WO2012090021A1 - Recombinant microorganism for the fermentative production of methionine - Google Patents

Abstract

The present 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. The invention is also related to a method for producing methionine by fermentation.

Description

RECOMBINANT MICROORGANISM FOR THE FERMENTATIVE

PRODUCTION OF METHIONINE

Field of the invention

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. In particular, the expression of the gene ybdL coding for an amino transferase is attenuated, preferentially the gene ybdL is deleted in the recombinant microorganism.

Prior art

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. In particular 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.

With the decreased use of animal-derived proteins as a result of BSE and chicken flu, the demand for pure methionine has increased. Chemically D,L-methionine is commonly produced from acrolein, methyl mercaptan and hydrogen cyanide. Nevertheless the racemic mixture does not perform as well as pure L-methionine, as for example in chicken feed additives (Saunderson, C.L., (1985) British Journal of Nutrition 54, 621-633). Pure L-methionine can be produced from racemic methionine e.g. through the acylase treatment of N-acetyl-D,L-methionine which increases production costs dramatically. The increasing demand for pure L-methionine coupled to environmental concerns render microbial production of methionine attractive.

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.

The pathway for L-methionine synthesis is well known in many microorganisms. 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. In 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). Under exponential growth conditions and glucose excess, 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. One of them, 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. In glycolysis 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. In 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. Other genes 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 C0₂, NH₃ 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. Thus the oxidation reaction competes with methionine biosynthesis for CI units. Formyl-THF or formate is required for the biosynthesis of purines and histidine. In E. coli 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).

The reduction of methylene-THF to methyl-THF is catalyzed by the MetF protein.

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. In the absence of vitamin Bi₂ and thus active MetH, 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. Thus a strong expression of the enzymes MetH and/or MetE is clearly required for efficient methionine production.

In 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 H₂S 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 H₂S and their integration in the organo-sulfur compound cysteine, which, as methionine, is an essential amino acid.

In the absence of cysteine, MetB catalyzes the conversion of the methionine- precursor O-succinyl homoserine into ammonia, a-ketobutyrate and succinate, a reaction called γ-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 γ-elimination activity or other means to keep isoleucine production low are important aspects for the industrial production of methionine. The provisional patent application US 60/650,124 describes how γ-elimination can be reduced by optimizing the enzyme MetB. Optimizing cysteine biosynthesis can also reduce γ- elimination and thus the production of the byproduct isoleucine and constitutes an embodiment of this invention.

Improving methionine production by fermentation, with recombinant microorganisms, has been a goal for multiple years; numerous patent applications were filed on the subject as for examples:

- WO2005/111202, WO2007/077041 and WO2009/043803 from Metabolic Explorer;

- WO2007/012078 and WO2007/135188 from BASF;

- WO2007/051725 and WO2007/020295 from EVONIK.

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.

General disclosure of the invention

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:

- An increased expression of at least one of the following genes : 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*), and/or

- An attenuated expression of one of the following genes: met J, pykA, pykF, purU, yncA. Detained description of the invention

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.

The term "microorganism" designates a bacterium, yeast or a fungus. Preferentially, 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. Such 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. The man skilled in the art knows how to modulate the expression of specific genes. 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.

According to the invention 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.

In a preferred aspect of the invention, 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.

In another aspect of 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.

In a specific embodiment of the invention, 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.

Efficient production of methionine requires the optimisation of the methionine specific pathway and several precursor-providing 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 .

In a specific embodiment of the invention, 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,

• pntAB encode subunits of a membrane-bound transhydrogenase, such as described in patent applications EP 10306164.4 and US61/406249, • cysP encodes a periplasmic sulphate binding protein, as described in WO2007/077041 and in WO2009/043803,

• 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,

• cysA encodes a sulphate permease, 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. Preferably 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. For increasing enzyme activity, 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.

In a preferred embodiment of the invention, the one carbon metabolism is increased by enhancing the expression and/or the activity of at least one of the following:

• glyA encoding a serine hydroxymethyltransferase;

• gcvT, gcvH, gcvP, and lpd, coding for the glycine cleavage complex, as described in patent application WO 2007/077041. The glycine-cleavage complex (GCV) 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;

• MetF encoding a methylenetetrahydro folate reductase, as described in patent application WO 2007/077041;

• MetE and MetH (B12-dependent homocysteine-N5-methyltetrahydrofolate transmethylase ) encoding methyltransferases.

The overexpression of at least one of the following genes involved in serine biosynthesis also reduces the production of the by-product isoleucine:

• serA which encodes a phosphoglycerate dehydrogenase, as described in WO2007/077041 and in WO2009/043803,

• serB which encodes a phosphoserine phosphatase, as described in WO2007/077041 and in WO2009/043803,

• serC which encodes a phosphoserine aminotransferase, as described in WO2007/077041 and in WO2009/043803.

The overexpression of the following genes has already been shown to improve the production of methionine:

• 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. Preferentially, 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.

In a specific embodiment of the invention, genes may be under control of an inducible promoter. In a preferred embodiment of the invention, at least one of these genes is under the control of a temperature inducible promoter. In a most preferred embodiment, 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 terms "increased expression" "enhanced expression" or "overexpression" are used interchangeably in the text and have similar meaning.

To increase the expression of a gene, the man skilled in the art knows different techniques: increasing the numbers of copies of the gene in the microorganism, using a promoter inducing a high level of expression of the gene, attenuating the activity or the expression of a transcription repressor, specific or non-specific of the gene.

The gene is encoded chromosomally or extrachromosomally. When 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). When 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. These 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).

In a specific embodiment of the invention, the gene is expressed using promoters with different strength. In one embodiment of the invention, 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.

To increase the expression of a protein, the man skilled in the art knows different means, such as gene overexpression or use of elements stabilizing the corresponding messenger R A (Carrier and Keasling (1998) Biotechnol. Prog. 15, 58-64) or elements stabilizing the protein (e.g. GST tags, Amersham Biosciences).

In another specific embodiment of the invention, 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 genes pykA and pykF code for the enzymes 'pyruvate kinase'. 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,

• purU codes for a formyltetrahydro folate deformylase, an enzyme that catalyzes the formyl-THF deformylase reaction. The attenuation of the deformylase activity increases the production of methyl-THF that is required for methylation of homocysteine. Loss of Cl metabolites by deformylation leads to an increased production of homocysteine that cannot be transformed into methionine. 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.

In a particular aspect of the invention, the recombinant microorganism comprises the following genetic modifications:

• the gene ybdl is deleted,

• the expression of the genes metA *, metH, cysPUWAM, cysJIH, gcvTHP, metF, serA, serB, serC, cysE, thrA * and pyc is increased, and

• the genes MetJ, pykA, pykF, purJ, yncA are attenuated.

In the description of the present invention, 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 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 (htlp^/multalin.toulouse.inra.fr/multalin/Q, with the default parameters indicated on those websites.

Using the references given in GenBank for known genes, those skilled in the art are able to determine the equivalent genes in other organisms, bacterial strains, yeasts, fungi, mammals, plants, etc. This routine work is advantageously done using consensus sequences that can be determined by carrying out sequence alignments with genes derived from other microorganisms, and designing degenerate probes to clone the corresponding gene in another organism. These routine methods of molecular biology are well known to those skilled in the art, and are claimed, for example, in Sambrook et al. (1989 Molecular Cloning: a Laboratory Manual. 2^nd ed. Cold Spring Harbor Lab., Cold Spring Harbor, New York.).

In a particular embodiment of the invention, the overexpressed genes are at their native position on the chromosome, or are integrated at a non-native position. For an optimal methionine production, 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.

For example, a locus into which a gene can be integrated without disturbing the metabolism of the cell is chosen among the following loci: accession

Locus number function

Pseudogene, phage terminase protein A homo log, N-terminal aaaD 87081759 fragment

Pseudogene, phage terminase protein A homo log, C-terminal aaaE 1787395 fragment

Pseudogene, ferric ABC family transporter permease; C-terminal afuB 1786458 fragment

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

appY 1786776 DNA-binding transcriptional activator

argF 1786469 ornithine carbamoyltransferase

argU none arginine tRNA

argW none Arginine tRNA(CCU) 5

arpB 87081959 Pseudogene reconstruction, ankyrin repeats

arrD 1786768 lysozyme

arrQ 1787836 Phage lambda lysozyme R protein homo log

arsB 87082277 arsenite transporter

arsC 1789918 arsenate reductase

arsR 1789916 DNA-binding transcriptional repressor

beeE 1787397 Pseudogene, N-terminal fragment, portal protein

borD 1786770 bacteriophage lambda Bor protein homo log

cohE 1787391 Cl-like repressor croE 87081841 Cro-like repressor

cspB 1787839 Cold shock protein

cspF 1787840 Cold shock protein homo log

cspl 1787834 Cold shock protein

cybC 1790684 Pseudogene, N-terminal fragment, cytochrome b562 dicA 1787853 Regulatory for dicB

dicB 1787857 Control of cell division

dicC 1787852 Regulatory for dicB

dicF none DicF antisense sRNA

eaeH 1786488 Pseudogene, intimin homolog

efeU 87081821 Pseudogene reconstruction, ferrous iron permease emrE 1786755 multidrug resistance pump

essD 1786767 predicted phage lysis protein

essQ 87081934 Phage lambda S lysis protein homolog

exoD 1786750 Pseudogene, C-terminal exonuclease fragment eyeA none novel sRNA, unknown function

flu 48994897 Antigen 43

flxA 1787849 unknown

gapC 87081902 Pseudogene reconstruction, GAP dehydrogenase gatR 87082039 Pseudogene reconstruction, repressor for gat operon glvC 1790116 Pseudogene reconstruction

glvG 1790115 Pseudogene reconstruction, 6-phospho-beta-glucosidase gnsB 87081932 Multicopy suppressor of secG(Cs) and fabA6(Ts) gtrA 1788691 Bactoprenol-linked glucose translocase

gtrB 1788692 Bactoprenol glucosyl transferase

gtrS 1788693 glucosyl transferase

hokD 1787845 Small toxic membrane polypeptide

icd 1787381 Isocitrate dehydrogenase

icdC 87081844 pseudogene

ilvG 87082328 Pseudogene reconstruction, acetohydroxy acid synthase II insA 1786204 IS1 gene, transposition function

insA 1786204 IS1 gene, transposition function

insB 1786203 IS1 insertion sequence transposase

insB 1786203 IS 1 insertion sequence transposase

insC 1786557 IS2 gene, transposition function

insD 1786558 IS2 gene, transposition function

insD 1786558 IS2 gene, transposition function

insE 1786489 IS3 gene, transposition function insF 1786490 IS3 gene, transposition function

insH 1786453 IS5 gene, transposition function

insH 1786453 IS5 gene, transposition function

insH 1786453 IS5 gene, transposition function

insl 1786450 IS30 gene, transposition function

insl(-l) 1786450 IS30 gene, transposition function

insM 87082409 Pseudogene, truncated IS600 transposase

insN 1786449 Pseudogene reconstruction, IS911 transposase ORFAB

insO none Pseudogene reconstruction, IS911 transposase ORFAB

insX 87081710 Pseudogene, IS3 family transposase, N-terminal fragment insZ 1787491 Pseudogene reconstruction, IS4 transposase family, in ISZ' intA 1788974 Integrase gene

intB 1790722 Pseudogene reconstruction, P4-like integrase

intD 1786748 predicted integrase

intE 1787386 el4 integrase

intF 2367104 predicted phage integrase

intG 1788246 Pseudogene, integrase homo log

intK 1787850 Pseudogene, integrase fragment

intQ 1787861 Pseudogene, integrase fragment

intR 1787607 Integrase gene

intS 1788690 Integrase

intZ 1788783 Putative integrase gene

isrC none Novel sRNA, function unknown

jayE 87081842 Pseudogene, C-terminal fragment, baseplate

kilR 87081884 Killing function of the Rac prophage

lafU none Pseudogene, lateral flagellar motor protein fragment

lfhA 87081703 Pseudogene, lateral flagellar assembly protein fragment lit 1787385 Cell death peptidase

Pseudogene reconstruction, lorn homo log; outer membrane protein lomR 1787632 interrupted by IS5Y, missing N-terminus

malS 1789995 a-amylase

mcrA 1787406 5-methylcytosine-specific DNA binding protein

mdtQ 87082057 Pseudogene reconstruction, lipoprotein drug pump OMF family melB 1790561 melibiose permease

mmuM 1786456 homocysteine methyltransferase

mmuP 870811708 S-methylmethionine permease

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

Pseudogene, phage lambda Nul homo log, terminase small subunit nohQ 1787830 family, putative DNA packaging protein

ogrK 1788398 Positive regulator of P2 growth

ompT 1786777 outer membrane protease VII

oweE none Pseudogene, lambda replication protein O homo log

oweS 1788700 Pseudogene, lambda replication protein O homo log

pauD none argU pseudogene, DLP12 prophage attachment site

pawZ none CPS-53 prophage attachment site attR, argW pseudogene pbl 87082169 Pseudogene reconstruction, pilT homo log

Pseudogene, phage lambda replication protein P family; C-terminal peaD 87081754 fragment

perR 1786448 predicted DNA-binding transcriptional regulator

outer membrane porin of poly-P-l,6-N-acetyl-D-glucosamine pgaA 1787261 (PGA) biosynthesis pathway

pgaB 1787260 PGA N-deacetylase

UDP-N-acetyl-D-glucosamine β- 1 ,6-N-acetyl-D-glucosaminyl pgaC 1787259 transferase

pgaD 1787258 predicted inner membrane protein

phnE 87082370 Pseudogene reconstruction, phosphonate permease

pinE 1787404 DNA invertase

pinH 1789002 Pseudogene, DNA invertase, site-specific recombination pinQ 1787827 DNA invertase

pinR 1787638 DNA invertase

prfH 1786431 Pseudogene, protein release factor homo log

psaA none ssrA pseudogene, CP4-57 attachment site duplication

ptwF none thrW pseudogene, CP4-6 prophage attachment site

quuD 1786763 predicted antitermination protein

quuQ 87081935 Lambda Q antitermination protein homo log

racC 1787614 unknown

racR 1787619 Rac prophage repressor, cl-like

ralR 1787610 Restriction alleviation gene

rbsA 1790190 D-ribose ABC transporter subunit (ATP -binding component) rbsD 87082327 D-ribose pyranase

recE 1787612 RecET recombinase

recT 1787611 RecET recombinase

relB 1787847 Antitoxin for RelE

relE 1787846 Sequence-specific mRNA endoribonuclease rem 1787844 unknown

Pseudogene reconstruction, lambda ren homo log, interrupted by renD 87081755 IS3C; putative activator of lit transcription

rhsE 1787728 Pseudogene, rhs family, encoded within RhsE repeat rnlA 1788983 RNase LS, endoribonuclease

rph 1790074 Pseudogene reconstruction, RNase PH

rusA 1786762 Endonuclease

rzoD 87081757 Probable Rzl-like lipoprotein

rzoQ none Probable Rzl-like lipoprotein

rzoR 87081890 Probable Rzl-like lipoprotein

rzpD 1786769 predicted murein endopeptidase

rzpQ 1787835 Rz-like equivalent

rzpR 87081889 Pseudogene, Rz homo log

sieB 87081885 Superinfection exclusion protein

sokA none Pseudogene, antisense sRNA blocking mokA/hokA translation

C-terminal Stf variable cassette, alternate virion-host specificity stfE 87081843 protein; Tail Collar domain, pseudogene

stfP 1787400 Predicted tail fiber protein

stfR 87081892 Side-tail fiber protein

tfaD 87081759 Pseudogene, tail fiber assembly gene, C-terminal fragment tfaE 1787402 Predicted tail fiber assembly gene

tfaP 1787401 Predicted tail fiber assembly gene

tfaQ 2367120 Phage lambda tail fiber assembly gene homo log

tfaR 1787637 Phage lambda tail fiber assembly gene homo log

tfaS 87082088 Pseudogene, tail fiber assembly gene, C-terminal fragment

Pseudogene reconstruction, tail fiber assembly gene, C-terminal tfaX 2367110 fragment

thrW none threonine tRNA (attachment site of the CP4-6 prophage) tori 87082092 CPS-53/KpLEl exisionase

treB 2367362 subunit of trehalose PTS permease (IIB/IIC domains) treC 1790687 trehalose-6-phosphate hydrolase

trkG 1787626 Major constitutive K+ uptake permease

ttcA 1787607 Integrase gene

ttcC none Pseudogene, prophage Rac integration site ttcA duplication uidB 1787902 Glucuronide permease, inactive point mutant

uxaA 1789475 altronate hydrolase

uxaC 2367192 uronate isomerase

wbbL 1788343 Pseudogene reconstruction, rhamnosyl transferase

wcaM 1788356 predicted colanic acid biosynthesis protein xisD none Pseudogene, exisionase fragment in defective prophage DLP12 xisE 1787387 el4 excisionase

yabP 1786242 Pseudogene reconstruction

yafF 87081701 Pseudogene, C-terminal fragment, H repeat-associated protein yafU 1786411 Pseudogene, C-terminal fragment

yafW 1786440 antitoxin of the Ykfl-YafW toxin-antitoxin system

yafX 1786442 unknown

predicted DNA-binding transcriptional regulator; inner membrane yafY 1786445 lipoprotein

yafZ 87081705 unknown

yagA 1786462 predicted DNA-binding transcriptional regulator

yagB 87081711 Pseudogene, antitoxin-related, N-terminal fragment

yagE 1786463 predicted lyase/synthase

yagF 1786464 predicted dehydratase

yagG 1786466 putative sugar symporter

yagH 1786467 putative β-xylosidase

yagi 1786468 predicted DNA-binding transcriptional regulator

yagj 1786472 unknown

yagK 1786473 unknown

yagL 1786474 DNA-binding protein

yagM 2367101 unknown

yagN 2367102 unknown

yagP 1786476 Pseudogene, LysR family, fragment

yaiT 1786569 Pseudogene reconstruction, autotransporter family

yaiX 87082443 Pseudogene reconstruction, interrupted by IS2A

ybbD 1786709 Pseudogene reconstruction, novel conserved family

ybcK 1786756 predicted recombinase

ybcL 1786757 predicted kinase inhibitor

ybcM 1786758 predicted DNA-binding transcriptional regulator

ybcN 1786759 DNA base-flipping protein

ybcO 1786761 unknown

ybcV 87081758 unknown

ybcW 1786772 unknown

ybcY 48994878 Pseudogene reconstruction, methyltransferase family

ybeM 1786843 Pseudogene reconstruction, putative CN hydrolase

ybfG 87081771 Pseudogene reconstruction, novel conserved family

ybfl none Pseudogene reconstruction, KdpE homo log

ybfL 87081775 Pseudogene reconstruction, H repeat-associated protein ybfO 1786921 Pseudogene, copy of Rhs core with unique extension ycgH 87081847 Pseudogene reconstruction

ycgl 1787421 Pseudogene reconstruction, autotransporter homo log ycjV 1787577 Pseudogene reconstruction, malK paralog

ydaC 1787609 unknown

ydaE 87081883 Metallothionein

ydaF 87081886 unknown

ydaG 87081887 unknown

ydaQ 87081882 Putative exisionase

ydaS 1787620 unknown

ydaT 1787621 unknown

ydaU 1787622 unknown

ydaV 1787623 unknown

ydaW 87081888 Pseudogene, N-terminal fragment

ydaY 1787629 pseudogene

ydbA 87081898 Pseudogene reconstruction, autotransporter homo log ydd 1787745 Pseudogene, C-terminal fragment, leucine-rich

yddL 1787746 Pseudogene, OmpCFN porin family, N-terminal fragment ydeT 1787782 Pseudogene, FimD family, C-terminal fragment

ydfA 1787854 unknown

ydfB 87081937 unknown

ydfC 1787856 unknown

ydfD 1787858 unknown

ydffi 1787859 Pseudogene, N-terminal fragment

ydfJ 1787824 Pseudogene reconstruction, MFS family

ydfK 1787826 Cold shock gene

ydfO 87081931 unknown

ydfR 1787837 unknown

ydfU 87081936 unknown

ydfV 1787848 unknown

ydfX 1787851 pseudogene

yedN 87082002 Pseudogene reconstruction, IpaH/Y opM family

yedS 87082009 Pseudogene reconstruction, outer membrane protein homo log yeeH none Pseudogene, internal fragment

yeeL 87082016 Pseudogene reconstruction, glycosyltransferase family yeeP 87082019 Pseudogene, putative GTP -binding protein

yeeR 87082020 unknown

yeeS 1788312 unknown yeeT 1788313 unknown

yeeU 1788314 Antitoxin component of toxin-antitoxin protein pair YeeV- YeeU yeeV 1788315 Toxin component of toxin-antitoxin protein pair YeeV- YeeU yeeW 1788316 pseudogene

yegZ none Pseudogene, gpD phage P2-like protein D; C-terminal fragment yehH 87082046 Pseudogene reconstruction

yehQ 87082050 Pseudogene reconstruction

yejO 1788516 Pseudogene reconstruction, autotransporter homo log

yfaH 1788571 Pseudogene reconstruction, C-terminal fragment, LysR homo log yfaS 87082066 Pseudogene reconstruction

yfcU 1788678 Pseudogene reconstruction, FimD family

yfdK 1788696 Unknown

yfdL 1788697 Pseudogene, tail fiber protein

Pseudogene, intact gene encodes a predicted DNA adenine yfdM 87082089 methyltransferase

yfdN 1788699 Unknown

yfdP 1788701 Unknown

yfdQ 1788702 Unknown

yfdR 87082090 Unknown

yfdS 1788704 Unknown

yfdT 1788705 Unknown

yffL 1788784 Unknown

yffM 1788785 Unknown

yffN 1788786 Unknown

yffO 1788787 Unknown

yffP 1788788 Unknown

yffQ 1788790 Unknown

yffR 1788791 Unknown

yffS 1788792 Unknown

1788976 Unknown

yiji 1788978 Unknown

yfiJ 1788979 Unknown

yfiK 1788980 Unknown

yQL 1788981 Unknown

yfjM 1788982 Unknown

y^o 87082140 Unknown

yQP 48994902 Unknown

y^Q 1788987 Unknown yfiR 1788988 Unknown

yQS 87082142 Unknown

yQ 1788990 Unknown

yfiu 1788991 Pseudogene

yfiv 1788992 Pseudogene reconstruction, arsB-like C-terminal fragment yfjW 2367146 Unknown

yfix 1788996 Unknown

yfiY 1788997 Unknown

yfiz 1788998 Antitoxin component of putative toxin-antitoxin YpjF-YfjZ ygaQ 1789007 Pseudogene reconstruction, has alpha-amylase-related domain ygaY 1789035 Pseudogene reconstruction, MFS family

ygeF 2367169 Pseudogene reconstruction, part of T3SS PAI ETT2 remnant ygeK 87082170 Pseudogene reconstruction, part of T3SS PAI ETT2 remnant ygeN 1789221 Pseudogene reconstruction, orgB homolog

ygeO 1789223 Pseudogene, orgA homolog, part of T3SS PAI ETT2 remnant ygeQ 1789226 Pseudogene reconstruction, part of T3SS PAI ETT2 remnant yghE 1789340 Pseudogene reconstruction, general secretion protein family yghF 1789341 Pseudogene, general secretion protein

ygho 1789354 Pseudogene, C-terminal fragment

yghX 1789373 Pseudogene reconstruction, S9 peptidase family

yhcE 1789611 Pseudogene reconstruction, interrupted by IS5R

yhdW 1789668 Pseudogene reconstruction

yhiL 87082275 Pseudogene reconstruction, FliA regulated

yhiS 1789920 Pseudogene reconstruction, interrupted by IS5T

yhjQ 1789955 Pseudogene reconstruction

yibJ 48994952 Pseudogene reconstruction, Rhs family

yibS none Pseudogene reconstruction, Rhs family, C-terminal fragment yibU none Pseudogene reconstruction, H repeat-associated protein yibW none Pseudogene reconstruction, rhsA-linked

yicT none Pseudogene, N-terminal fragment

yifN 2367279 Pseudogene reconstruction

yjbi 1790471 Pseudogene reconstruction

yjdQ none Pseudogene reconstruction, P4-like integrase remnant yjgx 1790726 Pseudogene reconstruction, EptAB family

yjhD 87082406 Pseudogene, C-terminal fragment

yjhE 87082407 Pseudogene, putative transporter remnant

yjhR 1790762 Pseudogene reconstruction, helicase family, C-terminal fragment yjhV 1790738 Pseudogene, C-terminal fragment yjhY none Pseudogene reconstruction, novel zinc finger family

yjhZ none Pseudogene reconstruction, rimK paralog, C-terminal fragment yjiP 1790795 Pseudogene reconstruction, transposase family

yjiT 87082428 Pseudogene, N-terminal fragment

yj v none Pseudogene reconstruction, helicase-like, C-terminal fragment yjj 87082432 predicted oxidoreductase

ykfA 87081706 putative GTP -binding protein

ykfB 1786444 Unknown

Pseudogene, retron-type reverse transcriptase family, N-terminal ykfC 87081707 fragment

ykfF 1786443 Unknown

ykfG 2367100 Unknown

ykfH 87081704 Unknown

ykfl 1786439 toxin of the Ykfl-YafW toxin-antitoxin system

ykfj 1786430 Pseudogene, N-terminal fragment

ykf 1786445 Pseudogene, N-terminal fragment

ykfL none Pseudogene, C-terminal fragment

ykfN none Pseudogene, N-terminal remnant, YdiA family

ykgA 87081714 Pseudogene, N-terminal fragment, AraC family

ykgP none Pseudogene, oxidoreductase fragment

ykgQ none Pseudogene, C-terminal fragment of a putative dehydrogenase ykgS none Pseudogene internal fragment

ykiA 1786591 Pseudogene reconstruction, C-terminal fragment

ylbE 1786730 Pseudogene reconstruction, yahG paralog

ylbG 87081748 Pseudogene reconstruction, discontinuous N-terminal fragment ylbH 1786708 Pseudogene, copy of Rhs core with unique extension

ylbl none Pseudogene, internal fragment, Rhs family

ylcG 87081756 Unknown

ylcH none Unknown

ylcl none Unknown

ymdE 87081823 Pseudogene, C-terminal fragment

ymfD 1787383 Putative SAM-dependent methyltransferase

ymffi 1787384 Unknown

ymfl 87081839 Unknown

ymfj 87081840 Unknown

ymfL 1787393 Unknown

ymfM 1787394 Unknown

ymfQ 1787399 Putative baseplate or tail fiber proteintt ymfPv 1787396 Unknown

ymjC none Pseudogene, N-terminal fragment

ymjD none Expressed deletion pseudogene fusion remnant protein ynaA 1787631 Pseudogene, N-terminal fragment

ynaE 1787639 Cold shock gene

ynaK 1787628 Unknown

ynci 1787731 Pseudogene reconstruction, H repeat-associated, RhsE-linked yncK none Pseudogene reconstruction, transposase homo log

yneL 1787784 Pseudogene reconstruction, C-terminal fragment, AraC family yneO 1787788 Pseudogene reconstruction, putative OM autotransporter adhesi ynfN 87081933 Cold shock gene

ynfO none Unknown

yoeA 87082018 Pseudogene reconstruction, interrupted by IS2F

yoeD none Pseudogene, C-terminal fragment of a putative transposase yoeF 87082021 Pseudogene, C-terminal fragment

yoeG none pseudogene, N-terminal fragment

yoeH none pseudogene, C-terminal fragment

ypdJ 87082091 Pseudogene, exisonase fragment

ypjc 1789003 Pseudogene reconstruction

ypjF 1788999 Toxin component of putative toxin-antitoxin pair YpjF-YfjZ ypji none Pseudogene reconstruction

ypjJ 87082144 Unknown

ypjK 87082141 Unknown

yqffi 1789281 Pseudogene reconstruction, C-terminal fragment, LysR family yqiG 48994919 Pseudogene reconstruction, FimD family, interrupted by IS2I yrdE none Pseudogene reconstruction, C-terminal fragment, yedZ paralog yrdF none Pseudogene, N-terminal fragment

yrhA 87082266 Pseudogene reconstruction, interrupted by IS IE

yrhC 87082273 Pseudogene reconstruction, N-terminal fragment

ysaC none Pseudogene, C-terminal remnant

ysaD none Pseudogene, internal sequence remnant

ytfA 1790650 Pseudogene, C-terminal fragment

yzgL 87082264 Pseudogene, putative periplasmic solute binding protein

According to the present invention, 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.

According to a preferred embodiment of the invention, the microorganism is an Escherichia coli.

The invention is also related to a method for the fermentative production of methionine, comprising the steps of:

- culturing a recombinant microorganism optimized for the fermentative production of methionine, and with an attenuated expression of the gene ybdL, in an appropriate culture medium comprising a fermentable source of carbon and a source of sulfur, and

recovering methionine from the culture medium.

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.

Those skilled in the art are able to define the culture conditions for the microorganisms according to the invention. In particular 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' according to the present invention 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. In a particular embodiment of the invention, 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.

The term "source of sulphur" according to the invention 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. The methods for the recovery and purification of the produced compounds are well known to those skilled in the art (WO 2005/007862, WO 2005/059155).

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.

Optionally, from 0 to 100%, 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.

Optionally, the methionine derivative N-acetyl-methionine is transformed into methionine by deacylation, before methionine is recovered.

In a specific aspect of the invention, 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.

"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.

Such limitation in microorganism growth has been described in the patent application WO 2009/043372.

EXAMPLES PROTOCOLES

Several protocols have been used to construct methionine producing strains and are described in the following examples.

Protocol 1: Chromosomal modifications by homologous recombination and selection of recombinants (Datsenko, K.A. & Wanner, B.L. (2000)

Allelic replacement or gene disruption in specified chromosomal locus was carried out by homologous recombination as described by Datsenko & Wanner (2000). The chloramphenicol (Cm) resistance cat, the kanamycin (Km) resistance kan, or the gentamycin (Gt) resistance gm genes, flanked by Flp recognition sites, were amplified by PCR by using pKD3 or pKD4 or p34S-Gm (Dennis et Zyltra, AEM july 1998, p 2710- 2715) plasmids as template respectively. The resulting PCR product was used to transform the recipient E. coli strain harbouring plasmid pKD46 that expresses the λ Red (γ, β,.εχο) recombinase. Antibiotic-resistant transformants were then selected and the chromosomal structure of the mutated loci was verified by PCR analysis with the appropriate primers. 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.

Preparation of the phage lysate

Inoculate 100 μΐ of an overnight culture of the strain MG1655 with the chromosomal modification of interest in 10 ml of LB + Km 50μg/ml or + glucose 0.2% + CaCl₂ 5 mM (antibiotinc solution is chosen according to the resistant cassette used for the construct).

- Incubate 30 min at 37°C with shaking.

Add 100 μΐ of PI phage lysate prepared on the donor strain MG1655 (approx. 1 x 10⁹ phage/ml). Shake at 37°C for 3 hours until the complete lysis of cells.

- Add 200 μΐ of chloroform, and vortex.

Centrifuge 10 min at 4500 g to eliminate cell debris.

Transfer supernatant to a sterile tube.

- Store the lysate at 4°C.

Transduction

Centrifuge 10 min at 1500 g 5 ml of an overnight culture of the E. coli recipient strain cultivated in LB medium.

- Suspend the cell pellet in 2.5 ml of MgS0₄ 10 mM, CaCl₂ 5 mM.

Infect 100 μΐ cells with 100 μΐ PI phage lysate of the chromosomal modification strain MG1655 (test tube) and as a control tubes 100 μΐ cells without PI phage lysate and 100 μΐ PI phage lysate without cells.

Incubate 30 min at 30°C without shaking.

Add 100 μΐ sodium citrate 1 M in each tube, and vortex.

- Add l ml of LB.

Incubate 1 hour at 37°C with shaking.

Centrifuge 3 min at 7000 rpm.

Plate on LB + Cm 30 μg/ml or Km 50 μg/ml (or other antibiotic according to the antibiotic resistant cassette present in the selected strain).

Incubate at 37°C overnight.

The resistant transductants are then selected and the chromosomal structure of the mutated locus is verified by PCR analysis with the appropriate primers.

Table 1 : Genotype and corresponding number of producer strains described in the following examples

Strain Genotype

number

1 MG1655 metA *ll Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36- ARNmstl 7-metF ¥trc07-serB AmetJ ApykF ApykA ApurUAyncA AmalS::TTadc-CI857- VlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE- PgapA-metA *ll AuxaCA : :TT07-TTadc-PlambdciR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *l 1 ACP4-6::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *ll AwcaM::TT02- TTadc-PlambdaR *(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 DtreBC: : TT02-serA-serC 2 MG1655 met A * 11 Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36- ARNmstl 7-metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- VlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE- PgapA-metA *l 1 AuxaCA ::TT07-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *ll ACP4-6::TT02-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *ll AwcaM::TT02- TTadc-PlambdaR *(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 DtreBC: : TT02-serA-serC

DybdL ::Km

3 MG1655 met A * 11 Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36- ARNmstl 7-metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- VlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE- PgapA-metA *l 1 AuxaCA : :TT07-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *l 1 ACP4-6::TT02-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *ll AwcaM::TT02- TTadc-PlambdaR *(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 DtreBC: : TT02-serA-serC

PCL1920-VgapA-pycRe-TT07

4 MG1655 met A * 11 Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36- ARNmstl 7-metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- VlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE- PgapA-metA *l 1 AuxaCA : :TT07-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *l 1 ACP4-6::TT02-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *ll AwcaM::TT02- TTadc-PlambdaR *(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 DtreBC: : TT02-serA-serC

DybdL r.Km _PCL1920-PgapA-pycRe-TT07

EXAMPLE 1:

Construction of strain 1, MG1655 metA *ll Vtrc-metH VtrcF-cysPUWAM VtrcF- cysJIH Vtrc09-gcvTHP Vtrc36-ARNmstl 7-metF Vtrc07-serB AmetJ ApykF ApykA

ApurU AyncA AmalS::TTadc-CI857-?lambdaR*(-35)-thrA*l-cysE ApgaABCD::TT02- TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AuxaCA ::TT07-TTadc- YlambdaR *(-35)-RBS01-thrA *l-cysE-YgapA-metA *11 ACP4-6::TT02-TTadc- VlambdaR *(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 A wcaM:: TT02-TTadc- PlambdaR *(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 DtreBC:: TT02-serA-serC Methionine producing strain 1 (Table 1) has been described in patent applications EP10306164.4 and US61/406249 (strain 10, Table 1) which is incorporated as reference into this application. EXAMPLE 2:

Construction of strain 2, MG1655 metA *ll Vtrc-metH VtrcF-cysPUWAM VtrcF- cysJIH Vtrc09-gcvTHP Vtrc36-ARNmstl 7-metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857-PlambdaR*(-35)-thrA*l-cysE ApgaABCD::TT02- TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AuxaCA ::TT07-TT dc- VlambdaR *(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 ACP4-6::TT02-TTadc- VlambdaR *(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 A wcaM:: TT02-TT dc- PlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 DtreBC::TT02-serA-serC

DybdL ::Km

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.

Ome 0589-DybdLF (SEQ ID N°l)

CACCGACAGCGGAATCGCCGCTACGCCGTGCTCCTGCGTCAGCCACTGGCAAA ACTCAACATCATCCAGGGTAGAAACCGTGTAGGCTGGAGCTGCTTCG

with

-upper case sequence homologous to sequence downstream ybdL gene (633791-633870, reference sequence on the website http://ecogene.org/)

- underlined upper case sequence corresponding to the primer site 1 of plasmid pKD4 (Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645)

Ome 0590-DybdLR (SEQ ID N°2)

GGTACAATAAAAATGACAAATAACCCTCTGATTCCACAAAGCAAACTTCCACA ACTTGGCACCACTATTTTCACCCAGCATATGAATATCCTCCTTAG

with:

- upper case sequence homologous to sequence upstream ybdL gene (632797- 632874,reference sequence on the website http://ecogene.org/)

- underlined upper case sequence corresponding to the primer site 2 of plasmid pKD4 (Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645)

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.

Ome 0591-ybdLR (SEQ ID N°3)

CGAAGTGCTGCGCCTGAAGC homologous to the sequence upstream of the ybdM gene (634054-634035, reference sequence on the website http://ecogene.org/)

Ome 0592-ybdLF (SEQ ID N°4)

GCCGGGCCGACGACCACGCGG homologous to sequence downstream of the ybdH gene (632663-632683, reference sequence on the website http://ecogene.org/)

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). The verified and selected strain MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36-ARNmstl 7-metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS: :TTadc-CI857-?lambdaR*(-35)- thrA *l-cysE ApgaABCD: :TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 AuxaCA : :TT07-TTadc-?lambdaR *(-35)-RBS01-thrA *l-cysE-FgapA-metA *11 ACP4-6: : TT02-TTadc-?lambdaR *(-35)-RBS01-thrA *l-cysE-PgapA-metA *11

AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-FgapA- metA *ll DtreBC: :TT02-serA-serC DybdL ::Km was called strain 2.

EXAMPLE 3:

Construction of strain 3, MG1655 metA *ll Vtrc-metH VtrcF-cysPUWAM VtrcF- cysJIH Vtrc09-gcvTHP Vtrc36-ARNmstl 7-metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857-VlambdaR*(-35)-thrA*l-cysE ApgaABCD: :TT02- TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AuxaCA ::TT07-TTadc- YlambdaR *(-35)-RBS01-thrA *l-cysE-YgapA-metA *11 ACP4-6::TT02-TTadc- VlambdaR *(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 A wcaM:: TT02-TTadc- PlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 DtreBC: :TT02-serA-serC pCL1920-?gapA-pycRe-TT07

The pCL1920-Pga/¾4-/?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-ΎΎΟΊ was introduced by electroporation into the strain 1 (Table 1). The presence of the pCL1920-P gap A-pycRe-ΊΊ 07 was verified by digestion and the selected strain MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09- gcvTHP Ptrc36-ARNmstl 7-metF Ptrc07-serB AmetJ ApykF ApykA Apurll AyncA AmalS::TTadc-CI857-PlambdaR*(-35)-thrA *l-cysE ApgaABCD: : TT02- TTadc-

PlambdaR *(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AuxaCA : :TT07-TTadc-

PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *ll ACP4-6::TT02-TTadc- PlambdaR *(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 Δ wcaM: : TT02- TTadc-Plam bdaR *(- 35)-RBS01-thrA *l-cysE-PgapA-metA *11 DtreBC: : TT02-serA-serC pCL1920-PgapA- pycRe- TT07 was called strain 3.

EXAMPLE 4:

Construction of strain 4, MG1655 metA*ll 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-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AuxaCA ::TT07-TTadc- PlambdaR *(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 ACP4-6:: TT02-TTadc- PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 A wcaM: : TT02- TTadc- PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 DtreBC: :TT02-serA-serC DybdL r.Km pCL1920-PgapA-pycRe-TT07

The pCL1920-Pga/?^-/?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-Pga/¾4-/?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-

PlambdaR *(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AuxaCA : :TT07-TTadc-

PlambdaR *(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 ACP4-6::TT02-TTadc- PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *ll A wcaM: : TT02- TTadc-

PlambdaR *(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 DtreBC: : TT02-serA-serC

DybdL r.Km pCLl 920-PgapA-pycRe-TT07 was called strain 4.

EXAMPLE 5

Production of methionine by fermentation

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. When it was necessary, 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. When the culture had reached an OD600 of 5 to 7, extracellular amino acids were quantified by HPLC after OPA/Fmoc derivatization and other relevant metabolites were analyzed using HPLC with refractometric detection (organic acids and glucose) and GC-MS after silylation. For each strain, several repetitions were made. Table 2: Minimal medium composition (PCI).

Table 3 : Methionine yield (Y_met), in % g of methionine per g of glucose produced in batch culture by the different strains. For the definition of methionine/glucose yield see below. SD denotes the standard deviation for the yields which was calculated on the basis of several repetitions (N = number of repetitions).

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:

Y methionine (g) ^ _m

m^et consummed glu cose (g)

As can be seen in table 3, yields of methionine/glucose are increased upon ybdL deletion in different genetic backgrounds (comparison of strains 2 and 4 versus 1 and 3 respectively).

REFERENCES

Saunderson, C.L., (1985) British Journal of Nutrition 54, 621-633

- Sauer & Eikmanns (2005) FEMS Microbiol Reviews 29 p765-94

- Figge RM (2006), "Amino acid biosynthesis" pi 64- 185, ed Wendisch VF, Microbiol Monogr (5)

- Nagy et al. (1995) J. Bacteriol 177 (5) p. 1292-98)

- Tuite et al, 2005 J. Bacteriol, 187, 13, 4362-4371

- 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"

- Aitken & Kirsch, 2005, Arch Biochem Biophys 433, 166-75

- Dolzan et al, FEBS Letters, 2004

Carrier and Keasling (1998) Biotechnol. Prog. 15, 58-64

- Dennis et Zyltra, AEM july 1998, p 2710-2715

Claims

Claims:

1) 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.

2) The microorganism of claim 1, wherein the gene ybdL is deleted.

3) The microorganism of anyone of claims 1 to 2, wherein the expression of at least one of the following genes is increased: pyc, pntAB, cysP, cysU, 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 *).

4) The microorganism of anyone of claims 1 to 3, wherein the expression of at least one of the following genes is attenuated: metJ, pykA, pykF, purJ, yncA.

5) The microorganism of anyone of claims 1 to 4, wherein :

o the gene ybdl is deleted,

o the expression of the genes metA *, metH, cysPUWAM, cysJIH, gcvTHP, metF, serA, serB, serC, cysE, thrA * and pyc is increased, and

o the genes MetJ, pykA, pykF, purJJ, yncA are attenuated.

6) The microorganism of anyone of claims 1 to 5, wherein said microorganism is an

Escherichia coli.

7) A method for the fermentative production of methionine, comprising the steps of :

• culturing a recombinant microorganism according to anyone of claims 1 to 6 in an appropriate culture medium comprising a fermentable source of carbon and a source of sulfur, and

• recovering methionine or its derivatives from the culture medium.

8) The method of claim 7 wherein 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.