METHOD FOR THE FERMENTATIVE PRODUCTION OF METHIONINE OR ITS HYDROXY ANALOG FORM BY MICROORGANISMS COMPRISING GENES CODING SUGAR PHOSPHOTRANSFERASE SYSTEM (PTS)

Abstract

The present invention relates to a new method for the production of methionine or its hydroxy analog form by conversion of a source of carbon in a fermentative process comprising culturing a microorganism genetically modified for the production of methionine orits hydroxy analog form, wherein said microorganism comprises functional genes coding PTS carbohydrate utilization system and wherein the expression of proteins regulated the expression of phosphoenolpyruvate synthase (PPS) is down-regulated. The present invention also relates to the genetically modified microorganism used in the method of the invention.

Claims

1. A method for the production of methionine or its hydroxy analog form by conversion of a source of carbon in a fermentative process, the method comprising the following: culturing a microorganism genetically modified for the production of methionine or its hydroxy analog form in a culture medium comprising a carbohydrate as a source of carbon, and recovering methionine or its hydroxy analog form from the culture medium, wherein said genetically modified microorganism comprises functional genes coding for a PTS carbohydrate utilization system, and wherein in said genetically modified microorganism, an expression of bifunctional ADP-dependent kinase-Pi-dependent pyrophosphorylase protein regulating an expression of phosphoenolpyruvate synthase (PPS) is decreased.

2. The method of claim 1, wherein the functional genes coding for a PTS carbohydrate utilization system are heterologous to the genetically modified microorganism.

3. The method of claim 1, wherein the functional genes coding for a PTS carbohydrate utilization system are native to the genetically modified microorganism.

4. The method of claim 3, wherein the native gene coding for the bifunctional ADP-dependent kinase-Pi-dependent pyrophosphorylase protein regulating the expression of the phosphoenolpyruvate synthase (PPS) is attenuated or deleted in said genetically modified microorganism.

5. The method of claim 1, wherein the microorganism is at least one selected from the group consisting of Enterobacteriaceae, Bacillaceae, Clostridiaceae, Streptomycetaceae, Corynebacteriaceae, Saccharomyceteceae and yeasts.

6. The method of claim 5, wherein the microorganism is at least one selected from the group consisting of Escherichia coli, Klebsiella pneumoniae, Thermoanaerobacterium thermosaccharolyticum, Clostridium sphenoides and Saccharomyces cerevisiae.

7. The method of claim 1, wherein the microorganism is E. coli.

8. The method of claim 7, wherein a gene ppsR coding for bifunctional ADP dependent kinase-Pi-dependent pyrophosphorylase DUF299 protein is attenuated or deleted.

9. The method of claim 1, wherein the microorganism is genetically modified for an enhanced production of methionine or its hydroxy analog form.

10. A genetically modified microorganism for an enhanced production of methionine or its hydroxy analog form from a carbohydrate as a source of carbon, wherein said genetically modified microorganism comprises: functional genes coding for a PTS carbohydrate utilization system, and wherein, in said genetically modified microorganism, an expression of bifunctional ADP-dependent kinase-Pi-dependent pyrophosphorylase protein regulating an expression of phosphenolpyruvate synthase (PPS) is decreased.

11. The genetically modified microorganism of claim 10, wherein the functional genes coding for a PTS carbohydrate utilization system are heterologous to the genetically modified microorganism.

12. The genetically modified microorganism of claim 10, wherein a gene ppsR coding for bifunctional ADP-dependent kinase-Pi-dependent pyrophosphorylase DUF229 protein is attenuated or deleted.

13. The genetically modified microorganism of claim 10, wherein the functional genes coding for a PTS carbohydrate utilization system are native to the genetically modified microorganism.

14. The genetically modified microorganism of claim 13, wherein the native gene coding for the bifunctional ADP-dependent kinase-Pi-dependent pyrophosphorylase protein regulating the expression of the phosphoenolpyruvate synthase (PPS) is attenuated or deleted in said genetically modified microorganism.

15. The genetically modified microorganism of claim 10, wherein the microorganism is at least one selected from the group consisting of Enterobacteriaceae, Bacillaceae, Clostridiaceae, Streptomycetaceae, Corynebacteriaceae, Saccharomyceteceae and yeasts.

16. The genetically modified microorganism of claim 15, wherein the microorganism is at least one selected from the group consisting of Escherichia coli, Klebsiella pneumoniae, Thermoanaerobacterium thermosaccharolyticum, Clostridium sphenoides and Saccharomyces cerevisiae.

17. The genetically modified microorganism of claim 10, wherein the microorganism is E. coli.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0021] 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.

[0022] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. However, publications mentioned herein are cited for the purpose of describing and disclosing the protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention.

[0023] Furthermore, the practice of the present invention employs, unless otherwise indicated, conventional microbiological and molecular biology techniques within the skill of the art. Such techniques are well-known to the skilled worker, and are explained fully in the literature.

[0024] 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, a reference to an enzyme is a reference to one or more enzymes, 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.

[0025] As used herein, the following terms may be used for interpretation of the claims and specification.

[0026] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word comprise or variations such as comprises or comprising is 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.

[0027] 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.

[0028] 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.

[0029] COGs (clusters of orthologous groups of proteins) are obtained by comparing protein sequences from 66 fully sequenced genomes representing 38 major phylogenetic lines. Each COG is defined from at least three lines, which permits the identification of former conserved domains.

[0030] The means of identifying homologous sequences and their percent homologies are well-known to those skilled in the art, and include, in particular, the BLAST programs (Altschul et al, 1990). The sequences obtained can then be exploited (e.g., aligned) using, for example, the programs CLUSTALW or MULTALIN.

[0031] 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 claimed, for example, in Sambrook et al. (2001).

[0032] As described above, the method of the present invention allows the production of methionine or its hydroxy analog form by conversion of a source of carbon in a fermentative process including the steps of: [0033] culturing a genetically modified microorganism for the production of methionine or its hydroxy analog form in an appropriate culture medium comprising a carbohydrate as source of carbon and [0034] recovering methionine or its hydroxy analog form from the culture medium, said genetically modified microorganism comprising functional genes coding for a PTS carbohydrate utilization system and a decreased expression of the bifunctional ADP-dependent kinase-Pi-dependent pyrophosphorylase protein regulating the expression of the phosphoenolpyruvate synthase (PPS).

[0035] The terms fermentative process, fermentation, or culture are used herein interchangeably to denote the growth of a microorganism. The fermentation is generally conducted in fermenters with an inorganic culture medium of a known, defined composition adapted to the microorganism being 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), an M63 medium (Miller, 1992) or a medium such as defined by Schaefer et al. (1999).

[0036] In the context of the present invention, by fermentative conversion, it is meant that the conversion of the carbon source into methionine or its hydroxy analog form occurs when the microorganism is cultured under appropriate fermentation conditions.

[0037] A culture medium means herein a medium (e.g., a sterile, liquid media) comprising nutrients essential or beneficial to the maintenance and/or growth of the microorganism 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.

[0038] The term source of carbon, carbon source, or carbon substrate according to the present invention refers to any molecule that a microorganism is capable of metabolizing and which contains at least one carbon atom. Examples of preferred carbon sources according to the invention include, without limitation, carbohydrates.

[0039] In a preferred embodiment of the invention, the carbon source is derived from renewable feed-stock. Renewable feed-stock is defined as raw material required for certain industrial processes that can be regenerated within a brief delay and in sufficient amount to permit its transformation into the desired product. Vegetal biomass pre-treated or not, is a particularly preferred renewable carbon source.

[0040] The term carbohydrate refers herein to any carbon source capable of being metabolized by a microorganism and containing at least one carbon atom, two atoms of hydrogen and one atom of oxygen. The carbohydrate of the invention is preferably selected from glucose, fructose, sucrose, mannose, chitobiose, cellobiose, trehalose, galactitol, mannitol, sorbitol, galactosamine, N-acetyl-D-galactosamine, N-acetylglucosamine, N-acetylmuramic acid, lactose, galactose, sorbose, maltose, N,N-diacetylchitobiose, ascorbate, -glucoside. In a more preferred embodiment of the invention, the source of carbon is selected from glucose, fructose, mannose, cellobiose, sucrose, and any combination thereof.

[0041] The person skilled in the art can easily determine the culture conditions necessary for growing the microorganisms in the method according to the invention. In particular, it is well-known that bacteria can be fermented at a temperature comprised between 20 C. and 55 C., preferentially between 25 C. and 40 C. E. coli can more particularly be cultured at a temperature comprised between about 30 C. and about 37 C.

[0042] This culturing process can be performed either in a batch process, in a fed-batch process or in a continuous process, and under aerobic, micro-aerobic or anaerobic conditions.

[0043] According to a particular embodiment of the method of the invention, the functional genes coding for a PTS carbohydrate utilization system are heterologous (recombinant microorganism) or native to the genetically modified microorganism (wild-type microorganism).

[0044] By gene, it is meant herein a nucleic acid molecule or polynucleotide that codes for a particular protein (i.e. polypeptide), or in certain cases, for a functional or structural RNA molecule. In the context of the present invention, the genes referred to herein encode proteins, such as enzymes, efflux systems or uptake transporters. Genes according to the invention are either endogenous genes or exogenous genes.

[0045] The term recombinant microorganism or genetically modified microorganism as used herein, refers to a bacterium, yeast, or a fungus that is not found in nature and is genetically different from equivalent microorganisms found in nature. According to the invention, the term modifications designate any genetic change introduced or induced in the microorganism. The microorganism may be modified through either the introduction of new genetic elements, the increase or the attenuation of the expression of endogenous or exogenous genes or the deletion of endogenous genetic elements. Further, a microorganism may be modified by forcing the development and evolution of new metabolic pathways by combining directed mutagenesis and evolution under specific selection pressure (see, for example, WO 2004076659).

[0046] In the context of the present invention, the term exogenous gene (or alternatively, heterologous gene or transgene) refers to a gene not naturally occurring in the microorganism. It may be artificial or it may originate from another microorganism.

[0047] It shall be further understood that, in the context of the present invention, should an exogenous gene encoding a protein of interest be expressed in a specific microorganism, a synthetic version of this gene is preferably constructed by replacing non-preferred codons or less preferred codons with preferred codons of said microorganism which encode the same amino acid. It is indeed well-known in the art that codon usage varies between microorganism species, which may impact the recombinant expression level of the protein of interest. To overcome this issue, codon optimization methods have been developed, and are extensively described in Graf et al. (2000), Deml et al. (2001) or Davis & Olsen (2011). Several softwares have been developed for codon optimization determination such as the GeneOptimizer software (Lifetechnologies) or the OptimumGene software (GenScript). In other words, the exogenous gene encoding a protein of interest is preferably codon-optimized for expression in a specific microorganism.

[0048] According to another embodiment of the method of the present invention, the genetically modified microorganism comprises a native gene coding for the bifunctional ADP-dependent kinase-Pi-dependent pyrophosphorylase protein regulating the expression of the phosphoenolpyruvate synthase (PPS) whose expression is attenuated or deleted. In other worlds, in said genetically modified microorganism, expression of the native gene coding for the bifunctional ADP-dependent kinase-Pi-dependent pyrophosphorylase protein is attenuated or deleted compared to the microorganism unmodified. Preferably in the microorganism of the invention, the native gene coding for the bifunctional ADP-dependent kinase-Pi-dependent pyrophosphorylase protein is deleted.

[0049] By native gene or endogenous gene it is meant herein that said gene is naturally present in the microorganism.

[0050] In the context of the present invention, should the microorganism be genetically modified to modulate the expression level of one or more endogenous genes, it is meant herein that the expression level of said gene is up-regulated, downregulated (i.e. attenuated), or even completely abolished by comparison to its natural expression level. Such modulation can therefore theoretically result in an enhancement of the activity of the gene product, or alternatively, in a lower or null activity of the endogenous gene product.

[0051] Endogenous gene activity and/or expression level can also be modified by introducing mutations into their coding sequence to modify the gene product. A deletion of an endogenous gene can also be performed to totally inhibit its expression within the microorganism. Another way to modulate the expression of an endogenous gene is to exchange its promoter (i.e. wild type promoter) with a stronger or weaker promoter to up- or down-regulate the expression level of this gene. Promoters suitable for such a purpose can be homologous or heterologous and are well-known in the art. It is within the skill of the person in the art to select appropriate promoters for modulating the expression of an endogenous gene.

[0052] According to another embodiment of the present invention, the microorganism is selected from microorganisms expressing a functional PTS sugar system. Preferentially, the microorganism is selected from the group comprising Enterobacteriaceae, Clostridiaceae, Bacillaceae, Streptomycetaceae, Deinococcaceae, Nitrosomonadaceae, Vibrionaceae, Pseudomonadaceae, Corynebacteriaceae, Saccharomyceteceae and yeasts. More preferentially, the microorganism is a species of Citrobacter, Corynebacterium, Deinococcus, Escherichia, Pantoea, Klebsiella, Nitrosomonas, Photorhabdus, Photobacterium, Pseudomonas, Salmonella, Serratia, Shigella and Yersinia. Even more preferentially, the microorganism is selected from Escherichia coli, Klebsiella pneumoniae, Klebisella oxytoca, Pseudomonas aeruginosa, Pseudomonas fluorescens, Salmonalla typhimurium, Salmonella enterica, Serratia marcescens, Pantoea ananatis, Corynebacterium glutamicum, Deinococcus radiodurans, Thermoanaerobacterium thermosaccharolyticum, Clostridium sphenoides, and Saccharomyces cerevisiae.

[0053] In particular, the examples show modified E. coli strains, but these modifications can easily be performed on other microorganisms of the same family.

[0054] E. coli belongs to the Enterobacteriaceae family which comprises members that are gram-negative, rod-shaped, non-spore forming and are typically 1-5 m in length. Most members use flagella 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 of 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 pneumoniae, and Salmonella.

[0055] According to another embodiment of the method of the present invention, the gene ppsR of SEQ ID NO:1 coding for the bifunctional ADP-dependent kinase-Pi-dependent pyrophosphorylase DUF299 protein of SEQ ID NO:2 is deleted (which can be referred to as ppsR).

[0056] The term deleted, as used herein, refers to the complete suppression of the expression of a gene. This suppression of expression can be either an inhibition of the expression of the gene, a deletion of all or part of the promoter region necessary for expression of the gene, or a deletion in the coding region of the gene. The deleted gene can be replaced by a selection marker gene that facilitates the identification, isolation and purification of the strains according to the invention. For example, suppression of gene expression may be achieved by the technique of homologous recombination (Datsenko & Wanner, 2000).

[0057] In another embodiment, the gene ppsR coding for the bifunctional ADP-dependent kinase-Pi-dependent pyrophosphorylase DUF299 protein may be attenuated.

[0058] The term attenuated, as used herein, refers to the partial suppression of the expression of a gene. This attenuation of expression can be either the exchange of the wild-type promoter for a weaker natural or synthetic promoter or the use of an agent reducing ppsR gene expression, including antisense RNA or interfering RNA (iRNA), and more particularly small interfering RNAs (siRNAs) or short hairpin RNAS (shRNAs). For example, promoter exchange may be achieved by the technique of homologous recombination (Datsenko & Wanner, 2000).

[0059] Any other methods known to those skilled in the art suitable for the inhibition of the expression or the function of a protein, and especially of this protein, may be used.

[0060] The method of the present invention may be used for producing methionine or its hydroxy analog form in high quantity. Thus, the method of the invention allows the production of methionine or its hydroxy analog form to be increased.

[0061] More particularly, the method of the invention allows improving the production of methionine or its derivatives.

[0062] The term improved methionine production refers to an increased productivity of methionine and/or an increased titer of methionine and/or an increased methionine/carbon source yield and/or an increased purity of methionine compared to its parent strain, i.e. the microorganism prior to the deletion or the attenuation of ppsR gene. The production of methionine by the microorganism in the culture broth can be recorded unambiguously by standard analytical means known by those skilled in the art and in particular with HPLC. Some genetically modified microorganisms increasing methionine production are disclosed in patent applications WO2016034536, WO2014029592 and WO2012091479 for examples of E. coli strains producing methionine and patent applications WO2008080900 and WO2012098042 for examples of Corynebacterium strains producing methionine. All of these disclosures are herein incorporated by reference.

[0063] Preferably, the microorganism producing methionine of the invention is an Escherichia coli strain and comprises at least: [0064] the expression of at least one gene selected from the genes metA of SEQ ID NO:3 or a mutant gene encoding an enzyme with reduced feed-back inhibition to methionine or its derivatives as disclosed in patent application WO2005108561, US2010041108 or WO2008127240, metH of SEQ ID NO: 5, cysPUWAM of SEQ ID NO:7-9-11-13-15, cysJIH of SEQ ID NO:17-19-21, gcvTHP of SEQ ID NO:23-25-27, metF of SEQ ID NO: 29, serA of SEQ ID NO:31, serB of SEQ ID NO:33, serC of SEQ ID NO:35, cysE of SEQ ID NO:37, thrA of SEQ ID NO:39 or a mutant gene encoding an enzyme with reduced feed-back sensitivity to threonine as described in patent application WO2005111202, ptsG of SEQ ID NO:41, ygaZH of SEQ ID NO:43 and 45 or their homologous genes disclosed in patent application WO2016034536 and pyc of SEQ ID NO:47 is enhanced, and [0065] the expression of at least one of the genes selected from metJ of SEQ ID NO:49, pykA of SEQ ID NO:51, pykF of SEQ ID NO:53, purU of SEQ ID NO:55, metE of SEQ ID NO:57, dgsA of SEQ ID NO:59 and yncA of SEQ ID NO:61 is attenuated.

TABLE-US-00001 TABLE 1 Enzymes and genes according to the invention (n/a: not available) Gene Gene Protein Micro- RefSeq or SEQ Enzyme SEQ organism GenBank ID Uniprot ID Name of origin Enzyme Function reference NO: reference NO: PpsR Escherichia bifunctional ADP-dependent NP_416218.1 1 P0A8A4 2 coli kinase-Pi-dependent pyrophosphorylase MetA Escherichia homoserine NP_418437.1 3 P07623 4 coli O-succinyltransferase MetH Escherichia Methionine Synthase NP_418443.1 5 P13009 6 coli CysP Escherichia periplasmic sulphate NP_416920.1 7 P16700 8 coli binding protein CysU Escherichia component of NP_416919.1 9 P16701 10 coli sulphate ABC transporter CysW Escherichia membrane bound YP_026168.2 11 P0AEB0 12 coli sulphate transport protein CysA Escherichia sulphate permease NP_416917.1 13 P16676 14 coli CysM Escherichia O-acetyl serine NP_416916.1 15 P16703 16 coli sulfhydralase CysJ Escherichia alpha subunits of NP_417244.1 17 P38038 18 coli sulfite reductase Cysl Escherichia beta subunits of NP_417243.1 19 P17846 20 coli sulfite reductase CysH Escherichia adenylylsulfate NP_417242.1 21 P17854 22 coli reductase GcvT Escherichia aminomethyltransferase NP_417381.1 23 P27248 24 coli GcvH Escherichia lipoyl-GcvH-protein NP_417380.1 25 P0A6T9 26 coli GcvP Escherichia glycine NP_417379.1 27 P33195 28 coli dehydrogenase MetF Escherichia methylenetetrahydrofolate NP_418376.1 29 P0AEZ1 30 coli reductase SerA Escherichia phosphoglycerate NP_417388.1 31 P0A9T0 32 coli dehydrogenase SerB Escherichia phosphoserine NP_418805.1 33 P0AGB0 34 coli phosphatase SerC Escherichia phosphoserine NP_415427.1 35 P23721 36 coli aminotransferase CysE Escherichia serine acyltransferase NP_418064.1 37 P0A9D4 38 coli ThrA Escherichia aspartokinase/ NP_414543.1 39 P00561 40 coli homoserine dehydrogenase PtsG Escherichia PTS enzyme IICB.sup.Glc NP_415619.1 41 P69786 42 coli YgaZ Escherichia inner membrane NP_417167 43 P76630 44 coli protein YgaZ YgaH Escherichia putative L-valine NP_417168 45 P43667 46 coli exporter, norvaline resistance protein Pyc Rhizobium Pyruvate Carboxylase WP_011427190.1 47 Q2K340 48 etli MetJ Escherichia repressor protein NP_418373.1 49 P0A8U6 50 coli MetJ PykA Escherichia pyruvate kinase NP_416368.1 51 P21599 52 coli PykF Escherichia pyruvate kinase NP_416191.1 53 P0AD61 54 coli PurU Escherichia formyltetrahydrofolate NP_415748.1 55 P37051 56 coli deformylase MetE Escherichia cobalamin-independent NP_418273.1 57 P25665 58 coli methionine synthase DgsA Escherichia transcriptional dual NP_416111.1 59 P50456 60 coli regulator YncA Escherichia N-acyltransferase NP_415965.1 61 P76112 62 coli

[0066] As discussed above, sugar is transported into bacterial cells and phosphorylated by the phosphoenolpyruvate: sugar phosphotransferase system (PTS)). Phosphorylated sugar and particularly, phosphorylated glucose is toxic to cells in high concentrations and as a result the PTS system is highly regulated. This, coupled with the fact that the system is complex, makes manipulation of the system very difficult. However, as described below, the inventors have surprisingly produced a genetically modified microorganism comprising functional genes coding for a PTS carbohydrate utilization system while lacking at least one protein regulating PPS expression.

[0067] In a second aspect, the present invention thus relates to a genetically modified microorganism for the enhanced production of methionine or its hydroxy analog form from a carbohydrate as source of carbon, said genetically modified microorganism comprising functional genes coding for the PTS carbohydrate utilization system and a decreased expression of the bifunctional ADP-dependent kinase-Pi-dependent pyrophosphorylase protein regulating the expression of the Phosphenolpyruvate synthase (PPS).

[0068] This genetically modified microorganism has the same genetic characteristics as those used in the method of the present invention. Particularly, in this microorganism, the gene ppsR coding for the bifunctional ADP-dependent kinase-Pi-dependent pyrophosphorylase DUF229 is deleted or attenuated. More preferentially the gene ppsR is deleted in the microorganism of the invention.

[0069] Consequently, it may be used in the fermentative method according to the invention for increasing the production of a methionine or its hydroxy analog form.

[0070] Preferably, said microorganism may be used in the fermentative method according to the invention for increasing production of methionine.

EXAMPLES

Example 1: Methods for Strain Construction

[0071] In the examples given below, methods well known in the art were used to construct E. coli strains containing replicating vectors and/or various chromosomal deletions, and substitutions using homologous recombination well described by Datsenko & Wanner, (2000) for E. coli. In the same manner, the use of plasmids or vectors to express or overexpress one or several genes in a recombinant microorganism are well-known by the man skilled in the art. Examples of suitable E. coli expression vectors include pTrc, pACYC184n pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, etc . . .

[0072] Several protocols have been used in the following examples. Protocol 1 (chromosomal modifications by homologous recombination, selection of recombinants), protocol 2 (transduction of phage P1) and protocol 3 (antibiotic cassette excision, the resistance genes were removed when necessary) used in this invention have been fully described in patent application EP 2532751. The antibiotic resistant cassette can be amplified on pKD3, pKD4, pKD13 or any other plasmid containing another antibiotic resistant gene surrounded by FRT sites. Chromosomal modifications were verified by PCR analysis with appropriate oligonucleotides that the person skilled in the art is able to design.

Example 2: Construction of Strains 1 and 2

Construction of Strain 1

[0073] The full protocol for constructing strain 1 is provided in patent application number WO2012055798: strain N 10.

Construction of Strain 2

[0074] To inactivate the PEP synthase regulatory protein PSRP encoded by the ppsR gene, the homologous recombination strategy was used (according to Protocols 1 and 3). Oligonucleotides for DppsR: SEQ ID N 63 and 64, were used to PCR amplify the resistance cassettes. The strains retained were designated MG1655 DppsR::Km or MG1655 DppsR::Gt. Finally, the DppsR::Km or MG1655 DppsR::Gt deletion was transferred by P1 phage transduction (according to Protocol 2) into strain 1.

Example 3: Shake Flask Cultures and Yields

[0075] Methionine production strains were assessed in small Erlenmeyer flasks. A 5.5 mL preculture was grown at 30 C. for 21 hours in a mixed medium (10% LB medium (Sigma 25%) with 2.5 g.L.sup.1 glucose and 90% minimal medium PC1, described in WO2012055798). It was used to inoculate a 50 mL culture of PC1 medium to an OD.sub.600 of 0.2. The temperature of the culture was 37 C. for two hours, 42 C. for two hours and 37 C. until the culture end. When the culture reached an OD.sub.600 of 5 to 7, Methionine (Met) and Homolanthionine (HLA) were quantified by HPLC after OPA/Fmoc derivatization and GCMS-silylation and glucose was quantified by HPLC with refractometric detection.

[0076] For all the cultures, when it was necessary, antibiotics were added at a concentration of 50 mg.L.sup.1 for kanamycin and spectinomycin, at a concentration of 30 mg.L.sup.1 for chloramphenicol and at a concentration of 10 mg.L.sup.1 for gentamycin.

TABLE-US-00002 TABLE 2 Yields (g product/g consumed sugar) of the strains described above Yield Culture (g product/g Strain Control strain conditions Product consumed sugar) 2 1 Glucose 37 C. Met + HLA +++ =: no difference with control strain, +: yield higher than control strain (110%-120%), ++: yield higher than control strain (120%-150%), +++: yield higher than control strain (>150%)

[0077] Strain 2 had better yield than corresponding control strains 1.

CONCLUSION

[0078] As demonstrated by the above examples, the deletion of ppsR coding for the bifunctional ADP-dependent kinase-Pi-dependent pyrophosphorylase DUF299 protein allows the production of methionine to be increased.

BIBLIOGRAPHY

[0079] Altschul S, Gish W, Miller W, Myers E, Lipman D J (1990). J. Mol. Biol; 215 (3): 403-410

[0080] Anderson, Proc. Natl. Acad. Sci. USA., 1946, 32:120-128.

[0081] Burnell J N (2010) BMC Biochemistry 11 (1)

[0082] Canovas J L, Kornberg H L (1965). Biochim Biophys Acta 96;169-72.

[0083] Cooper R A, Kornberg H L (1965). Biochim Biophys Acta 104(2);618-20.

[0084] Datsenko K A & Wanner B L, (2000), Proc Nati Acad Sci USA., 97: 6640-6645

[0085] Davis J J & Olsen G J. (2011). Mol. Biol. Evol.; 28(1):211-221

[0086] Deml L, Bojak A, Steck S, Graf M, Wild J, Schirmbeck R, Wolf H, Wagner R. (2011). J. Virol., 75(22): 10991-11001.

[0087] Gabor E, Ghler A K, Kosfeld A, Staab A, Kremling A, Jahreis K. (2011). Eur. J. Cell Biol., 90(9): 711-720

[0088] Graf M, Bojak A, Deml L, Bieler K, Wolf H, Wagner R. (2000). J. Virol.; 74(22): 10/22-10826

[0089] Gosset G, Yong-Xiao J, Berry A (1996). Journal of Industrial Microbiology 17: 47-52.

[0090] Kim P, Laivenieks M, Vieille C, Gregory Zeikus J. (2004). Applied and Environmental Microbiology 70(2):1238-1241.

[0091] Kornberg H L, Malcovati M (1973). FEBS Lett 32(2);257-9.

[0092] Kotrba P, Inui M, Yukawa H (2001). J. Bioscience & Bioeng. 92(6):502-517

[0093] Meza E, Becker J, Bolivar F, Gosset G, Wittmann C (2012). Microbial Cell Factories 11:127.

[0094] 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.

[0095] Patnaik R, Roof W D, Young R F, Liao J C. (1992). Journal of Bacteriology 174(23):7527-7532.

[0096] Pittard J, Wallace B J. (1966). Journal of Bacteriology 91(4):1494-1508.

[0097] Postma, P. W., Roseman, S. (1976). Biochim Biophys Acta 457(3-4);213-57.

[0098] Sambrook and Russell, (2001), Molecular Cloning: 3rd edition, Cold Spring Harbor Laboratory Press, N.Y., Vol 1, 2, 3

[0099] Schaefer U, Boos W, Takors R, Weuster-Botz D, (1999). Anal. Biochem. 270: 88-96.

[0100] Tatarko M, Romeo T (2001). Current Microbiology 43: 26-32.