BRPI0510819B1 - method for the preparation of methionine, its precursors or products derived therefrom and recombinant microorganism - Google Patents

Abstract

METHOD FOR THE PREPARATION OF METHIONIN, ITS PRECURSORS OR PRODUCTS DERIVED FROM THE SAME, HOMOSERINE TRANSSSUCINYLASE, MUTATED, NUCLEOTIDE SEQUENCE, S-ADENOSYLMethionine SYNTHESIS, AND, MICROORGANISM The present invention relates to the use of transgenic enzymes (MetA *) and, eventually, recombinant S-adenosyl methionine synthase enzymes with reduced activity (MetK *) for the production of methionine, its precursors or its derivatives.

Description

Field the Invention

[0001] The present invention relates to the use of recombinant homoserine trans-succinylase enzymes with altered feedback sensitivity (MetA *) and, eventually, recombinant S-adenosyl methionine synthase enzymes with reduced activity (MetK *) for the production of methionine, precursors or derivatives. Prior Art

[0002] Sulfur-containing compounds, such as cysteine, homocysteine, methionine or S-adenosylmethionine are fundamental for cellular metabolism and are industrially produced to be used as food or food additives and medicines. Methionine, in particular, an essential amino acid, which cannot be synthesized by animals, plays an important role in many functions of the body. Apart from its role in protein biosynthesis, methionine is involved in the transmethylation and bioavailability of selenium and zinc. Methionine is also directly used as a treatment for disorders such as allergy and rheumatic fever. Despite this, most of the methionine that is produced is added to animal feed.

[0003] With the reduced use of animal proteins, as a result of BSE and avian influenza, 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 food additives for chickens (Saunderson, C. L, (1985)

[0004] British Journal of Nutrition 54, 621-633). Pure L-methionine can be produced from racemic methionine, for example by treating N-acetyl-D-L-methionine acylase, which dramatically increases production costs. The growing demand for pure L-methionine, associated with environmental concerns, makes microbial production of methionine attractive.

[0005] Microorganisms have developed extremely complex regulatory mechanisms that precisely tune the biosynthesis of cellular components, thus allowing maximum growth rates. Consequently, only the necessary amounts of metabolites, such as amino acids, are synthesized and may usually not be detected in the supernatant of the wild type strain culture. The amino acid biosynthesis controlled by bacteria, mainly by inhibiting the feedback 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 the overproduction of amino acids in microorganisms require the deregulation of these control mechanisms.

[0006] The pathway for the synthesis of L-methionine in many microorganisms is well known (Figure 1 A / B). Methionine is derived from the amino acid aspartate, but its synthesis requires the convergence of two additional pathways, cysteine biosynthesis and C1 (N-methyltetrahydrofolate) metabolism. Aspartate is converted to homoserine by a sequence of three reactions. Homoserine can subsequently enter the threonine / isoleucine or methionine biosynthetic pathway. The entry of E. coli into the methionine pathway requires the acylation of homoserin in succinyl-homoserin. This activation step allows for subsequent condensation with cysteine, leading to cystathionine containing thioether, which is hydrolyzed to provide homocysteine. The final transfer of methyl which leads to methionine is carried out either by a B12-dependent or B12-independent methyltransferase.

[0007] Methionine biosynthesis in E. coli is regulated by repression and activation of methionine biosynthetic genes via the MetJ and MetR proteins, respectively (examined in Neidhardt, FC (Editor in Chief), R. Curtiss III, JL Ingraham, ECC Lin, KB Low, B. Magasanik, WS Reznikoff, M. Riley, M. Schaechtcr and HE Umbarger (editors) 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology; Weissbach et al., 1991 Mol.Microbiol., 5, 1593-1597). MetJ uses S-adenosylmethionine as a co-repressor, which is produced from methionine by the enzyme S-adenosylmethionine synthetase (EC 2.5.1.6) encoded by the essential metK gene. MetA encoding homoserin trans-succinylase (EC 2.3.1.46), the first unique enzyme for the synthesis of methionine, is another major control point for the production of methionine. In addition to the transcriptional regulation of metApor MetJ and MetR, the enzyme is also regulated by feedback by the end products methionine and S-adenosylmethionine (Lee, L.-W et al. (1966) Multimetabolite control of a biosynthetic pathway by sequential metabolites, JBC 241 (22), 5479-5780). The inhibition of feedback by these two products is synergistic, meaning that low concentrations of each metabolite alone are only slightly inhibitory, while in combination a strong inhibition is exerted.

[0008] Amino acid analogs inhibit bacterial growth through the synthesis of abnormal polypeptides and by interfering with the inhibition of feedback, which leads to harmful processes within the cell. Analog-resistant mutants were obtained, which present altered and deregulated enzymes, leading to an excess of synthesis of the corresponding metabolites that can compete with the analog. Several groups have used the methionine analogs, norleucine, ethionine and oc-methylmethionine, to generate microbial strains that overproduce methionine. It has been observed that oc-methylmethionine is a potent inhibitor of the enzyme homoserine trans-succinylase MetA (Rowbury, RJ, (1968) The inhibitory effect of a-methylmethionine on Escherichia coli, J. gen. Microbiol., 52, 223- 230) . The feedback-resistant mutants in Salmonella typhimurium that showed the metA site were isolated (Lawrence, DA, (1972) Regulation of the methionine feedback-sensitive enzyme in mutants of Salmonella typhimurium; Lawrence, DA, Smith, DA, Rowbury, RJ (1967 ) Regulation of methionine synthesis in Salmonella typhimurium: Mutants resistant to inhibition by analogues of methionine, Genetics 58, 473-492). Norleukin-resistant mutants were observed to evidence the metK site. (Chattopadhyay, M. K., Ghosh, A. K. and Sengupta, S. (1991), Control of methionine biosynthesis in Escherichia coli K12: a closer study with analogue resistant mutants, Journal of General Microbiology, 137, 685-691). The same authors reported the isolation of feedback-resistant metA mutants and norleukin-resistant mutants in E. coli, but the actual mutations in metA and metK have not been described.

[0009] The critical role of homoserin trans-succinylase in the production of bacterial methionine by fermentation has been demonstrated (Kumar, D., Garg, S., Bisaria, VS, Sreekrishnan, TR and Gomes, J. (2003) Production of methionine by a multi-analogue resistant mutant of Corynebacterium lilium, Process Biochemistry 38, 1165-1171). Patent application JP 2000139471 describes a process for the production of L-methionine using mutants in the metA and metK genes. In this case, the precise location of the mutations has been determined. The enzymes of the MetA mutant partially lost sensitivity to inhibition of feedback by methionine and S-adenosylmethionine. Although their initial activities have been reduced to about 25% compared to the wild type enzyme, and at 1 mM methionine concentrations for some mutants or 1 mM SAM for others, another 25 to 90% of enzyme activity is lost. The metK mutants were not characterized enzymatically, but used in fermentations to increase the amount of methionine produced.

General Description of the Invention

[0010] This invention relates to a method for the preparation of methionine, its precursors or products derived therefrom, in a fermentative process with a microorganism where L-homoserin is converted to O-succinyl-homoserine by a trans-succinylase homoserine, including the stage of cultivation of the mentioned microorganism in an appropriate medium, and the recovery of methionine, its precursors or products derived therefrom, in which the homoserin trans-succinylase is the mutant homoserin trans-succinylase with reduced sensitivity to the inhibitors feedback, S-adenosylmethionine and methionine.

[0011] The invention also concerns the same method with microorganisms, in which the activity of the enzyme S-adenosylmethionine synthase is reduced.

[0012] The present invention also relates to transformed enzymes, to nucleotide sequences encoding said enzymes with reduced sensitivity or activity, and microorganisms comprising said nucleotide sequences as described above and below.

[0013] Recombinant enzymes can be used together and can also be combined with several other changes in the corresponding microorganisms, such as the overexpression of genes or their suppression. Preferably, the gene encoding aspartokinase / homoserin dehydrogenase (metL or thrA) is overexpressed and the gene encoding the methionine repressor metJ is deleted.

[0014] In the description of the present invention, genes and proteins are identified using the names of the corresponding genes in E. coli. However, and unless otherwise specified, the use of these names has a more general meaning according to the invention, and covers all corresponding genes and proteins in other organisms, more particularly in microorganisms.

[0015] PFAM (database of protein families of hidden alignments and Markov models; http://www.sanger.ac.uk/Software/Pfam/) represents a large collection of protein sequence alignments. Each PFAM makes it possible to view multiple alignments, observe protein domains, assess the distribution between organisms, gain access to other databases, and view known protein structures.

[0016] COGs (groupings of orthologous protein groups; http // www.ncbi.nlm.nih.gov / COG /) are obtained by comparing protein sequences from 43 completely sequenced genomes representing 30 major phylogenetic strains. Each COG is defined from at least three strains, which allow the identification of the preceding conserved domains.

[0017] The means of identifying homologous sequences and their percentage homologies are well known to those skilled in the art, and include, in particular, BLAST programs, which can be used from the website httpZZwww.ncbi.nlm.nih.gov/BLAST / with the standard parameters indicated on that website. The strings obtained can then be used (for example, aligned) using, for example, the CLUSTALW programs (http://www.cbi.ac.uk/clustalw/) or MULTALIN (http://prodcs.Toulouse.infra.fr/multalin/cgi-bin/multalin.pl), with the standard parameters indicated on those websites.

[0018] 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 performing sequence alignments with genes derived from other microorganisms, and designing degenerate probes to clone the corresponding gene in another organism. These 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, 2nd edition. Cold Spring Harbor Lab., Cold Spring Harbor, New York).

Detailed Description of the Invention

[0019] The modified homoserine succinyltransferases having a reduced feedback sensitivity to methionine and S-adenosylmethionine, compared to the wild type enzyme according to the present invention, comprise at least one mutation of amino acids compared to the sequence of the wild type. The mutation is preferably selected in the conserved regions coding for amino acids 24 to 30 or in the region coding for amino acids 58 to 65, or in the region coding for amino acids 292 to 298 with the first amino acid proline after counting formylmethionine as number 1 .

[0020] All references to the amino acid positions are made based on the homoserin succinyltransferase encoded by the E. coli metA gene shown in figure 2. The relative positions of the corresponding conserved regions in other homoserin succinyltransferases of the different organisms can be found by one person versed in the technique by simple sequence alignment, as shown in figure 3, with the enzymes listed below: - gi | 20138683 | sp | Q97l29 | META Clostridium acetobutylicum Homoserina O-succinyltransferase - gi | 12230304 | sp | Q9PLV2 | META Campylobacter jejuni Homosser O- succinyltransferase - gi | 12230277 | sp | Q9KAK7 | META Bacillus halodurans Homoserin O- succinyltransferase - gi | 20138686 | sp | Q97PM9 | META Streptococcus pneumoniae Homoserina O-succinyltransferase - gi | 20138715 | succinyltransferase - gi | 20138656 | sp | Q92L99 | META Sinorhizobium meliloti Homoserina 0- succinyltransferase - gi | 20138618 | s p | Q8YBV5 | META Brucella melitensis Homoserin 0- succinyltransferase - gi | 20141549 | sp | P37413 | META Salmonella typhimurium Homoserina 0- succinyltransferase - gi | 20138601 | sp | Q8X610 | META Escherichia gi Ooss74 | | sp | P07623 | META Escherichia coli Homoserin 0- succinyltransferase - gi | 12230285 | sp | Q9KRM5 | META Vibrio cholerae Homoserin 0- succinyltransferase - gi | 38258142 | sp | Q8FWG9 | META Brucella suitisina Homitizistr | | sp | Q8ZAR4 | META Yersinia pestis Homoserina 0- succinyltransferase - gi | 1223101 O | sp | P54167 | META Bacillus subtilis Homoserina 0- succinyltransferase - gi | 12230320 | sp | Q9WZY3 | META Thermotoga maritima Homosserina 0- succinyltransferase - gi | 20138625 | sp | Q8Z1 W1 | META Salmonella ty | 31340217 | sp | Q8D937 | META Vibrio vulnificus Homoserina 0- succinyltransferase - gi | 31340213 | sp | Q87NW7 | META Vibrio parahaemolyticus Homoserina O-succinyltransferase

[0021] The modified homoserine succinyltransferases preferably have a specific activity that is at least ten times that of the wild-type enzyme in the presence of 10 mM methionine and 1 mM S-adenosylmethionine, and at least 80 times that of the wild-type enzyme in presence of 10 mM methionine and 0.1 mM S-adenosylmethionine. Preferred enzymes retain an activity of at least two percent of their initial activity in the presence of 100 mM methionine and 1 mM S-adenosylmethionine.

Preferably, the modified homoserine succinyltransferase protein sequence according to the invention contains the amino acid mutation of at least one of the conserved region sequences specified below.

[0023] Preferably, at least one mutation is present in conserved region 1 comprising the amino acid sequence defined below, in the N-terminal part of the wild type trans-succinylase homoserine, corresponding to amino acids 24 to 30 in the MctA amino acid sequence E. coli shown in SEQ ID NO: 1. This non-mutated conserved region 1 has the following sequence: X1-X2-X3-A-X4-X5-Q

[0024] where

[0025] X1 represents E, D, T, S, L, preferably T

[0026] X2 represents D, S, K, Q, E, A, R, preferably S

[0027] X3 represents R, E, D, preferably R

[0028] X4 represents Y, I, F, A, K, S, V, preferably S

[0029] X5 represents H, S, N, G, T, R, preferably G

[0030] In a preferred embodiment, conserved region 1 of the unmodified homoserine succinyltransferase has the following amino acid sequence: T-S-R-A-S-G-Q.

[0031] In another preferred embodiment of the invention, at least one mutation is present in conserved region 2, also in the N-terminal part of the wild type trans-succinylase homoserine, corresponding to amino acids 58 to 65 in the MetA amino acid sequence of E. coli shown in SEQ ID NO: 1. Conserved region 2 not mutated has the following formula: X1-X2-X3-PLQ-X4-X5

[0032] where

[0033] X1 represents G, A, S, preferably S

[0034] X2 represents N, A, preferably N

[0035] X3 represents S, T, preferably S

[0036] X4 represents V, L, I, preferably V

[0037] X5 represents N, E, H, D, preferably D.

[0038] In a preferred embodiment, conserved region 2 of the unmodified homoserine succinyltransferase has the following amino acid sequence: S-N-S-P-L-Q-V-D.

[0039] In a third embodiment of the invention, homoserine succinyltransferase comprises at least one mutation in a region conserved in its C-terminal part, corresponding to amino acids 292 to 298 in the E. coli MetA amino acid sequence shown in SEQ ID NO: 1. Conserved region 3 not mutated has the following formula: X1-YQ-X2-TP-X3

[0040] where

[0041] X1 represents V, I, M, preferably V

[0042] X2 represents E, K, G, I, Q, T, S, preferably I

[0043] X3 represents F, Y, preferably Y.

[0044] In a preferred embodiment, conserved region 3 of the unmodified homoserine succinyltransferase has the following amino acid sequence: V-Y-Q-1-T-P-Y.

[0045] In a preferred embodiment, the alanine conserved in conserved region 1 is replaced by another amino acid, more preferable by a valine. The conserved modified region most preferably has the following amino acid sequence: T-S-R-V-S-G-Q.

[0046] In another preferred embodiment, the conserved amino acids L and / or Q in conserved region 2, are replaced by other amino acids. Preferably, leucine is replaced by phenylalanine and / or glutamine is replaced by a glutamate or aspartate. Most preferably, the conserved modified region 2 has the following amino acid sequence: S-N-S-P-L-E-V-D.

[0047] In another preferred embodiment, the conserved amino acids L and / or Q in conserved region 3 are replaced by another amino acid.

[0048] In a preferred application, the metA gene is the E. coli K12 homoserin trans-succinylase enzyme represented by SEQ ID NO: 1 and sequences homologous to that sequence which has trans-succinylase homoserine activity and which shares at least 80% homology, preferably 90% homology to the amino acid sequence of SEQ ID NO: 1.

[0049] The modified homoserine succinyltransferases can be obtained, for example, by the selection of strains in cultivation in the presence of methionine analogs, such as oc-methylmethionine, norleucine or ethionine. Preferably these strains will be selected while cultivated in the presence of oc-methylmethionine.

The present invention furthermore relates to nucleotide sequences, DNA or RNA sequences, which encode a transformed homoserine succinyltransferase according to the invention, as defined above. In a preferred embodiment, the DNA sequence is characterized by the fact that it comprises at least one mutation in the coding regions of conserved regions 1 to 3 of the wild-type metA gene, represented in SEQ ID NO: 1, said mutation not being a silent mutation.

[0051] These DNA sequences can be prepared, for example, from strains in cultivation in the presence of methionine analogs. The starting DNA fragment, including the modified metA gene, is cloned into a vector using standard known techniques to prepare recombinant DNA.

[0052] These DNA sequences can also be prepared, for example, by non-specific methods or directed mutagenesis of the strains that harbor the DNA sequence encoding the wild type homoserin succinyltransferase.

[0053] Non-specific mutations within the said region of DNA can be produced, for example, by chemical agents (for example, nitrosoguanidine, ethyl methanesulfonic acid and others) and / or by physical methods and / or by PCR reactions, which are carried out under particular conditions.

[0054] Methods for introducing mutations at specific positions within a DNA fragment are known and described in Molecular Cloning: a Laboratory Manual, 1989, 2nd edition, Cold Spring Harbor Lab., Cold Spring Harbor, New York.

[0055] With the use of the methods mentioned above, one or more mutations that cause the modified homoserine succinyltransferase to have an amino acid sequence that leads to the insensitivity of methionine and S-adenosylmethionine, can be introduced in the said region of DNA of any MetA gene. Preferably, one or more nucleotides in the DNA sequence encoding the homoserine succinyltransferase are changed in such a way that the amino acid sequence that is encoded by the gene has at least one mutation.

[0056] Another method of producing metA alleles resistant to feedback is to combine different point mutations that lead to resistance to feedback, thereby giving rise to multiple mutants that have new properties.

[0057] The modified S-adenosylmethionine synthetase according to the invention has reduced activity compared to the wild type enzyme, and has at least one mutation in its protein sequence compared to the wild type sequence.

[0058] The mutation is preferably found in one of the conserved regions defined below.

[0059] All references to amino acid positions are made based on the S-adenosylmethionine synthetase encoded by the E. coli metK gene. The relative positions of the corresponding conserved regions in other S-adenosylmethionine synthetases from different organisms can be found by a person skilled in the art by simply aligning sequences as shown in Figure 4, with the enzymes listed below:> gi | 39574954 | emb | CAE78795 .11 methionine adenosyltransferase Bdellovibrio bacteriovorus HD100]> gi | 45657232 | ref] YP_001318.11 s-adenosylmetioniπa siπtetase protein Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130> gi | 28378057 | ref | NP_784949.11 methionine adenosyltransferase Lactobacillus plantarum WCFS1> gi | 26453553 | dbj | BAC43885.11 S-adenosylmethionine synthase Mycoplasma penetrans> gi | 24212014 | sp | Q9K5E4 | S-adenosylmethionine synthetase Corynebacterium glutamicum> gi | 18145842 | dbj | BAB81883.11 S-adenosylmethionine synthetase Clostridium perfringens str. 13> gi | 13363290 | dbj | BAB37241.11 methionine adenosyltransferase 1 Escherichia coli O157: H7> gi | 45443250 | ref | NP_994789.11 S-adenosylmethionine synthetase Yersinia pestis biovar Mediacvails str. 91001> gi | 44888151 | sp | Q7WQX8 | METK S-adenosylmethionine synthetase Borrelia burgdorferi> gi | 4488814 | sp | Q7U4S6 | S-adenosylmethionine synthase Synechococcus sp. WH8102> gi | 44888135 | sp | Q7MHK6 | S-adenosylmethionine synthetase Vibrio vulnificus YJ016> gi | 23466330 | ref | NP_696933.11 S-adenosylmethionine synthetase Bifidobacterium longum NCC2705]> gi | 21219978 | ref | NP_625757.11 S-adenosilmetionin 3 | | ref | NP_949352.11 methionine S-adenosyltransferase Rhodopseudomonas palustris CGA009> gi | 16766391 | ref | NP_462006.11 methionine adenosyltransferase 1 Salmonella typhimurium LT2> gi | 33594910 | ref | NP_8825ioni 44 | | sp | Q7VRG5 | S-adenosylmethionine synthetase Candidatus Blochmannia floridanus> gi | 44888147 | sp | Q7VNG7 | METK_HAEDU S-adenosylmethionine synthetase Haemophilus ducreyi> gi | 44888146 | sp | Q7VFY5 | Helicobacter hepaticus S-adenosylmethionine synthetase> gi | 44888145 | sp | Q7VDM7 | Prochlorococcus marinus S-adenosylmethionine synthetase> gi | 44888142 | sp | Q7URU7 | S-adenosylmethionine synthetase Pirellula spec. > gi | 44888138 | sp | Q7NHG0 | S-adenosylmethionine synthetase Gloeobacter violaceus> gi | 44888137 | sp | Q7N119 | S-adenosylmethionine synthetase Photorhabdus luminescens subsp. laumondii> gi | 44888136 | sp | Q7MTQ0 | S-adenosylmethionine synthase Porphyromonas gingivalis> gi | 39650934 | emb | CAE29457.11 methionine S-adenosyltransferase Rhodopseudomonas palustris CGA009> gi | 15792421 | ref | NP_282244.11 S-adenosylmethionine syntase Subsylobacter jej. jejuni NCTC 11168> gi | 39574954 | emb | CAE78795.11 methionine adenosyltransferase Bdellovibrio bacteriovorus HD100> gi | 45657232 | ref | YP_001318.11 s-adenosylmethionine synthetase protein Leptospira interrogans serovar Copenhageni str. Fiocruz LI-130> gi | 28378057 | ref | NP_784949.11 methionine adenosyltransferase Lactobacillus plantarum WCFS1> gi | 45600470 | gb | AAS69955.11 s-adenosylmethionine protein synthase Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130> gi | 26453553 | dbj | BAC43885.11 S-adenosylmethionine synthase Mycoplasma penetrans> gi | 18145842 | dbj | BAB81883.11 S-adenosylmethionine synthetase Clostridium perfringens str. 13> gi | 13363290 | dbj | BAB37241.11 methionine adenosyltransferase 1 Escherichia coli O157: H7> gi | 45443250 | ref | NP_994789.11 S- adenosylmethionine synthetase Yersinia pestis biovar Mediaevails str. 91001> gi | 44888153 | sp | Q8CXS7 | S-adenosylmethionine synthase Leptospira interrogans> gi | 44888151 | sp | Q7WQX8 | Bordetella bronchiseptica S-adenosylmethionine synthetase> gi | 44888150 | sp | Q7W200 | S-adenosylmethionine synthetase Bordetella parapertussis> gi | 44888149 | sp | Q7VUL5 | Bordetella pertussis S-adenosylmetionins synthetase

[0060] The modified S-adenosylmethionine synthetase preferably has a specific activity that is at least five times less than that of the wild type enzyme.

[0061] Preferably, the protein sequence of a modified S-adenosylmethionine synthetase contains the amino acid mutation of at least one of the sequences specified below.

[0062] In one embodiment of the invention, the amino acid changes concern cysteine at position 89 or cysteine at position 239 which both reduce MetK activity (Reczkowski and Markham, 1995, JBC 270, 31, 18484 - 18490).

[0063] In one embodiment, at least one mutation is present in conserved region 1, corresponding to amino acids 54 to 59 in the E. coli MetK amino acid sequence shown in SEQ ID NO: 2, the non-mutated conserved region 1 comprising the amino acid sequence defined below: GE-XI-X2-X3-X4

[0064] where

[0065] X1 represents I, V, L, T preferably I

[0066] X2 represents T, K, S, R, preferably T

[0067] X3 represents T, S, G, preferably T

[0068] X4 represents S, N, T, K, E, R, P, N, A preferably S.

[0069] Preferably the unmodified conserved region 1 of S-adenosylmethionine synthetase has the following amino acid sequence: G-E-1-T-T-S.

[0070] In another preferred embodiment at least one mutation is present in conserved region 2, corresponding to amino acids 98 to 107 in the E. coli MetK amino acid sequence shown in SEQ ID NO: 2, conserved region 2 not mutated comprising the amino acid sequence defined below: QS-X1-DI-X2-X3-GV-X4

[0071] where

[0072] X1 represents P, Q, A, S, preferably P

[0073] X2 represents A, N, Q, S, F, preferably N

[0074] X3 represents V, Q, Y, R, M, N, preferably Q

[0075] X4 represents K, D, N, T, S, A, E, preferably D.

[0076] Preferably the unmodified conserved region 2 of S-adenosylmethionine synthetase has the following amino acid sequence: Q-S-P-D-1-N-Q-G-V-D.

[0077] In another preferred embodiment at least one mutation is present in conserved region 3, corresponding to amino acids 114 to 121 in the E. coli MetK amino acid sequence shown in SEQ ID NO: 2, conserved region 3 does not mutated comprising the amino acid sequence defined below: X1-GAGDQG-X2

[0078] where

[0079] X1 represents Q, A, I, V, E, T, preferably Q

[0080] X2 represents L, I, S, V, M, preferably L.

[0081] Preferably the unmodified conserved 3 region of S-adenosylmethionine synthetase has the following amino acid sequence: Q-G-A-G-D-Q-G-L.

[0082] In another preferred embodiment, at least one mutation is present in conserved region 4, corresponding to amino acids 137 to 144 in the E. coli MetK amino acid sequence shown in SEQ ID NO: 2, conserved region 4 does not mutated comprising the amino acid sequence defined below: X1-I-X2-X3-X4-H-X5-X6

[0083] where

[0084] X1 represents S, P, T, A, preferably P

[0085] X2 represents A, T, W, Y, F, S, N, preferably T

[0086] X3 represents M, Y, L, V, preferably Y

[0087] X4 represents S, A, preferably A.

[0088] X5 represents K, R, E, D, preferably R

[0089] X6 represents L, I, preferably L.

[0090] Preferably, the conserved unmodified region of S-adenosylmethionine synthetase has the following amino acid sequence: P-1-T-Y-A-H-R-L.

[0091] In another preferred embodiment at least one mutation is present in conserved region 5, corresponding to amino acids 159 to 163 in the E. coli MetK amino acid sequence shown in SEQ ID NO: 2, the conserved region 5 not mutated comprising the amino acid sequence defined below: X1-L-X2-X3-D

[0092] where

[0093] X1 represents W, F, Y, V, E, preferably W

[0094] X2 represents R, G, L, K, preferably R

[0095] X3 represents P, L, H, V, preferably P.

[0096] Preferably the conserved 5 region of unmodified S-adenosylmethionine synthetase has the following amino acid sequence: W-L-R-P-D.

[0097] Preferably at least one mutation is present in conserved region 6, corresponding to amino acids 183 to 189 in the E. coli MetK amino acid sequence shown in SEQ ID NO: 2, the non-mutated conserved region 6 comprising the amino acid sequence defined below: X1-X2-X3-S-X4-QH

[0098] where

[0099] X1 represents V, I, preferably V

[0100] X2 represents V, L, I, preferably V

[0101] X3 represents V, L, I, M, preferably L

[0102] X4 represents T, V, A, S, H, preferably T.

[0103] In a preferred embodiment, the unmodified conserved region 6 of S-adenosylmethionine synthetase has the following amino acid sequence: V-V-L-S-T-Q-H.

[0104] Preferably the mutations are introduced into conserved region 7, corresponding to amino acids 224 to 230, in the E. coli MetK amino acid sequence shown in SEQ ID NO: 2, the non-mutated conserved region 7 comprising the defined amino acid sequence below: X1-NP-X2-G-X3-F

[0105] where

[0106] X1 represents I, V, preferably I

[0107] X2 represents T, G, S, preferably T

[0108] X3 represents R, T, Q, K, S, preferably R.

[0109] In a preferred embodiment, the unmodified conserved region 7 of S-adenosylmethionine synthetase has the following amino acid sequence: 1-N-P-T-G-R-F.

[0110] Preferably the mutations are introduced into conserved region 8, corresponding to amino acids 231 to 237 in the E. coli MetK amino acid sequence shown in SEQ ID NO: 2, the non-mutated conserved region 8 comprising the amino acid sequence defined below : X1-X2-G-X3-P-X4-X5

[0111] where

[0112] X1 represents T, V, I, Y, E, preferably V

[0113] X2 represents V, I, L, N, preferably I

[0114] X3 represents G, S, preferably G

[0115] X4 represents M, I, Q, A, H, D, preferably M.

[0116] X5 represents G, S, A, H, preferably G.

[0117] In a preferred embodiment, the conserved unmodified region of S-adenosylmethionine synthetase has the following amino acid sequence: V-1-G-G-P-M-G.

[0118] Preferably, mutations are introduced into conserved region 9, corresponding to amino acids 246 to 253 in the E. coli MetK amino acid sequence shown in SEQ ID NO: 2, the non-mutated conserved region 9 comprising the defined amino acid sequence below: X1-X2-DTYGG

[0119] where

[0120] X1 represents M, I, preferably I

[0121] X2 represents V, I, preferably I.

[0122] In a preferred embodiment, the unmodified conserved region 9 of S-adenosylmethionine synthetase has the following amino acid sequence: 1-V-D-T-Y-G-G.

[0123] Preferably the mutations are introduced into conserved region 10, corresponding to amino acids 269 to 275 in the E. coli MetK amino acid sequence shown in SEQ ID NO: 2, the unmuted conserved region 10 comprising the amino acid sequence defined below : KVDRS-X1-X2

[0124] where

[0125] X1 represents A, G, preferably A

[0126] X2 represents A, S, L, preferably A.

[0127] In a preferred embodiment, the unmodified conserved region 10 of S-adenosylmethionine synthetase has the following amino acid sequence: K-V-D-R-S-A-A.

[0128] In another preferred application of the invention, at least one mutation is present in the conserved region 11. The unmodified S-adenosylmethionine synthetase houses the conserved region in its C-terminal part with the following amino acid sequence: X1-X2-Q -X3-X4-YAIG-X5-X6

[0129] where

[0130] X1 represents L, E, I, Q, T, preferably E

[0131] X2 represents V, I, L, preferably I

[0132] X3 represents V, L, I, preferably V

[0133] X4 represents A, S, preferably S.

[0134] X5 represents V, I, R, K, A, preferably V

[0135] X6 represents A, V, T, S, preferably A.

[0136] This region corresponds to amino acids 295 to 305 in the E. coli MetK amino acid sequence shown in SEQ ID NO: 2.

[0137] In a preferred embodiment of the invention, the conserved unmodified region of S-adenosylmethionine synthetase has the following amino acid sequence: E-1-Q-V-S-Y-A-1-G-V-A.

[0138] In another preferred application of the invention, mutations are introduced at the N-terminus of the protein leading to a shift and changes in the last 6 amino acids.

[0139] In S-adenosylmethionine synthetase with reduced activity, preferably the serine in conserved region 1 is replaced by another amino acid. In a preferred application of the invention, serine is replaced with an asparagine.

[0140] In a preferred embodiment, the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 1: G-E-1-T-T-N.

[0141] In S-adenosylmethionine synthetase with reduced activity, preferably glycine conserved in conserved region 2 is replaced by another amino acid. In a preferred application of the invention, the conserved glycine is replaced with a serine.

[0142] In a preferred embodiment, the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 2: Q-S-P-D-1-N-Q-S-V-D.

[0143] In S-adenosylmethionine synthetase with reduced activity, preferably glycine conserved in conserved region 3 is replaced by another amino acid. In a preferred application of the invention, the conserved glycine is replaced with a serine.

[0144] In a preferred embodiment, the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 3: Q-S-A-G-D-Q-G-L.

[0145] In S-adenosylmethionine synthetase with reduced activity, preferably conserved histidine and / or semiconserved proline in conserved region 4 is / are replaced by other amino acids. In a preferred application of the invention, the conserved histidine is replaced by a tyrosine and / or the semi-preserved proline is replaced by a leucine.

[0146] In a preferred embodiment, the modified S-adenosylmethionine synthetase has one of the following amino acid sequences in conserved region 4: P-1-T-Y-A-Y-R-L, L-1-T-Y-A-H-R-L, L-1-T-Y-A-Y-R-L.

[0147] In S-adenosylmethionine synthetase with reduced activity, preferably semiconserved arginine in conserved region 5 is replaced by another amino acid. In a preferred application of the invention, arginine is replaced by a cysteine.

[0148] In a preferred embodiment, the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 5: W-L-C-P-D.

[0149] In S-adenosylmethionine synthetase with reduced activity, preferably conserved histidine or semiconserved valine in conserved region 6 is replaced by another amino acid. In a preferred application of the invention, the conserved histidine is replaced by tyrosine and / or valine by aspartate.

[0150] In a preferred embodiment, the modified S-adenosylmethionine synthetase has one of the following amino acid sequences in conserved region 6: V-V-L-S-T-Q-Y V-D-L-S-T-Q-H V-D-L-S-T-Q-Y.

[0151] In S-adenosylmethionine synthetase with reduced activity, preferably the threonine semiconserved in the cyberserved region 7 is replaced by another amino acid. In a preferred application of the invention, threonine is replaced by an isoleucine.

[0152] In a preferred embodiment, S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 7: 1-N-P-1-G-R-F.

[0153] In S-adenosylmethionine synthetase with reduced activity, preferably proline conserved in conserved region 8 is replaced by another amino acid. In a preferred application of the invention, proline is replaced by a serine.

[0154] In a preferred embodiment, the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 8: V-1-G-G-S-M-G.

[0155] In S-adenosylmethionine synthetase with reduced activity, preferably the second conserved glycine in conserved region 9 is replaced by another amino acid. In a preferred application of the invention, glycine is replaced with an aspartate.

[0156] In a preferred embodiment, S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 9: 1-V-D-T-Y-G-D.

[0157] In reduced activity S-adenosylmethionine synthetase, preferably the conserved serine in conserved region 10 is replaced by another amino acid. In a preferred application of the invention, serine is replaced with phenylalanine.

[0158] In a preferred embodiment, the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 10: K-V-D-R-F-A-A.

[0159] In S-adenosylmethionine synthetase with reduced activity, preferably the isoleucine conserved in the conserved region 11 is replaced by other amino acids. In a preferred application of the invention, isoleucine is replaced by leucine.

[0160] In a preferred embodiment, the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 11: E-1-Q-V-S-Y-A-L-G-V-A.

[0161] The present invention, furthermore, concerns nucleotide sequences, DNA or RNA sequences, which encode a transformed S-adenosylmethionine synthetase according to the invention, as defined above. In a preferred embodiment, these DNA sequences are characterized by the fact that they comprise at least one mutation in the regions of DNA sequences coding for conserved regions 1 to 11 of the wild type metK gene, represented as wild type in SEQ ID NO: 2, said mutation is not a silent mutation.

[0162] In a preferred application, the metK gene is the E. coli K12 S-adenosylmethionine synthetase represented by SEQ ID NO: 2 and sequences homologous to those sequences that have S-adenosylmethionine synthase activity and that share at least 80% homology, preferably 90% homology, with the amino acid sequence of SEQ ID NO: 2.

[0163] The transformed S-adenosylmethionine synthase genes described above can be obtained by conventional techniques known to the person skilled in the art presented above and below, including random or directed mutagenesis or the construction of synthetic DNA.

[0164] The metA gene encoding the modified homoserine succinyltransferase and / or the metK gene encoding the modified S-adenosylmethionine synthetase can be encoded chromosomally or extrachromosomally. Chromosomally, there may be one or more copies in the genome that can be introduced by recombination methods known to those experienced in the field. Extrachromosomally, both genes can be carried by different types of plasmids that differ in terms of their origin of replication and thus their number of copies in the cell. They can be present as 1 to 5 copies, about 20 or up to 500 copies corresponding to the low copy number plasmids with firm replication (pSC101, RK2), low copy number plasmids (pACYC, pRSF1010) or high number plasmids of copies (pSK bluescript II).

[0165] The metA and / or metK gene can be expressed with the use of promoters with different intensities that need or not to be induced by the inducing molecules. Examples are the Ptrc, Ptac, Plac promoters, the lambda cl promoter or other promoters known to those skilled in the art.

[0166] The expression of MetA and / or MetK can be enhanced or reduced by stabilizing or destabilizing elements of the corresponding messenger RNA (Carrier and Keasling (1998) Biotechnol. Prog.15, 58-64) or protein (for example, labels of GST, Amersham Biosciences).

[0167] The present invention also relates to microorganisms that contain a feedback-resistant metA allele according to the invention and, eventually, a metK allele with reduced activity according to the invention.

[0168] Such strains are characterized by the fact that they have a methionine metabolism that is disrupted by at least one feedback-resistant metA allele and / or reduced SAM production caused by a reduced activity MetK enzyme.

[0169] The new strains can be prepared from any microorganism in which the metabolism of methionine proceeds through the same metabolic pathway.

[0170] Gram-negative bacteria, in particular E. coli, are especially suitable.

[0171] In order to express the enzyme homoserine succinyltransferase and S-adenosylmethionine synthetase, the metA alleles resistant to feedback and the metK alleles with reduced activity, are transformed into a host strain using customary methods. Screening for strains that have modified properties of homoserine succinyltransferase and S-adenosylmethionine synthetase is, for example, carried out using enzymatic tests.

[0172] For example, homoserine succinyltransferase activity can be determined in an enzymatic test with homoserine and succinyl-CoA as substrates. The reaction is initiated by the addition of the protein extract containing the enzyme homoserin succinyltransferase, and the formation of O-succinyl homoserin is monitored by GC-MS after protein precipitation and derivation with a silylating reagent. The inhibition of feedback is tested in the presence of methionine and S-adenosylmethionine in the reaction mixture.

[0173] The activity of S-adenosylmethionine synthetase can be determined in an enzymatic test with methionine and ATP as substrates. The reaction is initiated by the addition of the protein extract containing the enzyme S-adenosylmethionine synthetase, and the formation of S-adenosylmethionine is monitored by FIA-MS / MS.

[0174] Preferably, E. coli strains are used in which the endogenous genes metA and metK are inactivated and complemented by new recombinant genes.

[0175] The metA and metK alleles resistant to feedback with reduced activity make it possible to abolish control at important biosynthetic control points, thereby expanding the production of a large number of compounds that are located downstream of the aspartate. These include, in particular, homoserin, O-succinyl-homoserine, cystathionine, homocysteine, methionine and S-adenosylmethionine.

[0176] In particular, the invention concerns the preparation of L-methionine, its precursors or compounds derived therefrom, by culturing new microorganisms. The products described above are classified below as compound (I).

[0177] An increase in the production of the compound (I) can be obtained by changing the levels of expression or suppression of the following genes involved in the production of aspartate, a precursor to the compound (I) fosfotransacetilase accessible g1790505 acetate synthase ACEA g1790445 isocitrate lyase ACEB g1790444 malate synthase LECA g1786304 pyruvate dehydrogenase E1 ACEF g1786305 pyruvate dehydrogenase E2 Ipd g1786307 pyruvate dehydrogenase E3 aceK g1790446 isocitrate dehydrogenase kinase / phosphatase succinate g1786948 succinyl-CoA synthetase, beta subunit sucD g1786949 succinyl-CoA synthetase , alpha subunit ppc g1790393 phosphoenolpyruvate carboxylase pck g1789807 phosphoenolpyruvate carboxicinase pykA g1788160 pyruvate kinase II pykF g1787965 pyruvate kinase I poxB g1787096 pyruvate oxidase synthase acid, g1787994 phosphoenolpyride10 major phosphoenolpyase small ilvG g1790202 acetohydroxy acid synthase II, subunit. Large g1790203 ilvM g1790204 acetohydroxy acid synthase II, subunit. small ilvl g1786265 acetohydroxy acid synthase III, subunit. large ilvH g1786266 acetohydroxy acid synthase III, subunit. small aroF g1788953 DAHP synthetase aroG g1786969 DAHP synthetase aroH g1787996 DAHP synthetase aspC g1787159 aspartate aminotransferase

[0178] In addition, Rhizobium etli pyruvate carboxylase (accession number U51439) can be introduced by genetic engineering into E. coli and overexpressed.

[0179] An additional increase in the production of compound (I) can be obtained by overexpression of genes in the lysine / methionine pathway, such as homoserin synthesizing enzymes encoded by the thrA (homoserine dehydrogenase / aspartocinase, g1786183) or metL (homoserine dehydrogepinase) genes / aspartokinase, g1790376) or lysC (aspartokinase, g1790455) or asd (semialdehyde aspartate dehydrogenase, g1789841), or a combination thereof.

[0180] Another increase in the production of (I) is made possible by the overexpression of genes involved in the assimilation of sulfate and in the production of cysteine. This can be achieved by overexpression of the following genes (see below) or by deregulation of the pathway by introducing a constitutive cysB allele as described by Colyer and Kredich (1994 Mol Microbiol 13 797-805) and by introducing a cysE allele encoding a serine acetyl transferase with reduced sensitivity to its L-cysteine inhibitor (US patent application 6,218,168, Denk & Bock 1987 J Gen Microbiol 133 515-525). The following genes need to be overexpressed. CysA g1788761 sulfate permease CysU g1788764 cysteine transport system CysW g1788762 sulphate transport system attached to CysZ membranes g1788753 ORF upstream of cysK CysN g1789108 ATP sulfurylase CysD g1789109 sulfate adenylsulfine c17 g17210 sulfate adenylsiline g17217 alpha CysJ g1789123 sulfite reductase, beta subunit cysE g1790035 serine acetyltransferase cysK g1788754 cysteine synthase cysM g2367138 O-acetyl sulfhydrolase

[0181] In addition, the genes involved in the production of C1 (methyl) groups can be enhanced by overexpression of the following genes. serA g1789279 phosphoglycerate dehydrogenase serB g1790849 phosphoserine phosphatase serC g1787136 phosphoserine aminotransferase glyA g1788902 serine hydroxymethyltransferase metF g1790377 5,10-methylenetetrahydrofolate reductase

[0182] In addition, the genes directly involved in the production of methionine may be overexpressed: metB g1790375 cystathionine gamma synthase metC g1789383 cystathionine beta-lyase metH g1790450 homocysteine-N5-methyltetrahydrofolate trans-methylase methane g1767304 transtrametetrahydrate g2367304 , 10-methylenetetrahydrofolate reductase metR g1790262 positive regulatory gene for metE and metH and autogenous regulation

[0183] Furthermore, the expression of genes in the pathways of methionine degradation or deviation from the methionine production pathway can be reduced or the genes can be suppressed. speD g1786311 S-adenosylmethionine decarboxylase speC g1789337 Ornithine decarboxylase thrB g1786184 Homoserine kinase astA g1788043 Arginine succinyltransferase dapA g1788823 Dihydrodipicolinato synthase

[0184] Another increase in (I) production is possible by suppressing the gene for the repressor protein MetJ, responsible for the methionine regulon infra-regulation as suggested in JP 2000157267-A / 3 (see also GenBank g1790373 ).

[0185] The production of (I) can be further increased by using an altered metB allele that preferentially or exclusively uses H2S and thus produces homocysteine from O-succinyl-homoserine as described in the PCT patent application PCT / FR04 / 00354, whose content is hereby incorporated by reference.

[0186] In another preferred embodiment, the gene encoding aspartokinase / homoserin dehydrogenase is overexpressed and / or the gene encoding the methionine repressor metJ is deleted in the microorganism of the present invention and in the microorganism used in the method according to the invention.

[0187] Preferably, the aspartokinase / homoserine dehydrogenase is encoded by a thrA allele deregulated from the feedback. Such deregulation of the feedback can be obtained by introducing the Phe318Ser mutation in the ThrA enzyme. The position of the enzyme is given here by reference to the ThrA sequence shown in the genbank, accession number V00361.1 (g1786183).

[0188] The metA and metB alleles described above can be used in eukaryotes and prokaryotes. Preferably, the organism used is a prokaryote. In a preferred application, the organism is either E. coli or C. glutamicum.

[0189] The invention also concerns a process for the production of compound (I). Compound (I) is usually prepared by fermenting the designated bacterial strain.

[0190] According to the invention, the terms 'culture' and 'fermentation' are used interchangeably to denote the growth of an appropriate microorganism or culture medium containing a simple carbon source.

[0191] In accordance with the invention, a simple carbon source is a carbon source that can be used by those skilled in the art to obtain the normal growth of a microorganism, in particular 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 source of simple carbon is glucose. Another simple preferred carbon source is sucrose.

[0192] 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, preferably between 25 ° C and 40 ° C, and more specifically about 30 ° C for C. glutamicum and about 37 ° C for E. coli.

[0193] Fermentation is generally carried out 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 necessary substrate for the production of the metabolite.

[0194] 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. 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 as defined by Schaefer et al. (1999, Anal. Biochem. 270: 88-96).

[0195] Similarly, the inorganic culture medium for C. glutamicum can be of identical or similar composition to the BMCG medium (Liebl et al., 1989, Appl. Microbiol. Biotechnol. 32: 205-210) or to a medium such as the one described by Riedel et al. (2001, J. Mol. Microbiol. Biotechnol. 3: 573-583). The medium can be supplemented to compensate for the auxotrophies introduced by the mutations.

[0196] After fermentation, compound (I) is recovered and purified, if necessary. The methods for the recovery and purification of compound (I), such as methionine in the culture medium, are well known to those skilled in the art. - Figure 1A / B: Methionine metabolism in Escherichia coli. - Figure 2: Alignment of the wild type and recombinant metA genes, obtained after selection in oc-methyl-methionyl. Conserved waste is represented by light gray boxes and processed waste is indicated by white boxes. - Figure 3: Alignment of the MetA sequences of different microorganisms. - Figure 4: Alignment of MetK sequences from different microorganisms. - Figure 5: Amino acid substitutions in E.coli's MetK protein. The amino acids above the coherent lines indicate the position and the replacement amino acid.

EXAMPLES Example 1

[0197] Isolation of E. coli mutants containing homoserine trans-succinylase enzymes which have reduced feedback sensitivity with respect to methionine and S-adenosylmethionine.

[0198] Isolation of E. coli strains growing in oc-methylmethionine

[0199] Oc-methylmethionine is a growth-inhibiting methionine analog, producing an immediate effect on the growth rate of E. coli at very low concentrations (minimum inhibitory concentration of 1 pg / ml and minimum concentration for maximum inhibition of 5 pig / ml, Rowbury et al., 1968). Analogs can mimic methionine in inhibiting homoserin trans-succinylase feedback and interfere with protein synthesis without being metabolized. Only mutant strains are able to grow in a medium containing the analog.

[0200] E. coli was routinely grown aerobically at 37 ° C in LB supplemented, when necessary, with the appropriate antibiotic. The medium used for the resistant oc-methylmethionine analogue was a minimal medium containing (per liter): 8 g K2HPO4, 2 g Na2HPO4, 0.75 g (NH ^ SCU, 8 g (NH ^ HPCU , 0.13 g NH4CI, 6.55 g citric acid, 2.05 g MgSCh, 40 mg CaCl2, 40 mg FeSCM, 20 mg MnSCU, 8 mg C0CI2, 4 mg ZnSCh, 2, 8 mg of (NH4) 2Mθ2θ ?, 2 mg of CuCl2, 1 mg of H3BO3. The pH was adjusted to 6.7 and the medium was sterilized. Before use, 10 g / liter of glucose and 10 mg / liter of thiamine have been added.

[0201] The oc-methylmethionine powder marketed by Sigma contains traces of methionine. A nocturnal liquid culture of an E. coli strain (MG1655 ΔmetE) unable to grow without the addition of methionine, was performed to eliminate methionine (from oc-methylmethionine) from the minimal medium. The culture was centrifuged for 10 minutes at 7000 rpm and the supernatant was filtered (Sartorius 0.2 pm). 15 g / liter of agar was added to this minimal medium.

[0202] 1 * 108 cells / ml of a night culture in minimal medium of the wild type strain (MG1655) were dispersed on plates with minimal medium and oc-methylmethionine after 4 washing steps in sterile water. The plates were incubated at 37 ° C until colonies appeared.

[0203] Evidence of mutations in the coding sequence for the metA gene encoding the homoserin trans-succinylase enzyme

[0204] Genomic DNA was prepared from 4 clones that had grown in 4 mg / ml oc-methylmethionine after cultivation in LB medium. The cell pellet was washed once with sterile water and resuspended in 30 pl of sterile water. The cells were disrupted by heating for 5 minutes at 95 ° C and the debris was pelleted. The metA gene was amplified by PCR using the Taq polymerase and the following primers: MetAF (SEQ ID NO: 3): tcaccttcaacatgcaggctcgacattggc (4211759- 4211788) MetAR (SEQ ID NO: 4): ataaaaaaggcacccgaaggt85 (12).

[0205] In three clones, point mutations were detected, which led to amino acid substitutions. In the metA11 clone, CAG was exchanged for a GAC leading to the replacement of Q by E. In the metA * 13, TTG was exchanged for a TTT, leading to the substitution of L for F. In the metA * 14, GCG was exchanged for GTG leading to the replacement of A by V (see figure 2).

[0206] As can be seen from the alignment shown in Figure 3, all three amino acids that are replaced are highly conserved in the MetA proteins of the various species.

[0207] The mutated homoserine trans-succinylase enzymes show reduced feedback sensitivity with respect to methionine and S-adenosylmethionine.

[0208] Homoserine trans-succinylase activity was determined in vitro. E. coli strains carrying either wild-type or mutant enzymes were grown in a medium rich with 2.5 g / liter of glucose and harvested in a late log phase. The cells were resuspended in cold potassium phosphate buffer and sonicated on ice (Branson sonifier, 70 W). After centrifugation, the proteins contained in the supernatants were quantified (Bradford, 1976).

[0209] Ten pl of the extracts were incubated for 30 minutes at 25 ° C with 30 mM homoserin and 4 mM succinyl-CoA. Methionine and / or S-adenosylmethionine were added as indicated. The succinyl-homoserin produced by the enzymes homoserine trans-succinylases was quantified by GC-MS after derivation with tert-butydimethylsilyItrifluoroacetamide (TBDMSTFA). L-serine [1 -13C] was included as an internal standard.

[0210] The results of the activities of homoserin trans-succinylase are reported in Table 1 below: Table 1. Specific activities of the homoserin trans-succinylase enzymes of the wild type and mutants after the addition of the inhibitors L-methionine and S-adenosylmethionine.

[0211] The mutated homoserine trans-succinylase enzymes thus exhibit reduced sensitivity to feedback in relation to methionine and S-adenosylmethionine.

Example 2

[0212] Isolation of E. coli mutants containing reduced activity S-adenosylmethionine synthetase enzymes

[0213] Isolation of E. coli strains that grow on norleukin

[0214] Norleukin is a growth inhibitor methionine analog. At the highest concentrations (50 mg / liter) only the mutant strains are able to grow in a medium containing the analog. Most of these mutations show the metK and metJ sites.

[0215] E. coli was routinely grown aerobically at 37 ° C in LB supplemented, when necessary, with the appropriate antibiotic. The medium used for the resistant norleukin analogue was a minimal medium containing (per liter): 8 g K2HPO4, 2 g Na2HPO4, 0.75 g (NH4) 2SO4, 8 g (NH4) 2HPO4, 0.13 g of NH4CI, 6.55 g of citric acid, 2.05 g of MgSCh, 40 mg of CaCI2, 40 mg of FeSCU, 20 mg of MnSO4, 8 mg of CoCI2, 4 mg of ZnSCh, 2.8 mg of ( NH4) 2Mo2O ?, 2 mg of CuCl2, 1 mg of H3BO3. The pH was adjusted to 6.7 and the medium was sterilized. Before use, 10 g / liter of glucose and 10 mg / liter of thiamine were added.

[0216] 1 * 108 cells / ml of a night culture in minimal medium of the wild type strain (MG1655) were dispersed on plates with minimal medium and norleukin (50 to 200 g / liter) after 4 washing steps in sterile water. The plates were incubated at 37 ° C until colonies appeared.

[0217] Evidence of mutations in the coding sequence of the metK gene encoding the S-adenosylmetionine synthase enzyme

[0218] Genomic DNA was prepared from cultures grown in LB liquid medium. The cell pellet was washed once with sterile water and resuspended in 30 µl of sterile water. The cells were disrupted by heating for 5 minutes at 95 ° C and debris was extracted. The DNA was amplified by PCR using Taq polymerase and using the following primers:

[0219] MetKpF: cccggctggaagtggcaacacg (3084372-3084393) (SEQ ID NO: 5)

[0220] MetKR: gccggatgcggcgtgaacgcctatcc (3085956-3085931) (SEQ ID NO: 6).

[0221] The metK gene has been sequenced. In 10 clones, point mutations were detected, which led to amino acid substitutions. In the clone metK * 59/105, AGC was replaced by AAC, leading to the replacement of S by N (position 59) and GGC was replaced by AGC leading to the replacement of G by S (position 105). In the metK * 105 clone, GGC was replaced by AGC, leading to the replacement of G by S. In metK * 115, GGC was replaced by AGC leading to the replacement of G by S. In metK * 137, CCT was replaced by CTT leading to the substitution of P for L. In metK * 142, CAC was replaced by TAC leading to the substitution of H for Y. In metK * 161/235, CGC was replaced by TGC leading to the substitution of R for C, and CCA was replaced by TCA leading to the replacement of P by S. In metK * 189, CAC was replaced by TAC leading to the replacement of H by Y. In metK * 227, ACC was replaced by ATC leading to the replacement of T by I. In metK * 253, GGC was replaced by GAC leading to the replacement of G by D. In metK * 273, TCC was replaced by TTC leading to the replacement of S by F (see figure 5).

[0222] As can be seen from the alignment shown in Figure 4, all the amino acids that are replaced are conserved in the MetK proteins of the various species.

[0223] Recombinant S-adenosylmethionine synthase enzymes with reduced activity

[0224] S-adenosylmethionine synthase activity was determined in vitro. E. coli strains carrying enzymes or wild type or mutants were grown in a minimum medium with 5 g / liter of glucose and harvested in a late log phase. The cells were resuspended in cold potassium phosphate buffer and sonicated on ice (Branson sonifier, 70 W). After centrifugation, the proteins contained in the supernatants were quantified (Bradford, 1976).

[0225] One hundred µl of the extracts were incubated for 30 minutes at 37 ° C with 10 mM methionine and 10 mM ATP. Potassium chloride was included to activate the enzymes. The adenosylmethionine produced by the enzymes methionine adenosyltransferase was quantified by FIA-MS / MS.

[0226] Results of S-adenosylmethionine synthase activities are reported in Table 2 below: Table 2: S-Adenosylmethionine Synthase (Mat) activities of Wild Type and Metk Mutants *

Example 3

[0227] Construction of E. Coli strains for the production of O-succinyl-homoserin or methionine by overexpression of altered homoserin trans-succinylase and other enzymes in the methionine biosynthesis pathway

[0228] For the construction of E. coli strains that enable the production of O-succinylhomoserin, a plasmid overexpressing the metL gene, encoding homoserin dehydrogenase and aspartocinase, it was introduced in the strains harboring different metA alleles. The overexpressing plasmid metL was constructed as follows:

[0229] The following two oligonucleotides were used: MetLF with 32 bases (SEQ ID NO: 7): TATTCatgagtgtgattgcgcaggcaggggcg com - a region (lower case) homologous to the sequence (4127415 to 4127441) of the metL gene (sequences 4127415 to 41298 reference on the website http://genolist.pasteur.fr/Colibri/). - a region (upper case) that, together with the sequence belonging to metL, forms a restriction site for the enzyme BspHI (underlined). - Metblar with 38 bases (SEQ ID NO 8) TATAAGCTTccataaacccgaaaacatgagtaccgggc

[0230] com - a region (lower case) homologous to the sequence (4129894 to 4129866) of the metL gene - a region (upper case) that houses the Hindlll restriction site.

[0231] The metL gene was amplified by PCR using the oligonucleotides MetLF and MetBLAR, and the BspHI and Hindlll restriction sites were introduced at the N and C ends of the MetL gene, respectively. The resulting PCR fragment was restricted by BspHI and Hindlll and cloned into the vector pTRC99A (Stratagene) previously cut by Ncol and Hindlll.

[0232] Plasmid preparations were examined for the presence of inserts of the correct size. The DNA sequence of metL was verified and the resulting pTRCmetL plasmid was transformed into the strains harboring the metA * 11 and metA * 13 alleles.

[0233] Homoserine dehydrogenase II (HDH) activity was determined in vitro. E. coli strains were grown in a minimum medium with 10 g.l'1 of glucose and harvested in the late log phase. The cells were resuspended in cold potassium phosphate buffer and sonicated on ice (Branson sonifier, 70 W). After centrifugation, the proteins contained in the supernatants were quantified (Bradford, 1976).

[0234] Thirty pl of the extracts were incubated at 30 ° C in a spectrophotometer, with 25 mM homoserin and 1 mM NADP. Potassium chloride was included to activate the enzyme. Considering that E. coli hosts a second gene encoding an activity of homoserine dehydrogenase (thrA), threonine was added, which inhibits this activity. The appearance of NADPH was monitored for 30 minutes at 340 nm.

[0235] Ptrc promoter metL expression dramatically increases HDH activity compared to a similar plasmid strain. Table 3: Homoserine dehydrogenase MetL protein activities in the E. coli strain harboring the metA * 11 mutation with or without overexpression of metL from the Ptrc promoter

[0236] In another application, the metJ gene regulating methionine was suppressed in the strains that harbored the metA * 11, metA * 13 and metA * 14 alleles. To inactivate the metJ gene, the homologous recombination strategy described by Datsenko and Wanner (2000) was used. This strategy allows the insertion of a chloramphenicol resistance cassette, while suppressing most of the genes involved. For this purpose, 2 oligonucleotides were used:

[0237] DmetJF with 100 bases (SEQ ID NO 9):

[0238] Caggcaccagagtaaacattgtgttaatggacgtcaatacatctggacatctaaacttctttgcgtatag attgagcaaaC AT AT G AAT ATC CTC CTT AG

[0239] com - a region (lower case) homologous to the sequences (4126216 to 4126137) of the metJ gene (sequences 4125658 to 4125975, reference sequence on the website http://genolist.pasteur.fr/Colibri/), - one region (upper case) for the amplification of the chloramphenicol resistance cassette (reference sequence in Datsenko, KA and Wanner, B. L, 2000, PNAS, 97: 6640-6645), - DmetJR with 100 bases (SEQ ID NO 10):

[0240] tgacgtaggcctgataagcgtagcgcatcaggcgattccactccgcgccgctcttttttgctttagtattcc cacgtctcTGTAGGCTGGAGCTGCTTCG

[0241] with - a region (lower case) homologous to the sequence (4125596 to 4125675) of the metJ gene - a region (upper case) for the amplification of the chloramphenicol resistance cassette.

[0242] Oligonucleotides DmetJR and DmetJF were used to amplify the chloramphenicol resistance cassette of the plasmid pKD3. The PCR product obtained was then introduced by electroporation into the MG1655 (pKD46) strain, in which the enzyme recombinase Red expressed allowed homologous recombination. The chloramphenicol resistant transformers were then selected and the insertion of the resistance cassette was verified by PCR analysis with the ooligonucleotides MetJR and MetJF defined below. The retained strain is designated MG1655 (ΔmetJ :: Cm) metA *

[0243] MetJR (SEQ ID NO 11): ggtacagaaaccagcaggctgaggatcagc (homologous to the sequence from 4125431 to 4125460).

[0244] MetBR (SEQ ID NO 12): ttcgtcgtcatttaacccgctacgcactgc (homologous to the sequence from 4126305 to 4126276).

[0245] The chloramphenicol resistance cassette was then eliminated. The plasmid pCP20 carrying recombinant FLP at the FRT sites of the chloramphenicol resistance cassette was introduced into the recombinant strains by electroporation. After a series of cultures at 42 ° C, the loss of the chloramphenicol resistance cassette was verified by a PCR analysis with the same oligonucleotides as those used previously. The strains retained were designated MG1655 (ΔmetJ) metA *.

[0246] Subsequently the plasmid pTRCmetL harboring the metL gene was introduced into these strains, giving rise to ΔmetJ metA * 11 pTRCmetL, ΔmetJ metA * 13 pTRCmetL and ΔmetJ metA * 14 pTRCmetL.

Example 4

[0247] Construction of alloserin succinyltransferase alleles expressing enzymes with still reduced feedback sensitivity

[0248] To further reduce the sensitivity of homoserin trans-succinylase to its inhibitors methionine and SAM, other metA mutants were constructed by site-directed mutagenesis.

[0249] Initially, the metA and metA * 11 alleles were cloned into the vector pTRC99A (Stratagene). The following two oligonucleotides were used: - MetA-Ncol with 49 bases (SEQ ID NO: 13):

[0250] TATTAAATTACCatggcaccgattcgtgtgccggacgagctacccgccg

[0251] com - a region (lower case) homologous to the sequence (4211862 to 4211892) of the metA gene (sequence 4211859 to 4212788, reference sequence on the website http://genolist.pasteur.fr/Colibri/), - a region (upper case) that, together with the sequence belonging to metA forms a restriction site for the enzyme Ncol (underlined), - MetA-EcoRI with 47 bases (SEQ ID NO 14):

[0252] TATTAAATTAGaattccgactatcacagaagattaatccagcgttgg

[0253] com - a region (lower case) homologous to the sequence (4212804 to 42127774) of the metA gene - a region (upper case) that houses the EcoRI restriction site.

[0254] The metA and metA * 11 alleles were amplified by PCR using the oligonucleotides MetAF and MetAR, and the restriction sites Ncol and EcoRI were introduced at the N and C ends of the metA gene, respectively. The resulting PCR fragments were restricted by Ncol and EcoRI and cloned into the vector pTRC99A (Stratagene) previously cut by Ncol and EcoRI.

[0255] Plasmid preparations were examined for the presence of inserts of the correct size and the DNA sequences of metA and metA * 11 were verified by sequencing.

[0256] Enzymatic analysis of clones expressing metA and metA * 11 gave very low MetA activity. We assume that the introduction of an alanine between the amino acids 1M and 2P, which was necessary for cloning the metA in the vector pTRC99A, caused this loss of activity. Therefore, using site-directed mutagenesis (Stratagene), alanine was eliminated with the use of oligonucleotides:

[0257] metA-alaF (Afllll) (SEQ ID NO 15): cacacaggaaacagaccatgccgatacgtgtgccggacgagctaccc

[0258] metA-alaR (Afllll) (SEQ ID NO 16): gggtagctcgtccggcacacgtatcggcatggtctgtttcctgtgtg

[0259] Subsequently, the mutants metA * 15 (A27V + Q64E), metA * 16 (Q64D) and metA * 17 (A27V + Q64D) were constructed by site-directed mutagenesis (Stratagene) using the correct metA sequence as a matrix. For the construction of pTRCmetA * 15 (A27V + Q64E), the following oligonucleotides were used:

[0260] MetAA28VF (Xbal) (SEQ ID NO 17): Gtgatgacaacttctagagtgtctggtcaggaaattcgtcc

[0261] MetAA28VR (Xbal) (SEQ ID NO 18): Ggacgaatttcctgaccagacactctagaagttgtcatcac

[0262] and pTRCmetA * 11 as a matrix.

[0263] For the construction of pTRCmetA * 16 (Q64D) the following oligonucleotides were used:

[0264] MetAQ64DF (EcoRV) (SEQ ID NO 19): Gctttcaaactcacctttggatgtcgatatccagctgttgc

[0265] MetAQ64DR (EcoRV) (SEQ ID NO 20) gcaacagctggatatcgacatccaaaggtgagtttgaaagc

[0266] and pTRCmetA as a matrix.

[0267] For the construction of pTRCmetA * 17 (A27V + Q64D) the following oligonucleotides were used: MetAA28VF (Xbal) (SEQ ID NO 17) and MetAA28VR (Xbal) (SEQ ID NO 18); and pTRCmetA * 16 as a matrix.

[0268] Plasmids were checked by sequencing.

[0269] To transfer the metA * 15, metA * 16 and metA * 17 alleles to the metA site on the chromosome, a metA suppression was constructed in the ΔmetJ feedback by applying the same strategy used for the metJ suppression. The following oligonucleotides were used to suppress metA:

[0270] DmetAF (4211866-4211945) (SEQ ID NO 21)

[0271] ttcgtgtgccggacgagctacccgccgtcaamcttgcgtgaagaaaacgtctttgtgatgacaacttct cgtgcgtctTGTAGGCTGGAGCTGCTTCG

[0272] DmetAR (4212785-4212706) (SEQ ID NO 22)

[0273] Atccagcgttggattcatgtgccgtagatcgtatggcgtgatctggtagacgtaatagttgagccagttg gtaaacagtaCATATGAATATCCTCCTTAG The region indicated in lower case corresponds to the sequence between metA and yjaB. The uppercase region is used to amplify the kanamycin resistance cassette (Datsenko and Wanner, 2000). (The numbers in parentheses correspond to the reference sequence on the website http://genolist.pasteur.fr/Colibri/).

[0274] The resulting deletion was verified using the following oligonucleotides (The numbers in parentheses correspond to the reference sequence on the website http://genolist.pasteur.fr/Colibri/).

[0275] MetAF (SEQ ID NO 3) and MetAR (SEQ ID NO 4).

[0276] DmetJ DmetA from the resulting strain was used to introduce the metA * 15, metA * 16 and metA * 17 alleles into the chromosome. To this end, the plasmid pKD46 was introduced into the DmetJ DmetA strain (Datsenko and Wanner, 2000). The alleles were amplified from the vectors pTRCmetA * 15, pTRCmetA * 16 and pTRCmetA * 17 using the following oligonucleotides:

[0277] MetArcF (4211786-4211884) (SEQ ID NO 23)

[0278] Ggcaaattttctggttatcttcagctatctggatgtctaaacgtataagcgtatgtagtgaggtaatcaggtt atgccgattcgtgtgccggacgagc

[0279] MetArcR (4212862-4212764) (SEQ ID NO 24)

[0280] Cggaaataaaaaaggcacccgaaggtgcctgaggtaaggtgctgaatcgcttaacgatcgactatc acagaagattaatccagcgttggattcatgtgc

[0281] The sequence in bold is homologous to the sequence of the metA gene; the rest of the sequence is adjacent to metA.

[0282] For PCR verification, the following oligonucleotides were used: MetAF (SEQ ID NO 3) and MetAR (SEQ ID NO 4).

[0283] As described above, the resulting strains were grown in minimal medium, the crude extracts were prepared, and the MetA activity was determined. As can be seen from Table 4, newly constructed alleles are less sensitive to inhibition by methionine and SAM than the alleles described above. Especially interesting is the metA * 15 allele with high intrinsic activity that cannot be inhibited by SAM or methionine. Table 4: Homoserinatrans-succinylase activities of MetA mutants in the absence and presence of 100 mm methionine and 1 mM SAM. The percentage values in parentheses indicate the amount of activity remaining after inhibition.

Example 5

[0284] Construction of E. coli strains for the production of homoserin or methionine O-succinyl by combining feedback-resistant target alleles with reduced activity metk alleles

[0285] In order to transfer the recombinant metK alleles to the strains that harbor the feedback-resistant metA alleles, a kanamycin resistance cassette was introduced between the metK gene and galP.

[0286] To introduce the cassette, the homologous recombination strategy described by Datsenko and Wanner (2000) is used. This strategy allows the insertion of a kanamycin resistance cassette, while suppressing most of the genes in question. For this purpose, 2 oligonucleotides are used: - DMetKFscr with 100 bases (SEQ ID NO 25):

[0287] ccgcccgcacaataacatcattcttcctgatcacgtttcaccgcagattatcatcacaactgaaaccgat tacaccaaccTGTAGGCTGGAGCTGCTTCG

[0288] com - a region (lower case) homologous to the region sequence between metK and galP (sequence 3085964 to 3086043, 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, KA and Wanner, B. L, 2000, PNAS, 97: 6640-6645), - DmetKRscr with 100 bases (SEQ ID NO 26):

[0289] gagttatatcatcatagattaaacgctgttatctgcaatiaagactttactgaaaagaaatgiaacaactgi gaaaaccgCATATGAATATCCTCCTTAG

[0290] com - a region (lower case) homologous to the region between the metK gene and galP (3086162 to 3086083) - a region (upper case) for the amplification of the kanamycin resistance cassette of plasmid pKD4. The PCR product obtained is then introduced by electroporation into the MG1655 (pKD46) strain in which the expressed recombinase enzyme Red allows homologous recombination. The kanamycin-resistant transformers are then selected and the insertion of the resistance cassette is verified by a PCR analysis with the oligonucleotides MetKFscr and MetKRscr defined below. The retained strain is called MG1655 (metK, Km).

[0291] MetKFscr (SEQ ID NO 27): gcgcccatacggtctgattcagatgctgg (homologous to the sequence from 3085732 to 3085760).

[0292] MetKRscr (SEQ ID NO 28): gcgccagcaattacaccgatatccaggcc (homologous to the sequence from 3086418 to 3086390).

[0293] To transfer the metK alleles, the P1 phage transduction method is used. The protocol followed is implemented in 2 stages with the preparation of the phage lysate of the MG1655 strain (metK, Km) and then the transduction in the MG1655 ΔmetJ metA * 1 IpTRCmetL strain.

[0294] The construction of the strain (metK, Km) is described above.

[0295] Preparation of phage P1 lysate: - Inoculation with 100 pL of a night culture of the MG1655 strain (metK, Km) of 10 ml LB + 50 pg / ml Km + 0.2% glucose + 5 mM CaCL. - Incubation for 30 minutes at 37 ° C with shaking. - Addition of 100 pl of P1 lysate of phage prepared on the wild strain MG 1655 (about 1,109 phage / ml) - Stirring at 37 ° C for 3 hours, until all cells are lysed - Addition of 200 pl of chloroform and whirlwind. - Centrifugation for 10 minutes at 4500 g to eliminate cell debris - Transfer the supernatant to a sterile tube and add 200 pl of chloroform - Store the lysate at 4 ° C.

[0296] Transduction - Centrifugation for 10 minutes in 1500 g of 5 ml of a culture of the MG1655 ΔmetJ metA * 11 pTRCmerL strain in LB medium - Suspension of the cell pellet in 2.5 ml of 10 mM MgSCM, 5 mM CaCh - Tubes control: 100 pl of cells 100 pl of P1 phage from the MG1655 strain (ΔmetK, Km) - Test tube: 100 pl of cells + 100 pl of P1 phage from the MG1655 strain (ΔmetK, Km) - Incubation for 30 minutes at 30 ° C without shaking - Addition of 100 pl of 1 M sodium citrate in each tube and swirling - Addition of 1 ml of LB - Incubation for 1 hour at 37 ° C with shaking - Spread on plates 50 gg / ml of LB + Km after centrifuging the tubes for 3 minutes at 7000 rpm - Incubation at 37 ° C overnight. Verification of the strain

[0297] The kanamycin-resistant transformants are then selected and the insertion of the region containing (metK, Km) is verified by a PCR analysis with the oligonucleotides MetKFscr and MetKRscr. The retained strain is designated MG1655 ΔmetJ metA * 11 pTRCmetL metK *

[0298] The kanamycin resistance cassette can then be disposed of. The plasmid pCP20 carrying the FLP recombinase acting on the FRT sites of the kanamycin resistance cassette is then introduced into the recombinant sites by electroporation. After a series of cultures at 42 ° C, the loss of the kanamycin resistance cassette is verified by a PCR analysis with the same oligonucleotides as used previously (MetKFscr and MetKRscr).

Example 6

[0299] Fermentation of E. coli production strains and production analysis

[0300] Production strains were initially analyzed in small cultures in an Erlenmeyer flask using modified M9 medium (Anderson, 1946, Proc. Natl. Acad. Sci. USA 32: 120-128), which was supplemented with 5 g / liter of MOPS and 5 g / liter of glucose. Carbenicillin was added, if necessary, at a concentration of 100 mg / liter. A night culture was used to inoculate a 30 ml culture at an OD600 of 0.2. After the culture had reached an OD600 of 4.5 to 5, 1.25 ml of a 50% glucose solution and 0.75 ml of a 2M MOPS (pH 6.9) were added and the culture was stirred for 1 hour. Subsequently, IPTG was added, if necessary.

[0301] Extracellular metabolites were analyzed during the batch phase. Amino acids were quantified by HPLC after the derivation of OPA / Fmoc and other relevant metabolites were analyzed using GC-MS after silylation.

[0302] The following results were obtained for the MG1655 strains. MG1655 pTRCmetL, MG1655 metA * 11 pTRCmetL, MG1655 metA * 13 pTRCmetL, MG1655 metA * 11 ΔmetJ pTRCmetL, MG1655 metA * 11Δ | metJ pTRCmetL metK * H142Y. Table 5. Specific concentrations of extracellular metabolites (mmol / g dry weight) after batch fermentation (Erlenmeyer flask). ND = not detected.

[0303] To further enhance the production of homoserin, aspartokinase / homoserin, a thrA * allele with reduced feedback resistance to threonine, is expressed from plasmid pCL1920 (Lerner and Inouye, 1990, NAR 18, 15 p. 4631) using the Ptrc promoter. For the construction of plasmid pME107, thrA was amplified by PCR from genomic DNA using the following oligonucleotides:

[0304] BspHIthrA (SEQ ID NO 29): ttaTCATGAgagtgttgaagttcggcggtacatcagtggc

[0305] SmalthrA (SEQ ID NO 30): ttaCCCGGGccgccgccccgagcacatcaaacccgacgc

[0306] The amplified PCR fragment is cut with the restriction enzymes BspHI and Smal and cloned into the Ncol / Smal sites of the vector pTRC99A (Stratagene). For the expression of a low copy vector, plasmid pME101 is constructed as follows. Plasmid pCL1920 is amplified in PCR using the PME101F and PME101R oligonucleotides and the BstZ17l-Xmnl fragment from the pTRC99A vector harboring the lacl gene, and the Ptrc promoter is inserted into the amplified vector. The resulting vector and the vector that houses the thrA gene are restricted by Apal and Smal and the fragment containing thrA is cloned into the vector pME101. To relieve ThrA from the inhibition of feedback, the F318S mutation is introduced by site-directed mutagenesis (Stratagene) with the use of the oligonucleotides ThrAF F318S and ThrAR F318S, resulting in the vector pME107. The pME107 vector was introduced in the ΔmetJ metA * 11 strains with different metK * alleles.

[0307] PME101F (SEQ ID NO 31): Ccgacagtaagacgggtaagcctg

[0308] PME101R (SEQ ID NO 32): Agcttagtaaagccctcgctag

[0309] ThrAF F318S (Smal) (SEQ ID NO 33): cccaatctgaataacatggcaatgtccagcgtttctggcccggg

[0310] ThrAR F318S (Smal) (SEQ ID NO 34): cccgggccagaaacgctggacattgccatgttattcagattgg

[0311] The strains produced substantial amounts of metabolites of interest were subsequently tested under production conditions in 300 ml of fermenters (DASGIP) using a fed batch protocol.

[0312] For this purpose, the fermenter was filled with 145 ml of modified minimum medium and inoculated with 5 ml of pre-culture to an optical density (QD600 nm) between 0.5 and 1.2.

[0313] The culture temperature was kept constant at 37 ° C and the pH was permanently adjusted to values between 6.5 and 8, using an NH4OH solution. The agitation index was maintained between 200 and 300 rpm during the batch phase and was increased to 1000 rpm at the end of the fed batch phase. The concentration of dissolved oxygen was maintained at values between 30 and 40% saturation using a gas controller. When the optical density reached a value between three and five, the fed batch was started with an initial flow rate between 0.3 and 0.5 ml / hour and a progressive increase to flow rate values between 2.5 and 3 , 5 ml / hour. At this point, the flow rate was kept constant for 24 to 48 hours. The average batch fed was based on the minimum medium containing glucose in concentrations between 300 and 500 g / liter.

[0314] Table 6 shows methionine concentrations for ΔmetJ metA * 11 pME107 metK * H142Y and ΔmetJ metA * l1 pME107 metK * T227l strains compared to the ΔmetJ metA * 11 pME107 reference layer after about 75 hours of operation . Both strains harboring the metK * alleles achieve an increased methionine concentration compared to the reference strain. Thus, metK mutations confer an industrial advantage over methionine production by increasing the productivity of the strains. Table 6. Production of methionine from the reference strain ΔmetJ metA * 11 pME107 and two strains harboring the metK * mutations AFTER the indicated time of operation including batch and fed batch

Claims (6)

1. Method for the preparation of methionine, its precursors or products derived therefrom, in a fermentation process with a microorganism in which L-homoserine is converted to O-succinyl-homoserine with a trans-succinylase homoserine, comprising the stage of cultivation the microorganism in an appropriate medium, and recover methionine, its precursors or products derived from it once produced, in which the homoserin trans-succinylase is mutated with a reduced sensitivity to the S-adenosylmethionine and methionine feedback inhibitors in comparison with the wild type enzyme, characterized by the fact that the mutated homoserin trans-succinylase is homoserin trans-succinylase having the amino acid sequence of SEQ ID No: 35 or SEQ ID No: 36. 2. Method, according to claim 1, characterized by the fact that the modified microorganism is selected from prokaryotes and eukaryotes. 3. Method, according to claim 2, characterized by the fact that the modified microorganism is selected from prokaryotes, especially Escherichia coli or Corynebacterium glutamicum. 4. Recombinant microorganism characterized by the fact that it comprises a nucleotide sequence, according to SEQ ID No: 37 or SEQ ID No: 38. 5. Recombinant microorganism, according to claim 4, characterized by the fact that the microorganism is selected from Escherichia coli or Corynebacterium glutamicum. 6. Recombinant microorganism according to either of claims 4 or 5, characterized in that the gene encoding aspartokinase / homoserin dehydrogenase is overexpressed and / or the gene encoding methionine methrepressor is deleted.

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