FIELD OF THE INVENTION

The present invention relates to a recombinant microorganism useful for the production of L-methionine and/or its derivatives and process for the preparation of L-methionine. The microorganism of the invention is modified in a way that the methionine/carbon source yield is increased by combining the attenuation of the L-methionine uptake system to the overexpression of a specific export system. In particular, the operon metNIQ is deleted and the genes ygaZ and ygaH or their homologous genes are overexpressed in the recombinant microorganism.

PRIOR ART

Sulphur-containing compounds such as cysteine, homocysteine, methionine or S- adenosylmethionine are critical to cellular metabolism. In particular L-methionine, an essential amino acid, which cannot be synthesized by animals, plays an important role in many body functions. Most of the methionine produced industrially is widely used as an animal feed and food additive.

With the decreased use of animal-derived proteins as a result of BSE and chicken flu, the demand for pure methionine has increased. Commonly, D,L-methionine is produced chemically from acrolein, methyl mercaptan and hydrogen cyanide. However, the racemic mixture does not perform as well as pure L-methionine (Saunderson, 1985). Additionally, although pure L-methionine can be produced from racemic methionine, for example, through the acylase treatment of N-acetyl-D,L-methionine, this dramatically increases production costs. Accordingly, the increasing demand for pure L-methionine coupled with environmental concerns render microbial production of methionine an attractive prospect.

Other important amino acids, such as lysine, threonine and tryptophan are produced via fermentation for use in animal feed. Therefore, these amino acids can be made using glucose and other renewable resources as starting materials. Industrial production of L- methionine via fermentation has not been successful yet, but the development of the technology is on going.

Different approaches for the optimisation of L-methionine production in microorganisms have been described previously (see, for example, Patents or patent applications US 7,790,424, US 7,611,873, WO 2002/10209, WO 2005/059093 and WO 2006/008097); however, industrial production of L-methionine from microorganisms requires further improvements. When L-methionine is synthesized at a certain level or higher, it inhibits its own further production via feedback loop and disturbs the physiology of the cell. Therefore one of these improvements is to reduce the L-methionine accumulation into the microorganism to ensure an efficient production by reducing the L-methionine import capability of the microorganism while enhancing the L-methionine efflux at the same time in a recombinant L-methionine overproduces

Early biochemical and kinetic studies demonstrated that methionine uptake in Escherichia coli involves at least two specific transporters: the high-affinity MetD and low-affinity MetP transport systems (Jones & George, 1999; Kadner, 1974). Both are regulated by the internal methionine pool size and, for MetD, MetJ-mediated repression has been inferred (Kadner, 1975; Kadner & Winkler, 1975). The MetD methionine uptake system was characterized as an ABC transporter. In 2002, Merlin et al, report that the genes abc, yaeC, and yaeE comprise metD, the locus encoding a methionine uptake system. They propose to rename abc, yaeE, and yaeC as metN, metl, and metQ, respectively.

Methionine export is mediated, in Escherichia coli by the complex YgaZH and in Corynebacterium glutamicum by the homologous complex BrnFE (Trotschel et al., 2005). YgaZ is a member of the branched chain amino acid exporter (LIV-E) family responsible for export of L-valine and L-methionine. YgaZ forms a complex with YgaH, a predicted inner membrane protein, to export amino-acids under conditions in which theirs levels would be toxic to the cell.

Patent applications WO 2002/097096 and WO 2005/085463 relate to reduction of the L-methionine uptake in Coynebacterium by attenuating the MetD2 methionine uptake system, especially by deleting one or more of the genes yaeC, abc and yaeE. In Corynebacterium, the attenuation of the MetD2 methionine uptake system leads to an improved production of methionine. The homologous MetD methionine uptake system, encoded by the metN, metl and metQ genes, has been also characterized in Escherichia coli (Jones & George, 1999; Kadner 1974, Merlin et al, 2002). Patent application WO 2008/127240 discloses that in Escherichia coli as in Corynebacterium the methionine production is increased when MetD methionine uptake system is attenuated.

Patent applications EP 1239041 and WO 2008/082211 describe the overexpression of a branched chain amino acid exporter (YgaZH) responsible for the export of L-valine and L- methionine in Escherichia coli. This overexpression leads to an improved production of methionine in E. coli.

Trotschel et al. overexpressed in Corynebacterium glutamicum brnF and brnE genes encoding the BrnFE methionine exporter and in the same time deleted the metD system (Trotschel et al., 2005). Nevertheless any evidence of the impact of these modifications on the methionine production in Corynebacterium glutamicum neither in Escherichia coli has been published.

Unlike prior art on C. glutamicum and on E. coli, inventors have shown that the deletion of only metD in E. coli (achieved either by the deletion of one of the gene from the operon metNIQ or by the deletion of the entire operon, deletion of any single gene of this operon leading to abolishment of high affinity methionine uptake) is not sufficient to improve the methionine production performances. This modification must be combined to the overexpression of an L-methionine export system.

This is then the first time that the combination of the deletion of the L-methionine uptake system with the overexpression of an L-methionine export is shown as being beneficial for the methionine production.

SUMMARY OF THE INVENTION The invention relates to a recombinant Escherichia coli strain and method for optimising the production of methionine and/or its derivatives, wherein the methionine import is attenuated and the methionine efflux is enhanced. In the recombinant microorganism, methionine import is attenuated by attenuating the expression or deleting at least one gene chosen among metN, metl or metQ whereas methionine efflux is enhanced by overexpressing the genes ygaZH or their homologous genes.

The recombinant microorganism may also comprise other genetic modifications such as:

an increased expression of at least one of the following genes: ptsG, pyc, pntAB, cysP, cysU, cysW, cysA, cysM, cysJ, cysl, cysH, gcvT, gcvH, gcvP, Ipd, serA, serB, serC, cysE, metF, metH, fldA, fpr, met A, met A* allele encoding for an enzyme with reduced feed-back sensitivity to S- adenosylmethionine and/or methionine, thrA, or a thrA * allele encoding for an enzyme with reduced feed-back inhibition to threonine and/or

an attenuated expression of one of the following genes: metJ, pykA, pykF, purU, ybdL, udhA, dgsA, metE oryncA.

In a particular embodiment, the present invention is related to a recombinant microorganism wherein: a) the genes metN, metl and metQ are deleted whereas the genes ygaZ and ygaH or their homologous genes originating from Citrobacter koseri, Shigella flexneri, Raoultella ornithinolytica, Enterobacter sp., Yersinia enter ocolitica, Photorhabdus luminescens, Citrobacter youngae or Citrobacter freundii are overexpressed, and b) the expression of the genes metA *, metH, cysPUWAM, cysJIH, gcvTHP, metF, serA, serB, serC, cysE, thrA * and pyc are enhanced; and c) the expression of the genes metJ, pykA, pykF, purU, ybdL, yncA, dgsA, metE and udhA are attenuated. DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified methods and may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting, which will be limited only by the appended claims.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

Furthermore, the practice of the present invention employs, unless otherwise indicated, conventional microbiological and molecular biological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a microorganism" includes a plurality of such microorganisms, and a reference to "an endogenous gene" is a reference to one or more endogenous genes, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are now described.

In the claims that follow and in the consecutive description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise", "contain", "involve" or "include" or variations such as "comprises", "comprising", "containing", "involved", "includes", "including" are used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

The term "methionine" and "L-methionine" designate the essential sulphur-containing amino-acid with chemical formula H0 ₂ CCH(NH ₂ )CH ₂ CH ₂ SCH ₃ and CAS number 59-51- 8 or 63-68-3 for the specific L-isomer.

"Derivatives of methionine" refers to molecules analogs to methionine which present the same chemical backbone but differ from methionine with at least one chemical group. In this invention, preferred methionine derivatives are N-acetyl methionine (NAM), S- adenosyl methionine (SAM) and hydroxy-methionine (or methionine hydroxy analogue or MHA).

The term "microorganism", as used herein, refers to a bacterium, yeast or fungus which is not modified artificially. Preferentially, the microorganism is selected among Enterobacteriaceae, Bacillaceae, Streptomycetaceae and Corymb acteriaceae. More preferentially the microorganism is a species of Escherichia, Klebsiella, Pantoea, Salmonella, or Corynebacterium. Even more preferentially the microorganism of the invention is either the species Escherichia coli or Corynebacterium glutamicum.

The term "recombinant microorganism" or "genetically modified microorganism", as used herein, refers to a bacterium, yeast or fungus that is not found in nature and is genetically different from its equivalent found in nature. It means, it is modified either by introduction or by deletion or by modification of genetic elements. It can also be transformed by forcing the development and evolution of new metabolic pathways by combining directed mutagenesis and evolution under specific selection pressure (see, for example, WO 2004/076659 or WO 2007/011939).

A microorganism may be modified to express exogenous genes if these genes are introduced into the microorganism with all the elements allowing their expression in the host microorganism. The modification or "transformation" of microorganisms with exogenous DNA is a routine task for those skilled in the art.

A microorganism may be modified to modulate the expression level of an endogenous gene.

The term "endogenous gene" means that the gene was present in the microorganism before any genetic modification. Endogenous genes may be overexpressed by introducing heterologous sequences in addition to, or to replace endogenous regulatory elements, or by introducing one or more supplementary copies of the gene into the chromosome or a plasmid. Endogenous genes may also be modified to modulate their expression and/or activity. For example, mutations may be introduced into the coding sequence to modify the gene product or heterologous sequences may be introduced in addition to or to replace endogenous regulatory elements. Modulation of an endogenous gene may result in the up- regulation and/or enhancement of the activity of the gene product, or alternatively, down regulate and/or lower the activity of the endogenous gene product.

Another way to modulate their expression is to exchange the endogenous promoter of a gene (e.g., wild type promoter) with a stronger or weaker promoter to up or down regulate expression of the endogenous gene. These promoters may be homologous or heterologous. It is well within the ability of the person skilled in the art to select appropriate promoters.

Contrariwise, "exogenous gene" means that the gene was introduced into a microorganism, by means well known by the man skilled in the art whereas this gene is not naturally occurring in the microorganism. Exogenous genes may be integrated into the host chromosome, or be expressed extra-chromosomally by plasmids or vectors. A variety of plasmids, which differ with respect to their origin of replication and their copy number in the cell, are well known in the art. These genes may be homologous. In the context of the invention, the term "homologous gene" is not limited to designate genes having a theoretical common genetic ancestor, but includes genes which may be genetically unrelated that have, none the less, evolved to encode protein which perform similar functions and/or have similar structure. Therefore the term 'functional homolog" for the purpose of the present invention relates to the fact that a certain enzymatic activity may not only be provided by a specific protein of defined amino acid sequence, but also by proteins of similar sequence from other (un)related microorganisms.

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, yeast, 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.

The terms "improved methionine production", "improve methionine production" and grammatical equivalents thereof, as used herein, refer to an increased methionine/carbon source yield (ratio of gram/mol methionine produced per gram/mol carbon source consumed that it can be expressed in percent). Methods for determining the amount of carbon source consumed and of methionine produced are well known to those in the art. The yield is higher in the recombinant microorganism compared to the corresponding unmodified microorganism.

The terms "microorganism optimised for the fermentative production of methionine" refers to microorganisms evolved and/or genetically modified to present an improved methionine production in comparison with the endogenous production of the corresponding wild-type microorganisms. Such microorganisms "optimised" for methionine production are well known in the art, and have been disclosed in particular in patent applications WO 2005/111202, WO 2007/077041, WO 2009/043803 and WO 2012/098042.

According to the invention the terms "fermentative production", "culture" or "fermentation" are used to denote the growth of bacteria. This growth is generally conducted in fermenters with an appropriate culture medium adapted to the microorganism being used and containing at least one simple carbon source, and if necessary co- substrates.

An "appropriate culture medium" designates a medium (e.g., a sterile, liquid media) comprising nutrients essential or beneficial to the maintenance and/or growth of the cell such as carbon sources or carbon substrates, nitrogen sources, for example, peptone, yeast extracts, meat extracts, malt extracts, urea, ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate; phosphorus sources, for example, monopotassium phosphate or dipotassium phosphate; trace elements (e.g., metal salts), for example magnesium salts, cobalt salts and/or manganese salts; as well as growth factors such as amino acids and vitamins.

The term "carbon source" or "carbon substrate" or "source of carbon" according to the present invention denotes any source of carbon that can be used by those skilled in the art to support the normal growth of a microorganism, including monosaccharides (such as glucose, galactose, xylose, fructose or lactose), oligosaccharides, disaccharides (such as sucrose, cellobiose or maltose), molasses, starch or its derivatives, hemicelluloses and combinations thereof. An especially preferred simple carbon source is glucose. Another preferred simple carbon source is sucrose. The carbon source can be derived from renewable feed-stock. Renewable feed-stock is defined as raw material required for certain industrial processes that can be regenerated within a brief delay and in sufficient amount to permit its transformation into the desired product. Vegetal biomass treated or not, is an interesting renewable carbon source.

The term "source of sulphur" according to the invention refers to sulphate, thiosulfate, hydrogen sulphide, dithionate, dithionite, sulphite, methylmercaptan, dimethylsulfide and other methyl capped sulphides or a combination of the different sources. More preferentially, the sulphur source in the culture medium is sulphate or thiosulfate or a mixture thereof.

The terms "source of nitrogen" corresponds to either an ammonium salt or ammoniac gas. The nitrogen source is supplied in the form of ammonium or ammoniac.

The terms "attenuation" or "expression attenuated" mean in this context that the expression of a gene or the production of an enzyme is decreased or suppressed compared to the non modified microorganism leading to a decrease in the intracellular concentration of a ribonucleic acid, a protein or an enzyme compared to the non modified microorganism. The man skilled in the art knows different means and methods to measure ribonucleic acid concentration or protein concentration in the cell including for instance use of Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Real-time Polymerase Chain Reaction (qPCR) to determine ribonucleic acid concentration and use of specific antibody to determine concentration of specific protein.

Decrease or suppression of the production of an enzyme is obtained by the attenuation of the expression of gene encoding said enzyme.

Attenuation of genes may be achieved by means and methods known to the man skilled in the art. Generally, attenuation of gene expression may be achieved by:

- Mutating the coding region or the promoter region or,

- Deleting of all or a part of the promoter region necessary for the gene expression or, - Deleting of all or a part of the coding region of the gene by homologous recombination or,

- Inserting an external element into coding region or into promoter region or,

- Expressing the gene under control of a weak promoter or an inducible promoter.

The man skilled in the art knows a variety of promoters which exhibit different strength and which promoter to use for a weak or an inducible genetic expression.

The term "activity" of an enzyme is used interchangeably with the term "function" and designates, in the context of the invention, the reaction that is catalyzed by the enzyme. The man skilled in the art knows how to measure the enzymatic activity of said enzyme.

The terms "attenuated activity" or "reduced activity" of an enzyme mean either a reduced specific catalytic activity of the protein obtained by mutation in the aminoacids sequence and/or decreased concentrations of the protein in the cell obtained by mutation of the nucleotidic sequence or by deletion of the coding region of the gene.

The terms "enhanced activity" or "increased activity'Of an enzyme designate either an increased specific catalytic activity of the enzyme, and/or an increased quantity/availability of the enzyme in the cell, obtained for example by overexpressing the gene encoding the enzyme.

The terms "increased expression", "enhanced expression" or "overexpression" and grammatical equivalents thereof, are used interchangeably in the text and have a similar meaning. These terms mean that the expression of a gene or the production of an enzyme is increased compared to the non modified microorganism leading to an increase in the intracellular concentration of a ribonucleic acid, a protein or an enzyme compared to the non modified microorganism. The man skilled in the art knows different means and methods to measure ribonucleic acid concentration or protein concentration in the cell including for use of Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Real-time Polymerase Chain Reaction (qPCR) to determine ribonucleic acid concentration and use of specific antibody to determine concentration of specific protein.

Increase production of an enzyme is obtained by increasing expression of the gene encoding said enzyme.

To increase the expression of a gene, the man skilled in the art knows different techniques such as:

- Increasing the number of copies of the gene in the microorganism. The gene is encoded chromosomally or extrachromosomally. When the gene is located on the chromosome, several copies of the gene can be introduced on the chromosome by methods of recombination, known by the expert in the field (including gene replacement). When the gene is located extra-chromosomally, it 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. These plasmids are present in the microorganism in 1 to 5 copies, or about 20 copies, or up to 500 copies, depending on the nature of the plasmid : low copy number plasmids with tight replication (pSClOl , RK2), low copy number plasmids (pACYC, pRSFlOlO) or high copy number plasmids (pSK bluescript II).

- Using a promoter leading to a high level of expression of the gene. The man skilled in the art knows which promoters are the most convenient, for example promoters Ptrc, Ptoc, P/ac, or the lambda promoter cl are widely used. These promoters can be "inducible" by a particular compound or by specific external condition like temperature or light. These promoters may be homologous or heterologous.

- Attenuating the activity or the expression of a transcription repressor, specific or non-specific of the gene.

- Using elements stabilizing the corresponding messenger RNA (Carrier and Keasling, 1999) or elements stabilizing the protein (e.g., GST tags, GE Healthcare).

The terms "encoding" or "coding" refer to the process by which a polynucleotide, through the mechanisms of transcription and translation, produces an amino-acid sequence. The gene(s) encoding the enzyme(s) can be exogenous or endogenous.

The terms "feed-back sensitivity" or "feed-back inhibition" refer to a cellular mechanism control in which an or several enzyme that catalyse the production of a particular substance in the cell are inhibited or less active when that substance has accumulated to a certain level. So the terms "reduced feed-back sensitivity" or "reduced feed-back inhibition" mean that the activity of such a mechanism is decreased or suppressed compared to a non modified microorganism. The man skilled in the art knows how to modify the enzyme to obtain this result. Such modifications have been described in the patent application WO 2005/111202 or in the patent US 7,611,873.

In a first aspect of the invention, a recombinant Escherichia coli strain is optimised for the fermentative production of methionine and/or its derivatives by attenuating the methionine uptake and by enhancing the methionine efflux in said microorganism..

As described above, methionine import is mediated by the MetD methionine uptake system encoded by the metN, metl and metQ genes, formerly named abc, yaeE, and yaeC respectively. These genes have been identified in several microorganisms included E. coli and C. glutamicum. MetNIQ belongs to the famous ABC-transporter family.

In one embodiment of the invention, the expression of at least one gene chosen among metN, metl and metQ is attenuated in the recombinant microorganism. The man skilled in the art knows different means to attenuate gene expression like cloning the gene to be attenuated under control of an inducible or weak promoter, deleting all or part of the promoter region or coding region of the gene to be attenuated. Preferably, at least one of the genes metN, metl and metQ is deleted. More preferably, the three genes metN, metl and metQ are deleted in the recombinant microorganism of the invention. In amino-acid producer microorganisms, methionine is excreted by a specific efflux transporter. Notably, in E. coli, this transporter is called YgaZH and is encoded by the ygaZ and ygaH genes whereas in C. glutamicum, it is named BrnFE and is encoded by the brnF and brnE genes. Functional homologues of this methionine efflux system have been identified in several other microorganisms. Alternatively, the recombinant microorganism of the invention may overexpress functional homologues of YgaZH or BrnFE systems. YgaZ and YgaH homologous protein are presented respectively in Table 1 and Table 2.

Table 1 : YgaZ homologous proteins

Table 2: YgaH homologous proteins

Acession

Number Name Organism

YP 001455540.1 hypothetical protein CKO 04032 [Citrobacter koseri

Citrobacter koseri ABV15104.1 ATCC BAA-895]

WP 005122930.1 branched-chain amino acid ABC transporter

Shigella flexneri

EIQ78634.1 permease [Shigella flexneri]

YP 007877062.1

L-valine exporter [Raoultella ornithinolytica B6] Raoultella ornithinolytica AGJ89510.1

YP 008107734.1

branched-chain amino acid ABC transporter

WP 020454910.1 Enterobacter sp.

permease [Enterobacter sp. R4-368]

AGN85394.1

WP 004959351.1 branched-chain amino acid ABC transporter

Serratia odorifera

EFE95944.1 permease [Serratia odorifera]

YP 003884335.1 hypothetical protein Dda3937_00895 [Dickeya

Dickeya dadantii

ADM99778.1 dadantii 3937]

hypothetical protein PCC21_033290 Pectobacterium

YP 006647985.1

[Pectobacterium carotovorum subsp. carotovorum carotovorum subsp.

AFR04732.1

PCC21] carotovorum

YP 001007413.1 hypothetical protein YE3239 [Yersinia enterocolitica Yersinia enterocolitica CAL 13269.1 subsp. enterocolitica 8081] subsp. enterocolitica

NP 928589.1 hypothetical protein plu1278 [Photorhabdus Photorhabdus luminescens CAE 13572.1 luminescens subsp. laumondii TT01] subsp. laumondii With accession number disclosed in the tables for each homo log the man skilled in the art is able to obtain the amino acid sequence and its nuceotidic coding sequence on NCBI databases for instance.

From the amino acid sequence or nucleotidic sequence, it is a routine task for the man skilled in the art to obtain genes encoding these homologues. It can be done either by artificial synthesis of the gene coding the protein of interest from its amino acid sequence or by PCR amplification of the coding region of interest from the corresponding genomic DNA. In the context of the invention, these genes are called "j aZ or ygaH homologous genes". The sequences of these ygaZH homologous genes may be adjusted to the codon bias of the host microorganism.

In a specific embodiment of the invention, the recombinant microorganism overexpresses the genes ygaZ and ygaH coding the proteins whose sequences are respectively disclosed in SEQ ID NO: 1 and SEQ ID NO: 2 or their homologous genes. Preferably, ygaZ and ygaH homologous genes are composed by the gene pair originating from the same organism and composed by the homologous gene of ygaZ and the homologous gene of ygaH. However mismatch pair of an ygaZ homologous gene from a first organism and an ygaH homologous gene from a second organism coud be used.

YgaZH homologous genes are chosen among genes encoding the YgaZ and YgaH homologues disclosed respectively in table 1 and in table 2. Preferably, ygaZH homologous genes are chosen among genes encoding YgaZH homologues from Citrobacter species, Shigella species, Raoultella species, Enterobacter species, Yersinia species and Photorhabdus species. More preferably ygaZH homologous genes originate from Citrobacter koseri, Shigella flexneri, Raoultella ornithinolytica, Enterobacter sp., Yersinia enter ocolitica, Photorhabdus luminescens, Citrobacter youngae or Citrobacter freundii. Most preferably, ygaZH homologous genes originate from Citrobacter koseri, Citrobacter youngae, Citrobacter freundii or Enterobacter sp..

Therefore, ygaZH homologous genes are preferably chosen among genes coding the pair of YgaZ homologue and YgaH homologue defined respectively by: SEQ ID NO: 3 and SEQ ID NO: 4 from Citrobacter koseri, SEQ ID NO: 5 and SEQ ID NO: 6 from Shigella flexneri, SEQ ID NO: 7 and SEQ ID NO: 8 from Raoultella ornithinolytica, SEQ ID NO: 9 and SEQ ID NO: 10 from Enterobacter sp. (R4-368), SEQ ID NO: 11 or 12 and SEQ ID NO: 13 or 14 from Yersinia enterocolitica subsp. enter ocolitica, SEQ ID NO: 15 and SEQ ID NO: 16 from Photorhabdus luminescens subsp. laumondii, SEQ ID NO: 17 and SEQ ID NO: 18 from Citrobacter youngae, SEQ ID NO: 19 and SEQ ID NO: 20 from Citrobacter freundii. In a specific embodiment, the recombinant microorganism is characterized by:

attenuation of at least one of the gene metN, metl or metQ; and overexpression of the genes ygaZH or their homologous genes originating from Citrobacter koseri, Shigella flexneri, Raoultella ornithinolytica, Enterobacter sp., Yersinia enter ocolitica, Photorhabdus luminescens, Citrobacter youngae or Citrobacter freundii.

In another specific embodiment, the recombinant microorganism is characterized by:

attenuation of the gene metN; and

overexpression of the genes ygaZH or their homologous genes Citrobacter koseri, Shigella flexneri, Raoultella ornithinolytica, Enterobacter sp., Yersinia enter ocolitica, Photorhabdus luminescens, Citrobacter youngae or Citrobacter freundii.

In another specific embodiment, the recombinant microorganism is characterized by:

attenuation of the gene metl; and

overexpression of the genes ygaZH or their homologous genes Citrobacter koseri, Shigella flexneri, Raoultella ornithinolytica, Enterobacter sp., Yersinia enter ocolitica, Photorhabdus luminescens, Citrobacter youngae or Citrobacter freundii.

In another specific embodiment, the recombinant microorganism is characterized by:

attenuation of the gene metQ; and

overexpression of the genes ygaZH or their homologous genes Citrobacter koseri, Shigella flexneri, Raoultella ornithinolytica, Enterobacter sp., Yersinia enter ocolitica, Photorhabdus luminescens, Citrobacter youngae or Citrobacter freundii.

In another specific embodiment, the recombinant microorganism is characterized by:

attenuation of the genes metN, metl and metQ; and

overexpression of the genes ygaZH or their homologous genes Citrobacter koseri, Shigella flexneri, Raoultella ornithinolytica, Enterobacter sp., Yersinia enter ocolitica, Photorhabdus luminescens, Citrobacter youngae or Citrobacter freundii.

In a preferred embodiment of the invention, these genes are overexpressed under the control of an inducible promoter. The man skilled in the art knows such inducible promoters. For instance, promoters like P _R or XP _L may be used to overexpress ygaZH genes or ygaZH homologous genes originating from Citrobacter koseri, Shigella flexneri, Raoultella ornithinolytica, Enterobacter sp., Yersinia enter ocolitica, Photorhabdus luminescens, Citrobacter youngae or Citrobacter freundii in the recombinant microorganism of the invention. It is another object of the invention to identify ygaZH homologous genes and to overexpress said genes in amino-acid producer microorganism, alone or in combination with other genetic modifications as disclosed below. Optimisation of methionine biosynthesis pathway

The recombinant microorganism according to the invention is modified for improving the production of methionine. Genes involved in methionine production are well known in the art, and comprise genes involved in the methionine specific biosynthesis pathway as well as genes involved in precursor-providing pathways and genes involved in methionine consuming pathways.

Efficient production of methionine requires the optimisation of the methionine specific pathway and several precursor-providing pathways. Methionine producing strains have already been described, in particular in patent applications WO 2005/111202, WO 2007/077041 and WO 2009/043803. These applications are incorporated as reference into this application.

Except otherwise stated, all the genes mentioned below concerning optimisation of methionine biosynthesis pathway are referring to those from E. coli.

In a specific embodiment of the invention, the recombinant microorganism is modified as described below: the expression of at least one gene chosen among ptsG, pyc, pntAB, cysP, cysll, cysW, cysA, cysM, cysJ, cysl, cysH, gcvT, gcvH, gcvP, Ipd, serA, serB, serC, cysE, metF, metH, fldA, fpr, met A, met A * allele encoding for an enzyme with reduced feedback sensitivity to S-adenosylmethionine and/or methionine, thrA, and thrA * allele encoding for an enzyme with reduced feed-back inhibition to threonine is increased.

• ptsG encodes the PTS enzyme IICB ^Glc as described in patent application WO 2013/001055.

• pyc encodes a pyruvate carboxylase as described in patent application WO2013/001055. In a preferred embodiment, the pyc gene is heterologous and is chosen from pyc genes from Rhizobium etli, Bacillus subtilis, Lactococcus lactis, Pseudomonas fluorescens or Corynebacterium species,

· pntAB encode subunits of a membrane-bound transhydrogenase, such as described in patent application WO 2012/055798,

• cysP encodes a periplasmic sulphate binding protein, as described in WO 2007/077041 and in WO 2009/043803,

• cysll encodes a component of sulphate ABC transporter, as described in WO 2007/077041 and in WO 2009/043803,

• cysW encodes a membrane bound sulphate transport protein, as described in WO 2007/077041 and in WO 2009/043803, • cysA encodes a sulphate permease, as described in WO 2007/077041 and in WO 2009/043803,

• cysM encodes an O-acetyl serine sulfhydralase, as described in WO 2007/077041 and in WO 2009/043803,

· cysl and cysJ encode respectively the alpha and beta subunits of a sulfite reductase as described in WO 2007/077041 and in WO 2009/043803. Preferably cysl and cysJ are overexpressed together,

• cysH encodes an adenylylsulfate reductase, as described in WO2007/077041 and in WO 2009/043803.

Increasing CI metabolism is also a modification that leads to improved methionine production. It relates to the increase of the activity of at least one enzyme involved in the CI metabolism chosen among GcvTHP, Lpd, MetF or MetH. In a preferred embodiment of the invention, the one carbon metabolism is increased by enhancing the expression and/or the activity of at least one of the following:

· gcvT, gcvH, gcvP, and Ipd, coding for the glycine cleavage complex, as described in patent application WO 2007/077041. The glycine-cleavage complex (GCV) is a multienzyme complex that catalyzes the oxidation of glycine, yielding carbon dioxide, ammonia, methylene-THF and a reduced pyridine nucleotide. The GCV complex consists of four protein components, the glycine dehydrogenase said P-protein (GcvP), the lipoyl- GcvH-protein said H-protein (GcvH), the aminomethyltransferase said T-protein (GcvT), and the dihydrolipoamide dehydrogenase said L-protein (GcvL or Lpd). P-protein catalyzes the pyridoxal phosphate-dependent liberation of C02 from glycine, leaving a methylamine moiety. The methylamine moiety is transferred to the lipoic acid group of the H-protein, which is bound to the P-protein prior to decarboxylation of glycine. The T- protein catalyzes the release of NH3 from the methylamine group and transfers the remaining CI unit to THF, forming methylene-THF. The L protein then oxidizes the lipoic acid component of the H-protein and transfers the electrons to NAD ⁺ , forming NADH;

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

· MetH (B12-dependent homocysteine-N5-methyltetrahydrofolate transmethylase) encoding methyltransferases.

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

• serA which encodes a phosphoglycerate dehydrogenase, as described in WO 2007/077041 and in WO 2009/043803,

• serB which encodes a phosphoserine phosphatase, as described in WO 2007/077041 and in WO 2009/043803, • serC which encodes a phosphoserine aminotransferase, as described in WO 2007/077041 and in WO 2009/043803.

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

· cysE encodes a serine acyltransferase; its overexpression allows an increase in methionine production, as described in WO 2007/077041;

• metA encodes a homoserine succinyltransferase. The allele metA * codes for an enzyme with reduced feed-back sensitivity to S-adenosylmethionine and/or methionine. Preferentially, the allele metA* described in the patent application WO 2005/111202 is used;

• thrA encodes an aspartokinase /homoserine dehydrogenase; the thrA * allele codes for an enzyme with reduced feed-back inhibition to threonine, as described in WO 2005/111202.

In a specific embodiment of the invention, genes may be under control of an inducible promoter. In a preferred embodiment of the invention, at least one of these genes is under the control of a temperature inducible promoter. Preferably, the expression of at least one of the genes: thrA, cysE, metA, is under the control of an inducible promoter, directly or indirectly. More preferably, the genes thrA, cysE and metA are under control of an inducible promoter, directly or indirectly. In a preferred embodiment of the invention, expression of thrA gene is under direct control of an inducible promoter and expression of cysE gene is under polar effect of inducible expression of thrA gene. In another preferred embodiment of the invention, expression of thrA gene is under direct control of an inducible promoter and expressions of cysE and metA genes are under polar effect of inducible expression of thrA gene.

In a most preferred embodiment, the temperature inducible promoter belongs to the family of P _R promoters. A methionine producing strain having genes under control of inducible promoters is described in patent application WO 2011/073122.

In another specific embodiment of the invention, the microorganism has been further modified, and the expression of at least one of the following genes is attenuated: metJ, pykA, pykF, purU, ybdL, yncA, metE, dgsA or udhA.

• the gene metJ codes for the repressor protein MetJ (GenBank 1790373), responsible for the down-regulation of the methionine regulon as was suggested in patent application JP 2000/157267,

• The genes pykA and pykF code for the enzymes 'pyruvate kinase'. The attenuation of the expression of at least one or both of the pyruvate kinases decrease the consumption of phosphoenol pyruvate (PEP). Increased availability of PEP can increase the production of oxaloacetate, an important precursor of aspartate, which in turn is a precursor of methionine, as described in WO 2007/077041 and in WO 2009/043803, • purU codes for a formyltetrahydro folate deformylase, an enzyme that catalyzes the formyl-THF deformylase reaction. The attenuation of the deformylase activity increases the production of methyl-THF that is required for methylation of homocysteine. Loss of CI metabolites by deformylation leads to an increased production of homocysteine that cannot be transformed into methionine. Homocysteine can then be a substrate for the enzyme cystathionine gamma synthase (MetB) that can catalyze the reaction between O- succinylhomoserine and homocysteine resulting in the production of homolanthionine, as described in WO 2007/077041 and in WO 2009/043803,

• ybdL encodes an aminotransferase as described in patent application WO 2012/090021,

• yncA encodes a N-acyltransferase, as described in patent application WO 2010/020681,

• metE encodes a cobalamin-independent methionine synthase, as described in patent application PCT/IB2012/001336,

· dgsA, better known as Mlc, encodes a transcriptional dual regulator that controls the expression of genes encoding enzymes of the phosphotransferase (PTS) and phosphoenolpyruvate (PEP) systems as described in patent application WO 2013/001055,

• udhA encodes soluble pyridine nucleotide transhydrogenase, as described in patent application WO 2012/055798.

In a more preferred embodiment of the invention, the fermentative production of methionine and/or its derivatives by a recombinant microorganism, wherein the methionine import is attenuated and the methionine efflux is enhanced, from glucose as a main carbon source, may be achieved through a combination of the above discussed modifications in said microorganism, for example:

the expression of the gene metJ is attenuated and the expression of a met A * allele encoding for an enzyme with reduced feed-back sensitivity to S- adenosylmethionine and/or methionine (MetA *) is enhanced;

the expression of the gene metJ is attenuated; the expression of a metA * allele encoding for an enzyme with reduced feed-back sensitivity to S- adenosylmethionine and/or methionine (MetA *) is enhanced; and the expression of a thrA * allele encoding for an enzyme with reduced feed-back inhibition to threonine (thrA *) is enhanced;

the expression of the gene metJ is attenuated; the expression of a metA * allele encoding for an enzyme with reduced feed-back sensitivity to S- adenosylmethionine and/or methionine (MetA *) is enhanced; the expression of a thrA * allele encoding for an enzyme with reduced feed-back inhibition to threonine (thrA *) is enhanced; and the expression of the gene cysE is enhanced; the expression of the gene metJ is attenuated; the expression of a metA * allele encoding for an enzyme with reduced feed-back sensitivity to S- adenosylmethionine and/or methionine (MetA *) is enhanced; the expression of a thrA * allele encoding for an enzyme with reduced feed-back inhibition to threonine (thrA *) is enhanced; the expression of the gene cysE is enhanced; and the expression of the genes metF and/or metH is enhanced.

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

· the genes metN, metl, metQ are deleted and the genes ygaZ and ygaH or their homologous genes originating from Citrobacter koseri, Shigella flexneri, Raoultella ornithinolytica, Enterobacter sp., Yersinia enter ocolitica, Photorhabdus luminescens, Citrobacter youngae or Citrobacter freundii are overexpressed,

· the expression of the genes metA *, metH, cysPUWAM, cysJIH, gcvTHP, metF, serA, serB, serC, cysE, thrA *, ptsG and pyc are enhanced, and • the expression of the genes met J, pykA, pykF, purJ, metE, dgsA and yncA are attenuated.

In a particular embodiment of the invention, the microorganism is from the bacterial family Enterobacteriaceae or Corynebacteriaceae.

Preferentially, the microorganism is Escherichia coli or Corynebacterium glutamicum. More preferentially the microorganism of the invention is E. coli.

Culture conditions

In a second aspect of the invention, a method is optimised for the fermentative production of methionine and/or its derivatives. It comprises the followings steps:

Culturing a recombinant microorganism wherein the methionine import is attenuated by attenuating the expression of at least one gene among metN, metl, metQ and the methionine efflux is enhanced by overexpressing the genes ygaZH or their homologous genes in an appropriate culture medium comprising a fermentable source of carbon and a source of sulphur, and,

Recovering methionine and/or its derivatives from the culture medium.

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. For E. coli, the culture medium can be of identical or similar composition to an M9 medium (Anderson, 1946), an M63 medium (Miller, 1992); or a medium such as defined by Schaefer et al, (1999).

For C. glutamicum, the culture medium can be of identical or similar composition to BMCG medium (Liebl et al., 1989) or to a medium such as described by Riedel et al., (2001).

In the method of the invention, the ygaZH homologous genes which are overexpressed in the recombinant microorganism are preferably chosen among the group consisting in homologous genes from Citrobacter species, Shigella species, Raoultella species, Enterobacter species, Yersinia species and Photorhabdus species, and more preferably originate from Citrobacter koseri, Shigella flexneri, Raoultella ornithinolytica, Enterobacter sp., Yersinia enterocolitica, Photorhabdus luminescens, Citrobacter youngae or Citrobacter freundii.

According to a specific aspect of the invention, the method is performed with a recombinant microorganism that comprises :

a. deletion of at least one gene chosen among metN, metl or metQ, and b. overexpression of the genes ygaZH or their homologous genes.

In this specific aspect of the method of the invention, said ygaZH homologous genes are preferably chosen among the group consisting in homologous genes from Citrobacter species, Shigella species, Raoultella species, Enterobacter species, Yersinia species and Photorhabdus species, and more preferably chosen among the groups consisting in homologous genes from Citrobacter koseri, Shigella flexneri, Raoultella ornithinolytica, Enterobacter sp., Yersinia enterocolitica, Photorhabdus luminescens, Citrobacter youngae or Citrobacter freundii.

In the method of the invention, the ygaZH homologous genes which are overexpressed in the recombinant microorganism are most preferably originating from Citrobacter koseri, Citrobacter youngae, Citrobacter freundii or Enterobacter sp..

In some embodiment of the invention, the growth of the recombinant microorganism is subjected to a limitation or starvation for one or several inorganic substrate, in particular phosphate and/or potassium, in the culture medium. It refers to condition under which growth of the microorganisms is governed by the quantity of an inorganic chemical supplied that still permits weak growth. Such limitation in microorganism growth has been described in the patent application WO 2009/043372. In a preferred embodiment of the invention, the culture is subjected to phosphate limitation.In a particular embodiment of the method of the invention, the recombinant microorganism is from the bacterial family Enterobacteriaceae or Corynebacteriaceae. Preferentially, the recombinant microorganism is Escherichia coli or Cory neb acterium glutamicum, and more preferentially the recombinant microorganism of the invention is E. coli. The action of "recovering methionine and/or its derivatives from the culture medium" designates the action of recovering L-methionine and/or one of its derivatives, in particular N-acetyl methionine (NAM) and S-adenosyl methionine (SAM) and all other derivatives that may be useful such as hydroxy-methionine (or methionine hydroxy analogue or MHA). The methods for the recovery and purification of the produced compounds are well known to those skilled in the art (see in particular WO 2005/007862, WO 2005/059155). Preferably, the step of recovering methionine and/or its derivatives comprises a step of concentration of methionine and/or its derivatives in the fermentation broth.

The amount of product in the fermentation medium can be determined using a number of methods known in the art, for example, high performance liquid chromatography (HPLC) or gas chromatography (GC). For example the quantity of methionine obtained in the medium is measured by HPLC after OPA/Fmoc derivatization using L-methionine (Fluka, Ref 64319) as a standard. The amount of NAM is determinated using refractometric HPLC using NAM (Sigma, Ref 01310) as a standard.

EXAMPLES

The present invention is further defined in the following examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From above disclosure and these examples, the man skilled in the art can make various changes of the invention to adapt it to various uses and conditions without modify the essentials means of the invention.

In particular, examples show modified Escherichia coli (E. coli) strains, but these modifications can easily be performed in other microorganisms of the same family.

Escherichia coli belongs to the Enterobacteriaceae family, which comprises members that are Gram-negative, rod-shaped, non- spore forming and are typically 1-5 μιη in length. Most members have flagella used to move about, but a few genera are non-motile. Many members of this family are a normal part of the gut flora found in the intestines of humans and other animals, while others are found in water or soil, or are parasites on a variety of different animals and plants. E. coli is one of the most important model organisms, but other important members of the Enterobacteriaceae family include Klebsiella, in particular Klebsiella terrigena, Klebsiella planticola or Klebsiella oxytoca, and Salmonella.

Moreover, several patent applications point out that optimisation for methionine production can easily be applied in E. coli and in Corynebacterium glutamicum without undue experimentation. PROTOCOLS

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

Protocol 1 (Chromosomal modifications by homologous recombination and selection of recombinants) and protocol 2 (Transduction of phage PI) used in this invention have been fully described in patent application WO 2013/001055.

Protocol 3: Construction of recombinant plasmids

Recombinant DNA technology is well described and known by the man skilled in the art. Briefly, the DNA fragments were PCR amplified using oligonucleotides (that the person skilled in the art will be able to define) and MG1655 genomic DNA as matrix. The DNA fragments and chosen plasmid were digested with compatible restriction enzyme (that the person skilled in the art is able to define), then ligated and transformed in competent cells. Trans formants were analysed and recombinant plasmid of interest were verified by DNA sequencing.

Table 3 : oligonucleotides sequences cited in the following examples

Protocol 4: Plasmid curing

This plasmid curing method is based on the high- voltage electroporation which is usually used to transform DNA. For plasmid curing, the principle is rather the same except that no DNA is added to cell before the electric shock (Heery et al, 1989).

DNA transformation technologies are well described and known by the man skilled in the art.

Briefly, the strain for which the plasmid has to be removed was cultured until exponential growth phase. Then, the cells were pelleted and washed three times in sterile deionised water. The cells were incubated on ice for five to ten minutes before to go through one electric pulse at 2.50kV, 25μΡ (time constant approximate 4.5ms). One mL of SOC buffer was added immediately after pulsing and the cells were grown at appropriate temperature for one to two hours before plating on non-selective media for plasmid to get rid (antibiotics are added according to the other plasmids to keep into the strain). After isolation of the cured cells, the absence of plasmid was verified. EXAMPLE 1: Overexpression of a L-methionine secretion system in a L-methionine overproducer E.coli recombinant strain - Construction of strain 1

Methionine producing strains 16 described in patent application WO 2013/001055 (which is incorporated as reference into this application) was used as recipient strain. This strain contains the mutation in metE gene disclosed in patent application WO2013/190343.

The gene encoding the cobalamin-dependent methionine synthase, metH, was overproduced by using the same promoter and ribosome binding site as described in patent application WO 2007/077041 and a bacterial artificial chromosome (pCClBAC, Epicentre). More precisely, metH gene and the artificial promoter were cloned into the pCClBAC type plasmid contained in strain 17 described in patent application WO 2013/001055. This plasmid was named pMEl 109.

In parallel, genes fldA and fpr encoding for the reactivation system of MetH, were overexpressed from the moderate plasmid copy number pCL1920 (Lerner & Inouye, 1990) by using their natural promoters. This plasmid was named pME1089.

Thirdly, the genes ygaZH encoding the exporter of methionine, were overexpressed. They were cloned on the moderate plasmid copy number pCL1920 (Lerner & Inouye, 1990) with the use of the natural promoter of ygaZ. More precisely, ygaZH operon and its promoter were cloned into the pME1089 described above. This plasmid was named pME1219.

Finally, the plasmids pME1109 and pME1219 were transformed into the Methionine producing strain 16 of patent application WO 2013/001055, giving the strain 1.

EXAMPLE 2: Deletion of the L-methionine uptake system in a L-methionine overproducer E.coli strain - Construction of strains 2, 3, 4 and 5

The methionine producing strain 16 of patent application WO2013/001055 was transformed with plasmids pME1109 and pME1089 (described in Example 1), giving the rise to strain 2.

To inactive the methionine importer encodes by the metNIQ operon in strain 2, the homologous recombination strategy described by Datsenko & Wanner, 2000 (according to Protocol 1) was used. Thus the oligonucleotides, Ome0233/Ome0232 (SEQ ID N° 21 and 22 listed in table 1) were used to PCR amplified the chloramphenicol resistance cassette from the plasmid pKD3. The PCR product obtained was then introduced by electroporation into the strain MG1655 metA* \ \ (pKD46). The chloramphenicol resistant transformants were then selected and the insertion of the resistance cassette was verified by a PCR analysis with appropriate oligonucleotides. The strain retained is designated MG1655 metA* \ \ AmetNIQ::Cm. Finally, the AmetNIQ::Cm deletion was transferred by PI phage transduction (according to Protocol 2) from the MG1655 metA* \ \ AmetNIQ::Cm strain to strain 2. Chloramphenicol resistant transductants were selected and the presence of AmetNIQwCm chromosomal deletion was verified by PCR with appropriate oligonucleotides. The strain retained was called strain 3.

As the same manner, the 3 genes, metN, metl and metQ were deleted in strain 1 described in patent application WO2013/001055. This strain 1 from patent application WO 2013/001055, is re-named herein as strain 4 to be the reference of strain 5.

The deletion of metNIQ performed into strain 4 as described above, gave rise to strain 5.

EXAMPLE 3: Combination of the overexpression of a L-methionine secretion system with the deletion of the L-methionine uptake system in a L-methionine overproducer E.coli strain - Construction of strain 6

To inactivate the methionine importer encodes by metNIQ operon in the strain overproducing the methionine exporter encodes by ygaZH operon, the AmetNIQ::Cm deletion was transferred by PI phage transduction (according to Protocol 2) from the MG1655 metA* \ \ AmetNIQ::Cm strain to methionine producing strain 1. Chloramphenicol resistant transductants were selected and the presence of AmetNIQ::Cm chromosomal deletion was verified by PCR with appropriate oligonucleotides. The strain retained was called strain 6.

EXAMPLE 4: Production of L-methionine by fermentation in bio-reactor

Strains that produced L-methionine were tested under production conditions in 2.5

L reactors (Pierre Guerin) using a fedbatch strategy.

Briefly, an 24 hours culture grown in 10 mL LB medium with 2.5 g.L ^"1 glucose was used to inoculate a 24 hours preculture in minimal medium (Bla). These incubations were carried out in 500 mL baffled flasks containing 50 mL of minimal medium (Bla) in a rotary shaker (200 RPM). The first preculture was realized at a temperature of 30°C, the second one at a temperature of 34°C.

A third preculture step was carried out in bio-reactors (Sixfors) filled with 200 mL of minimal medium (Bib) inoculated to a biomass concentration of 1.2 g.L ^"1 with 5 mL concentrated preculture. The preculture temperature was maintained constant at 34°C and the pH was automatically adjusted to a value of 6.8 using a 10 % NH ₄ OH solution. The dissolved oxygen concentration was continuously adjusted to a value of 30 % of the partial air pressure saturation with air supply and /or agitation. After glucose exhaustion from the batch medium, the fedbatch was started with an initial flow rate of 0.7 mL.h ^"1 , before increasing exponentially for 26 hours with a growth rate of 0.13 h ^"1 in order to obtain a final cellular concentration of about 20 g.L ^"1 . Table 4: Preculture batch mineral medium composition (Bla and Bib)

Table 5: Preculture fedbatch mineral medium composition (Fl)

Subsequently, 2.5 L fermentors (Pierre Guerin) were filled with 600 or 620 mL of minimal medium (B2) and were inoculated to a biomass concentration of 3,2 g.L ^"1 with a preculture volume ranging between 80 to 100 mL.

Cell growth is controlled by phosphate, that is why the final phosphate concentration in batch medium B2 was adjusted to a value comprised between 0 to 20 mM, by addition of different concentrations of KH ₂ PO ₄ , K ₂ HPO ₄ and (TNiH ₄ ) ₂ HP0 ₄ . In the same manner, the final phosphate concentration of F2 medium was adjusted to a value comprise between 5 to 30 mM, by addition of different concentrations of KH ₂ PO ₄ , K ₂ HPO ₄ and (TNiH ₄ ) ₂ HP0 ₄ . Thiosulfate concentration in fedbatch medium can be adjusted in order to prevent a starvation of this compound during the culture.

Table 6: Culture batch mineral medium composition (B2)

Table 7: Culture fedbatch medium composition (F2)

The culture temperature was maintained constant at 37 °C and pH was maintained to the working value (6.8) by automatic addition of NH ₄ OH solutions (10 % and 28 %). The initial agitation rate was set at 200 RPM during the batch phase and was increased up to 1000 RPM during the fedbatch phase. The initial airflow rate was set at 40 NL.h ^"1 during the batch phase and was augmented to 100 NL.h ^"1 at the beginning of the fedbatch phase. The dissolved oxygen concentration was maintained at values between 20 and 40%, preferentially 30% saturation by increasing the agitation.

IPTG was added in batch and fedbatch media when it was necessary at a final concentration of 20 μΜ. When it was needed, antibiotics were added at a concentration of 50 mg.L ^"1 for spectinomycin, 30 mg.L ^"1 for chloramphenicol and 100 mg.L ^"1 for ampicillin.

When the cell mass reached a concentration close to 5 g.L ^"1 , the fedbatch was started with an initial flow rate of 5 mL.h ^"1 . Feeding solution was injected with a sigmoid profile with an increasing flow rate that reached 24 mL.h ^"1 after 25 hours. The precise p2

feeding conditions were calculated by the equation: Q(t) = ρ\ λ _ ₃₍ ,■, . where Q(t) is the feeding flow rate in mL.h ^"1 with pi = 1.80, p2 = 22.4, p3 = 0.27, p4 = 6.50. This flow rate was increased from 10 to 50 %, preferentially between 20 and 30 % throughout the entire culture.

After 25 hours fedbatch, feeding solution pump was stopped and culture was finalized after glucose exhaustion.

Extracellular amino acids were quantified by HPLC after OPA/Fmoc derivatization and other relevant metabolites were analyzed using HPLC with refractometric detection (organic acids and glucose) and GC-MS after silylation.

Impact of combination of deletion of the metNIQ operon and/or overexpression of the ygaZH operon on methionine production was tested. The results are presented in Table 8 and Table 9.

Table 8: Maximal and final methionine yields produced in fedbatch cultures by the different strains. The performances of the strains of interest, strain 3, 1, and 6 are compared to the reference strain, strain 2 cultivated in the same conditions. The symbol ~ indicates that there is no difference between the strains, the symbol + indicates an increase between 1 to 3 % and symbol ++ indicates an increase greater than 3 %. For the definition of methionine/glucose yield see below.

The results presented on table 8 show that the deletion of metNIQ operon is of no benefit to the production of methionine (strain 3) in the genetic background of strain 2. Therefore, this genetic modification was tested in strain 4 with a different genetic backgroung than strain 2. This assay shows a negative effect of the deletion of metNIQ operon on the methionine production (see table 9 below). Strains 4 and 5 were cultivated in 2L reactors as described in patent application WO2013/001055.

Table 9: Maximal and final methionine yields produced in fedbatch cultures by the strain 5. The performances of the strain of interest (strain 5) are compared to the reference strain, strain 4 cultivated in the same conditions. The symbol - indicates a decrease greater than 4% compared to the reference strain. For the definition of methionine/glucose yield see below.

These results show that unlike prior art described for C. glutamicum, the deletion of metNIQ operon alone in E. coli does not enhance methionine production whatever the genetic background (strain 3 and 5). Moreover, performances of strain 5 are below performances of its mother strain (strain 4). Even if the overexpression ofygaZH leads to an increased production of methionine (strain 1, Table 8) at the end of the culture, surprisingly the combination of deletion of metNIQ operon and overexpression of ygaZH enhance the averall performances of methionine production from strain 6. This result was not expected since the deletion of metNIQ operon was shown as to be negative or neutral on the L-methionine production performances in different genetic backgrounds including the mother strain. Determination of methionine/glucose yield (Y _met )

The reactor volume was calculated by adding to the initial volume the amount of solutions added to regulate the pH and to feed the culture and by subtracting the volume used for sampling and lost by evaporation.

The fedbatch volume was followed continuously by weighing the feeding stock.

The amount of injected glucose was then calculated on the basis of the injected weight, the density of the solution and the glucose concentration determined by the method of Brix ([Glucose]). The methionine yield was expressed as followed: Methionine _t V _T - Methionine^ VQ 100

met ⁼

Consumed glu cos e _t

With Methionineo and Methionine _t respectively the initial and final methionine concentrations and Vo and V _t the initial and the instant t volumes.

The consumed glucose was calculated as follows: , , fed weighty - fed weight,

fed volume _t = 2—^— - — - density fed solution

Injected Glucose _t = fed volume _t * [Glucose]

Consumed glucose _t = [Glucose]o * Vo + Injected Glucose - [Glucose] _res iduai * V _t With [Glucose]o, [Glucose], [Glucose] _res iduai respectively the initial, the fed and the residual glucose concentrations.

EXAMPLE 5: Combination of the deletion of the L-methionine uptake system with the overproduction of different L-methionine secretion systems from various microorganims in an E.coli strain overproducer of L-methionine - Construction of strains 7 to 15

The ygaZH homologous genes from Citrobacter species, Raoultella species, Shigella species, Enterobacter species, Yersinia species and Photorhabdus species were overexpressed in genetic background of strain 3.

Before using strain 3, the plasmid pME1089 was removed from this strain using a curing plasmid method as described by Heery et al, 1989 (according to Protocol 4). The cured cells without plasmid pME1089 but having retained the plasmid pME1109 were selected. The resulting strain was named strain 7.

Construction of strains 8 to 15 - Overproduction of homologue L-methionine secretion systems, overexpression ofygaZH from genus and species listed in table 10. To overexpress the ygaZH homologous genes listed in table 10, each couple of genes was cloned, as for ygaZH genes of E. coli, on the moderate copy number plasmid pCL1920 (Lerner & Inouye, 1990) with the use of the natural promoter and natural ribosome binding site of E. coli ygaZ gene. More precisely, ygaZH homologous genes were cloned into the pMEl 089 plasmid described above. As specified in table 11 , the ygaZH homologous genes were either amplified from genomic DNA of the corresponding strain or chemically synthesized, with or without optimizing the codon usage to E. coli (as proposed by Gene Art® Gene Synthesis service with GeneOptimizer® software - Lifetechnologies). The amplified DNA fragments comprising the ygaZH homologous genes are disclosed in SEQ ID indicated in the Table 1 1. The resulting plasmids were named as mentioned in table 11. Finally each plasmid was transformed into strain 7, giving rise to strains 8 to 15 listed as "strain name" in table 11. Table 10: YgaZH homologue proteins

reun

Table 11 : Plasmids and strains carrying ygaZH homo logue genes

Chemical Codon usage SEQ ID N° Plasmid

Microorganism Strain name synthesis optimisation name

Citrobacter

no no 23 pME1277a Strain 8 koseri

Shigella flexneri yes no 24 pME1274a Strain 9

Raoultella

yes yes 25 pME1275a Strain 10 ornithinolytica

Enterobacter sp. yes yes 26 pME1283a Strain 1 1

Yersinia

enterocolitica

no no 27 pME1287a Strain 12 subsp.

Enterocolitica

Photorhabdus

luminescens

no no 28 pME1281a Strain 13 subsp.

Laumondii

Citrobacter

yes yes 29 pME131 1a Strain 14 youngae

Citrobacter

yes yes 30 pME1307a Strain 15 freundii EXAMPLE 6: Production of L-methionine by fermentation in flask experiments

Recombinant L-methionine producers having the deletion of metNIQ operon combined to the overexpression of different L-methionine secretion systems from various microorganisms (homologous to YgaZH from E.coli) were evaluated in small Erlenmeyer flasks.

A 5.5 mL preculture was grown at 30°C for 21 hours in a mixed medium (10 % LB medium (Sigma 25 %) with 2.5 g.L ^"1 glucose and 90 % minimal medium PCI, Table 12). It was used to inoculate a 50 mL culture to an OD ₆ oo of 0.2 in medium PCI . Spectinomycin and kanamycin were added at a concentration of 50 mg.L ^-1 , chloramphenicol at 30 mg.L ¹ and gentamycin at 10 mg.L ^"1 when it was necessary. The temperature of the cultures was 37°C. When the culture had reached an OD ₆ oo of 5 to 7, extracellular amino acids were quantified by HPLC after OPA/Fmoc derivatization and other relevant metabolites were analyzed using HPLC with refractometric detection (organic acids and glucose) and GC- MS after silylation.

Table 12: Minimal medium composition (PCI)

MOPS 20.00

IPTG 0.0048 Table 13: Methionine yield (Y _met ) in g methionine / % g of glucose produced in flask culture by the strains of interest, carrying homologues ofygaZH genes and the deletion of metNIQ operon. For the precise definition of methionine/glucose yield see below, "n" indicates the number of repeats.

As can be seen in table 13, overexpression of ygaZH homologous genes from various microorganisms in the L-methionine producer carrying the deletion of metNIQ operon leads to equivalent or better performances than those obtained with strain 6 which overexpresses ygaZH from E.coli. The homologous L-methionine secretion systems from other microorganisms than E. coli can replace the endogenous proteins of the bacterium.

The homologous proteins YgaZH from Citrobacter Koseri (strain 8, Ymet=19,6g/g),

Citrobacter youngae (strain 14, Ymet=19,6g/g), Citrobacter freundii (strain 15,

Ymet=19,6g/g) and Enterobacter sp. (Strain 11 , Ymet=19,4g/g) showed the best L- methionine yields of production compared to strain 6 (Ymet=18.7g/g).

The methionine yield was expressed as followed:

methionine (g) REFERENCES

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