the end product of the pathway.
  • ⁇ -ketobutyrate is transformed to isoleucine by a set of 4 additional reactions catalyzed by the enzymes acetohydroxyacid synthase (encoded by ilvB, ilvN, ilvG, ilvM, ilvL, ilvH) acetohydroxy acid isomeroreductase (ilvC), dihydroxyacid dehydratase (ilvD) and branched chained amino acid aminotransferase (ilvE), all of which are shared with valine biosynthesis.
  • acetohydroxyacid synthase encoded by ilvB, ilvN, ilvG, ilvM, ilvL, ilvH
  • ilvC acetohydroxy acid isomeroreductase
  • ilvD dihydroxyacid dehydratase
  • ilvE branched chained amino acid aminotransferase
  • coli three acetohydroxyacid synthases exist that are each preferentially feedback regulated by one of the three end-products isoleucine, valine and leucine (reviewed in Neidhardt, F. C. (Ed. in Chief), R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (eds). 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology).
  • ⁇ -ketobutyrate can also be derived from activated homoserine by ⁇ -elimination, a side reaction of cystathionine- ⁇ - synthase and/or acylhomoserine sulfhydrylase and/or phosphohomoserine sulfhydrylase.
  • Cystathionine- ⁇ -synthase and/or acylhomoserine sulfliydrylase and/or phosphohomoserine sulfhydrylase from different organisms will preferentially accept activated homoserines in the form of O-acetylhomoserine, O-succinylhomoserine and phosphohomoserine.
  • activated homoserines in the form of O-acetylhomoserine, O-succinylhomoserine and phosphohomoserine.
  • methionine and threonine are synthesized from phosphohomoserine, which is thus the branchpoint between the two biosyntheses.
  • bacteria homoserine iulfils this role.
  • homoserine is either activated to acetyl- or succinyl-homoserine.
  • activated homoserine is the substrate of cystathionine- ⁇ -synthase and/or acylhomoserine sulfliydrylase and/or phosphohomoserine sulfliydrylase that can catalyze one of the three reactions: (i) the synthesis of ⁇ -cystathionine with cysteine, (ii) the sulfhydrylation to homocysteine with hydrogen sulfide or (iii) the ⁇ -elimination to ammonia, phosphate and/or acylate and ⁇ -ketobutyrate in the absence of sulfur containing substrates.
  • Cystathionine- ⁇ -synthase and/or acylhomoserine sulfliydrylase and/or phosphohomoserine sulfhydrylase vary with respect to their ratios between the three reactions. Relatively low ⁇ - eliminase activity has been observed in plant enzymes, whereas higher activity is found in the E. coli enzyme, ⁇ -elimination activity also depends on the presence and the accessibility of the enzyme for the sulfur containing compounds.
  • Object of the invention is a process for the enzymatic production of isoleucine in which ⁇ - ketobutyrate is produced from activated homoserine. This can be accomplished by either reducing the amount of intracellular H 2 S and/or cysteine, limiting access of these substrates to the active site of cystathionine- ⁇ -synthase and/or acylhomoserine sulfhydrylase and/or phosphohomoserine sulfhydrylase or by using cystathionine- ⁇ -synthase and/or acylhomoserine sulfhydrylase and/or phosphohomoserine sulfhydrylase with intrinsically high ⁇ -elimination and low cystathionine- ⁇ -synthase and/or acylhomoserine sulfhydrylase and/or phosphohomoserine sulfhydrylase activity.
  • the invention provides a new process for the production of ⁇ -ketobutyrate and its derivatives, in particular isoleucine by utilizing cystathionine- ⁇ -synthase and/or acylhomoserine sulfliydrylase and/or phosphohomoserine sulihydrylase, enzymes that normally are utilized in methionine biosynthesis. Enzymes that have high ⁇ -elimination activity are preferred, since they will convert phospho and/or acylhomoserine into ⁇ - ketobutyrate.
  • limiting conditions for the sulfur containing substrates H 2 S and cysteine as well as reduced access to the active site of the acyl and/or phosphohomoserine cystathionine- ⁇ -synthase and/or acylhomoserine sulihydrylase and/or phosphohomoserine sulihydrylase favor ⁇ -elimination.
  • the invention provides a new pathway for the synthesis of isoleucine that deviates from the classical known pathway that relies on the synthesis of threonine and its subsequent deamination to ⁇ -ketobutyrate. As a consequence the invention makes the requirement for feedback resistant threonine deaminases obsolete.
  • the invention describes a process for the enzymatic production of ⁇ -ketobutyrate and its derivatives in particular isoleucine, in which ⁇ -ketobutyrate is derived from activated homoserine by ⁇ -elimination.
  • ⁇ -ketobutyrate is derived from activated homoserine by ⁇ -elimination.
  • the implicated enzymes have to be produced and active in an appropriate medium.
  • An appropriate medium can be any solvent or mixture of solvents containing ions that allows the enzymes to be active in the production of ⁇ - ketobutyrate from activated homoserine.
  • activated homoserine can be phosphohomoserine or acylhomoserine, preferentially acetyl or succinylhomoserine.
  • the ⁇ -elimination reaction can be performed using phosphohomoserine accepting cystathionine- ⁇ -synthases and/or sulfhydrylases that are known in plants.
  • phosphohomoserine accepting cystathionine- ⁇ -synthases and/or sulfhydrylases that are known in plants.
  • Several amino acid positions are known to be relevant for the binding of the phosphogroup (Steegborn et ah, 1999, J. MoI. Biol. 290, 983-996). According to these conserved residues the inventors have deduced that cystathionine- ⁇ -synthases and/or sulfhydrylases from the bacterial family Chloroflexaceae should also preferentially accept phosphohomoserine as a substrate.
  • cystathionine- ⁇ -synthases and/or sulfhydrylases may be derived from any of the organismal groups described above.
  • Phosphohomoserine is obtained by phosphorylation of homoserine using homoserine kinase, which is preferentially homoserine kinase from E. coli or Corynebacterium glutamicum.
  • activated homoserine is acylhomoserine, preferentially acetylhomoserine.
  • the ⁇ -elimination reaction is catalyzed by acetylhomoserine sulfhydrylases and/or cystathionine- ⁇ -synthases that are preferentially derived from Corynebacterium glutamicum, Bacillus subtilis or Saccharomyces cerevisiae.
  • Acetylhomoserine is obtained by acetylation of homoserine, a reaction catalyzed by homoserine acetyltransferase.
  • homoserine transacetylases are employed that are feedback resistant to methionine and S-adenosylmethionine.
  • homoserine transacetylases are obtained from Corynebacterium glutamicum, Bacillus subtilis or Saccharomyces cerevisiae or a spirochaete.
  • activated homoserine is succinylhomoserine.
  • Succinylhomoserine is converted into succinate, ammonia and ⁇ -ketobutyrate by the ⁇ - elimination reaction catalyzed by succinylhomoserine sulfhydrylase and/or cystathionine- ⁇ - synthase.
  • succinylhomoserine sulfhydrylase and/or cystathionine- ⁇ -synthase is obtained from enterobacteriaceae, specifically from E. coli.
  • Homoserine is activated to succinylhomoserine by homoserine succinyltransferase.
  • Homoserine succinyltransferases that are feedback resistant to methionine and S-adenosylmethionine are preferred and have been described in patent application PCT IB 2004/001901.
  • Homoserine succinyltransferases are derived from Enterobacteriaceae, preferentially from E. coli.
  • enzymes are identified by their specific activities. This definition thus includes all polypeptides that have the defined specific activity also present in other organisms, more particularly in other microorganisms. Often enzymes with similar activities can be identified by their grouping to certain families defined as PFAM or COG.
  • PFAM protein families database of alignments and hidden Markov models; http://www.sanger.ac.uk/Soflware/Pfam/) represents a large collection of protein sequence alignments. Each PFAM makes it possible to visualize multiple alignments, see protein domains, evaluate distribution among organisms, gain access to other databases, and visualize known protein structures.
  • COGs clusters of orthologous groups of proteins; http://www.ncbi.nlm.nih.gov/CQG/) are obtained by comparing protein sequences from 43 fully sequenced genomes representing 30 major phylogenic lines. Each COG is defined from at least three lines, which permits the identification of former conserved domains.
  • the means of identifying homologous sequences and their percentage homologies are well known to those skilled in the art, and include in particular the BLAST programs, which can be used from the website http://www.ncbi.nlm.nih.gov/BLAST/ with the default parameters indicated on that website.
  • the sequences obtained can then be exploited (e.g., aligned) using, for example, the programs CLUSTALW (http://www.ebi.ac.uk/clustalw/) or MULTALIN (http://prodes.toulouse.mra.fr/multalm/cgi-bm/multalin..pl), with the default parameters indicated on those websites.
  • the invention also related to enzymes that have been modified, especially optimized for the desired activity.
  • this optimization concerns specifically enzymes that activate homoserine, in particular the reduction of feedback by end- products can increase their activity. Equally important is the optimization or an increase in ⁇ - eliminase activity while at the same time the cystathionine- ⁇ -synthase and/or acylhomoserine sulfhydrylase and/or phosphohomoserine sulfhydrylase activities are reduced.
  • This optimization can be related to the amount of isoleucine versus methionine produced in a strain in which the enzymes normally producing isoleucine are lacking (*7vA, tdcB); an example is the strain MGl 655 AmetBJ met All AiIvA Atdc ABCDEFG in which the meB alleles can be introduced on a low copy plasmid (Details for these experiments are given in example 1).
  • these strains produce a ratio of isoleucine to methionine that is at least of a factor of two or higher.
  • the wildtype MetB allele could be shown to produce only a ratio of 1.4.
  • Modified enzymes may be obtained by directed evolution, preferentially coupled with enzyme modeling, site directed mutagenesis or in vivo or in vitro evolution of the enzyme as described in (Directed Enzyme Evolution Screening and Selection Methods and Directed Enzyme Evolution Library Creation Methods and Protocols, 2003, eds Arnold, F. and Georgiou G., Humana Press).
  • the inventors have identified mutated versions of cystathionine- ⁇ -synthase and/or acylhomoserine sulfhydrylase and/or phosphohomoserine sulfliydrylase with an increased ratio between in vivo ⁇ -elimination activity and cystathionine- ⁇ -synthase and/or acylhomoserine sulfliydrylase and/or phosphohomoserine sulfhydrylase.
  • These mutated MetB enzymes are also object of the invention. Mutated MetB enzymes have preferentially at least one mutation in the following three conserved regions or combinations thereof.
  • COG2873 O-acetylhomoserine sulfliydrylase, Oenococcus oeni PSU-I
  • MetB has one or several amino acid changes in conserved region 1 comprising the following amino acids: Xl -X2-X3-Y-X4-R-X5-X6-N-P-T in which
  • Xl represents S, R, G or is missing X2 represents F, E, N, H, Y, P X3 represents E, I, V, D, R X4 represents G, A, S, T X5 represents Y, F, R, S, T, L, I X6 represents G, T, A, S, M
  • MetB has one or several amino acid changes in conserved region 2 comprising the following sequence: Xl -X2-X3-X4-X5-G-X6-X7-X8
  • Xl represents I, H, L, N, R
  • X2 represents A, S, T, G, L, V,
  • X3 represents P, N, T, G, E, A, V
  • X4 represents S, N
  • X5 represents F, L, I, V
  • X6 represents G, D
  • X7 represents C, V, S, T, A,
  • X8 represents E, K, R
  • MetB has one or several amino acid changes in conserved region 3 comprising the following sequence: Xl -X2-V/I-X3-X4-P/A-X5-X6-X7-X8 In which
  • Xl represents S
  • T X2 represents I
  • T X3 represents D
  • E represents T
  • A S
  • C S
  • I X4 represents Q
  • H V
  • I X5 represents A
  • S K
  • X6 represents I
  • T S
  • R V
  • X7 M
  • T X8 represents S, T
  • Modified cystathionine- ⁇ -synthase and/or acylhomoserine sulfliydrylase and/or phosphohomoserine sulfliydrylase comprises at least one of the following mutations and combinations thereof.
  • valine at position X3 of conserved region 1 a leucine at position X5 of conserved region 1 a leucine or asparagine at position Xl of conserved region 2 an alanine, threonine or valine at position X3 of conserved region 2 an asparagine at position X4 of conserved region 2 a proline at the conserved position P/A an aspartate at position X6 of conserved region 2 a lysine or arginine at position X8 of conserved region 2 a threonine at position X7 of conserved region 3.
  • the meiB/Y/Z genes encoding modified cystathionine- ⁇ -synthase and/or acylhomoserine sulfliydrylase and/or phosphohomoserine sulfliydrylase may be encoded chromosomally or extrachromosomally. Chromosomally there may be one or several copies on the genome that can be introduced by methods of recombination known to the expert in the field. Extrachromosomally the genes may be carried by different types of plasmids that differ with respect to their origin of replication and thus their copy number in the cell.
  • the meiB gene may be expressed using promoters with different strength that need or need not to be induced by inducer molecules. Examples are the promoter Ptrc, Ptac, Plac, the lambda promoter cl or other promoters known to the expert in the field.
  • MetB expression may be boosted or reduced by elements stabilizing or destabilizing the corresponding messenger RNA (Carrier and Keasling (1998) Biotechnol. Prog. 15, 58-64) or the protein (e.g. GST tags, Amersham Biosciences)
  • the method as claimed by the invention also includes the conduction of the above described enzymatic reaction in cells comprising genes encoding the required enzymes to produce ⁇ -ketobutyrate from activated homoserine.
  • these cells may express genes that are required for the subsequent conversion of ⁇ -ketobutyrate to isoleucine.
  • Cells used in the method according to the invention are eukaryotes or prokaryotes.
  • cells are microorganisms, preferably selected among S. cerevisiae, E. coli, or C. glutamicum.
  • the enzymes with ⁇ -eliminase activity expressed in said cells are preferably different from the native enzymes present in the same organism. They may be either mutated genes of the same species or native or mutated genes of other species.
  • the cell is transformed to introduce a gene coding for an enzyme with said ⁇ -eliminase activity.
  • Such gene may be introduced by different means available to the man skilled in the art: modification of the native gene by homologous recombination to introduce mutations in the enzyme encoded by the said gene to enhance ⁇ -eliminase activity; integrating into the genome of the microorganism a foreign gene coding for the selected enzyme known to have high ⁇ -eliminase activity; said foreign gene being under control of regulatory elements functional in the host microorganism; introducing a plasmid comprising a foreign gene coding for the selected enzyme known to have high ⁇ -eliminase activity under control of regulatory elements functional in the host microorganism.
  • the gene When the gene is integrated into the genome of the microorganism, it may advantageously be introduced in a locus selected to replace the native gene.
  • Production of ⁇ -ketobutyrate or the derived isoleucine may be further increased by enhancing the expression of one or the following genes. Enhancing in this context means increasing the expression of a said gene and thus the activity of the corresponding enzyme:
  • Production of ⁇ -ketobutyrate or the derived isoleucine may be further increased by decreasing or completely eliminating the expression of one or the following genes: Gene genbank entry name asp A gl 790581 aspartate ammonia lyase pck gl 789807 phosphoenolpyruvate carboxykinase ackA gl 788633 acetate kinase pta gl788635 phosphotransacetylase acs gl790505 acetate synthase aceE gl786304 pyruvate deydrogenase El aceF gl786305 pyruvate deydrogenase E2 lpd gl786307 pyruvate deydrogenase E3 sucC gl786948 succinyl-CoA synthetase, beta subunit sucD gl786949 succinyl-CoA synth
  • the invention also concerns the process for the production of ⁇ -ketobutyrate or its derivatives in particular isoleucine.
  • ⁇ -ketobutyrate or its derivatives are usually prepared by fermentation of the designed bacterial strain.
  • the terms 'culture' and 'fermentation' are used indifferently to denote the growth of a microorganism on an appropriate culture medium containing a simple carbon source.
  • a simple carbon source is a source of carbon that can be used by those skilled in the art to obtain normal growth of a microorganism, in particular of a bacterium.
  • it can be an assimilable sugar such as glucose, galactose, sucrose, lactose or molasses, or by-products of these sugars.
  • An especially preferred simple carbon source is glucose.
  • Another preferred simple carbon source is sucrose.
  • the bacteria are fermented at a temperature between 20 0 C and 55°C, preferentially between 25°C and 40 0 C, and more specifically about 30 0 C for C. glutamicum and about 37°C for E. coli.
  • the fermentation is generally conducted in fermenters with an inorganic culture medium of known defined composition adapted to the bacteria used, containing at least one simple carbon source, and if necessary a co-substrate necessary for the production of the metabolite.
  • the inorganic culture medium for E. coli can be of identical or similar composition to an M9 medium (Anderson, 1946, Proc. Natl. Acad. ScL USA 32:120-128), an M63 medium (Miller, 1992; A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) or a medium such as defined by Schaefer et al. (1999, Anal. Biochem. 270: 88-96).
  • the inorganic culture medium for C. glutamicum can be of identical or similar composition to BMCG medium (Liebl et al., 1989, Appl. Microbiol. Biotechnol. 32: 205-210) or to a medium such as that described by Riedel et al. (2001, J. MoI. Microbiol. Biotechnol. 3: 573-583).
  • the media can be supplemented to compensate for auxotrophies introduced by mutations.
  • ⁇ -ketobutyrate or its derivatives After fermentation ⁇ -ketobutyrate or its derivatives are recovered and purified if necessary.
  • the methods for the recovery and purification of ⁇ -ketobutyrate or its derivatives such as isoleucine in the culture media are well known to those skilled in the art.
  • Fig. 1 Metabolic pathway used for isoleucine production. Four different pathways are indicated: the original pathway that starts with the production of threonine, three new pathways that utilize activated homoserines, i.e. phosphohomoserine, acetylhomoserine and succinylhomoserine.
  • homoserine can be activated by homoserine-transsuccinylase to succinylhomoserine. Subsequently succinylhomoserine is cleaved by cystathionine- ⁇ -synthase/sulfhydrylase to succinate, NH 3 , and ⁇ -ketobutyrate. In Escherichia coli this reaction sequence requires the expression of the genes metA and meiB, encoding homoserine-transsuccinylase and cystathionine- ⁇ -synthase/sullhydrylase.
  • threonine is deaminated to ⁇ -ketobutyrate by the enzyme threonine deaminase encoded by the UvA gene.
  • threonine deaminase encoded by the UvA gene.
  • the strain ⁇ metBJ metA ® * was constructed starting with the metA & ⁇ already described (metA* 11 in this case).
  • the homologous recombination strategy described by Datsenko & Wanner (2000) was used. This strategy allowed the insertion of a chloramphenicol resistance cassette, while deleting most of the genes concerned.
  • DmetJR (SEQ ID NO 2) tgacgtaggcctgataagcgtagcgcatcaggcgattccactccgcgcctctttttgctttagtattcccacg tctcTGTAGGCTGGAGCTGCTTCG with
  • the oligonucleotides DmetJBR and DmetJBF were used to amplify the chloramphenicol resistance cassette from the plasmid pKD3.
  • the PCR product obtained was then introduced by electroporation into the strain MGl 655 metA & ⁇ (pKD46), described in patent application PCT IB2004/001901, in which the expressed Red recombinase enzyme permited the homologous recombination.
  • the chloramphenicol resistant transformants were then selected and the insertion of the resistance cassette was verified by a PCR analysis with the oligonucleotides MetJR and MetJF defined below.
  • the strain retained was designated MG1655 ⁇ metJB::Cm metA & ⁇
  • MetJR SEQ ID NO 4: ggtacagaaaccagcaggctgaggatcagc (homologous to the sequence from 4125431 to 4125460).
  • MetLR (SEQ ID NO 5): aaataacacttcacatcagccagactactgccaccaaattt (homologous to the sequence from 4127500 to 4157460).
  • the chloramphenicol resistance cassette was then eliminated.
  • the plasmid pCP20 carrying recombinase FLP acting at the FRT sites of the chloramphenicol resistance cassette was introduced into the recombinant strains by electroporation. After a series of cultures at 42°C, the loss of the chloramphenicol resistance cassette was verified by a PCR analysis with the same oligonucleotides as those used previously.
  • the oligonucleotides DiIvAR and DiIvAF were used to amplify the chloramphenicol resistance cassette from the plasmid pKD3.
  • the PCR product obtained was then introduced by electroporation into the strains MGl 655 ⁇ metBJ metK ⁇ (pKD46) and ⁇ metJ metK ⁇ (pKD46) in which the expressed Red recombinase enzyme permitted the homologous recombination.
  • the chloramphenicol resistant transformants were then selected and the insertion of the resistance cassette was verified by a PCR analysis with the oligonucleotides ilvAR and ilvAF defined below.
  • ilvAR (3954693-3954670) (SEQ ID NO 8): gccccgaaccggtgcgtaaccgcg ilvAF (3952775-3952795) (SEQ ID NO 9): ggtaagcgatgccgaactggc
  • TdcB threonine dehydratase
  • DtdcGR and DtdcAF were used to amplify the cassette and tdcGR and tdcGF for the verification.
  • DtdcGR (3255915-3255993) (SEQ ID NO 10) gctgacagcaatgtcagccgcagaccactttaatggccagtcctccgcgtgatgtttcgcggtatttatcgttcatatcCAT ATGAA TATCCTCCTTAG
  • DtdcAF (3264726-3264648) (SEQ ID NO 11) GgtaattaacgtaggtcgttatgagcactattcttcttcccgaaaacgcagcacctggtagtctttcaggaagtcattagTGTAGGCTGGAG
  • CTGCTTCG tdcGR (3255616-3255640) (SEQ ID NO 12)
  • gcgtctgcaatgacgcctttattcg tdcAF (3264922-3264899) (SEQ ID NO 13)
  • thrA* allele with reduced feed-back resistance to threonine was expressed from the plasmid pCL1920 (Lerner & Inouye, 1990, NAR 18, 15 p 4631) using the promoter Vtrc.
  • thrA was PCR amplified from genomic DNA using the following oligonucleotides: AspHlthrA (SEQ ID NO 14): ttaTCATGAgagtgttgaagttcggcggtacatcagtggc Smal ⁇ xA (SEQ ID NO 15): ttaCCCGGGccgccgcccgagcacatcaaacccgacgc
  • SEQ ID NO 15 ttaCCCGGGccgccgccccgagcacatcaaacccgacgc
  • the PCR amplified fragment was cut with the restriction enzymes Bsp ⁇ U and Sma ⁇ and cloned into the Nco ⁇ I Sma ⁇ sites of the vector pTRC99A (Stratagene).
  • the plasmid pMElOl was constructed as follows.
  • the plasmid pCL1920 was PCR amplified using the oligonucleotides PMElOlF and PMElOlR and the BstZll ⁇ - Xmn ⁇ fragment from the vector pTRC99A harboring the lac ⁇ gene and the Ptrc promoter was inserted into the amplified vector.
  • the resulting vector and the vector harboring the thrA gene were restricted by Apa ⁇ and Sma ⁇ and the thrA containing fragment was cloned into the vector pMElOl.
  • ThrAF F318S (Smal) (SEQ ID NO 18): Ccaatctgaataacatggcaatgtccagcgtttctggccggg ThrAR F318S (Smal) (SEQ ID NO 19): Cccgggccagaaacgctggacattgccatgttattcagattgg
  • Extracellular metabolites were analyzed during the batch phase. Amino acids were quantified by HPLC after OPA/Fmoc derivatization and other relevant metabolites were analyzed using GC-MS after silylation.
  • the strain MG 1655 metA ⁇ AmetB] AiIvA pSBl was grown in the presence of either 2 mM methionine or 2 mM methionine and 2 mM isoleucine. In the absence of isoleucine the strain failed to grow demonstrating that meiB is required for the production of isoleucine if the threonine deaminase encoding gene UvA is deleted.
  • DASGIP 300 ml fermentors
  • the fermentor was filled with 145 ml of modified minimal medium containing 10 ⁇ M IPTG and inoculated with 5 ml of preculture to an optical density (OD600nm) between 0.5 and 1.2.
  • the temperature of the culture was maintained constant at 37 0 C and the pH was permanently adjusted to values between 6.5 and 8 using an NH 4 OH solution.
  • the agitation rate was maintained between 200 and 300 rpm during the batch phase and was increased to up to 1000 rpm at the end of the fed-batch phase.
  • the concentration of dissolved oxygen was maintained at values between 30 and 40% saturation by using a gas controller.
  • the optical density reached a value between three and five the fed-batch (medium containing 10 ⁇ M IPTG) was started with an initial flow rate between 0.3 and 0.5 ml/h and a progressive increase up to flow rate values between 2.5 and 3.5 ml/h. At this point the flow rate was maintained constant for 24 to 48 hours.
  • the media of the fed was based on minimal media containing glucose at concentrations between 300 and 500 g/1.
  • cystathionine- ⁇ -synthase/ sulfliydrylase (MetB) for ⁇ - elimination, several mutations were introduced into regions that are involved in the binding of the substrate cysteine.
  • Escherichia coli metB was PCR-amplified from genomic DNA using the oligonucleotides MetBF and MetBR (numbers in parentheses correspond to positions on the E. coli genome). The PCR fragment was restricted by Pst ⁇ and Hindl ⁇ and cloned into pUC18 into the same restriction sites.
  • MetBF (4125957 ⁇ 125982)
  • SEQ ID NO 20 Ttagacagaactgcagcgccgctccattcagccatgagatac MetBR (4127500 ⁇ 127469)
  • SEQ ID NO 21 Cgtaacgcccaagcttaaataacacttcacatcagccagactactgcc
  • E325PF (SEQ ID NO 22) Cgttgtttacgctggcgccgtcattagggggagtggaaag E325PR (SEQ ID NO 23) ctttccactcccctaatgacggcgccagcgtaaacaacg E32LF (SEQ ID NO 24) Cgttgtttacgctggcgctgtcattagggggagtggaaag E325LR (SEQ ID NO 25) Ctttccactccccctaatgacagcgccagcgtaaacaacg E325VF (SEQ ID NO 26) cgttgtttacgctggcggtgtcattagggggagtggaaag E325VF (SEQ ID NO 27) ctttccactccccctaatgacaccgccagcgta
  • the strains MGl 655 AmetBJ metAM AiIvA ⁇ fcfcABCDEFG expressing the different metB alleles were analyzed for isoleucine production as described in example 2.
  • the strains with the mutant metB alleles showed an increased ratio of isoleucine to methionine produced, indicating that ⁇ -elimination activity of the corresponding enzymes has been increased while cystathionine- ⁇ -synthase and sulfliydrylase activities remain equal or decrease.
  • Fermentation under sulfur limiting conditions ⁇ -ketobutyrate production can be farther increased by fermenting strains with high ⁇ -eliminase activity under suliur limiting conditions. These can either be achieved by reducing the assimilation of inorganic sulfur through mutations in the cys operon or by fermenting the strains under sulfur limiting conditions. Under these conditions ⁇ -elimination will be favored and higher isoleucine titers can be obtained.

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Abstract

Cette invention concerne un procédé de production enzymatique d'a-cétobutyrate et de ses dérivés, en particulier l'isoleucine. Dans le procédé décrit, l'a-cétobutyrate est obtenu par ?-élimination à partir d'homosérine activée. L'homosérine est activée à la fois en acylhomosérine ou phosphohomosérine à l'aide respectivement d'une homosérine transacylase et d'une homosérine kinase. La phosphohomosérine et/ou l'acylhomosérine est/sont alors transformées en phosphate ou/et en acylate, ammoniac et a-cétobutyrate grâce à l'activité de ?-éliminase de la cystathionine- ?-synthase et/ou de la sulfhydrylase de l'acylhomosérine et/ou de la sulfhydrylase de la phosphohomosérine. Cette réaction est favorisée lorsque (i) l'on réduit les concentrations intracellulaires de H2S ou de cystéine, (ii) lorsque l'on restreint l'accès de ces deux sulfures qui contiennent des substrats à l'enzyme de la cystathionine-?- synthase et/ou de la sulfhydrylase d'acylhomosérine et/ou de la sulfhydrylase de phosphohomosérine ou (iii) lorsque l'on utilise la cystathionine-?-synthase et/ou la sulflhydrylase d'acylhomosérine et/ou la sulfhydrylase de phosphohomosérine avec une forte activité de ?-élimination et une faible activité de cystathionine-?-synthase et/ou une faible activité de sulfhydrylase d'acylhomosérine et/ou une faible activité de sulfhydrylase de phosphohomosérine. L'une de ces mesures, au moins, est appliquée aux micro-organismes, en particulier aux bactéries corynéformes ainsi qu'aux entérobactéries, dans lesquelles l'a-cétobutyrate et/ou ses dérivés peuvent alors être produits en grandes quantités.
EP06708069A 2005-02-07 2006-02-07 Procede destine a la production enzymatique d'alpha-cetobutyrate Withdrawn EP1846564A2 (fr)

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KR100905381B1 (ko) 2006-07-28 2009-06-30 씨제이제일제당 (주) Ｌ-메치오닌 전구체 생산 균주 및 상기 ｌ-메치오닌전구체로부터의 ｌ-메치오닌 및 유기산의 생산방법
CN102341502B (zh) 2008-12-31 2015-07-01 代谢探索者公司 用于制备二醇的方法
KR102076532B1 (ko) * 2009-06-05 2020-02-13 에보니크 오퍼레이션즈 게엠베하 2-케토 카르복실산의 제조 방법
US8283152B2 (en) * 2009-08-28 2012-10-09 Cj Cheiljedang Corporation Microorganism producing O-acetyl-homoserine and the method of producing O-acetyl-homoserine using the microorganism
US8911978B2 (en) 2010-07-02 2014-12-16 Metabolic Explorer Method for the preparation of hydroxy acids
TWI500768B (zh) 2010-07-05 2015-09-21 Metabolic Explorer Sa 由蔗糖製備１，３－丙二醇之方法
EP2540834A1 (fr) 2011-06-29 2013-01-02 Metabolic Explorer Procédé de préparation de 1,3-propanediol
US9410164B2 (en) 2011-10-11 2016-08-09 Metabolic Explorer Biosynthesis pathway for prenol in a recombinant microorganism
EP2647718A3 (fr) 2012-04-06 2014-12-24 Metabolic Explorer Procédé de production de 5-aminopentanoate en utilisant un micro-organisme recombinant
WO2014049382A2 (fr) 2012-09-26 2014-04-03 Metabolic Explorer Production de fermentation d'éthylènediamine par un micro-organisme recombinant
EP3504318B1 (fr) 2016-08-24 2025-04-09 Danmarks Tekniske Universitet Procédé d'amélioration de l'activité de la méthyltransférase

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CN111471638A (zh) * 2020-05-22 2020-07-31 江南大学 一株产l-高丝氨酸的谷氨酸棒杆菌突变株的构建与应用
CN111471638B (zh) * 2020-05-22 2021-11-23 江南大学 一株产l-高丝氨酸的谷氨酸棒杆菌突变株的构建与应用

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