# Patent

## Title
Recombinant enzyme with altered feedback sensitivity

## Abstract

The present invention relates to the use of recombinant homoserine transsuccinylase enzymes with altered feedback sensitivity (MetA*) and possibly recombinant S-adenosyl methionine synthetase enzymes with reduced activity (MetK*) for the production of methionine, its precursors or derivatives thereof.

## Background
SEQUENCE LISTING
This application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 15, 2010, is named REG06146.txt, and is 196,993 bytes in size.
FIELD OF THE INVENTION
The present invention relates to the use of recombinant homoserine transsuccinylase enzymes with altered feedback sensitivity (MetA*) and eventually, recombinant S-adenosyl methionine synthetase enzymes with reduced activity (MetK*) for the production of methionine, its precursors or derivatives thereof.
PRIOR ART
Sulfur-containing compounds such as cysteine, homocysteine, methionine or S-adenosylmethionine are critical to cellular metabolism and are produced industrially to be used as food or feed additives and pharmaceuticals. In particular methionine, an essential amino acid, which cannot be synthesized by animals, plays an important role in many body functions. Aside from its role in protein biosynthesis, methionine is involved in transmethylation and in the bioavailability of selenium and zinc. Methionine is also directly used as a treatment for disorders like allergy and rheumatic fever. Nevertheless most of the methionine which is produced is added to animal feed.
With the decreased use of animal-derived proteins as a result of BSE and chicken flu, the demand for pure methionine has increased. Chemically D,L-methionine is commonly produced from acrolein, methyl mercaptan and hydrogen cyanide. Nevertheless the racemic mixture does not perform as well as pure L-methionine, as for example in chicken feed additives (Saunderson, C. L., (1985) British Journal of Nutrition 54, 621-633). Pure L-methionine can be produced from racemic methionine e.g. through the acylase treatment of N-acetyl-D,L-methionine which increases production costs dramatically. The increasing demand for pure L-methionine coupled to environmental concerns render microbial production of methionine attractive.
Microorganisms have developed highly complex regulatory mechanisms that fine-tune the biosynthesis of cell components thus permitting maximum growth rates. Consequently only the required amounts of metabolites, such as amino acids, are synthesized and can usually not be detected in the culture supernatant of wild-type strains. Bacteria control amino acid biosynthesis mainly by feedback inhibition of enzymes, and repression or activation of gene transcription. Effectors for these regulatory pathways are in most cases the end products of the relevant pathways. Consequently, strategies for overproducing amino acids in microorganisms require the deregulation of these control mechanisms.
The pathway for L-methionine synthesis is well known in many microorganisms (FIG. 1A/B). Methionine is derived from the amino acid aspartate, but its synthesis requires the convergence of two additional pathways, cysteine biosynthesis and C1 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 succinylhomoserine. 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 B12-dependent or a B12-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 Mol. Microbiol., 5, 1593-1597). MetJ uses S-adenosylmethionine as a corepressor, that is made from methionine by the enzyme S-adenosylmethionine synthetase (EC 2.5.1.6) encoded by the essential gene metK. metA encoding homoserine transsuccinylase (EC 2.3.1.46), the first enzyme unique to the synthesis of methionine is another major control point for methionine production. Besides the transcriptional regulation of metA by MetJ and MetR the enzyme is also feedback regulated 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). Feedback inhibition 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.
Amino acid analogues inhibit bacterial growth through the synthesis of abnormal polypeptides and by interfering with feedback inhibition, which lead to detrimental processes inside the cell. Analogue-resistant mutants have been obtained which show altered and deregulated enzymes leading to excess synthesis of the corresponding metabolites that can outcompete the analogue. Several groups have used the methionine analogues norleucine, ethionine, and α-methylmethionine to generate microbial strains that overproduce methionine. It was shown that α-methylmethionine is a potent inhibitor of the homoserine transsuccinylase enzyme MetA (Rowbury R J, (1968) The inhibitory effect of α-methylmethionine on Escherichia coli, J. gen. Microbiol., 52, 223-230). Feedback resistant mutants in Salmonella typhimurium that mapped to the metA locus were isolated (Lawrence D. A., (1972) Regulation of the methionine feedback-sensitive enzyme in mutants of Salmonella typhimurium; Lawrence, D. A., Smith, D. A., Rowbury, R. J. (1967) Regulation of methionine synthesis in Salmonella typhimurium: Mutants resistant to inhibition by analogues of methionine; Genetics 58, 473-492). Norleucine resistant mutants were shown to map to the metK locus. (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 have reported the isolation of feedback resistant metA mutants and norleucine resistant mutants in E. coli, but the actual mutations in metA and metK have not been described.
The critical role of homoserine transsuccinylase for bacterial methionine production by fermentation has been demonstrated (Kumar, D. Garg, S. Bisaria V. S., Sreekrishnan, T. R. and Gomes, J. (2003) Production of methionine by a multi-analogue resistant mutant of Corynebacterium lilium, Process Biochemistry 38, 1165-1171). The patent application JP2000139471 describes a process for the production of L-methionine using mutants in the genes metA and metK. In this case the precise location of the mutations has been determined. The MetA mutant enzymes have partially lost the sensitivity to feed-back inhibition by methionine and S-adenosyl-methionine. Nevertheless their initial activities are decreased down to about 25% when compared to the wildtype enzyme, and at concentrations of 1 mM methionine for some mutants or 1 mM SAM for others another 25-90% of the enzyme activity is lost. The meiK mutants were not characterized enzymatically but used in fermentations to increase the amount of methionine produced.
GENERAL DISCLOSURE OF THE INVENTION
This invention relates to a method for the preparation of methionine, its precursors or products derived thereof in a fermentative process with a microorganism where L-homoserine is converted into O-succinyl-homoserine with a homoserine transsuccinylase, comprising the step of culturing the said microorganism on an appropriate medium and recovering methionine, its precursors or products derived thereof once produced, wherein the homoserine transsuccinylase is a mutated homoserine transsuccinylase with reduced sensitivity for the feedback inhibitors S-adenosylmethionine and methionine.
The invention also relates to the same method with microorganisms where the S-adenosyl-methionine synthetase enzyme activity is reduced.
The present invention also concerns the mutated enzymes, nucleotide sequences coding for the said enzymes with reduced sensitivity or activity and microorganisms comprising the said nucleotide sequences as disclosed above and below.
The recombinant enzymes can be used together and can also be combined with several other changes in the corresponding microorganisms such as overexpression of genes or their deletion. Preferentially the gene encoding aspartokinase/homoserine dehydrogenase (metL or thrA) is over expressed and the gene encoding the methionine repressor metJ is deleted.
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) 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) are obtained by comparing protein sequences from 43 fully sequenced genomes representing 30 major phylogenic lines. Each COG is defined from at least three lines, which permits the identification of former conserved domains.
The means of identifying homologous sequences and their percentage homologies are well known to those skilled in the art, and include in particular the BLAST programs, which can be used with the default parameters. The sequences obtained can then be exploited (e.g., aligned) using, for example, the sequence alignment programs with the default parameters.
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. 2nd ed. Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.).
DETAILED DESCRIPTION OF THE INVENTION
The modified homoserine succinyltransferases showing a decreased feed-back sensitivity to methionine and S-adenosylmethionine in comparison to the wild-type enzyme, according to the present invention, comprises at least one amino acid mutation when compared with the wild-type sequence. The mutation is preferentially selected in the conserved rcgions 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 the formylmethionine counting as number 1.
All references to amino acid positions are made based on the homoserine succinyltransferase encoded by the metA gene of E. coli represented in FIG. 2. The relative positions of corresponding conserved regions in other homoserin succinyltransferases from different organisms can be found by a person skilled in the art by simple sequence alignment as represented on FIG. 3 with the enzymes listed below:

    
    
        gi|20138683|sp|Q97I29|META Clostridium acetobutylicum Homoserine O-succinyltransferase
        gi|12203304|sp|Q9PLV2|META Campylobacter jejuni Homoserine O-succinyltransferase
        gi|12230277|sp|Q9KAK7|META Bacillus halodurans Homoserine O-succinyltransferase
        gi|20138686|sp|Q97PM9|META Streptococcus pneumoniae Homoserine O-succinyltransferase
        gi|20138715|sp|Q9CEC5|META Lactococcus lactis Homoserine O-succinyltransferase
        gi|20138656|sp|Q92L99|META Sinorhizobium meliloti Homoserine O-succinyltransferase
        gi|20138618|sp|Q8YBV5|META Brucella melitensis Homoserine O-succinyltransferase
        gi|20141549|sp|P37413|META Salmonella typhimurium Homoserine O-succinyltransferase
        gi|20138601|sp|Q8X610|META Escherichia coli O157:H7 Homoserine O-succinyltransferase
        gi|12231004|sp|P07623|META Escherichia coli Homoserine O-succinyltransferase
        gi|12230285|sp|Q9KRM5|META Vibrio cholerae Homoserine O-succinyltransferase
        gi|38258142|sp|Q8FWG9|META Brucella melitensis biovar suis Homoserine O-succinyltransferase
        gi|20138631|sp|Q8ZAR4|META Yersinia pestis Homoserine O-succinyltransferase
        gi|12231010|sp|P54167|META Bacillus subtilis Homoserine O-succinyltransferase
        gi|12230320|sp|Q9WZY3|META Thermotoga maritima Homoserine O-succinyltransferase
        gi|20138625|sp|Q8Z1W1|META Salmonella typhi Homoserine O-succinyltransferase
        >gi|31340217|sp|Q8D937|META Vibrio vulnificus Homoserine O-succinyltransferase
        gi|31340213|sp|Q87NW7|META Vibrio parahaemolyticus Homoserine O-succinyltransferase
    
    


The modified homoserine succinyltransferases preferentially exhibit a specific activity which 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 tunes that of the wild-type enzyme in the 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.
Preferentially, the protein sequence of the modified homoserine succinyltransferase according to the invention contains the amino acid mutation of at least one of the conserved regions sequences specified below.
Preferentially at least one mutation is present in the conserved region 1 comprising the amino acid sequence defined below, in the N-terminal part of the wild type homoserine transsuccinylases, corresponding to amino acid 24 to 30 in the amino acid sequence of E. coli MetA shown in SEQ ID NO 1. This non mutated conserved region I has the following sequence:

X1-X2-X3-A-X4-X5-Q  (SEQ ID NO: 35)

In which

X1 represents E, D, T, S, L, preferentially T

X2 represents D, S, K, Q, E, A, R, preferentially S

X3 represents R, E, D, preferentially R

X4 represents Y, I, F, A, K, S, V, preferentially S

X5 represents H, S, N, G, T, R, preferentially G.

In a preferred embodiment the unmodified homoserine succinyltransferase conserved region 1 has the following amino acid sequence:

T-S-R-A-S-G-Q  (SEQ ID NO: 36).

In another preferred embodiment of the invention, at least one mutation is present in the conserved region 2, also in the N-terminal part of the wild type homoserine transsuccinylase, corresponding to amino acid 58 to 65 in the amino acid sequence of E. coli MetA shown in SEQ ID NO 1. The non mutated conserved region 2 has the following formula:

X1-X2-X3-P-L-Q-X4-X5  (SEQ ID NO: 37)

In which

X1 represents G, A, S, preferentially S

X2 represents N, A, preferentially N

X3 represents S, T, preferentially S

X4 represents V, L, I, preferentially V

X5 represents N, E, H, D, preferentially D.

In a preferred embodiment the unmodified homoserine succinyltransferase conserved region 2 has the following amino acid sequence:

S-N-S-P-L-Q-V-D  (SEQ ID NO: 38).

In a third embodiment of the invention the homoserine succinyltransferase comprises at least one mutation in a conserved region in its C-terminal part, corresponding to amino acid 292 to 298 in the amino acid sequence of E. coli MetA shown in SEQ ID NO 1. The non mutated conserved region 3 has the following formula:

X1-Y-Q-X2-T-P-X3  (SEQ ID NO: 39)

In which

X1 represents V, I, M, preferentially V

X2 represents E, K, G, I, Q, T, S, preferentially I

X3 represents F, Y, preferentially Y.

In a preferred embodiment, the unmodified homoserine succinyltransferase conserved region 3 has the following amino acid sequence:

V-Y-Q-I-T-P-Y  (SEQ ID NO: 40).

In a preferred embodiment, the conserved alanine in conserved region 1 is replaced with another amino acid, more preferentially with a valine. The modified conserved region has most preferentially the following amino acid sequence:

T-S-R-V-S-G-Q  (SEQ ID NO: 41).

In another preferred embodiment, the conserved amino acids L and/or Q in conserved region 2 are replaced with other amino acids. Preferentially, leucine is replaced by phenylalanine and/or glutamine is replaced with a glutamate or aspartate. Most preferentially, the modified conserved region 2 has the following amino acid sequence:

S-N-S-P-L-E-V-D  (SEQ ID NO: 42).

In a further preferred embodiment, the conserved amino acids L and/or Q in conserved region 3 are replaced with another amino acid.
In a preferred application the metA gene is the homoserine transsuccinylase enzyme of E. coli K12 represented by the SEQ ID NO 1 and sequences homologous to that sequence that have homoserine transsuccinylase activity and that share at least 80% homology, preferentially 90% homology with the amino acid sequence of SEQ ID NO 1.
Modified homoserine succinyltransferases, may be obtained, for example, by selecting strains growing in the presence of methionine analogues such as α-methylmethionine, norleucine or ethionine. Preferentially these strains will be selected while growing in the presence of α-methylmethionine.
The present invention furthermore relates to nucleotide sequences, DNA or RNA sequences, which encode a mutated 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 the conserved regions 1 to 3 of the wild type metA gene, represented in SEQ ID NO 1, the said mutation being not a silent mutation.
These DNA sequences can be prepared, for example, from the strains growing in the presence of methionine analogues. The starting DNA fragment encompassing the modified metA gene, is cloned in a vector using standard known techniques for preparing recombinant DNA.
These DNA sequences can also be prepared, for example, by non-specific or by targeted mutagenesis methods from strains harboring the DNA sequence encoding wild-type homoserine succinyltransferase.
Non-specific mutations within the said DNA region may be produced, for example, by chemical agents (e.g. nitrosoguanidine, ethylmethanesulfonic acid and the like) and/or by physical methods and/or by PCR reactions, which are carried out under particular conditions.
Methods for introducing mutations at specific positions within a DNA fragment are known and are described in Molecular Cloning: a Laboratory Manual, 1989, 2nd ed. Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.
Using the afore mentioned methods, one or more mutations which cause the modified homoserine succinyltransferase to possess an amino acid sequence which leads to methionine and S-adenosylmethionine insensitivity can be introduced in the said DNA region of any metA gene. Preferentially, one or more nucleotides in the DNA sequence encoding homoserine succinyltransferase are changed such that the amino acid sequence that is encoded by the gene exhibits at least one mutation.
Another method of producing feedback-resistant metA alleles consists in combining different point mutations which lead to feedback resistance, thereby giving rise to multiple mutants possessing new properties.
The modified S-adenosylmethionine synthetase according to the invention has decreased activity in comparison to the wild-type enzyme, and has at least one mutation in its protein sequence when compared to the wild-type sequence.
The mutation is preferentially in one of the conserved regions defined below.
All references to amino acid positions are made based on the S-adenosylmethionine synthetase encoded by the metK gene of E. coli. The relative positions of corresponding conserved regions in other S-adenosylmethionine synthetases from different organisms can be found by a person skilled in the art by simple sequence alignment as represented in FIG. 4 with enzymes listed below:
>gi|39574954|emb|CAE78795.1| methionine adenosyltransferase Bdellovibrio bacteriovorus HD100]
>gi|45657232|ref|YP—001318.1| s-adenosylmethionine synthetase protein Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130
>gi|28378057|ref|NP—784949.1| methionine adenosyltransferase Lactobacillus plantarum WCFS1
>gi|26453553|dbj|BAC43885.1| S-adenosylmethionine synthetase Mycoplasma penetrans 
>gi|24212014|sp|Q9K5E4| S-adenosylmethionine synthetase. Corynebacterium glutamicum 
>gi|18145842|dbj|BAB81883.1| S-adenosylmethionine synthetase Clostridium perfringens str. 13
>gi|13363290|dbj|BAB37241.1| methionine adenosyltransferase 1 Escherichia coli O157:H7
>gi|45443250|ref|NP—994789.1| S-adenosylmethionine synthetase Yersinia pestis biovar Mediacvails. str. 91001
>gi|44888151|sp|Q7WQX8|METK S-adenosylmethionine synthetase Borrelia burgdorferi 
>gi|44888141|sp|Q7U4S6| S-adenosylmethionine synthetase Synechococcus sp. WH8102
>gi|44888135|sp|Q7 MHK6| S-adenosylmethionine synthetase Vibrio vulnificus YJ016
>gi|23466330|ref|NP—696933.1| S-adenosylmethionine synthetase Bifidobacterium longum NCC2705]
>gi|21219978|ref|NP—625757.1| S-adenosylmethionine synthetase Streptomyces coelicolor A3(2)]
>gi|39937076|ref|NP—949352.1| methionine S-adenosyltransferase Rhodopseudomonas palustris CGA009
>gi|16766391|ref|NP—462006.1| methionine adenosyltransferase 1 Salmonella typhimurium LT2
>gi|33594910|ref|NP—882553.1| S-adenosylmethionine synthetase Bordetella parapertussis 12822
>gi|44888148|sp|Q7VRG5| S-adenosylmethionine synthetase Candidatus Blochmannia floridanus 
>gi|44888147|sp|Q7VNG71METK_HAEDU S-adenosylmethionine synthetase Haemophilus ducreyi 
>gi|448881146|sp|Q7VFY5| S-adenosylmethionine synthetase Helicobacter hepaticus 
>gi|44888145|sp|Q7VDM7| S-adenosylmethionine synthetase Prochlorococcus marinus 
>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 synthetase Porphyromonas gingivalis 
>gi|39650934|emb|CAE29457.1| methionine S-adenosyltransferase Rhodopseudomonas palustris CGA009
>gi|15792421|ref|NP—282244.1| S-adenosylmethionine synthetase Campylobacter jejuni subsp. jejuni NCTC 11168
>gi|39574954|cmb|CAE78795.1| methionine adenosyltransferase Bdellovibrio bacteriovorus HD100
>gi|45657232|ref|YP—001318.1| s-adenosylmethionine synthetase protein Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130
>gi|28378057|ref|NP—784949.1| methionine adenosyltransferase Lactobacillus plantarum WCFS1
>gi|45600470|gb|AAS69955.1| s-adenosylmethionine synthetase protein Leptospira interrogans serovar Copenhageni sr. Fiocruz L1-130
>gi|264535531 dbj|BAC43885.1| S-adenosylmethionine synthetase Mycoplasma penetrans 
>gi|18145842|dbj|BAB81883.1| S-adenosylmethionine synthetase Clostridium perfringens str. 13
>gi|13363290|dbj|BAB37241.1| methionine adenosyltransferase I Escherichia coli O157:H7
>gi|45443250|ref|NP—994789.1| S-adenosylmethionine synthetase Yersinia pestis biovar. Mediaevails str. 91001
>gi|44888153|sp|Q8CXS7| S-adenosylmethionine synthetase Leptospira interrogans 
>gi|44888151|sp|Q7WQX8| S-adenosylmethionine synthetase Bordetella bronchiseptica 
>gi|44888150|sp|Q7W200| S-adenosylmethionine synthetase Bordetella parapertussis
>gi|44888149|sp|Q7VUL5| S-adenosylmethionine synthetase Bordetella pertussis 
The modified S-adenosylmethionine synthetase preferentially exhibits a specific activity which is at least five times less than that of the wild-type enzyme.
Preferentially, the protein sequence of a modified S-adenosylmethionine synthetase contains the amino acid mutation of at least one of the sequences specified below.
In one embodiment of the invention the amino acid changes concern the cystein at position 89 or the cystein at position 239 that both reduce the activity of MetK (Reczkowski and Markham, 1995, JBC 270, 31, 18484-18490).
In a preferred embodiment at least one mutation is present in the conserved region 1, corresponding to amino acid 54 to 59 in the amino acid sequence of E. coli MetK shown in SEQ ID NO 2, the non mutated conserved region I comprising the amino acid sequence defined below:

G-E-X1-X2-X3-X4  (SEQ ID NO: 43)

wherein

X1 represents I, V, L, T preferentially I

X2 represents T, K, S, R, preferentially T

X3 represents T, S, G, preferentially T

X4 represents S, N, T, K, E, R, P, N, A preferentially S.

Preferentially the unmodified S-adenosylmethionine synthetase conserved region I has the following amino acid sequence:

G-E-I-T-T-S  (SEQ ID NO: 44).

In another preferred embodiment at least one mutation is present in the conserved region 2, corresponding to amino acid 98 to 107 in the amino acid sequence of E. coli MetK shown in SEQ ID NO 2, the non mutated conserved region 2 comprising the amino acid sequence defined below:

Q-S-X1-D-I-X2-X3-G-V-X4  (SEQ ID NO: 45)

wherein

X1 represents P, Q, A, S, preferentially P

X2 represents A, N, Q, S, F, preferentially N

X3 represents V, Q, Y, R, M, N preferentially Q

X4 represents K, D, N, T, S, A, E preferentially D.

Preferentially the unmodified S-adenosylmethionine synthetase conserved region 2 has the following amino acid sequence:

Q-S-P-D-I-N-Q-G-V-D  (SEQ ID NO: 46).

Preferentially the unmodified S-adenosylmethionine synthetase conserved region 2 has the following amino acid sequence: Q-S-P-D-I-N-Q-G-V-D.
In another preferred embodiment at least one mutation is present in the conserved region 3, corresponding to amino acid 114 to 121 in the amino acid sequence of E. coli MetK shown in SEQ ID NO 2, the non mutated conserved region 3 comprising the amino acid sequence defined below:

X1-G-A-G-D-Q-G-X2  (SEQ ID NO: 47)

wherein

X1 represents Q, A, I, V, E, T, preferentially Q

X2 represents L, I, S, V, M, preferentially L.

Preferentially the unmodified S-adenosylmethionine synthetase conserved region 3 has the following amino acid sequence:

Q-G-A-G-D-Q-G-L  (SEQ ID NO: 48).

In another preferred embodiment at least one mutation is present in the conserved region 4, corresponding to amino acid 137 to 144 in the amino acid sequence of E. coli MetK shown in SEQ ID NO 2, the non mutated conserved region 4 comprising the amino acid sequence defined below:

X1-I-X2-X3-X4-H-X5-X6  (SEQ ID NO: 49)

wherein

X1 represents S, P, T, A, preferentially P

X2 represents A, T, W, Y, F, S, N preferentially T

X3 represents M, Y, L, V preferentially Y

X4 represents S, A, preferentially A

X5 represents K, R, E, D, preferentially R

X6 represents L, I, preferentially L.

Preferentially the unmodified S-adenosylmethionine synthetase conserved region 4 has the following amino acid sequence:

P-I-T-Y-A-H-R-L  (SEQ ID NO: 50).

In another preferred embodiment at least one mutation is present in the conserved region 5, corresponding to amino acid 159 to 163 in the amino acid sequence of E. coli MetK shown in SEQ ID NO 2, the non mutated conserved region 5 comprising the amino acid sequence defined below:

X1-L-X2-X3-D  (SEQ ID NO: 51)

wherein

X1 represents W, F, Y, V, E preferentially W

X2 represents R, G, L, K, preferentially R

X3 represents P, L, H, V, preferentially P.

Preferentially the unmodified S-adenosylmethionine synthetase conserved region 5 has the following amino acid sequence:

W-L-R-P-D  (SEQ ID NO: 52).

Preferentially at least one mutation is present in the conserved region 6, corresponding to amino acid 183 to 189 in the amino acid sequence of E. coli MetK shown in SEQ ID NO 2, the non mutated conserved region 6 comprising the amino acid sequence defined below.

X1-X2-X3-S-X4-Q-H  (SEQ ID NO: 53)

In which

X1 represents V, I, preferentially V

X2 represents V, L, I, preferentially V

X3 represents V, L, I, M, preferentially L

X4 represents T, V, A, S, H, preferentially T.

In a preferred embodiment the unmodified S-adenosylmethionine synthetase conserved region 6 has the following amino acid sequence:

V-V-L-S-T-Q-H  (SEQ ID NO: 54).

Preferentially the mutations are introduced in the conserved region 7, corresponding to amino acid 224 to 230 in the amino acid sequence of E. coli MetK shown in SEQ ID NO 2, the non mutated conserved region 7 comprising the amino acid sequence defined below:

X1-N-P-X2-G-X3-F  (SEQ ID NO: 55)

In which

X1 represents I, V preferentially I

X2 represents T, G, S, preferentially T

X3 represents R, T, Q, K, S, preferentially R.

In a preferred embodiment the unmodified S-adenosylmethionine synthetase conserved region 7 has the following amino acid sequence:

I-N-P-T-G-R-F  (SEQ ID NO: 56).

Preferentially the mutations are introduced into the conserved region 8, corresponding to amino acid 231 to 237 in the amino acid sequence of E. coli MetK 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  (SEQ ID NO: 57)

In which

X1 represents T, V, I, Y, E, preferentially V

X2 represents V, I, L, N, preferentially I

X3 represents G, S, preferentially G

X4 represents M, I, Q, A, H, D preferentially M

X5 represents G, S, A, H preferentially G.

In a preferred embodiment the unmodified S-adenosylmethionine synthetase conserved region 8 has the following amino acid sequence: V-I-G-G-P-M-G (SEQ ID NO: 58).
Preferentially the mutations are introduced in the conserved region 9, corresponding to amino acid 246 to 253 in the amino acid sequence of E. coli MetK shown in SEQ ID NO 2, the non mutated conserved region 9 comprising the amino acid sequence defined below:

X1-X2-D-T-Y-G-G  (SEQ ID NO: 59)

In which

X1 represents M, I, preferentially I

X2 represents V, I preferentially I.
In a preferred embodiment the unmodified S-adenosylmethionine synthetase conserved region 9 has the following amino acid sequence:

I-V-D-T-Y-G-G  (SEQ ID NO: 60).

Preferentially the mutations are introduced into the conserved region 10, corresponding to amino acid 269 to 275 in the amino acid sequence of E. coli MetK shown in SEQ ID NO 2, the non mutated conserved region 10 comprising the amino acid sequence defined below:

K-V-D-R-S-X1-X2  (SEQ ID NO: 61)

In which

X1 represents A, G, preferentially A

X2 represents A, S, L, preferentially A.

In a preferred embodiment the unmodified S-adenosylmethionine synthetase conserved region 10 has the following amino acid sequence:

K-V-D-R-S-A-A  (SEQ ID NO: 62).

In another preferred application of the invention at least one mutation is present in the conserved region 11. The unmodified S-adenosylmethionine synthetase harbors the conserved region in its C-terminal part with the following amino acid sequence:

X1-X2-Q-X3-X4-Y-A-I-G-X5-X6  (SEQ ID NO: 63)

In which
X1 represents L, E, I, Q, T preferentially E
X2 represents V, I, L preferentially I
X3 represents V, L, I preferentially V
X4 represents A, S preferentially S
X5 represents V, I, R, K, A preferentially V
X6 represents A, V, T, S, preferentially A.
This region corresponds to amino acid 295 to 305 in the amino acid sequence of E. coli MetK shown in SEQ ID NO 2.
In a preferred embodiment the unmodified S-adenosylmethionine synthetase conserved region 11 has the following amino acid sequence:

E-I-Q-V-S-Y-A-I-G-V-A  (SEQ ID NO: 64).

In another preferred application of the invention the mutations are introduced into the N-terminus of the protein leading to a frameshift and changes in the last 6 amino acids.
In the S-adenosylmethionine synthetase with decreased activity preferentially the serine in conserved region 1 is replaced with another amino acid. In a preferred application of the invention the serine is replaced with a asparagine.
In a preferred embodiment the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 1:

G-E-I-T-T-N  (SEQ ID NO: 65).

In the S-adenosylmethionine synthetase with decreased activity preferentially the conserved glycine in conserved region 2 is replaced with another amino acid. In a preferred application of the invention the conserved glycine is replaced with a serine.
In a preferred embodiment the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 2:

Q-S-P-D-I-N-Q-S-V-D  (SEQ ID NO: 66).

In the S-adenosylmethionine synthetase with decreased activity preferentially the conserved glycine in conserved region 3 is replaced with another amino acid. In a preferred application of the invention the conserved glycine is replaced with a serine.
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  (SEQ ID NO: 67).

In the S-adenosylmethionine synthetase with decreased activity preferentially the conserved histidine and/or the semi-conserved proline in conserved region 4 is/are replaced with other amino acids. In a preferred application of the invention the conserved histidine is replaced with a tyrosine and/or the semi-conserved proline is replaced with a leucine.
In a preferred embodiment the modified S-adenosylmethionine synthetase has one of the following amino acid sequences in conserved region 4:

P-I-T-Y-A-Y-R-L  (SEQ ID NO: 68),

L-I-T-Y-A-H-R-L  (SEQ ID NO: 69),

L-I-T-Y-A-Y-R-L  (SEQ ID NO: 70).

In the S-adenosylmethionine synthetase with decreased activity preferentially the semi-conserved arginine in conserved region 5 is replaced with another amino acid. In a preferred application of the invention the arginine is replaced with a cysteine.
In a preferred embodiment the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 5:

W-L-C-P-D  (SEQ ID NO: 71).

In the S-adenosylmethionine synthetase with decreased activity preferentially the conserved histidine or semi-conserved valine in conserved region 6 is replaced with another amino acid. In a preferred application of the invention the conserved histidine is replaced with a tyrosine and/or the valine with aspartate.
In a preferred embodiment the modified S-adenosylmethionine synthetase has one of the three following amino acid sequences in conserved region 6:

V-V-L-S-T-Q-Y  (SEQ ID NO: 72)

V-D-L-S-T-Q-H  (SEQ ID NO: 73)

V-D-L-S-T-Q-Y  (SEQ ID NO: 74).

In the S-adenosylmethionine synthetase with decreased activity preferentially the semi-conserved threonine in conserved region 7 is replaced with another amino acid. In a preferred application of the invention the threonine is replaced with an isoleucine.
In a preferred embodiment the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 7:

I-N-P-I-G-R-F  (SEQ ID NO: 75).

In the S-adenosylmethionine synthetase with decreased activity preferentially the conserved proline in conserved region 8 is replaced with another amino acid. In a preferred application of the invention the proline is replaced with a serine.
In a preferred embodiment the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 8:

V-I-G-G-S-M-G  (SEQ ID NO: 76).

In the S-adenosylmethionine synthetase with decreased activity preferentially the second conserved glycine in conserved region 9 is replaced with another amino acid. In a preferred application of the invention the glycine is replaced with an aspartate.
In a preferred embodiment the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 9:

I-V-D-T-Y-G-D  (SEQ ID NO: 77).

In the S-adenosylmethionine synthetase with decreased activity preferentially the conserved serine in conserved region 10 is replaced with another amino acid. In a preferred application of the invention the serine is replaced with a phenylalanine.
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  (SEQ ID NO: 78).

In the S-adenosylmethionine synthetase with decreased activity preferentially the conserved isoleucine in conserved region 11 is replaced with other amino acids. In a preferred application of the invention isoleucine is replaced by leucine.
In a preferred embodiment the modified S-adenosylmethionine synthetase has the following amino acid sequence in conserved region 11:

E-I-Q-V-S-Y-A-L-G-V-A  (SEQ ID NO: 79).

The present invention furthermore relates to nucleotide sequences, DNA or RNA sequences, which encode a mutated 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 coding DNA sequence regions for the conserved regions 1 to 11 of the wild type metK gene, represented as wild type in SEQ ID NO. 2, the said mutation being not a silent mutation.
In a preferred application the metK gene is the S-adenosylmethionine synthetase of E. coli K12 represented by the SEQ ID NO 2 and sequences homologous to that sequence that have S-adenosylmethionine synthetase activity and that share at least 80% homology, preferentially 90% homology with the amino acid sequence of SEQ ID NO 2.
The mutated S-adenosylmethionine synthetase genes described above may be obtained by conventional techniques known to the person skilled in the art disclosed above and below, including random or targeted mutagenesis or synthetic DNA construction.
The metA gene encoding modified homoserine succinyltransferase and/or the metK gene encoding modified S-adenosylmethionine synthetase 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 both 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 (pSC101, RK2), low copy number plasmids (pACYC, pRSF1010) or high copy number plasmids (pSK bluescript II).
The metA and/or metK gene may be expressed using promoters with different strength that need or need not to be induced by inducer molecules. Examples are the promoter Ptrc, Ptac, Plac, the lambda promoter c1 or other promoters known to the expert in the field.
MetA and/or MetK expression may be boosted or reduced by elements stabilizing or destabilizing the corresponding messenger RNA (Carrier and Keasling (1998) Biotechnol. Prog. 15, 58-64) or the protein (e.g. GST tags; Amersham Biosciences).
The present invention also relates to microorganisms which contain a feedback-resistant metA allele according to the invention and eventually a metK allele with reduced activity according to the invention.
Such strains are characterized by the fact that they possess a methionine metabolism which is deregulated by at least one feedback-resistant metA allele and/or by a reduced production of SAM caused by a MetK enzyme with reduced activity.
Novel strains may be prepared from any microorganism in which methionine metabolism proceeds by the same metabolic pathway.
Gram-negative bacteria, in particular E. coli, are especially suitable.
For the purpose of expressing the modified homoserine succinyltransferase enzyme and S-adenosylmethionine synthetase, the feedback-resistant metA alleles and metK alleles with reduced activity are transformed in a host strain using customary methods. The screening for strains possessing modified homoserine succinyltransferase and S-adenosylmethionine synthetase properties is, for example, realized using enzymatic tests.
For example, the homoserine succinyltransferase activity can be determined in an enzymatic test with homoserine and succinyl-CoA as substrates. The reaction is started by adding the protein extract containing the homoserine succinyltransferase enzyme, and the formation of O-succinylhomoserine is monitored by GC-MS after protein precipitation and derivatization with a silylating reagent. Feedback inhibition is tested in the presence of methionine and S-adenosylmethionine in the reaction mixture.
S-adenosylmethionine synthetase activity can be determined in an enzymatic test with methionine and ATP as substrates. The reaction is started by adding the protein extract containing the S-adenosylmethionine synthetase enzyme, and the formation of S-adenosylmethionine is monitored by FIA-MS/MS.
Preferentially, use is made of E. coli strains in which the endogenous metA and metK genes are inactivated and complemented by novel recombinant genes.
The feedback-resistant metA alleles and the metK alleles with reduced activity render it possible to abolish the control at important biosynthetic control points, thereby amplifying the production of a large number of compounds which are situated downstream of aspartate. These include, in particular, homoserine, O-succinylhomoserine, cystathionine, homocysteine, methionine and S-adenosylmethionine.
In particular the invention relates to the preparation of L-methionine, its precursors or compounds derived thereof, by means of cultivating novel microorganisms. The above-described products are classified below as compound (I).
An increase in the production of compound (I) can be achieved by changing the expression levels or deleting the following genes implicated in the production of aspartate, a precursor of compound (I).












Gene
genbank entry
name







ackA
g1788633
acetate kinase


pta
g1788635
phosphotransacetylase


acs
g1790505
acetate synthase


aceA
g1790445
isocitrate lyase


aceB
g1790444
malate synthase


aceE
g1786304
pyruvate deydrogenase E1


aceF
g1786305
pyruvate deydrogenase E2


lpd
g1786307
pyruvate deydrogenase E3


aceK
g1790446
isocitrate dehydrogenase kinase/phosphatase


sucC
g1786948
succinyl-CoA synthetase, beta subunit


sucD
g1786949
succinyl-CoA synthetase, alpha subunit


ppc
g1790393
phosphoenolpyruvate carboxylase


pck
g1789807
phosphoenolpyruvate carboxykinase


pykA
g1788160
pyruvate kinase II


pykF
g1787965
pyruvate kinase I


poxB
g1787096
pyruvate oxidase


pps
g1787994
phosphoenolpyruvate synthase


ilvB
g1790104
acetohydroxy acid synthase I, large subunit


ilvN
g1790103
acetohydroxy acid synthase I, small subunit


ilvG
g1790202
acetohydroxy acid synthase II, large subunit



g1790203



ilvM
g1790204
acetohydroxy acid synthase II, small subunit


ilvI
g1786265
acetohydroxy acid synthase III, large subunit


ilvH
g1786266
acetohydroxy acid synthase III, small subunit


aroF
g1788953
DAHP synthetase


aroG
g1786969
DAHP synthetase


aroH
g1787996
DAHP synthetase


aspC
g1787159
aspartate aminotransferase









In addition pyruvate carboxylase, from Rhizobium etli, (accession number U51439) may be introduced by genetic engineering into E. coli and overexpressed.
An additional increase in the production of compound (I) can be achieved by overexpressing genes of the lysine/methionine/pathway, such as the homoserine synthesizing enzymes encoded by the genes thrA (homoserine dehydrogenase/aspartokinase, gI786183) or metL (homoserine dehydrogenase/aspartokinase, gI790376) or lysC (apartokinase, gI790455) or asd (aspartate semialdehyde dehydrogenase, gI789841) or a combination thereof.
A further increase in the production of (I) is possible by overexpressing genes involved in sulfate assimilation and production of cysteine. This can be achieved by overexpressing the following genes (see below) or by deregulating the pathway through the introduction of 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 decreased sensitivity for its inhibitor L-cysteine (U.S. Pat. No. 6,218,168, Denk & Bock 1987 J Gen Microbiol 133 515-25). The following genes need to be overexpressed.














CysA
g1788761
sulfate permease


CysU
g1788764
cysteine transport system


CysW
g1788762
membrans bound sulphate transport system


CysZ
g1788753
ORF upstream of cysK


cysN
g1789108
ATP sulfurylase


cysD
g1789109
sulfate adenylyltransferase


cysC
g1789107
adenylylsulfate kinase


cysH
g1789121
adenylylsulfate reductase


cysI
g1789122
sulfite reductase, alpha subunit


cysJ
g1789123
sulfite reductase, beta subunit


cysE
g1790035
serine acetyltransferase


cysK
g1788754
cysteine synthase


cysM
g2367138
O-acetyl-sulfhydrolase









In addition genes involved in the production of C1 (methyl) groups may be enhanced by overexpressing 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









In addition genes directly involved in the production of methionine may be overexpressed:














metB
g1790375
Cystathionine gamma-synthase


metC
g1789383
Cystathionine beta-lyase


metH
g1790450
B12-dependent homocysteine-N5-




methyltetrahydrofolate transmethylase


metE
g2367304
Tetrahydropteroyltriglutamate methyltransferase


metF
g1790377
5,10-Methylenetetrahydrofolate reductase


metR
g1790262
Positive regulatory gene for metE and metH and




autogenous regulation.









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
g1786311
S-Adenosylmethionine decarboxylase



speC
g1789337
Ornithine decarboxylase



thrB
g1786184
Homoserine kinase



astA
g1788043
Arginine succinyltransferase



dapA
g1788823
Dihydrodipicolinate synthase.









A further increase in the production of (1) is possible 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 gI790373).
Production of (I) may be further increased by using an altered metB allele that uses preferentially or exclusively H2S and thus produces homocysteine from O succinylhomoserine as has been described in the patent application PCT No PCT/FR04/00354, which content is incorporated herein by reference.
In another preferred embodiment, the gene encoding aspartokinase/homoserine dehydrogenase is over expressed and/or the gene encoding methionine repressor metJ is deleted in the microorganism of the present invention and in the microorganism used in the method according to the invention.
Preferably, the aspartokinase/homoserine dehydrogenase is encoded by a feed-back deregulated ThrA allele. Such feed-back deregulation may be obtained by introducing the mutation Phe318Ser in the ThrA enzyme. Enzyme position is given here by reference to the ThrA sequence disclosed in genebank accession number V00361.1 (gI786183).
The metA and metK alleles described above may be used in eukaryotes or prokaryotes. Preferentially the organism used is a prokaryote. In a preferred application the organism is either E. coli or C. glutamicum. 
The invention also concerns the process for the production of compound (I). Compound (I) is usually prepared by fermentation of the designed bacterial strain.
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 20° C. and 55° C., preferentially between 25° C. and 40° C., and more specifically about 30° C. for C. glutamicum and about 37° C. for E. coli. 
The fermentation is generally conducted in fermenters with an inorganic culture medium of known defined composition adapted to the bacteria used, containing at least one simple carbon source, and if necessary a co-substrate necessary for the production of the metabolite.
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, N.Y.) or a medium such as defined by Schaefer et al. (1999, Anal. Biochem. 270: 88-96).
Analogously, the inorganic culture medium for C. glutamicum can be of identical or similar composition to BMCG medium (Liebl et al., 1989, Appl. Microbiol. Biotechnol. 32: 205-210) or to a medium such as that described by Riedel et al. (2001, J. Mol. Microbiol. Biotechnol. 3: 573-583). The media can be supplemented to compensate for auxotrophies introduced by mutations.
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 media are well known to those skilled in the art.



FIG. 1A/B: Methionine metabolism in Escherichia coli. 
FIG. 2: Alignment of wildtype and recombinant metA genes obtained upon selection on α-methyl-methionine. Conserved residues are represented by light grey boxes and mutated residues are indicated by white boxes. The figure discloses SEQ ID NO:s 84-88, respectively, in order of appearance.
FIG. 3A: Alignment of MetA sequences from different microorganisms. The figure discloses SEQ ID NO:s 89-106, respectively, in order of appearance.
FIG. 3B: Continuation of the alignment of MetA sequences from different microorganisms depicted in FIG. 3A.
FIG. 3C: Continuation of the alignment of MetA sequences from different mircoorganisms depicted in FIG. 3B.
FIG. 4A: Alignment of MetK sequences from different microorganisms. The figure discloses SEQ ID NO:s 107-136, respectively, in order of appearance.
FIG. 4B: Continuation of alignment of MetK sequences from different microorganisms depicted in FIG. 4A.
FIG. 4C: Continuation of alignment of MetK sequences from different microorganisms depicted in FIG. 4B.
FIG. 4D: Continuation of alignment of MetK sequences from different microorganisms depcited in FIG. 4C.
FIG. 5: Amino acid replacements in the E. coli MetK protein. Amino acids above the coherent lines indicate the position and the replacing amino acid. The figure discloses SEQ ID NO: 137.



EXAMPLES
Example 1
Isolation of E. coli Mutants Containing Homoserine Transsuccinylase Enzymes which Show Decreased Feedback-Sensitivity Towards Methionine and S-Adenosylmethionine
Isolation of E. Coli Strains Growing on α-Methylmethionine
α-methylmethionine is a growth-inhibitory analogue of methionine producing an immediate effect on the growth rate of E. coli at very low concentrations (minimal inhibitory concentration of 1 μg/ml and minimal concentration for a maximal inhibition 5 μg/ml, Rowbury et al., 1968). Analogues can mimic methionine in the feedback inhibition of homoserine transsuccinylase and interfere with protein synthesis without being metabolized. Only mutant strains are able to grow in a medium containing the analogue.
E. coli was routinely grown aerobically at 37° C. in LB supplemented when needed with the appropriate antibiotic. The medium used for the α-methyl-methionine analogue resistant was a minimal medium containing (per liter): K2HPO4 8 g, Na2HPO4 2 g, (NH4)2SO4 0.75 g, (NH4)2HPO4 8 g, NH4Cl 0.13 g, citric acid 6.55 g, MgSO4 2.05 g, CaCl2 40 mg, FeSO4 40 mg, MnSO4 20 mg, CoCl2 8 mg, ZnSO4 4 mg, (NH4)2Mo2O7 2.8 mg, CuCl2 2 mg, H3BO3 1 mg. The pH was adjusted to 6.7 and the medium sterilized. Before use, glucose 10 g/l and thiamine 10 mg/l were added.
The α-methylmethionine powder commercialized by Sigma contains methionine traces. An overnight liquid culture of an E. coli strain (MG1655 ΔmetE) unable to grow without methionine addition was carried out to eliminate the methionine (coming from α-methylmethionine) from the minimal medium. The culture was centrifuged 10 minutes at 7000 rpm and the supernatant was filtered (Sartorius 0.2 μm). Agar 15 g/l was added to this minimal medium.
1*108 cells/ml from an overnight culture in minimal medium of the wild type strain (MG1655) were spread onto plates with minimal medium and α-methylmethionine after 4 washing steps in sterile water. The plates were incubated at 37° C. until colonies appeared.

Evidence of Mutations in the Coding Sequence of the metA Gene Coding for the Homoserine Transsuccinylase Enzyme.

Genomic DNA was prepared from 4 clones that grew on α-methyl-methionine at 4 mg/ml after cultivation in LB medium. The cell pellet was washed once with sterile water and resuspended in 30 μl of sterile water. Cells were broken by heating 5 minutes at. 95° C. and the debris were pelleted. The metA gene was PCR-amplified using Taq polymerase and the following primers:








MetAF (SEQ ID NO 3):



tcaccttcaacatgcaggctcgacattggc (4211759-4211788)


 


MetAR (SEQ ID NO 4):


ataaaaaaggcacccgaaggtgcctgaggt (4212857-4212828).






In three clones point mutations were detected which led to amino acid substitutions. In clone metA11 CAG was exchanged for a GAG leading to the replacement of Q by E. In metA*13 TTG was exchanged for a TTT leading to the replacement of L by F. In metA*14 GCG was exchanged for GTG leading to the replacement of A by V (see FIG. 2).
As can be seen from the alignment shown in FIG. 3 all three amino acid that are replaced are highly conserved in MetA proteins from various species.
The Mutated Homoserine Transsuccinylase Enzymes Show Decreased Feedback-Sensitivity Towards Methionine and S-adenosylmethionine.
The activity of homoserine transsuccinylase was determined in vitro E. coli strains carrying either wild-type or mutant-enzymes were cultured in rich medium with 2.5 g/l glucose and harvested at late log phase. Cells were resuspended in cold potassium phosphate buffer and sonicated on ice (Branson sonifier, 70W). After centrifugation, proteins contained in the supernatants were quantified (Bradford, 1976).
Ten μl of the extracts were incubated for 30 minutes at 25° C. with 30 mM homoserine and 4 mM succinyl-CoA. Methionine and/or S-adenosylmethionine were added as indicated. The succinylhomoserine produced by homoserine transsuccinylase enzymes was quantified by GC-MS after derivatization with tert-butyldimethylsilyltrifluoroacetamide (TBDMSTFA). L-Serine[1-13C] was included as an internal standard.
Results of homoserine transsuccinylase activities are reported in table 1 below:







TABLE 1







Specific activities of wild-type and mutant homoserine


transsuccinylase enzymes upon the addition of


inhibitors L-methionine and S-adenosyl methionine.












S-adenosylmethionine
Specific activity


Strain
Methionine mM
mM
mUI/mg proteins














metA
0
0
36.7
(100%)



0
0.1
27.9
(76%)



0
1
4.7
(12.8%)



10
0
0.4
(1.1%)



10
0.1
0.2
(0.5%)



10
1
0.1
(0.3%)


metA*11
0
0
19.2
(100%)



0
0.1
21.7
(113%)



0
1
18.5
(96%)



10
0
18.7
(97%)



10
0.1
16.9
(88%)



10
1
2.7
(14.1%)


metA*13
0
0
10.9
(100%)



0
1
12.7
(117%)



10
0
13.7
(126%)



10
1
1.0
(9.2%)


metA*14
0
0
18.0
(100%)



0
1
15.1
(83.9%)



10
0
20.8
(116%)



10
1
2.0
(11.1%)









The mutated homoserine transsuccinylase enzymes thus show decreased feedback-sensitivity towards methionine and S-adenosylmethionine.
Example 2
Isolation of E. Coli Mutants Containing S-adenosylmethionine Synthetase Enzymes with Reduced Activity
Isolation of E. Coli Strains Growing on Norleucine
Norleucine is a growth-inhibitory analogue of methionine. At higher concentrations (50 mg/l) only mutant strains are able to grow in a medium containing the analogue. Most of these mutations map to the metK and metJ loci.
E. coli was routinely grown acrobically at 37° C. in LB supplemented when needed with the appropriate antibiotic. The medium used for the norleucine analogue resistant was a minimal medium containing (per liter): K2HPO4 8 g, Na2HPO4 2 g, (NH4)2SO4 0.75 g, (NH4)2HPO4 8 g, NH4Cl 0.13 g, citric acid 6.55 g, MgSO4 2.05 g, CaCl2 40 mg, FeSO4 40 mg, MnSO4 20 mg, CoCl2 8 mg, ZnSO4 4 mg, (NH4)2Mo2O7 2.8 mg, CuCl2 2 mg, H3BO3 1 mg. The pH was adjusted to 6.7 and the medium sterilized. Before use, glucose 10 g/l and thiamine 10 mg/l were added.
1*108 cells/ml from an overnight culture in minimal medium of the wild type strain (MG1655) were spread onto plates with minimal medium and norleucine (50 to 200 g/l) after 4 washing steps in sterile water. The plates were incubated at 37° C. until colonies appeared.
Evidence of Mutations in the Coding Sequence of the metK Gene Coding for the S-Adenosylmethionine Synthetase Enzyme
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. Cells were broken by heating 5 minutes at 95° C. and the debris was extracted.
DNA was PCR-amplified with Taq polymerase using the following primers:










MetKpF:









(SEQ ID NO 05)











cccggctggaagtggcaacacg (3084372-3084393)




 



MetKR:








(SEQ ID NO 06)











gccggatgcggcgtgaacgcctatcc (3085956-3085931).







The metK gene was sequenced. In 10 clones point mutations were detected which led to amino acid substitutions. In clone metK*59/105 AGC was exchanged for AAC leading to the replacement of S by N (position 59) and GGC was exchanged for AGC leading to the replacement of G by S (position 105). In clone metK*105 GGC was exchanged for AGC leading to the replacement of G by S. In metK*115 GGC was exchanged for AGC leading to the replacement of G by S. In metK*137 CCT was replaced by CTT leading to the replacement of P by L. In metK*143 CAC was replaced by TAC leading to the replacement of H by Y. In metK*161/235 CGC was replaced by TGC leading to the replacement of R by 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 FIG. 5).
As can be seen from the alignment shown in FIG. 4 all amino acids that are replaced are conserved in MetK proteins from various species.
Recombinant S-adenosylmethionine Synthetase Enzymes with Decreased Activity
The activity of S-adenosylmethionine synthetase was determined in vitro. E. coli strains carrying either wild-type or mutant enzymes were cultured in minimal medium with 5 g/l glucose and harvested at late log phase. Cells were resuspended in cold potassium phosphate buffer and sonicated on ice (Branson sonifier, 70W). After centrifugation, proteins contained in the supernatants were quantified (Bradford, 1976).
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 methionine adenosyltransferase enzymes was quantified by FIA-MS/MS.
Results of S-adenosylmethionine synthetase activities are reported in Table 2 below:







TABLE 2







S-adenosylmethionine synthetase activities


(MAT) of WT and MetK* mutants.













MAT



Strain
Mutations
mUI/mg prot






WT
—
7.5



MetK*
H189Y
0.6



MetK*
S273F
2.2



MetK*
T227I
4.9



MetK*
G115S
0.4



MetK*
G105S
2.8



MetK*
H143T
2.5



MetK*
R161C + P235S
0.7



MetK*
S59N + G105S
2.9



MetK*
P137L
1.2



MetK*
G253D
0.2









Example 3
Construction of E. Coli Strains for the Production of O-succinylhomoserine or Methionine by Overexpressing Altered Homoserine Transsuccinylase and Other Enzymes of the Methionine Biosynthesis Pathway
For the construction of E. coli strains that allow the production of O-succinylhomoserine, a plasmid overexpressing the metL gene, coding for homoserine dehydrogenase and aspartokinase was introduced into strains harboring different alleles of metA. The plasmid overexpressing metL was constructed as follows:
The following two oligonucleotides were used:










MetLF with 32 bases (SEQ ID NO 7):




TATTCatgagtgtgattgcgcaggcaggggcg






    
    
        with
    
    


a region (lower case) homologous to the sequence (4127415 to 4127441) of the gene metL (sequence 4127415 to 4129847, reference sequence on the website http://genolist.pasteur.fr/Colibri/),
a region (upper case) that together with the sequence pertaining to metL forms a restriction site for the enzyme BspHI (underlined),










MetBLAR with 38 bases (SEQ ID NO 8):




TATAAGCTTccataaacccgaaaacatgagtaccgggc






    
    
        with
    
    


a region (lower case) homologous to the sequence (4129894 to 4129866) of the gene metL
a region (upper case) that harbors the restriction site HindIII.
The gene metL was amplified by PCR using the oligonucleotides MetLF and MetBLAR and the restriction sites BspHI and HindIII were introduced at the N- and C-terminus of the metL gene, respectively. The resulting PCR fragment was restricted by BspHI and HindIII and cloned into the vector pTRC99A (Stratagene) previously cut by NcoI and HindIII.
Plasmid preparations were examined for the presence of inserts of the correct size. The DNA sequence of metL was verified and the resulting plasmid pTRCmetL transformed into the strains harboring the alleles metA*11 and metA*13.
The activity of Homoserine dehydrogenase II (HDH) was determined in vitro. E. coli strains were cultured in minimal medium with 10 g·l−1 glucose and harvested at late log phase. Cells were resuspended in cold potassium phosphate buffer and sonicated on ice (Branson sonifier, 70W). After centrifugation, proteins contained in the supernatants were quantified (Bradford, 1976).
Thirty μl of the extracts were incubated at 30° C. in a spectrophotometer, with 25 mM homoserine and 1 mM NADP+. Potassium chloride was included to activate the enzyme. Since E. coli harbors a second gene encoding a homoserine dehydrogenase activity (thrA), threonine was added which inhibits this activity. The appearance of NADPH was monitored for 30 minutes at 340 nm.
Expression of metL from the Ptrc promoter increases drastically the HDH activity when compared to a similar strain with the plasmid.







TABLE 3







Homoserine dehydrogenase activities of the MetL


protein in the MG1655 E. coli strain harbouring


the metA*11 mutation with or without overexpressing


metL from the Ptrc promoter.










Strain
HDH in mUI/mg protein













MG1655 metA*11
11



MG1655 metA*11 pTRCmetL
117









In another application the methionine regulatory gene metJ was deleted in the strains harboring the metA*11, metA*13 and metA*14 alleles. To inactivate the metJ gene the homologous recombination strategy described by Datsenko & Wanner (2000) was used. This strategy allows the insertion of a chloramphenicol resistance cassette, while deleting most of the gene concerned. For this purpose 2 oligonucleotides were used:








DmetJF with 100 bases (SEQ ID NO 9):



Caggcaccagagtaaacattgtgttaatggacgicaatacatctggacat


 


ctaaacttctttgcgtatagattgagcaaaCATATGAATATCCTCCTTAG






    
    
        with
    
    


a region (lower case) homologous to the sequence (4126216 to 4126137) of the gene metJ (sequence 4125658 to 4125975, reference sequence on the website http://genolist.pasteur.fr/Colibri/),
a region (upper case) for the amplification of the chloramphenicol resistance cassette (reference sequence in Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645),








DmetJR with 100 bases (SEQ ID NO 10):



tgacgtaggcctgataagcgtagcgcatcaggcgattccactccgcgccg


 


ctcttttttgctttagtattcccacgtctcTGTAGGCTGGAGCTGCTTCG






    
    
        with
    
    


a region (lower case) homologous to the sequence (4125596 to 4125675) of the gene merJ
a region (upper case) for the amplification of the chloramphenicol resistance cassette.
The oligonucleotides DmetJR and DmctJF were used to amplify the chloramphenicol resistance cassette from the plasmid pKD3. The PCR product obtained was then introduced by electroporation into the strain MG1655 (pKD46) in which the Red recombinase enzyme expressed permitted the homologous recombination. The chloramphenicol resistant transformants were then selected and the insertion of the resistance cassette was verified by a PCR analysis with the oligonucleotides MetJR and MetJF defined below. The strain retained is designated MG1655 (ΔmetJ::Cm) metA*.








MetJR (SEQ ID NO 11):



ggtacagaaaccagcaggctgaggatcagc (homologous to the


 


sequence from 4125431 to 4125460).


 


MetBR (SEQ ID NO 12):


ttcgtcgtcatttaacccgctacgcactgc (homologous to the


 


sequence from 4126305 to 4126276).






The chloramphenicol resistance cassette was then eliminated. The plasmid pCP20 carrying recombinase FLP acting at the FRT sites of the chloramphenicol resistance cassette was introduced into the recombinant strains by electroporation. After a series of cultures at 42° C., the loss of the chloramphenicol resistance cassette was verified by a PCR analysis with the same oligonucleotides as those used previously. The strains retained were designated MG1655 (ΔmetJ) metA*.
Subsequently the plasmid pTRCmetL harbouring 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
Construction of Homoserine Succinyltransferase Alleles Expressing Enzymes with Further Reduced Feed-Back Sensitivity
To further reduce the sensitivity of homoserine transsuccinylase to its feed-back inhibitors methionine and SAM other metA mutants were constructed by site directed mutagenesis.
Initially the metA and metA*11 allele were cloned into the vector pTRC99A (Stratagene). The following two oligonucleotides were used:








MetA-NcoI with 49 bases (SEQ ID NO 13):



TATTAAATTACCatggcaccgattcgtgtgccggacgagctacccgccg






    
    
        with
    
    


a region (lower case) homologous to the sequence (4211862 to 4211892) of the gene metA (sequence 4211859 to 4212788, reference sequence on the website http://genolist.pasteur.fr/Colibri/),
a region (upper case) that together with the sequence pertaining to metA forms a restriction site for the enzyme NcoI (underlined),










MetA-EcoRJ with 47 bases (SEQ ID NO 14):




TATTAAATTAGaattccgactatcacagaagattaatccagcgttgg






    
    
        with
    
    


a region (lower case) homologous to the sequence (4212804 to 42127774) of the gene metA
a region (upper case) that harbors the restriction site EcoRI.
The alleles metA and metA*11 were amplified by PCR using oligonucleotides MctAF and MetAR and the restriction sites NcoI and EcoRJ were introduced at the N- and C-terminus of the metA gene, respectively. The resulting PCR fragments were restricted by NcoI and EcoRI and cloned into the vector pTRC99A (Stratagene) previously cut by NcoI and EcoRI.
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.
Enzymatic analysis of the clones expressing metA and metA*11 gave vary low MetA activity. We presumed that introducing an alanine between amino acid 1M and 2P, which was required for cloning of metA into the vector pTRC99A, caused this loss of activity. Therefore using site directed mutagenesis (Stratagene) the alanine was eliminated using the oligonucleotides:
metA-alaF (AflIII) (SEQ ID NO 15): cacacaggaaacagaccatgccgatacgtgtgccggacgagctaccc
metA-alaR (AflIII) (SEQ ID NO 16): gggtagctcgtccggcacacgtatcggcatggtctgtttcctgtgtg
Subsequently the mutants metA*15 (A27V+Q64E), metA*16 (Q64D) and metA*17 (A27V+Q64D) were constructed by site directed mutagenesis (Stratagene) using the corrected metA sequence as matrix. For the construction of pTRCmetA*15 (A27V+Q64E) the following oligonucleotides were used:










MetAA28VF (XbaI) (SEQ ID NO 17):




Gtgatgacaacttctagagtgtctggtcaggaaattcgtcc



 



MetAA28VR (XbaI) (SEQ ID NO 18):



Ggacgaatttcctgaccagacactctagaagttgtcatcac






and pTRCmetA*11 as matrix.

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










MetAQ64DF (EcoRV) (SEQ ID NO 19):




Gctttcaaactcacctttggatgtcgatatccagctgttgc



 



MetAQ64DR (EcoRV)








(SEQ ID NO 20)











gcaacagctggatatcgacatccaaaggtgagtttgaaagc







and pTRCmetA as matrix.

For the construction of pTRCmetA*17 (A27V+Q64D) the following oligonucleotides were used: MetAA28VF (XbaI) (SEQ ID NO. 17) and MetAA28VR (XbaI) (SEQ ID NO 18); and pTRCmetA*16 as matrix.
Plasmids were verified by sequencing.
To transfer the alleles metA*15, metA*16 and metA*17 onto the metA locus on the chromosome, a metA deletion was constructed in the ΔmetJ background applying the same strategy used for the metJ deletion. The following oligonucleotides were used for deleting metA:








DmetAF (4211866-4211945)









(SEQ ID NO 21)









ttcgtgtgccggacgagctacccgccgtcaatttcttgcgtgaagaaaac



 


gtctttgtgatgacaacttctcgtgcgtctTGTAGGCTGGAGCTGCTTCG


 


DmetAR (4212785-4212706)








(SEQ ID NO 22)









Atccagcguggattcatgtgccgtagatcgtatggcgtgatctggtagac



 


gtaatagttgagccagttggtaaacagtaCATATGAATATCCTCCTTAG






The region indicated in lower case corresponds to the sequence between metA and yjaB. The region in upper case is used to amplify the kanamycin resistance cassette (Datsenko & Wanner, 2000). (Numbers in parentheses correspond to the reference sequence on the website http://genolist.pasteur.fr/Colibri/).
The resulting deletion was verified using the following oligonucleotides. (Numbers in parentheses correspond to the reference sequence on the website http://genolist.pasteur.fr/Colibri/).
MetAF (SEQ ID NO 3) and MetAR (SEQ ID NO 4).
The resulting strain DmetJ DmetA was used to introduce the alleles metA*15, metA*16 and metA*17 onto the chromosome. For this purpose the plasmid pKD46 was introduced into the strain DmetJ DmetA (Datsenko and Wanner, 2000). The alleles were amplified from the vectors pTRCmetA*15, pTRCmetA*16 and pTRCmetA*17 using the following oligonucleotides:








MetArcF (4211786-4211884)









(SEQ ID NO 23)









Ggcaaattttctggttatcttcagctatctggatgtctaaacgtataagc



 


gtatgtagtgaggtaatcaggttatgccgattcgtgtgccggacgagc


 


MetArcR (4212862-4212764)








(SEQ iD NO 24)









Cggaaataaaaaaggcacccgaaggtgcctgaggtaaggtgctgaatcgc



 


ttaacgatcgactatcacagaagattaatccagcgttggattcatgtgc






The sequence in bold is homologous to the sequence of the gene metA; the rest of the sequence is adjacent to metA.
For verification by PCR the following oligonucleotides were used: MetAF (SEQ ID NO 3) and MetAR (SEQ ID NO 4).
As described above, the resulting strains were cultivated in minimal medium, crude extracts were prepared and the activity of MetA was determined. As can be seen from Tab. 4 the newly constructed alleles are less sensitive to the inhibition by methionine and SAM than the alleles described above. Especially interesting is the allele metA*15 with high intrinsic activity that cannot be inhibited by SAM and methionine.







TABLE 4







Homoserinetranssuccinylase activities of MetA


mutants in absence and presence of 100 mM methionine


and 1 mM SAM. Percentages values in parentheses


indicate the amount of remaining activity upon inhibition.











Methionine
S-adenosyl-
Specific activity


Strain
mM
methionine mM
mUI/mg proteins














metA*11 ΔmetJ
0
0
492
(100%)



100
1
14
(2.8%)


metA*11rc ΔmetJ
0
0
531
(100%)



100
1
18
(3.4%)


metA*15rc ΔmetJ
0
0
610
(100%)



100
1
733
(100%)


metA*16rc ΔmetJ
0
0
294
(100%)



100
1
143
(48.6%)


metA*17rc ΔmetJ
0
0
436
(100%)



100
1
305
(70%)









Example 5
Construction of E. coli Strains for the Production of O-Succinyl Homoserine or Methionine by Combining Feed-Back Resistant MetA Alleles with MetK Alleles with Decreased Activity
In order to transfer the recombinant metK alleles into strains harboring feedback resistant metA alleles, a kanamycin resistance cassette was introduced between the metK and galP gene.
To introduce the cassette the homologous recombination strategy described by Datsenko & Wanner (2000) is used. This strategy allows the insertion of a kanamycin resistance cassette, while deleting most of the gene concerned. For this purpose 2 oligonucleotides are used:








DMetKFscr with 100 bases (SEQ ID NO 25):



ccgcccgcacaataacatcattcttcctgatcacgtttcaccgcagatta


 


tcatcacaactgaaaccgattacaccaaccTGTAGGCTGGAGCTGCTTCG






    
    
        with
    
    


a region (lower case) homologous to the sequence of the region, 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, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645),








DmetKRscr with 100 bases (SEQ ID NO 26):



gagttatatcatcatagattaaacgctgttatctgcaattaagactttac


 


tgaaaagaaatgtaacaactgtgaaaaccgCATATGAATATCCTCCTTAG






    
    
        with
    
    


a region (lower case) homologous to the region between, the gene metK and galP (3086162 to 3086083)
a region (upper case) for the amplification of the kanamycin resistance cassette.
The oligonucleotides DMetKFscr and DmetKRscr are used to amplify the kanamycin resistance cassette from the plasmid pKD4. The PCR product obtained is then introduced by electroporation into the strain MG1655 (pKD46) in which the expressed Red recombinase enzyme allows the homologous recombination. The kanamnycin resistant transformants 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 strain retained is designated MG1655 (metK, Kin).
MetKFscr (SEQ ID NO 27): gcgcccatacggtctgattcagatgctgg (homologous to the sequence from 3085732 to 3085760).
MctKRscr (SEQ ID NO 28): gcgccagcaattacaccgatatccaggcc (homologous to the sequence from 3086418 to 3086390).
To transfer the metK-alleles, the method of phage P1 transduction is used. The protocol followed is implemented in 2 steps with the preparation of the phage lysate of the strain MG1655 (metK, Km) and then transduction into strain MG1655 ΔmetJ metA*11 pTRCmetL.
The construction of the strain (metK, Kin) is described above.
Preparation of Phage Lysate P1:

    
    
        Inoculation with 100 μl of an overnight culture of the strain MG1655 (metK, Km) 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 P1 prepared on the wild strain MG1655 (about 1·109 phase/ml)
        Shaking at 37° C. for 3 hours until all the cells were lysed
        Addition of 200 μl chloroform and vortexing
        Centrifugation for 10 min at 4500 g to eliminate cell debris
        Transfer of supernatant to a sterile tube and addition of 200 μl chloroform
        Storage of lysate at 4° C.
    
    


Transduction

    
    
        Centrifuging for 10 min at 1500 g of 5 ml of an overnight culture of the strain MG1655 ΔmetJ metA*11 pTRCmetL 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 P1 of strain MG1655 (ΔmetK, Km)
        
        
        Test tube: 100 μl of cells+100 μl of phages P1 of the strain MG1655 (ΔmetK, Km)
        Incubation for 30 min at 30° C. 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
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 strain retained is designated MG1655 ΔmetJ metA*11 pTRCmetL metK*.
The kanamycin resistance cassette can then be eliminated. The plasmid pCP20 carrying FLP recombinase acting at 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
Fermentation of E. Coli Production Strains and Analysis of Yield
Production strains were initially analyzed in small Erlenmeyer flask cultures using modified M9 medium (Anderson, 1946, Proc. Natl. Acad. Sci. USA 32:120-128) that was supplemented with 5 g/l MOPS and 5 g/l glucose. Carbenicillin was added if necessary at a concentration of 100 mg/l. An overnight culture was used to inoculate a 30 ml culture to 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 culture was agitated for 1 hour. Subsequently IPTG was added if necessary.
Extracellular metabolites were analyzed during the batch: phase. Amino acids were quantified by HPLC after OPA/Fmoc derivatization and other relevant metabolites were analysed using GC-MS after silylation.
The following results were obtained for the strains MG1655, MG1655 pTRCmetL MG1655 metA*11 pTRCmetL, MG1655 metA*13 pTRCmetL, MG1655 metA*11 ΔmetJ pTRCmetL, MG1655 metA*11 ΔmetJ pTRCmetL metK*H143Y.







TABLE 5







Specific concentrations of extracellular metabolites (mmol/g dry weight) after batch


(Erlenmeyer flask) fermentation.























MG1655










metA*11









MG1655
metJ


Product



MG1655
MG1655
MG1655
metA*11
pTRCmetL


(mmol/g)

MG1655
MG1655
metA*11
metA*13
metA*11
metJ
metK*


Strain
MG1655
metA*11
pTRCmetL
pTRCmetL
pTRCmetL
pTRCmetL
pTRCmetL
H143Y


















O-succinyl
>0.01
 0.02
0.05
0.31
0.27
0.03
0.03
0.39


homoserine










homoserine
0.01
ND
0.17
0.07
0.09
0.01
ND
0.01


threonine
>0.01
ND
0.37
0.6
0.59
0.01
0.02
0.13


methionine
>0.01
>0.01
>0.01
0.06
0.03
0.2
0.26
0.31


isoleucine
ND
ND
0.01
0.01
0.02
0.14
0.22
0.49





ND not detected.






To further boost the production of homoserine the aspartokinase/homoserine a thrA* allele with reduced feed-back resistance to threonine is expressed from the plasmid pCL1920 (Lerner & Inouyc, 1990, NAR 18, 15 p 4631) using the promoter Ptrc. For the construction of plasmid pME 107 thrA was PCR amplified from genomic DNA using the following oligonucleotides:










BspH1thrA (SEQ ID NO 29):




ttaTCATGAgagtgttgaagttcggcggtacatcagtggc



 



Sma1thrA (SEQ ID NO 30):



ttaCCCGGGccgccgccccgagcacatcaaacccgacgc






The PCR amplified fragment is cut with the restriction enzymes BspHI and SmaI and cloned into the NcoI/SmaI sites of the vector pTRC99A (Stratagene). For the expression from a low copy vector the plasmid pME101 is constructed as follows. The plasmid pCL1920 is PCR amplified using the oligonucleotides PME101F and PME101R and the BstZ171-XmnI fragment from the vector pTRC99A harboring the lacI gene and the Ptrc promoter is inserted into the amplified vector. The resulting vector and the vector harboring the thrA gene are restricted by ApaI and SmaI and the thrA containing fragment is cloned into the vector pME101. To relieve ThrA from feed-back inhibition the mutation F318S is introduced by site-directed mutagenesis (Stratagene) using the oligonucleotides ThrAF F318S for and ThrAR F318S, resulting in the vector pME107. The vector pME107 was introduced into the ΔmetJ metA*11 strains with differing metK* alleles.










PME101F (SEQ ID NO 31):




Ccgacagtaagacgggtaagcctg



 



PME101R (SEQ ID NO 32):



Agcttagtaaagccctcgctag



 



ThrAF F318S (SmaI) (SEQ ID NO 33):



Ccaatctgaaaacatggcaatgtccagcgtttctggcccggg



 



ThrAR F318S (SmaI) (SEQ ID NO 34):



Cccgggccagaaacgctggacattgccatgttattcagattgg






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 the fermentor was filled with 145 ml of modified minimal medium and inoculated with 5 ml of preculture to an optical density (OD600 nm) between 0.5 and 1.2.
The temperature of the culture was maintained constant at 37° C. and the pH was permanently adjusted to values between 6.5 and 8 using an NH4OH solution. The agitation rate was maintained between 200 and 300 rpm during the batch phase and was increased to up to 1000 rpm at the end of the fed-batch phase. The concentration of dissolved oxygen was maintained at values between 30 and 40% saturation by using a gas controller. 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/h and a progressive increase up to flow rate values between 2.5 and 3.5 ml/h. At this point the flow rate was maintained constant for 24-o 48 hours. The media of the fed was based on minimal media containing glucose at concentrations between 300 and 500 g/l.
Tab. 6 shows methionine concentrations for the strains ΔmetJ metA*11 pME107 metK*H143Y and ΔmetJ metA*11 pME107 metK*T227I compared to the reference strain ΔmetJ metA*11 pME107 after about 75 h of operation. Both strains harboring the metK* alleles attain an increased methionine concentration when compared to the reference strain. Thus the metK mutations confer an industrial advantage on methionine production by increasing the productivity of the strains.







TABLE 6







Methionine production of the reference strain


ΔmetJ metA*11 pME107 and two strains harboring the


metK* mutations after indicated time of operation including


batch and fed-batch.











Methionine


Strain
Time of operation
produced





ΔmetJ metA*11 pME107
77 h
75.5 mM


ΔmetJ metA*11 pME107 metK*H143Y
74.8 h 
87.3 mM


ΔmetJ metA*11 pME107 metK*T227I
75 h
95.0 mM

## Claims

1. A method for the preparation of methionine, its precursors or products derived thereof, in a fermentative process with a microorganism where L-homoserine is converted into O-succinylhomoserine with a homoserine transsuccinylase comprising the step of culturing the microorganism on an appropriate culture medium and recovering methionine, its precursors or products derived thereof once produced, wherein the homoserine transsuccinylase is a mutated homoserine transsuccinylase with reduced sensitivity for the feedback inhibitors S-adenosylmethionine and methionine, wherein the homoserine transsuccinylase has the sequence as shown in SEQ ID NO:1 and wherein the conserved amino acid Q at position 64 is replaced with a glutamic acid (E) or an aspartic acid (D).

2. The method as claimed in claim 1, wherein the microorganisms comprise a S-adenosylmethionine synthetase enzyme having the sequence show in SEQ ID NO: 2 with reduced S-adenosylmethionine synthetase enzymatic activity, and wherein the conserved amino acid H at position 143 is replaced with a tyrosine (Y).

3. The method as claimed in claim 1, wherein the microorganism is selected from the group consisting of prokaryotes and eukaryotes.

4. The method as claimed in claim 3 wherein the prokaryotic microorganism is Escherichia coli or Coryneybacterium glutamicum.

5. The method of claim 2, wherein the S-adenosylmethionine synthetase comprises the amino acid sequence of SEQ ID NO: 2 having a substitution at His 143.