WO2007077041A1

WO2007077041A1 - Process for the preparation of methionine and its precursors homoserine or succinylhomoserine employing a microorganism with enhanced sulfate permease expression

Info

Publication number: WO2007077041A1
Application number: PCT/EP2006/050033
Authority: WO
Inventors: Rainer Figge; Fabien Lux; Céline RAYNAUD; Michel Chateau; Philippe Soucaille
Original assignee: Metabolic Explorer
Priority date: 2006-01-04
Filing date: 2006-01-04
Publication date: 2007-07-12
Also published as: US20130183726A1; EP2314710A2; ES2459617T3; JP5172697B2; BRPI0620880A2; BRPI0620880B1; AR058914A1; MY148979A; US8389250B2; DK2314710T3; US20080311632A1; CN101351558A; EP1969130A1; PL1969130T3; CN101351558B; EP2314710B1; JP2009521939A; EP1969130B1; US9187775B2; EP2314710A3

Abstract

The present invention relates to a process for the production of methionine or its derivatives by culturing a microorganism in an appropriate culture medium comprising a source of carbon and a source of sulfur. The microorganism claimed is modified in a way that the production of cysteine and/or C1 units is enhanced and/or the transfer potential of the C1 units on 10 homocysteine is increased or optimized. The isolation of methionine or its derivates from the fermentation medium is also claimed.

Description

PROCESS FOR THE PREPARATION OF METHIONINE AND ITS PRECURSORS HOMOSERINE OR SUCCINYLHOMOSERINE EMPLOYING A MICROORGANISM WITH ENHANCED SULFATE PERMEASE EXPRESSION

5 Field of the invention

The present invention relates to a process for the production of methionine or its derivatives by culturing a microorganism in an appropriate culture medium comprising a source of carbon and a source of sulfur. The microorganism claimed is modified in a way that the production of cysteine and/or Cl units is enhanced and/or the transfer potential of the Cl units on 10 homocysteine is increased or optimized. The isolation of methionine or its derivates from the fermentation medium is also claimed.

Prior art

Sulfur-containing compounds such as cysteine, homocysteine, methionine or S-

15 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

20 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

25 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.

30 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.

35 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 Cl metabolism (N-methyltetrahydrofolate). Aspartate is converted into homoserine by a sequence of three reactions. Homoserine can subsequently enter the threonine/isoleucine 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 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; Weissbach et al., 1991 MoI. Microbiol., 5, 1593-1597). MetJ together with its corepressor S-adenosylmethionine is known to regulate the genes met A, metB, metC, metE and metF. Other genes encoding enzymes implicated in methionine production, such as glyA, metE, metR and metF are activated by MetR whereas met A is repressed by MetR. The corresponding enzymes are all involved in the production and the transfer of Cl units from serine to methionine. GIyA encoding serine hydroxymethyltransferase catalyzes the conversion of serine to glycine and the concomitant transfer of a Cl unit on the coenzyme tetrahydrofolate (THF). The Cl unit in form of methylene-THF needs to be reduced to methyl-THF before it can be transferred on homocysteine to yield methionine. This reaction is catalyzed by the MetF protein. Transfer of the methylgroup is either catalyzed by MetH via vitamin B 12 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 B 12 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 by not expressing large quantities of MetE. An accumulation of homocysteine is toxic for E. coli (Tuite et al., 2005 J. Bacterid, 187, 13, 4362-4371.) and at the same time has a negative, regulatory effect on met A 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, α-ketobutyrate and succinate, a reaction called γ-elimination (Aitken & Kirsch, 2005, Arch Biochem Biophys 433, 166-75). α-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 is an important aspect for the industrial production of methionine. The provisional patent application US 60/650,124 filed on February 7, 2005 describes how γ-elimination can be reduced by optimizing the enzyme MetB. Optimizing the flow of cysteine biosynthesis can also reduce γ- elimination and thus the production of the byproduct isoleucine and constitutes an embodiment of this invention.

General disclosure of the invention

The invention relates to a process for the production of methionine, its precursors or products derived thereof in a fermentative process using microorganisms that have an increased production of cysteine and that grow on a defined carbon and sulfur source.

Precursors of methionine are defined as metabolites that are part of the methionine specific metabolic pathway or can be derived of these metabolites. The methionine specific pathway starts with the transformation of homoserine to succinylhomoserine by the enzyme homoserine succinyl transferase (MetA). Products derived of methionine originate from methionine transforming and/or degrading pathways.

To increase cysteine production the inventors have enhanced the expression of genes involved in cysteine production.

The term enhanced in this context describes the increase in the intracellular activity of an enzymatic activity which is encoded by the corresponding DNA, for example by increasing the number of copies of the gene, using a strong promoter or using an allele with increased activity and possibly combining these measures.

The terms "increased expression" or "enhanced expression" are both used in the text and have similar meaning. To increase the expression of a gene it may be encoded chromosomally or extrachromosomally. Chromosomally there may be one or several copies on the genome that can be introduced by methods of recombination known to the expert in the field. Extrachromosomally genes may be carried by different types of plasmids that differ with respect to their origin of replication and thus their copy number in the cell. They may be present as 1-5 copies, ca 20 or up to 500 copies, corresponding to 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 preferred embodiment of the invention the gene may be expressed using promoters with different strength that need or need not to be induced by inducer molecules. These promoters may be homologous or heterologous. Examples are the promoters Ptrc, Ptac, Plac, the lambda promoter cl or other promoters known to the expert in the field.

Expression of the target genes may be boosted or reduced by elements stabilizing or destabilizing the corresponding messenger RNA (Carrier and Keasling (1998) Biotechnol. Prog. 15, 58-64) or the protein (e.g. GST tags, Amersham Biosciences).

The present invention also relates to microorganisms that contain one or several alleles of the gene to be enhanced according to the invention.

In a particular embodiment of the invention the expression of genes involved in cysteine production is enhanced.

Genes involved in cysteine production comprise genes encoding proteins required for the import of a sulfur source, the transformation of that sulfur source into hydrogen sulfide and the assimilation of hydrogen sulfide or the sulfur source into cysteine or its derivatives.

In E. coli these proteins are encoded by the following genes (followed by accession numbers and function of the corresponding polypeptide): gene accession number function cysA 1788761 sulfate permease cysU, cysT 1788764 component of sulfate ABC transporter cysVf 1788762 membrane bound sulfate transport protein ccyyssLZ 1 1778888775533 ORF upstream of cysK cysN 1789108 ATP sulfurylase cysD 1789109 sulfate adenylyltransferase cysC 1789107 adenylylsulfate kinase cysH 1789121 adenylylsulfate reductase ccyyssll 1 1778899112222 sulfite reductase, alpha subunit cysJ 1789123 sulfite reductase, beta subunit cysE 1790035 serine acetyltransferase cysK. 1788754 cysteine synthase cysM 2367138 O-acetyl serine sulfhydrylase ccyyssZZ 1 1778888775533 sulfate transport sbp 1790351 Periplasmic sulfate-binding protein 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://www.sanger.ac.uk/Soflware/Pfam/) represents a large collection of protein sequence alignments. Each PFAM makes it possible to visualize multiple alignments, see protein domains, evaluate distribution among organisms, gain access to other databases, and visualize known protein structures. COGs (clusters of orthologous groups of proteins; http://www.ncbi.nlm.nih.gov/COG/) are obtained by comparing protein sequences from 66 fully sequenced genomes representing 30 major phylogenic lines. Each COG is defined from at least three lines, which permits the identification of former conserved domains.

The means of identifying homologous sequences and their percentage homologies are well known to those skilled in the art, and include in particular the BLAST programs, which can be used from the website http://www.ncbi.iilm.nih.eov/BLAST/ with the default parameters indicated on that website. The sequences obtained can then be exploited (e.g., aligned) using, for example, the programs CLUSTALW (http://www.ebi.ac.uk/clustalw/) or MULTALIN

(http://prodes.toulouse.im-a.fr/multalin/cgi-bin/multalm.pl), with the default parameters indicated on those websites.

Using the references given on 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 described, for example, in Sambrook et al. (1989 Molecular Cloning: a Laboratory Manual. 2^nd ed. Cold Spring Harbor Lab., Cold Spring Harbor, New York.).

As one preferred embodiment, the microorganism used in the method of the present invention is modified to increase the expression of cysE encoding serine transacetylase.

The present invention also relates to microorganisms that contain one or several alleles encoding serine transacetylase according to the invention.

Such strains are characterized by the fact that they possess a cysteine metabolism which permits an increased flux towards methionine by providing an increased substrate concentration for the synthesis of γ-cystathionine, a reaction catalyzed by MetB. At low cysteine concentrations the MetB enzyme produces ammonia, succinate and α-ketobutyrate from succinyl-homoserine, a reaction called γ-elimination. An increased cysteine concentration reduces the amount of α- ketobutyrate produced and thus increases the flow towards methionine. Enhanced expression of serine transacetylase activities can be validated in enzymatic tests with serine and acetyl-CoA. The reaction is started by adding the protein extract containing serine transacetylase activity, and the formation of O-acetyl-serine is monitored by GC-MS after protein precipitation and derivatization with a silylating reagent. The present invention also relates to the enhanced expression of the cysM gene encoding

O-acetylserine sulfhydrylase which allows increased integration of thiosulfate into sulfocysteine boosting the production of cysteine.

The present invention thus claims a process in which γ-elimination and thus the production of isoleucine is reduced by optimizing the production of cysteine. A heterologous promoter according to the invention is understood as the modified wildtype promoter or any promoter from another organism or an entirely synthetic promoter. Preferentially the heterologous promoter is a strong promoter, such as Ptrc, Ptac, lamda cl or other promoters known to the expert in the field.

In another preferred embodiment of the invention a process is claimed in which a microorganism is used for the production of methionine or its derivatives in which the expression of genes involved in the production of Cl units and/or their transfer potential onto homocysteine is/are increased or optimized.

According to the invention, optimization is accomplished by adapting the expression level of the concerned gene in a way to obtain the highest methionine production. In most cases this is done by creating expression libraries of the concerned gene using for example heterologous promoters and screening for the best producers.

According to the invention the term Cl unit describes single carbon atoms that are bound to the carrier molecule tetrahydrofolate as methyl, methylene, methenyl or formyl groups.

The term "transfer potential" describes the capability of the microorganisms to transfer Cl units onto homocysteine. This potential is determined by the activities of MetF and/or MetH that have been enhanced and/or optimized by the inventors.

Genes involved in the production of Cl units are listed below: serA 1789279 phosphoglycerate dehydrogenase sseerrBB 1 1779900884499 phosphoserine phosphatase serC 1787136 phosphoserine aminotransferase

1788902 serine hydroxymethyltransferase gcvT 1789272 Tetrahydrofolate dependent aminomethyl transferase gcvH 1789271 Glycine cleavage, carrier of aminomethyl group ggccvvPP 1 1778899226699 Glycine dehydrogenase (decarboxylating) lpd 1786307 Lipoamide dehydrogenase Genes involved in the transfer of Cl units onto homocysteine are listed below: metF 1790377 5, 10-Methylenetetrahydrofolate reductase metH 1790450 B12-dependent homocysteine-N5- methyltetrahydrofolate transmethylase metE 2367304 Tetrahydropteroyltriglutamate methyltransferase

In a specially preferred embodiment of the invention the microorganism used for the production of methionine is modified to increase the expression of metF or metH, or both or to express metF from a heterologous promoter. Enhanced vitamin B 12 dependent methionine synthase (MetH) activity can be validated in enzymatic tests with methyl-THF and homocysteine in the presence of vitamin B12 and SAM. The reaction is started by adding the protein extract containing the methylene tetrahydrofolate reductase activity, and the formation of methionine is monitored by GC-MS after protein precipitation and derivatization with a silylating reagent. Methionine production can be further increased by increasing the expression of additional genes involved in methionine biosynthesis, which is also object of the invention.

These genes are listed below: met A 1790443 Homoserine succinyltransferase metB 1790375 Cystathionine-γ-synthase metC 1789383 Cystathionine-β-lyase metF 1790377 5, 10-Methylenetetrahydrofolate reductase metR 1790262 Positive regulatory gene for metE, metH and metF

Furthermore expression of genes in pathways degrading methionine or deviating from the methionine production pathway may be reduced or the genes may be deleted. speD 1786311 S-Adenosylmethionine decarboxylase speC 1789337 Ornithine decarboxylase astA 1788043 Arginine succinyltransferase dapA 1788823 Dihydrodipicolinate synthase

Anaplerotic reactions may be boosted by expressing ppc 1790393 phosphoenolpyruvate carboxylase pps 1787994 phosphoenolpyruvate synthase

Acetate consuming reactions may be boosted by over expressing acs 1790505 acetyl-CoA synthetase

An additional increase in the production of L-methionine, its precursors or compounds derived thereof can be achieved by overexpressing one or several of the following genes: pyruvate carboxylases, e.g. from Rhizobium etli (pyc, U51439), or one of its homologs, the homoserine synthesizing enzymes encoded by the genes thrA (homoserine dehydrogenase/ aspartokinase,

1786183), preferably with reduced feed-back sensitivity, metL (homoserine dehydrogenase/aspartokinase, gl790376) or lysC (aspartokinase, 1790455) and asd (aspartate semialdehyde dehydrogenase).

A further increase in the production of L-methionine, its precursors or compounds derived thereof, is achieved by means of deleting the gene for the repressor protein MetJ, responsible for the down-regulation of the methionine regulon as was suggested in JP 2000157267-A/3 (see also GenBank 1790373).

Methionine production is further increased by using homoserine succinyltransferase alleles with reduced feed-back sensitivity to its inhibitors SAM and methionine as described in patent application WO 2005/111202 that is incorporated into this document.

An increase in the production of L-methionine, its precursors or compounds derived thereof, can be achieved by attenuating the activity or deleting one of the following genes.

Attenuation in this context describes the reduction of the intracellular activity of an enzyme by measures such as reducing its expression, reducing the stability of the enzyme, increasing its degradation and/or other solutions known to the expert in the field.

Gene Genbankentry activity ackA 1788633 acetate kinase pta 1788635 phosphotransacetylase aceΕ 1786304 pyruvate deydrogenase El aceF 1786305 pyruvate deydrogenase E2 lpd 1786307 pyruvate deydrogenase E3 sucC 1786948 succinyl-CoA synthetase, beta subunit sucD 1786949 succinyl-CoA synthetase, alpha subunit pck 1789807 phosphoenolpyruvate carboxykinase pykA 1788160 pyruvate kinase II pykF 1787965 pyruvate kinase I poxB 1787096 pyruvate oxidase

UvB 1790104 acetohydroxy acid synthase I, large subunit

UvN 1790103 acetohydroxy acid synthase I, small subunit

UvG 1790202 acetohydroxy acid synthase II, large subunit

1790203

UvM 1790204 acetohydroxy acid synthase II, small subunit ilvl 1786265 acetohydroxy acid synthase III, large subunit

HvΑ 1786266 acetohydroxy acid synthase III, small subunit arόF 1788953 DAHP synthetase aroG 1786969 DAHP synthetase arόΑ 1787996 DAHP synthetase thrB 1786184 homoserine kinase thrC 1786185 threonine synthase sdaA 1788116 serine deaminase sdaB 1789161 serine deaminase

Production of methionine may be further increased by using an altered metB allele that uses preferentially or exclusively H₂S and thus produces homocysteine from O-succinyl- homoserine as has been described in the patent application WO 2004/076659, which content is incorporated herein by reference.

The sulfur source used for the fermentative production of L-methionine, its precursors or compounds derived thereof, may be any of the following or a combination thereof: sulfate, thiosulfate, hydrogen sulfide, dithionate, dithionite, sulfite.

In a preferred embodiment of the invention the sulfur source is sulfate and/or thiosulfate. The invention also concerns the process for the production of L-methionine, its precursors or compounds derived thereof, comprising the fermentation of the methionine producing microorganism described above, the concentration of methionine, its precursors or derivatives and the isolation of the desired product of the fermentation broth.

According to the invention, the terms 'culture' and 'fermentation' are used indifferently to denote the growth of a microorganism on an appropriate culture medium containing a simple carbon source. According to the invention a simple carbon source is a source of carbon that can be used by those skilled in the art to obtain normal growth of a microorganism, in particular of a bacterium. In particular it can be an assimilable sugar such as glucose, galactose, sucrose, lactose or molasses, or by-products of these sugars. An especially preferred simple carbon source is glucose. Another preferred simple carbon source is sucrose. 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 20⁰C and 55°C, preferentially between 25°C and 40⁰C, and more specifically about 30⁰C for C. glutamicum and about 37°C for E. coli.

The fermentation is generally conducted in fermenters with an inorganic culture medium of known defined composition adapted to the bacteria used, containing at least one simple carbon source, and if necessary a co-substrate necessary for the production of the metabolite.

In particular, the inorganic culture medium for E. coli can be of identical or similar composition to an M9 medium (Anderson, 1946, Proc. Natl. Acad. ScL USA 32:120-128), an M63 medium (Miller, 1992; A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) or a medium such as defined by Schaefer et al. (1999, Anal. Biochem. 270: 88- Analogously, the inorganic culture medium for C. glutamicum can be of identical or similar composition to BMCG medium (Liebl et ah, 1989, Appl. Microbiol. Biotechnol. 32: 205-

210) or to a medium such as that described by Riedel et a (2001, J. MoI. Microbiol. Biotechnol. 3:

573-583). The media can be supplemented to compensate for auxotrophies introduced by mutations.

After fermentation L-methionine, its precursors or compounds derived thereof, is/are recovered and purified if necessary. The methods for the recovery and purification of the produced compound such as methionine in the culture media are well known to those skilled in the art.

Optionally from 0 to 100% of the biomass may be retained during the purification of the fermentation product.

The invention also relates to a microorganism that is optimized for the fermentative production of methionine.

The term "optimized microorganism" describes the microorganism into which the above described modifications are integrated leading to the best industrial performance for the production of the desired metabolite(s) and possibly the lowest production of sideproducts..

In a preferred application the organism is either E. coli or C. glutamicum or Saccharomyces cerevisiae.

In the most preferred application the organisms is E. coli.

Detailed description of the invention

An E. coli strain in which the methionine repressor encoded by the metJ gene has been replaced by a chloramphenicol cassette (AmetJy.Cm) and that harbors a met A allele with reduced feed-back sensitivity to methionine and SAM (met A* I I) has been described in PCT N° PCT/IB04/001901 filed on May 12, 2004. Into this strain the following genetic modifications were introduced.

Construction of MG1655 metA*ll AmetJr.Cm ¥trc-met¥ ::Km

To clone the metF gene under the control of the heterologous Vtrc promoter, the homologous recombination strategy described by Datsenko & Wanner (2000) was used. This strategy allows the insertion of a chloramphenicol or a kanamycin resistance cassette near the genes concerned. For this purpose the following oligonucleotides were used: PtrcmetF R (SEQ ID NO 1 )

GCCAGGCTCTGATTCAGGGCATCCCGCTGGCTGGCGTGAAAAAAGCTCATααtatacc tccttattccacacattatacgagccggatgattaattgtcaacagctcΥGΥAGGCΥGGAGCΥGCTTCG with : a region (upper case) homologous to the sequence (4130259-4160195) of the gene metF (reference sequence on the website http://genolist.pasteur.fr/Colibri/) a region (italic case) homologous to the promoter Ptrc sequence with the RBS (bold) the -35 and -10 boxes (bold) a region (upper case) for the amplification of the kanamycin resistance cassette (reference sequence in Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645) Ptrc-metF F (SEQ ID NO 2 ) ccttcatctttacatctggacgtctaaacggatagatgtgcacaacacaacatataactacaagcgattgatgaggtaaggttcflcflctggctcflc cttcgggtgggcctttctgcCATATGAATATCCTCCTTAG with : a region (lower case) homologous to the sequence (4130114-4130195) of the region of gene metF (reference sequence on the website http://genolist.pasteur.fr/Colibri/) a region (italics, lower case) homologous to the sequence of the bacteriophage T7 terminus

(Genbank V01146) a region (upper case) for the amplification of the kanamycin resistance cassette (reference sequence in Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645). The oligonucleotides Ptrc-metF F and Ptrc-metF R were used to amplify the kanamycin resistance cassette from the plasmid pKD4. The PCR product obtained was then introduced by electroporation into the strain MG1655 met A*ll AmetJ (pKD46), in which the Red recombinase enzyme expressed permits the homologous recombination. The kanamycin resistant transformants were selected and the insertion of the resistance cassette was verified by a PCR analysis with the oligonucleotides Ptrc-metFv F and Ptrc-metFv R defined below.

Ptrc-metFv F (SEQ ID NO 3) : GCCCGGTACTCATGTTTTCGGGTTTATGG (homologous to the sequence from 4129866 to 4129894).

Ptrc-metFv R (SEQ ID NO 4): CCGTTATTCCAGTAGTCGCGTGCAATGG (homologous to the sequence from 4130524 to 4130497). The resulting strain was called MG1655 metA*l 1 AmetJ Vtrc-metF-.Km.

Construction of plasmid pME101-thrA*l-cysE pME101-thrA*l

To boost the production of homoserine thrA* encoding aspartokinase/homoserine with reduced feed-back resistance to threonine was expressed from the plasmid pCL1920 (Lerner &

Inouye, 1990, NAR 18, 15 p 4631) using the promoter Vtrc. For the construction of the plasmid pME101-thrA*l thrA was PCR amplified from genomic DNA using the following oligonucleotides:

SψHlthrA (SEQ ID NO 5): ttaTCATGAgagtgttgaagttcggcggtacatcagtggc SmαlthrA (SEQ ID NO 6): ttaCCCGGGccgccgccccgagcacatcaaacccgacgc

The PCR amplified fragment was cut with the restriction enzymes BspRI and Smal and cloned into the Ncol / Smal sites of the vector pTRC99A (Stratagene). For the expression from a low copy vector the plasmid pMElOl was constructed as follows. The plasmid pCL1920 was PCR amplified using the oligonucleotides PMElOlF and PMElOlR and the BstZ\ll-Xmnl fragment from the vector pTRC99A harboring the lad gene and the Pfrc promoter were inserted into the amplified vector. The resulting vector and the vector harboring the thrA gene were restricted by Apal and Smal and the thrA containing fragment was cloned into the vector pMElOl. To relieve ThrA from feed-back inhibition the mutation F318S was introduced by site-directed mutagenesis (Stratagene) using the oligonucleotides ThrAF F318S and ThrAR F318S, resulting in the vector pME101-thrA*l.

PMElOlF (SEQ ID NO 7): Ccgacagtaagacgggtaagcctg

PMElOlR (SEQ ID NO 8): Agcttagtaaagccctcgctag ThrAF F318S (Smal) (SEQ ID NO 9): Ccaatctgaataacatggcaatgtccagcgtttctggcccggg

ThrAR F318S (Smal) (SEQ ID NO 10): Cccgggccagaaacgctggacattgccatgttattcagattgg

pME101-thrA*l-cysE

For the construction of pME101-thrA*l-cysE the cysE gene was amplified by PCR using oligonucleotides Ome BOOl and Ome B002, the PCR-product was cut with the restriction enzyme PVMII and cloned into the Smal site of the vector pME101-thrA*l resulting in the vector pMElOl- thrA*l-cysE.

Ome BOOl cysER-PvuII (SEQ ID NO 11) GGAGGGAC4GCTGATACGAAAGAAGTCCGCGAACTGGCGC

Ome B002 cysEF-PvuII (SEQ ID NO 12) Atacgcαgcfeggacattagatcccatccccatactcaaatgtatgg

PVMII site are underlined. The sequence in bold corresponds to cysΕ (Colibri) (3780423-3780397)

Construction of MG1655 metA*U Δmetiv.Cm Ftrc-metH::Km

To boost the production of methionine the metH gene was overexpressed using the Pfrr promoter. For the construction the following oligonucleotides were used: DiclR-metHF (SEQ ID NO 13) gcaccagaatacgttcatttaactgcgcacgcagttgttccactttgctgctcatGrcrGrCCrCC4Gr^C4rGC^4CCCC4

CACATTATACGAGCCGGATGA TTAATTGTCAA CΛGCΓCTGTAGGCTGGAGCTGCTTCG with : a region (lower case) homologous to the sequence (4221461-4221408) of the gene metΑ (reference sequence on the website http://geiiolisLpasteur.fr/Colibri/) a region (italics, upper case) homologous to the promoter Ptrc sequence with the RBS (bold) the -35 and -10 boxes (bold) a region (upper case) for the amplification of the kanamycin resistance cassette (reference sequence in Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645).

iclR-metHF (SEQ ID NO 14) GCTTTTACCACAGATGCGTTTATGCCAGTATGGTTTGTTGAATTTTTATTAAATCTGGGTTGAG CGTGTCGGGAGCAAGTCATATGAATATCCTCCTTAG with : a region (italics, upper case) homologous to the sequence (4221327-4221406) of the region of gene metR (reference sequence on the website http://genolist.pasteur.fr/Colibri/), - a region (upper case) for the amplification of the kanamycin resistance cassette (reference sequence in Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645).

The oligonucleotides DiclR-metHF and iclR-metHF were used to amplify the kanamycin resistance cassette from the plasmid pKD4. The PCR product obtained is then introduced by electroporation into the strain MG 1655 met A* H AmetJ (pKD46), in which the Red recombinase enzyme was expressed permitting the homologous recombination. Kanamycin resistant transformants are selected and the insertion of the resistance cassette was verified by a PCR analysis with the oligonucleotides iclF and iclR defined below. iclF (SEQ ID NO 15): CCTTTGAGGTCGCATGGCCAGTCGGC (homologous to the sequence from 4221558 to 4221533). iclR (SEQ ID NO 16): GCTTTTTAATAGAGGCGTCGCCAGCTCCTTGCC (homologous to the sequence from 4219917 to 4219949).

The resulting strain was called MG1655 metA*l 1 AmetJ ¥trc-metR:Km

Construction of MG1655 metA*ll AmetJ::Cm ¥trc-metF:Km Vtrc-metΑ

For the construction of the strain MG1655 metA*\ 1 AmetJ::Cm Vtrc-metF-.Km Vtrc-metΗ. the chloramphenicol and the kanamycin resistance cassette was eliminated from the strain MG 1655 met A* W AmetJ ::Cm ¥trc-metK:Km. The plasmid pCP20 carrying FLP recombinase acting at the FRT sites of the chloramphenicol resistance cassette was introduced into the recombinant strain by electroporation. After a series of cultures at 42°C, the loss of the two cassettes was verified by PCR analysis. The strain retained was designated MG 1655 metA*ll AmetJ Vtrc-meM.

To transfer the promoter construct ¥trc::metF: Km into the strain MG1655 metA*ll AmetJ

^~Ptrc-meiΑ, the method of phage Pl transduction was used. The protocol followed was implemented in 2 steps with the preparation of the phage lysate of the strain MG1655 MG1655 metA*ll AmetJ Vtrc-metF-.Km and the subsequent transduction into strain MG1655 metA*ll AmetJ Vtrc-meiΑ

Preparation of phage lysate Pl:

Inoculation with 100 μl of an overnight culture of the strain MG 1655 met A* 11 AmetJ ¥trc-metF-Xm of 10 ml of LB + Km 50 μg/ml + glucose 0.2% + CaCl₂ 5 mM.

Incubation for 30 min at 37°C with shaking.

Addition of 100 μl of phage lysate Pl prepared on the strain MG 1655 (about 1.10⁹ phage/ml).

Shaking at 37°C for 3 hours until all cells were lysed. - Addition of 200 μl chloroform and vortexing.

Centrifugation for 10 min at 4500 g to eliminate cell debris. Transfer of the supernatant to a sterile tube and addition of 200 μl chloroform. Storage of lysate at 4°C. Transduction - Centrifugation for 10 min at 1500 g of 5 ml of an overnight culture of the strain

MG 1655 met A* 11 AmetJ Vtrc-metH in LB medium.

Suspension of the cell pellet in 2.5 ml of 10 mM MgSO₄, 5 mM CaCl₂ Control tubes: 100 μl cells

100 μl phages Pl of strain MG1655 metA*ll AmetJ ¥trc-metF:Km - Test tube: 100 μl of cells + 100 μl of phages Pl of the strain MG1655 metA*ll AmetJ Vtrc-metF :Km

Incubation for 30 min at 30⁰C without shaking. Addition of 100 μl of 1 M sodium citrate in each tube and vortexing. Addition of 1 ml of LB. - Incubation for 1 hour at 37°C with shaking.

Spreading on dishes LB + Km 50 μg/ml after centrifuging of tubes for 3 min at 7000 rpm.

Incubation at 37°C overnight. Verification of the strain Kanamycin resistant transformants were selected and the presence of the promoter construct Vtrc-metF :Km was verified by PCR analysis with the oligonucleotides Ptrc-metFv F and Ptrc-metFv R, described above. The strain retained was designated MG 1655 metA*ll AmetJ ::Cm Vtrc-metK Vtrc-metF :Km.

Construction of MG1655 metA*U Ametiv.Cm Ftrc-cysM::Km

To clone the cysM gene under the control of the heterologous Pfrr promoter, the homologous recombination strategy described by Datsenko & Wanner (2000) was used. This strategy allows the insertion of a chloramphenicol or a kanamycin resistance cassette near the genes concerned. For this purpose the following oligonucleotides were used: Ptrc-cysM F gcctgatgcgacgcttgcgcgtcttatcaggtctacaggttacaaaccttgccataatatacctccttaccacacattatacgagccggatgatt ααtfgfcααcαgcfcCATATGAATATCCTCCTTAG (SEQ ID NO 17) with : a region (lower case) homologous to the sequence (2537627-2537681) of the gene cysM

(reference sequence on the website http://genolist.pasteur.fr/Colibri/) a region (italics, lower case) homologous to the promoter Vtrc sequence with the RBS (bold) the -35 and -10 boxes (bold) a region (upper case) for the amplification of the kanamycin resistance cassette (reference sequence in Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645), Ptrc-cysM R ggttgagtgaatgttaaacgcccggaggcgcttcccgcgatccgggcttttr^rC4 CACTGGCTCACCTTCGGGTGGGC C7TΓCΓGCTGTAGGCTGGAGCTGCTTCG (SEQ ID NO 18) with : a region (lower case) homologous to the sequence (2537734-2537684) of the region of gene cysM (reference sequence on the website http://eenolist.pasteur.fr/Colibri/) a region (italics, upper case) homologous to the sequence of the bacteriophage T7 terminus (Genbank V01146) a region (upper case) for the amplification of the kanamycin resistance cassette (reference sequence in Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645).

The oligonucleotides Ptrc-cysM F and Ptrc-cysM R were used to amplify the kanamycin resistance cassette from the plasmid pKD4. The PCR product obtained was then introduced by electroporation into the strain MG1655 met A*ll AmetJ (pKD46), in which the Red recombinase enzyme was expressed permitting homologous recombination. Kanamycin resistant transformants were then selected and the insertion of the resistance cassette was verified by PCR analysis with the oligonucleotides Ptrc-cysMv F and Ptrc-cysMv R defined below.

Ptrc-cysMv F : ggtgacaagaatcagttccgc (homologous to the sequence from 2537262 to 2537282). (SEQ ID NO 19)

Ptrc-cysMv R : GCGTTTATTCGTTGGTCTGC (homologous to the sequence from 2537833 to

2537814). (SEQ ID NO 20)

The resulting strain is called MG 1655 met A* Il AmetJ J Ptrc-cy,sM::Km.

Construction of MG1655 metA*ll AmetJ Vtrc-metF Vtrc-metΑ ¥trc-cysM:Km

For the construction of the strain MG 1655 met A* 11 AmetJ Vtrc-metF Vtrc-metH Vtrc- cy,sM:Km the chloramphenicol and kanamycin resistance cassettes were eliminated from the strain MG1655 metA*\ 1 AmethCm Vtrc-metF-.Km Vtrc-metH using plasmid pCP20, as described above. The strain retained was designated MG 1655 met A* 11 Ante tJ Ytrc-meiF Ϋtrc-meiΑ

To transfer the promoter construct Vtrc-cysM: Km into the strain MG1655 metA*\ 1 AmetJ Ytrc-meiF ^~Ptrc-meiΑ, the method of phage Pl transduction was used. The protocol followed was implemented in 2 steps with the preparation of the phage lysate of the strain MG1655 MG1655 metA*ll AmetJ Vtrc-cysM-.Km and the subsequent transduction into strain MG1655 metA*\ \ AmetJ Vtrc-metF Vtrc-metH as described above. The strain retained was designated MG 1655 met A* 11 AmetJ Ytrc-mei? Ytrc-meiΑ Ϋtrc-cysM'.Km.

Combining enhanced expression of cjsE and metW and optimized expression of metF and cysM with alleles metA*ll and ΔmetJ

For the construction of the strains MG1655 metA*ll AmetJv.Cra (pME101-fλrA*l), MG1655 metA*ll AmetJwCra Vtrc-meiΑ-Xm ($ME\Q\-thrA*\-cysE), MG1655 metA*ll AmetJv.Cm Vtrc-meiΑ ¥trc-meiF:Km (jpMEl0l-thrA*l-cysE) and MG1655 metA*U AmetJ Vtrc- metF Vtrc-metΑ Vtrc-cysM'Xm (pME101-fλrA*l-cy,sE) the plasmids (pME101-fλrA*l) or (pME101-fλrA*l-cy,sE) were introduced into the strains MG1655 metA*ll AmetJwCra, MG1655 metA*ll AmetJwCm ¥trc-metH:Km, MG1655 metA*ll AmetJwCm Vtrc-metΑ ¥trc-metF:Km and MG1655 metA*\ \ AmetJ Ytrc-mei? Ytrc-meiΑ ¥trc-cysM:Km by transformation.

Evaluation of methionine producing strains with enhanced expression of cjsE, metW and/or cysM and/or metF under the control of a heterologous promoter

Production strains were initially evaluated in small Erlenmeyer flasks. A preculture was grown in LB medium with 2.5 g/1 glucose and used to inoculate an overnight culture in minimal medium PCl. This culture served to inoculate a 50 ml culture to an OD600 of 0.2 in medium PCl supplemented with 0.01 g.L^"1 vitamin B12. If indicated ammonium sulfate was replaced by 5.6 g/1 ammonium thiosulfate. Spectinomycin was added if necessary at a concentration of 100 mg/1. At an OD600 of 4.5 to 5 extracellular amino acids were quantified by HPLC after OPA/Fmoc derivatization and other relevant metabolites were analyzed using GC-MS after silylation.

Table 1. Composition of minimal medium PCl

As can be seen in table 2 the amount of methionine is increases upon overexpression of cysE, cysE and metH or cysE, metH and altered expression of metF altogether. Enhanced expression of cysM can further increase methionine production. Certain strains produced higher amounts of methionine in the presence of thiosulfate. The highest methionine production is obtained, when cysE, cysM and metH are overexpressed and metF expression is under the control of the Pfrr promoter in the presence of thiosulfate. Isoleucine production is drastically reduced upon expression of cysE and metH, indicating reduced γ-elimination activity. Overexpression of cysM reduces γ-elimination in a strain overexpressing cysE and metH and expressing metF from a heterologous promoter. Table 2. Methionine and isoleucine in mmol/g DW produced in batch culture with sulfate (S) or thiosulfate (T) as sulphur source by strains described above, n.d., not determined

3. Determination of changes in enzyme activities of CysE and MetH

To validate the changes in the expression of cysE and meiΑ expression, the activities of the corresponding enzymes were determined in crude extracts.

For the determination of enzyme activities in vitro, E. coli strains were cultured in minimal

10 medium as described above and harvested at mid log phase. Cells were resuspended in cold potassium phosphate buffer and sonicated on ice (Branson sonifϊer, 70W). After centrifugation, proteins contained in the supernatants were quantified (Bradford, 1976).

For the determination of serine acetyltransferase activity (CysE) 10 μl extract were assayed in 100 mM Potassium phosphate pH 7.5, 4 πoM Acetyl-CoA, 30 mM L-serine for 10 minutes at

15 25⁰C. Protein was precipitated with acetone and O-acetyl-serine was detected by GC-MS after derivatization with a silylating reagent.

For the determination of vitamin B12-dependent methionine synthase activity (MetH), 100 μl extract were assayed in 100 mM potassium phosphate pH 7.2, 1 mM homocysteine, 0.25 mM methyltetrahydrofolate, 50 μM vitamin B12, 20 μM S-adenosyl-methionine and 25 mM DTT for

20 10 minutes at 37°C. Protein was precipitated with acetone and the produced methionine was detected by GC-MS after derivatization with a silylating reagent.

As can be seen in table 3 overexpression of the genes cysE and meiΑ increases the corresponding enzyme activity. Thus the increased activity of these genes leads to increased methionine production.

25 Table 3. Activities in mUI/g DW of serine acetyltransferase (CysE) and methionine synthase (MetH) in methionine producing strains cultivated in the presence of thiosulfate.

Validation of methionine production under fermentation conditions

Strains that produced substantial amounts of metabolites of interest were subsequently tested under production conditions in 300 ml fermentors (DASGIP) using a fed batch protocol.

For this purpose an 8 hours culture grown in LB medium with 2.5 g/1 glucose was used to inoculate an overnight preculture in minimal medium PCl (see above). Fermentors were filled with 150 ml of minimal medium (Bl) and inoculated to a biomass concentration of nearly 0.09 g/1 with 1.5 ml concentrated preculture (between 9 and 12 g/1).

Table 4. Composition of minimal medium Bl

Table 5. Minimal medium FB type Tl

The temperature of the culture was maintained constant at 37 ⁰C and the pH was permanently adjusted to values between 6.5 and 8, preferentially 6.7 using an NH₄OH solution. The agitation rate was maintained at 600 rpm during the batch phase and was increased to up to 1000 rpm at the end of the fed-batch phase. The concentration of dissolved oxygen was maintained at values between 20 and 40%, preferentially 30% saturation by using a gas controller. When the cell mass reached a concentration of 0.9 to 1.2 g/1 the fed-batch was started with an initial flow rate between 0.1 and 1.5 ml/h, preferentially 0.43 ml/h and a sigmoidal (24h) increase up to flow rate values between 0.5 and 5.8 ml/h, preferentially 1.7 ml/h. The precise feeding conditions were calculated by the formula below:

where Q(t) is the feeding flow rate in mL/h for a batch volume of 15OmL Pl is between 0.025 and 0.35, preferentially 0.100. P2 is between 0.400 and 5.600, preferentially 1.600. P3 is between 0.068 and 0.95, preferentially 0.270. P4 is between 1.250 and 17.5, preferentially 5.000.

In this case FB medium containing glucose at concentrations between 300 and 800 g/1 (preferentially 500g/L) was used.

When the concentration of biomass had reached values between 20 and 50 g/1 (preferentially 35g/L, between 40 and 80h) the fermentation was stopped and the extracellular methionine and isoleucine concentrations were determined using HPLC.

Table 7. Methionine titers obtained in Fed-batch fermentations of strains overexpressing cysE, and metR or metF under a heterologous promoter or a combination of the three. Ref corresponds to MG1655 meth*\ \ AmetJ. Strains were grown in the presence of thiosulfate (T) or sulfate (S).

As can be seen in table 7, enhanced expression of cysE, cysE and metR, cysE, metR and metF under the control of a heterologous promoter or growing the stains in the presence of thiosulfate can significantly increase methionine production. Isoleucine production is significantly reduced by overexpressing cysE and/or metR.

The strain that produced the highest amount of methionine in the 300 mL fermentor was subsequently tested under production conditions in a 2.5L fermentor (PIERRE GUERIN) using a fed batch protocol.

For this purpose an 8 h culture grown in LB medium with 2.5 g/1 glucose was used to inoculate an overnight preculture in minimal medium PCl. Fermentors were filled with 600 ml of minimal medium (B2) and inoculated to a biomass of 0.9 g/1 with 6 ml concentrated preculture (between 9 and 12 g/1).

The temperature of the culture was maintained constant at 37 ⁰C and the pH was permanently adjusted to values between 6.3 and 8, preferentially 6.8 using an NH₄OH 28% solution. The initial agitation rate was set at 200 rpm during the batch phase and was increased to up to 1200 rpm during the fed-batch phase. The initial airflow rate was set at 40Nl/h during the batch phase and was increased to up to 250Nl/h during the fed-batch phase. The concentration of dissolved oxygen was maintained at values between 20 and 40% saturation, preferentially 30% by increasing the agitation rate and the airflow rate. When the biomass concentration reached 1.2 to 1.5 g/1, the fed-batch was started with an initial flow rate between 0.5 and 4 ml/h, preferentially 1.0 ml/h and an exponential increase (15h) up to flow rate values between 3 and 35ml/h, preferentially 20.1 ml/h. At this point, the flow rate was maintained constant for 10 to 45 hours, preferentially 30 h. For the feeding FB type T2 was used (See table 8) containing glucose at concentrations between 300 and 800 g/1, preferentially 750 g/1. When the concentration of biomass had reached values between 40 and 110 g/1, preferentially 90g/l the fermentation was stopped and the extracellular methionine concentration was determined using HPLC. The strain MG1655 metA*ll AmetJ Vtrc-metR Vtrc-metF (pMElOl- thrA*l-cysE) produced 169 πoM methionine under these conditions.

Table 8. Minimal medium FB T2

Table 9. Composition of minimal medium B2

Claims

CLAIMS:

1) A method for the production of methionine, its derivatives, or precursors by culturing a microorganism in an appropriate culture medium comprising a source of carbon and a source of sulfur and recovery of methionine form the culture medium, wherein the microorganism is modified to enhance the production of cysteine.

2) The method of claim 1 wherein the expression of at least one gene involved in cysteine production is increased.

3) The method of claim 2 wherein at least one gene involved in cysteine production is selected among the group consisting of: • cysA sulfate permease

• cysU, cysT component of sulfate ABC transporter

• cysW membrane bound sulfate transport protein

• cysL ORF upstream of cysK

• cysN ATP sulfurylase • cysO sulfate adenylyltransferase

• cysC adenylylsulfate kinase

• cysE. adenylylsulfate reductase

• cysl sulfite reductase, alpha subunit

• cysJ sulfite reductase, beta subunit • cysE serine acetyltransferase

• cysK cysteine synthase

• cysM O-acetyl-sulfhydrylase

• cysL sulfate transport

• sbp Periplasmic sulfate-binding protein. 4) The method of claim 2, wherein at least the expression of cysE is increased.

5) The method of claim 2, wherein at least the expression of cysM is increased.

6) The method of one of claims 1 to 5 wherein the expression of at least one gene, which is involved in the production of Cl units and the transfer potential onto homocysteine, is increased and/or driven by a heterologous promoter. 7) The method of claim 6 wherein at least one gene, which is involved in the production of Cl units and/or the transfer potential onto homocysteine, is selected among the group consisting of:

• metE, metH encoding methionine synthase

• metF encoding 5,10-methylenetetrahydrofolate reductase • glyA encoding serine hydroxymethyltransferase

• gcvTHP, lpd encoding the glycine cleavage complex. 8) The method of claim 7 wherein the expression of metE, metR and/or metF is increased and/or at least one of the genes is expressed from a heterologous promoter.

9) The method of one of claims 1 to 8 wherein γ-elimination activity is kept low by optimizing the flow of cysteine biosynthesis and thus the production of the byproduct isoleucine is reduced. 10) The method of one of claims 1 to 9 wherein the expression of additional genes involved in the production of methionine is increased.

11) The method of one of claims 1 to 10 wherein the expression of genes decreasing methionine production is attenuated.

12) The method of one of claims 1 to 11 wherein the methionine repressor encoded by the met J gene is deleted or mutated and/or homoserine succinyltransferase (MetA) alleles encoding enzymes with reduced feed-back sensitivity to S-adenosylmethionine and/or methionine is/are integrated into the strain.

13) The method of one of claims 1 to 12 wherein the sulfur source in the culture medium is sulfate, thiosulfate, hydrogen sulfide, dithionate, dithionite, sulfite or a combination of the different sources.

14) The method of claim 13 wherein the sulfur source in the culture medium is sulfate or thiosulfate, or a mixture of the two.

15) The method of one of claims 1 to 14 comprising the step of isolation of the desired amino acids/ constituents of the fermentation broth and/or the biomass optionally remaining in portions or in the total amount (0-100%) in the end product.

16) A microorganism used for the fermentative production of methionine or its derivatives in which the production of methionine or derivatives, wherein the microorganism is optimized to enhance the production of cysteine.

17) The microorganism of claim 16 wherein the expression of at least one gene involved in cysteine production is increased.

18) The microorganism of claim 17 wherein the at least one gene involved in cysteine production is selected among the group consisting of:

• cysA sulfate permease

• cysU, cysT component of sulfate ABC transporter • cysW membrane bound sulfate transport protein

• cysZ ORF upstream of cysK.

• cysN ATP sulfurylase

• cysD sulfate adenylyltransferase

• cysC adenylylsulfate kinase • cysΑ adenylylsulfate reductase

• cysl sulfite reductase, alpha subunit

• cysJ sulfite reductase, beta subunit

• cysE serine acetyltransferase • cysK. cysteine synthase

• cysM 0-acetyl-sulfhydrolase

• cysZ sulfate transport

• sbp Periplasmic sulfate-binding protein.

19) The microorganism of claim 17 wherein at least the expression of cysE is increased. 20) The microorganism of claim 17, wherein at least the expression of cysM is increased.

21) The microorganism of one of claims 16 to 20 wherein the expression of at least one gene, which is involved in the production of Cl units and the transfer potential onto homocysteine, is increased and/or driven by a heterologous promoter.

22) The microorganism of claim 21 wherein at least one gene, which is involved in the production of Cl units and the transfer potential onto homocysteine, is selected among the group consisting of:

• metE, metR encoding methionine synthase

• metF encoding 5,10-methylenetetrahydrofolate reductase

• glyA encoding serine hydroxymethyltransferase • gcvTHP, lpd encoding the glycine cleavage complex.

23) The microorganism of claim 21 wherein the expression of metE, meiΑ and/or metF is increased and/or at least one of the genes is expressed from a heterologous promoter.

24) The microorganism of one of claims 16 to 23 wherein γ-elimination activity is kept low by optimizing the flow of cysteine biosynthesis and thus the production of the byproduct isoleucine is reduced.

25) The microorganism of one of claims 16 to 24 wherein the expression of additional genes involved in the production of methionine is increased.

26) The microorganism of one of claims 16 to 25 wherein the expression of genes decreasing methionine production is attenuated. 27) The microorganism of one of claims 16 to 26 wherein the methionine repressor encoded by the me tJ gene is deleted or mutated and/or homoserine succinyltransferase (MetA) alleles encoding enzymes with reduced feed-back sensitivity to S-adenosylmethionine and/or methionine are integrated.