WO2012090021A1 - Micro-organisme recombinant pour la production par fermentation de méthionine - Google Patents

Micro-organisme recombinant pour la production par fermentation de méthionine Download PDF

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WO2012090021A1
WO2012090021A1 PCT/IB2010/003515 IB2010003515W WO2012090021A1 WO 2012090021 A1 WO2012090021 A1 WO 2012090021A1 IB 2010003515 W IB2010003515 W IB 2010003515W WO 2012090021 A1 WO2012090021 A1 WO 2012090021A1
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pseudogene
methionine
microorganism
gene
thra
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PCT/IB2010/003515
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Gwénaëlle BESTEL-CORRE
Cédric BOISART
Rainer Figge
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Metabolic Explorer
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Priority to PCT/IB2010/003515 priority Critical patent/WO2012090021A1/fr
Priority to ARP110104937A priority patent/AR084595A1/es
Publication of WO2012090021A1 publication Critical patent/WO2012090021A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
    • 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
    • C12P13/12—Methionine; Cysteine; Cystine

Definitions

  • the present invention relates to a recombinant microorganism for the production of methionine and to a method for producing methionine, by culturing the recombinant microorganism in an appropriate culture medium comprising a source of carbon and a source of sulfur.
  • the microorganism is modified in a way that the methionine/ carbon source yield is increased.
  • the expression of the gene ybdL coding for an amino transferase is attenuated, preferentially the gene ybdL is deleted in the recombinant microorganism.
  • Sulphur-containing compounds such as cysteine, homocysteine, methionine or S- adenosylmethionine are critical to cellular metabolism and are produced industrially to be used as food or feed additives and pharmaceuticals.
  • methionine an essential amino acid, which cannot be synthesized by animals, plays an important role in many body functions. Aside from its role in protein biosynthesis, methionine is involved in transmethylation and in the bioavailability of selenium and zinc. Methionine is also directly used as a treatment for disorders like allergy and rheumatic fever. Nevertheless most of the methionine which is produced is added to animal feed.
  • Microorganisms have developed highly complex regulatory mechanisms that fine- tune the biosynthesis of cell components thus permitting maximum growth rates. Consequently only the required amounts of metabolites, such as amino acids, are synthesized and can usually not be detected in the culture supernatant of wild-type strains. Bacteria control amino acid biosynthesis mainly by feedback inhibition of enzymes, and repression or activation of gene transcription. Effectors for these regulatory pathways are in most cases the end products of the relevant pathways. Consequently, strategies for overproducing amino acids in microorganisms require the deregulation of these control mechanisms.
  • Methionine is derived from the amino acid aspartate, but its synthesis requires the convergence of two additional pathways, cysteine biosynthesis and CI metabolism.
  • Aspartate is synthesized from oxaloacetate.
  • E. coli a stable oxaloacetate pool is required for the proper functioning of the citric acid cycle. Therefore the transformation of oxaloacetate into aspartate requires reactions that compensate for oxaloacetate withdrawal from this pool.
  • Several pathways, called anaplerotic reactions, fulfill these functions in E. coli (Sauer & Eikmanns (2005) FEMS Microbiol Reviews 29 p765-94).
  • PEP carboxylase catalyzes the carboxylation of PEP yielding oxaloacetate. Carboxylation efficiency depends among other on the intracellular PEP concentration.
  • PEP is a central metabolite that undergoes a multitude of reactions.
  • glycolytic transformation of PEP to pyruvate is not essential for E. coli, since the import of glucose via the PTS system transforms one of two PEP molecules generated from glucose into pyruvate.
  • the enzyme pyruvate kinase which in E. coli is encoded by two isoenzymes encoded by the genes pykA and pykF, catalyzes the transformation of PEP to pyruvate.
  • Aspartate is converted into homoserine by a sequence of three reactions.
  • Homoserine can subsequently enter the threonine/iso leucine or methionine biosynthetic pathway.
  • E. coli entry into the methionine pathway requires the acylation of homoserine to succinyl-homoserine. This activation step allows subsequent condensation with cysteine, leading to the thioether-containing cystathionine, which is hydrolyzed to give homocysteine.
  • the final methyl transfer leading to methionine is carried out by either a Bi2-dependent or a Bi2-independent methyltransferase.
  • Methionine biosynthesis in E. coli is regulated by repression and activation of methionine biosynthetic genes via the MetJ and MetR proteins, respectively (reviewed in Figge RM (2006), ed Wendisch VF, Microbiol Monogr (5) Amino acid biosynthesis pi 64- 185).
  • MetJ together with its corepressor S-adenosylmethionine is known to regulate the genes metA, metB, metC, metE and metF.
  • GlyA encoding enzymes involved in methionine production, such as glyA, metE, metH and metF are activated by MetR in presence of its co-activator homocysteine, whereas metA is only activated by MetR in the absence of homocysteine. All these enzymes are involved in the production and the transfer of CI units from serine to methionine.
  • GlyA encoding serine hydroxymethyltransferase catalyzes the conversion of serine to glycine and the concomitant transfer of a CI unit on the coenzyme tetrahydro folate (THF). Glycine can then be transformed into C0 2 , NH 3 while another CI unit is transferred onto THF. This reaction is catalyzed by the glycine cleavage complex encoded by the genes gcvTHP and Ipd.
  • CI units produced by the two reactions in form of methylene-THF can subsequently either be reduced to methyl-THF or further oxidized to formyl-THF.
  • Methionine biosynthesis requires the reduction to methyl-THF.
  • the oxidation reaction competes with methionine biosynthesis for CI units.
  • Formyl-THF or formate is required for the biosynthesis of purines and histidine.
  • formyl-THF can be transformed into THF and free formate in a reaction catalyzed by formyl-THF deformylase encoded by the purU gene (Nagy et al. (1995) J. Bacteriol 177 (5) p. 1292-98).
  • Transfer of the methyl group onto homocysteine is either catalyzed by MetH via vitamin B12 or directly by MetE.
  • the MetH enzyme is known to have a catalytic rate that is hundred times higher than the MetE enzyme.
  • MetE can compose up to 5% of the total cellular protein.
  • the presence of active MetH reduces MetE activity probably by reducing the amount of homocysteine that normally activates the transcription of metE via MetR. Therefore the production of methionine via MetH saves important resources for the cell, since MetE is not expressed in large quantities.
  • the accumulation of homocysteine is toxic for E. coli (Tuite et al., 2005 J. Bacteriol, 187, 13, 4362-4371.) and at the same time has a negative, regulatory effect on metA expression via MetR.
  • a strong expression of the enzymes MetH and/or MetE is clearly required for efficient methionine production.
  • E. coli reduced sulfur is integrated into cysteine and then transferred onto the methionine precursor O-succinyl-homoserine, a process called transulfuration (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). Cysteine is produced from O-acetylserine and H 2 S by sulfhydrylation.
  • the process is negatively feed-back regulated by the product, cysteine, acting on serine transacetylase, encoded by cysE.
  • N-acetyl-serine which is spontaneously produced from O-acetyl-serine, together with the transcription factor CysB activates genes encoding enzymes involved in the transport of sulfur compounds, their reduction to H 2 S and their integration in the organo-sulfur compound cysteine, which, as methionine, is an essential amino acid.
  • MetB catalyzes the conversion of the methionine- precursor O-succinyl homoserine into ammonia, a-ketobutyrate and succinate, a reaction called ⁇ -elimination (Aitken & Kirsch, 2005, Arch Biochem Biophys 433, 166-75). cc- ketobutyrate can subsequently be converted into isoleucine. This side reaction is not desirable for the industrial production of methionine, since the two amino acids are difficult to separate. Thus low ⁇ -elimination activity or other means to keep isoleucine production low are important aspects for the industrial production of methionine.
  • the gQUQ ybdL from Escherichia coli was identified during the whole sequencing of the genome.
  • the function of this gene was unknown up to 2004, when its crystal structure and reactivity identified it as an aminotransferase (Dolzan et al., FEBS Letters, 2004). It appears that this amino transferase had a preference for methionine, followed by histidine and phenylalanine.
  • the invention relates to a recombinant microorganism for the production of methionine, wherein the expression of the gene ybdL is attenuated.
  • the invention also relates to a method for the production of methionine, wherein the recombinant microorganism with an attenuated expression of the gene ybdL is cultivated in a medium with a source of carbon and a source of sulphur.
  • the recombinant microorganism may also comprise other genetic modifications, such as:
  • the invention is related to a recombinant microorganism for the fermentative production of methionine, wherein in said microorganism the expression of the gene ybdL encoding an amino transferase is attenuated.
  • microorganism designates a bacterium, yeast or a fungus.
  • the microorganism is selected among Enterobacteriaceae, Bacillaceae, Streptomycetaceae and Cory b acteriaceae. More preferentially the microorganism is a species of Escherichia, Klebsiella, Pantoea, Salmonella or Corynebacterium. Even more preferentially the microorganism is either the species Escherichia coli or Corynebacterium glutamicum.
  • a "recombinant microorganism for the fermentative production of methionine” denotes a microorganism that has been genetically modified with the goal to increase the methionine/carbon source (ratio of gram/mol methionine produced per gram/mol carbon source); after the modifications, the yield is higher in the recombinant microorganism compared to the corresponding unmodified microorganism. Indeed, the unmodified microorganisms produce methionine only for endogenous needs, when the modified microorganism produces more methionine than needed by the microorganism's metabolism.
  • microorganisms "optimized" for methionine production are well known in the art, and have been disclosed in particular in patent applications WO2005/111202, WO2007/077041 and WO2009/043803.
  • Usual modifications include deletions of genes by transformation and recombination, gene or promoter replacements, and introduction of vectors for the overexpression of endogenous genes or the expression of heterologous genes.
  • the term "fermentative production” is used to denote the growth of bacteria on an appropriate growth medium containing a simple carbon source.
  • Attenuation of the expression of a gene means, according to the invention, that the gene has a partial or complete suppression of its expression, i.e. of the translation of the encoded protein, product of the gene.
  • This suppression of expression is the result, either of the inhibition of the expression of the gene, of the deletion of all or part of the promoter region necessary for the gene expression, of a deletion in the coding region of the gene, or of the replacement of the wild-type promoter with a weaker, natural or synthetic, promoter.
  • the gene ybdL is deleted.
  • Deletion means, for the man skilled in the art, that the coding sequence of the gene is removed from the genome, partially or totally, in a way to cancel the expression of the encoded protein.
  • the deleted gene can be replaced with a selection marker gene that facilitates the identification, isolation and purification of the recombinant microorganisms according to the invention.
  • the activity of the YbdL protein may be attenuated.
  • the term "attenuated activity" designates an enzymatic activity that is inferior to the enzymatic activity of the non modified enzyme.
  • the man skilled in the art knows how to measure the enzymatic activity of said enzyme. This attenuation might be obtained by mutating specific aminoacids present in the catalytic site of the enzyme, introducing additional or deleting certain aminoacids.
  • the microorganism is furthermore modified for improving the production of methionine.
  • Genes involved in methionine production are well known in the art, and comprise genes involved in the methionine specific biosynthesis pathway as well as genes involved in precursor-providing pathways and genes involved in methionine consuming pathways.
  • Methionine producing strains have already been described in patent applications WO2005/111202, WO2007/077041 and WO2009/043803. These applications are incorporated as reference into this application.
  • the patent application WO2005/111202 describes a methionine producing strain that overexpresses homoserine succinyltransferase alleles with reduced feed-back sensitivity to its inhibitors SAM and methionine. This application describes also the combination of theses alleles with a deletion of the methionine repressor MetJ responsible for the down-regulation of the methionine regulon. In addition, the application describes the combination of the two modifications with the overexpression of aspartokinase/homo serine dehydrogenase .
  • the recombinant microorganism is modified as described below : the expression of at least one of the following genes is increased: pyc, pntAB, cysP, cysll, cysW, cysA, cysM, cysJ, cysl, cysH, gcvT, gcvH, gcvP, Ipd, serA, serB, serC, cysE, metF, metH, metA allele encoding for an enzyme with reduced feed-back sensitivity to S-adenosylmethionine and/or methionine ⁇ MetA *), thrA, and thrA allele encoding for an enzyme with reduced feed-back inhibition to threonine ⁇ thrA *).
  • pyc encodes a pyruvate carboxylase.
  • a heterologous pyc gene is introduced on the chromosome in one or several copies by recombination, or is carried by a plasmid present at least at one copy in the modified microorganism.
  • the heterologous pyc gene originates from Rhizobium etli, Bacillus subtilis, Lactococcus lactis, Pseudomonas fluorescens or Corymb acterium species,
  • cysU encodes a component of sulphate ABC transporter, as described in WO2007/077041 and in WO2009/043803,
  • cysW encodes a membrane bound sulphate transport protein, as described in WO2007/077041 and in WO2009/043803,
  • cysM encodes an O-acetyl serine sulfhydralase, as described in WO2007/077041 and in WO2009/043803,
  • cysl and cysJ encode respectively the alpha and beta subunits of a sulfite reductase as described in WO2007/077041 and in WO2009/043803.
  • cysl and cysJ are overexpressed together
  • cysH encodes an adenylylsulfate reductase, as described in WO2007/077041 and in WO2009/043803.
  • Increasing CI metabolism is also a modification that leads to improved methionine production. It relates to the increase of the activity of at least one enzyme involved in the CI metabolism chosen among GlyA, GcvTHP, Lpd; MetF, MetE or MetH.
  • the corresponding genes of these different enzymes may be overexpressed or modified in their nucleic sequence to expressed enzyme with improved activity or their sensitivity to feed-back regulation may be decreased.
  • the one carbon metabolism is increased by enhancing the expression and/or the activity of at least one of the following:
  • the glycine-cleavage complex is a multienzyme complex that catalyzes the oxidation of glycine, yielding carbon dioxide, ammonia, methylene-THF and a reduced pyridine nucleotide.
  • the GCV complex consists of four protein components, the glycine dehydrogenase said P-protein (GcvP), the lipoyl-GcvH-protein said H-protein (GcvH), the aminomethyltransferase said T-protein (GcvT), and the dihydrolipoamide dehydrogenase said L-protein (GcvL or Lpd).
  • P-protein catalyzes the pyridoxal phosphate-dependent liberation of C02 from glycine, leaving a methylamine moiety. The methylamine moiety is transferred to the lipoic acid group of the H-protein, which is bound to the P-protein prior to decarboxylation of glycine.
  • the T-protein catalyzes the release of NH3 from the methylamine group and transfers the remaining CI unit to THF, forming methylene-THF.
  • the L protein then oxidizes the lipoic acid component of the H-protein and transfers the electrons to NAD + , forming NADH;
  • cysE encodes a serine acyltransferase; its overexpression allows an increase in methionine production, as described in WO 2007/077041;
  • metA encodes a homoserine succinyltransferase.
  • the allele MetA* codes for an enzyme with reduced feed-back sensitivity to S-adenosylmethionine and/or methionine.
  • the allele MetA* described in the patent application WO 2005/111202 is used;
  • thrA encodes an aspartokinase /homoserine dehydrogenase; the thrA * allele codes for an enzyme with reduced feed-back inhibition to threonine, as described in WO 2005/111202.
  • genes may be under control of an inducible promoter.
  • at least one of these genes is under the control of a temperature inducible promoter.
  • the temperature inducible promoter belongs to the family of P R promoters. These promoters may be homologous or heterologous. The man skilled in the art knows which promoters are the most convenient, for example promoters Vtrc, Vtac, Viae or the lambda promoter cl are widely used.
  • a methionine producing strain having genes under control of inducible promoters is described in patent application PCT/IB2009/056033.
  • the gene is encoded chromosomally or extrachromosomally.
  • the gene is located on the chromosome, several copies of the gene can be introduced on the chromosome by methods of recombination, known to the expert in the field (including gene replacement).
  • the gene is located extra-chromosomally, the gene is carried by different types of plasmids that differ with respect to their origin of replication and thus their copy number in the cell.
  • plasmids are present in the microorganism in 1 to 5 copies, or about 20 copies, or up to 500 copies, depending on the nature of the plasmid : low copy number plasmids with tight replication (pSClOl, RK2), low copy number plasmids (pACYC, pRSFlOlO) or high copy number plasmids (pSK bluescript II).
  • the gene is expressed using promoters with different strength.
  • the promoters are inducible. These promoters are homologous or heterologous. The man skilled in the art knows which promoters are the most convenient, for example promoters Vtrc, Vtac, Viae or the lambda promoter cl are widely used.
  • the microorganism has been further modified, and the expression of at least one of the following genes is attenuated: met J, pykA, pykF, purU, yncA.
  • MetJ codes for the repressor protein MetJ (GenBank 1790373), responsible for the down-regulation of the methionine regulon as was suggested in patent application JP 2000/157267,
  • the attenuation of the expression of at least one or both of the pyruvate kinases decrease the consumption of phosphoenol pyruvate (PEP).
  • Increased availability of PEP can increase the production of oxaloacetate, an important precursor of aspartate, which in turn is a precursor of methionine, as described in WO2007/077041 and in WO2009/043803,
  • Homocysteine can then be a substrate for the enzyme cystathionine gamma synthase (MetB) that can catalyze the reaction between O- succinylhomoserine and homocysteine resulting in the production of homolanthionine, as described in WO2007/077041 and in WO2009/043803, • yncA encodes a N-acyltransferase, as described in patent application WO 2010/020681.
  • MethodB cystathionine gamma synthase
  • the recombinant microorganism comprises the following genetic modifications:
  • genes and proteins are identified using the denominations of the corresponding genes in E. coli. However, and unless specified otherwise, use of these denominations has a more general meaning according to the invention and covers all the corresponding genes and proteins in other organisms, more particularly microorganisms.
  • PFAM protein families database of alignments and hidden Markov models; http://wmv.sanger.ac.uk/Software/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/COG/ are obtained by comparing protein sequences from 66 fully sequenced genomes representing 38 major phylogenic lines. Each COG is defined from at least three lines, which permits the identification of former conserved domains.
  • the overexpressed genes are at their native position on the chromosome, or are integrated at a non-native position.
  • the overexpressed genes are at their native position on the chromosome, or are integrated at a non-native position.
  • several copies of the gene may be required, and these multiple copies are integrated into specific loci, whose modification does not have a negative impact on methionine production.
  • locus into which a gene can be integrated without disturbing the metabolism of the cell is chosen among the following loci: accession
  • afuC 87081709 predicted ferric ABC transporter subunit (ATP -binding component) agaA 48994927 Pseudogene, C-terminal fragment, GalNAc-6-P deacetylase agaW 1789522 Pseudogene, N-terminal fragment, PTS system EIICGalNAc alpA 1788977 protease
  • exoD 1786750 Pseudogene, C-terminal exonuclease fragment eyeA none novel sRNA, unknown function
  • gapC 87081902 Pseudogene reconstruction GAP dehydrogenase gatR 87082039 Pseudogene reconstruction, repressor for gat operon glvC 1790116 Pseudogene reconstruction
  • mcrA 1787406 5-methylcytosine-specific DNA binding protein
  • mokA none Pseudogene, overlapping regulatory peptide, enables hokB ninE 1786760 unknown nmpC 1786765 Pseudogene reconstruction, OM porin, interrupted by IS5B nohD 1786773 DNA packaging protein
  • pinH 1789002 Pseudogene, DNA invertase, site-specific recombination pinQ 1787827 DNA invertase
  • wcaM 1788356 predicted colanic acid biosynthesis protein xisD none Pseudogene, exisionase fragment in defective prophage DLP12 xisE 1787387 el4 excisionase
  • yagA 1786462 predicted DNA-binding transcriptional regulator
  • ybcM 1786758 predicted DNA-binding transcriptional regulator
  • Antitoxin component of putative toxin-antitoxin YpjF-YfjZ ygaQ 1789007 Pseudogene reconstruction has alpha-amylase-related domain ygaY 1789035 Pseudogene reconstruction, MFS family
  • yjhR 1790762 Pseudogene reconstruction, helicase family, C-terminal fragment yjhV 1790738 Pseudogene, C-terminal fragment yjhY none Pseudogene reconstruction, novel zinc finger family
  • extra copies of genes are preferentially integrated in the following loci: malS, pgaA, pgaB, pgaC, pgaD, uxaC, uxaA, wcaM, treB, treC.
  • the microorganism of the invention is selected among Enterobacteriaceae, Bacillaceae, Streptomycetaceae and Corymb acteriaceae. More preferentially the microorganism is a species of Escherichia, Klebsiella, Pantoea, Salmonella or Corynebacterium. Even more preferentially the microorganism is either the species Escherichia coli or Corynebacterium glutamicum.
  • the microorganism is an Escherichia coli.
  • the invention is also related to a method for the fermentative production of methionine, comprising the steps of:
  • the fermentation is generally conducted in fermenters with an appropriate culture medium adapted to the microorganism being used, containing at least one simple carbon source, and if necessary co-substrates for the production of metabolites.
  • the bacteria are fermented at a temperature between 20°C and 55°C, preferentially between 25°C and 40°C, and more specifically between 30°C and 37°C.
  • An appropriate culture medium for E. coli can be of identical or similar composition to an M9 medium (Anderson, 1946, Proc. Natl. Acad. Sci. 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).
  • An appropriate 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 described by Riedel et al. (2001, J. Mol. Microbiol. Biotechnol. 3: 573-583).
  • the term 'carbon source' denotes any source of carbon that can be used by those skilled in the art to support the normal growth of a microorganism, which can be hexoses (such as glucose, galactose or lactose), pentoses, monosaccharides, disaccharides such as sucrose (molasses), cellobiose or maltose, oligosaccharides, starch or its derivatives, hemicelluloses, glycerol and combinations thereof.
  • An especially preferred simple carbon source is glucose.
  • Another preferred simple carbon source is sucrose.
  • the carbon source is derived from renewable feed-stock.
  • Renewable feed-stock is defined as raw material required for certain industrial processes that can be regenerated within a brief delay and in sufficient amount to permit its transformation into the desired product.
  • An example of renewable feedstock is vegetal biomass, such as molasse from sugarcane.
  • source of sulphur refers to sulphate, thiosulfate, hydrogen sulphide, dithionate, dithionite, sulphite, methylmercaptan, dimethylsulfide and other methyl capped sulphides or a combination of the different sources. More preferentially, the sulphur source in the culture medium is sulphate or thiosulfate or a mixture thereof.
  • the action of "recovering methionine from the culture medium” designates the action of recovering L-methionine and/or one of its derivatives, in particular N-acetyl methionine (NAM) and S-adenosyl methionine (SAM) and all other derivatives that may be useful.
  • NAM N-acetyl methionine
  • SAM S-adenosyl methionine
  • the quantity of methionine obtained in the medium is measured by HPLC after OPA/Fmoc derivatization using L-methionine (Fluka, Ref 64319) as a standard.
  • the amount of NAM is determined using refractometric HPLC using NAM (Sigma, Ref 01310) as a standard.
  • the present invention is also related to a method for the production of methionine, comprising the step of isolation of methionine or its derivatives, of the fermentation broth and/or the biomass, optionally remaining in portions or in the total amount (0-100%) in the end product.
  • preferentially at least 90 %, more preferentially 95 %, even more preferentially at least 99% of the biomass may be retained during the purification of the fermentation product.
  • the methionine derivative N-acetyl-methionine is transformed into methionine by deacylation, before methionine is recovered.
  • the growth of the recombinant microorganism is subjected to limitation or deficiency for one or several inorganic substrate(s), in particular phosphate and/or potassium, in the culture medium.
  • inorganic substrate(s) in particular phosphate and/or potassium
  • Subjecting an organism to a limitation of an inorganic substrate defines a condition under which growth of the microorganisms is governed by the quantity of a nonorganic chemical supplied that still permits weak growth.
  • examples for these substrates are phosphate, potassium, magnesium or a combination of these.
  • Starving a microorganism for an inorganic substrate defines the condition under which growth of the microorganism stops completely due to the absence of the inorganic substrate.
  • examples for these substrates are phosphate, potassium, magnesium or a combination of these.
  • Protocol 1 Chromosomal modifications by homologous recombination and selection of recombinants (Datsenko, K.A. & Wanner, B.L. (2000)
  • Protocol 2 Transduction of PI phage
  • Chromosomal modifications were transferred to a given E. coli recipient strain by PI transduction.
  • the protocol is composed of 2 steps: (i) preparation of the phage lysate on a donor strain containing the resistance associated chromosomal modification and (ii) infection of the recipient strain by this phage lysate.
  • the resistant transductants are then selected and the chromosomal structure of the mutated locus is verified by PCR analysis with the appropriate primers.
  • Protocol 1 To delete the ybdL gene into the strain MG1655 metA *ll pKD46, Protocol 1 has been used with primers Ome 0589-DybdLF (SEQ ID N°l) and Ome 0590-DybdLR (SEQ ID N°2) to amplify the kanamycin resistance cassette from plasmid pKD4.
  • Kanamycin resistant recombinants were selected.
  • the insertion of the resistance cassette was verified by PCR with primers Ome 0591-ybdLR (SEQ ID N°3) and Ome 0592-ybdLF (SEQ ID N°4) and by DNA sequencing.
  • the verified and selected strain was called MG1655 metA *ll AybdL::Km pKD46.
  • the AybdL::Km deletion was then transduced into the strain 1 (Table 1) by using a PI phage lysate from the strain MG1655 metA *ll pKD46 AybdL::Km described above according to Protocol 2.
  • Kanamycin resistant transductants were selected and the presence of the AybdL::Km chromosomal modification was verified by PCR with Ome 0591-ybdLR (SEQ ID N°3) and Ome 0592-ybdLF (SEQ ID N°4).
  • DtreBC: :TT02-serA-serC DybdL ::Km was called strain 2.
  • the pCL1920-Pga/3 ⁇ 44-/?yci?e-TT07 plasmid has been described in patent applications EP 10306164.4 and US61/406249 which are incorporated as reference into this application.
  • the pCL1920-P gapA-pycRe- ⁇ was introduced by electroporation into the strain 1 (Table 1).
  • ACP4-6 :TT02-TTadc- PlambdaR *(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 ⁇ wcaM: : TT02- TTadc-Plam bdaR *(- 35)-RBS01-thrA *l-cysE-PgapA-metA *11
  • the pCL1920-Pga/3 ⁇ 44-/?yci?e-TT07 was introduced by electroporation into the strain 2 (Table 1). The presence of the pCL1920-Pga/? ⁇ -/?yci?e-TT07 was verified by digestion and the selected strain MG1655 met A * 11 Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09- gcvTHP Ptrc36-ARNmstl 7-metF Ptrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS: : TTadc-CI857-PlambdaR *(-35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-
  • DybdL r.Km pCLl 920-PgapA-pycRe-TT07 was called strain 4.
  • Production strains were assessed in small Erlenmeyer flasks.
  • a 5.5 mL preculture was grown at 30°C for 21 hours in a mixed medium (10 % LB medium (Sigma 25 %) with 2.5 g.L-1 glucose and 90 % minimal medium PCI). It was used to inoculate a 50 mL culture of PCI medium to an OD600 of 0.2.
  • antibiotics were added at a concentration of 50 mg.L-1 for kanamycin and spectinomycin.
  • the culture was grown at the following temperatures: 37°C for two hours, 42°C for two hours and 37°C until the culture end.
  • Methionine yield (Y met ), in % g of methionine per g of glucose produced in batch culture by the different strains.
  • Extracellular methionine concentration was quantified by HPLC after OPA/FMOC derivatization.
  • the residual glucose concentration was analyzed using HPLC with refractometric detection.
  • the methionine yield was expressed as followed:

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Abstract

Cette invention concerne un micro-organisme recombinant pour la production par fermentation de méthionine dont l'expression du gène ybdL codant pour une amino transférase est atténuée. Cette invention concerne également un procédé de production de méthionine par fermentation.
PCT/IB2010/003515 2010-12-30 2010-12-30 Micro-organisme recombinant pour la production par fermentation de méthionine WO2012090021A1 (fr)

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US20140134680A1 (en) * 2011-06-29 2014-05-15 Metabolic Explorer Microorganism for methionine production with enhanced glucose import
WO2015028674A1 (fr) 2013-08-30 2015-03-05 Metabolic Explorer Micro-organismes pour la production de méthionine ayant une activité de méthionine synthase améliorée et sortie de méthionine
WO2016034536A1 (fr) * 2014-09-01 2016-03-10 Metabolic Explorer Procédé et micro-organisme pour la production de méthionine par fermentation ayant un efflux de méthionine amélioré
CN105658785A (zh) * 2013-08-30 2016-06-08 代谢探索者公司 具有增强的甲硫氨酸流出的用于甲硫氨酸生产的微生物
US9506093B2 (en) 2012-06-18 2016-11-29 Metabolic Explorer Recombinant microorganism for the fermentative production of methionine
EP3296404A1 (fr) 2016-09-15 2018-03-21 Evonik Degussa GmbH Micro-organisme modifie pour production de methionine
WO2019011942A1 (fr) 2017-07-11 2019-01-17 Adisseo France S.A.S. Levure produisant de la méthionine
EP3362573A4 (fr) * 2015-10-14 2019-09-04 CJ Cheiljedang Corporation N-acétyl-l-méthionine d'origine biologique et son utilisation
CN112779200A (zh) * 2021-01-12 2021-05-11 浙江工业大学 高产l-甲硫氨酸的基因工程菌及其构建与应用
CN113755411A (zh) * 2020-06-04 2021-12-07 苏州华赛生物工程技术有限公司 高产β-烟酰胺单核苷酸的重组微生物及其生产β-烟酰胺单核苷酸的方法

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US20140134680A1 (en) * 2011-06-29 2014-05-15 Metabolic Explorer Microorganism for methionine production with enhanced glucose import
US9506093B2 (en) 2012-06-18 2016-11-29 Metabolic Explorer Recombinant microorganism for the fermentative production of methionine
JP2016529899A (ja) * 2013-08-30 2016-09-29 メタボリック エクスプローラー メチオニンシンターゼ活性及びメチオニン流出が改善された、メチオニン生産のための微生物
CN105658785A (zh) * 2013-08-30 2016-06-08 代谢探索者公司 具有增强的甲硫氨酸流出的用于甲硫氨酸生产的微生物
JP2016529900A (ja) * 2013-08-30 2016-09-29 メタボリック エクスプローラー メチオニン流出が強化された、メチオニン生産のための微生物
KR102270626B1 (ko) * 2013-08-30 2021-06-30 에보니크 오퍼레이션즈 게엠베하 메티오닌 신타제 활성 및 메티오닌 유출이 개선된 메티오닌 생산용 미생물
KR20160044037A (ko) * 2013-08-30 2016-04-22 메타볼릭 익스플로러 메티오닌 신타제 활성 및 메티오닌 유출이 개선된 메티오닌 생산용 미생물
WO2015028674A1 (fr) 2013-08-30 2015-03-05 Metabolic Explorer Micro-organismes pour la production de méthionine ayant une activité de méthionine synthase améliorée et sortie de méthionine
WO2016034536A1 (fr) * 2014-09-01 2016-03-10 Metabolic Explorer Procédé et micro-organisme pour la production de méthionine par fermentation ayant un efflux de méthionine amélioré
US10329591B2 (en) 2014-09-01 2019-06-25 Evonik Degussa Gmbh Method and microorganism for methionine production by fermentation with improved methionine efflux
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EP3362573A4 (fr) * 2015-10-14 2019-09-04 CJ Cheiljedang Corporation N-acétyl-l-méthionine d'origine biologique et son utilisation
US10750762B2 (en) 2015-10-14 2020-08-25 Cj Cheiljedang Corporation Bio-based N-acetyl-L-methionine and use thereof
EP3296404A1 (fr) 2016-09-15 2018-03-21 Evonik Degussa GmbH Micro-organisme modifie pour production de methionine
WO2019011942A1 (fr) 2017-07-11 2019-01-17 Adisseo France S.A.S. Levure produisant de la méthionine
US11162122B2 (en) 2017-07-11 2021-11-02 Adisseo France S.A.S. Methionine-producing yeast
CN113755411A (zh) * 2020-06-04 2021-12-07 苏州华赛生物工程技术有限公司 高产β-烟酰胺单核苷酸的重组微生物及其生产β-烟酰胺单核苷酸的方法
CN113755411B (zh) * 2020-06-04 2023-08-01 苏州华赛生物工程技术有限公司 高产β-烟酰胺单核苷酸的重组微生物及其生产β-烟酰胺单核苷酸的方法
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