FIELD OF THE INVENTION

The present invention relates to a method for increasing intracellular NADPH availability produced from NADH to enhance methionine production. Advantageously, the method for increasing levels of NADPH is associated to an increase of the one carbon metabolism to improve methionine production.

BACKGROUND OF THE INVENTION

Sulphur-containing compounds such as cysteine, homocysteine, methionine or S- adenosylmethionine are critical to cellular metabolism and are produced industrially to be used as food or feed additives and pharmaceuticals. In particular methionine, an essential amino acid, which cannot be synthesized by animals, plays an important role in many body functions. Aside from its role in protein biosynthesis, methionine is involved in transmethylation and in the bioavailability of selenium and zinc. Methionine is also directly used as a treatment for disorders like allergy and rheumatic fever. Most of the methionine that is produced is added to animal feed.

With the decreased use of animal-derived proteins as a result of BSE and chicken flu, the demand for pure methionine has increased. Chemically D,L-methionine is commonly produced from acrolein, methyl mercaptan and hydrogen cyanide. Nevertheless the racemic mixture does not perform as well as pure L-methionine, as for example in chicken feed additives (Saunderson, C.L., (1985) British Journal of Nutrition 54, 621-633). Pure L-methionine can be produced from racemic methionine e.g. through the acylase treatment of N-acetyl-D,L-methionine, which increases production costs dramatically. The increasing demand for pure L-methionine, coupled to environmental concerns, render microbial production of methionine attractive.

The cofactor pairs NADPH/NADP ⁺ and NADH/NAD ⁺ are essential for all living organisms. They are donor and/or acceptors of reducing equivalents in many oxidation- reduction reactions in living cells. Although chemically very similar, the redox cofactors NADH and NADPH serve distinct biochemical functions and participate in more than 100 enzymatic reactions (Ouzonis, C. A., and Karp, P. D. (2000) Genome Res. 10, 568-576). Catabolic reactions are normally linked to NADVNADH, and anabolic reactions are normally linked to NADPVNADPH. Together, these nucleotides have a direct impact on virtually every oxidation-reduction metabolic pathway in the cell.

Many industrially useful compounds require the cofactor NADPH for their biosynthesis, as for instance: production of ethanol on xylose in yeast (Verho et al, (2003) Applied and Environmental Microbiology 69, 5892-5897), high-yield production of (+)- catechins (Chemler et al, (2007) Applied and Environmental Microbiology 77, 797-807), lycopene biosynthesis in E.coli (Alper, H. et al, (2005) Metab. Eng. 7, 155-164) or lysine production in Corynebacterium glutamicum (Kelle T et al., (2005) L-lysine production; In: Eggeling L, Bott M (eds) Handbook of corynebacterium glutamicum. CRC, Boca Raton, pp467-490).

The biotechnological production of L-methionine with Escherichia coli requires a sufficient pool of NADPH. Methionine is derived from the amino acid aspartate, but its synthesis requires the convergence of two additional pathways, cysteine biosynthesis and CI metabolism (N-methyltetrahydrofolate). Asparate is converted into homoserine by a sequence of three reactions. Homoserine can subsequently enter the threonine/isoleucine or the methionine biosynthetic pathway.

Production of one molecule of L-methionine requires 8.5 moles of NADPH. To meet the NADPH requirement for both growth of E.coli and L-methionine production on minimal medium, the inventors propose here to increase the transhydrogenase activity, in the aim to increase the pool of NADPH in the cell.

The transhydrogenase reaction (below) may be catalyzed by either a membrane- bound, proton-translocating enzyme (PntAB) or a soluble, energy-independent isoform enzyme (SthA).

[NADPH] + [NAD ⁺ ] + [H ⁺ in] ¾ [NADP ⁺ ] + [NADH] + [H ⁺ out]

E.coli possesses two nicotinamide nucleotide transhydrogenases encoded by the genes sthA (also called udhA) and pntAB (Sauer U. et al, 2004, JBC, 279:6613-6619). The physiological function of the transhydrogenases PntAB and SthA in microorganisms is the generation and the re-oxidation of NADPH, respectively (Sauer U. et al, 2004, JBC, 279:6613-6619). The membrane-bound transhydrogenase PntAB uses the electrochemical proton gradient as driving force for the reduction of NADP ⁺ to NADPH by oxidation of NADH to NAD ⁺ (Jackson JB, 2003, FEBS Lett 545: 18-24).

Several strategies have been employed to improve the availability of NADPH in whole cells. Moreira dos Santos et al. (2004) report the use of NADP+-dependent malic enzymes to increase cytosolic levels of NADPH within Saccharomyces cerevisiae. Weckbecker and Hummel (2004) describe the overexpression of pntAB in E.coli to improve the NADPH-dependent conversion of acetophenone to (R)-phenylethanol. Sanchez et al. (2006) describe the overexpression of the soluble transhydrogenase SthA in E.coli to improve the NADPH-dependent production of poly(3-hydroxybutyrate).

The inventors have surpri singly found that, by increasing PntAB activity and reducing SthA catalyzed reaction in the bacterium, the methionine production is significantly increased.

Moreover, in combining a high transhydrogenase activity and an increase of the methylenetetrahydrofolate reductase (MetF) activity, to boost the one carbon metabolism in the cell, L-methionine production by fermentation of the modified microorganisms is greatly improved.

SUMMARY OF THE INVENTION

The present invention is related to a microorganism producing methionine, wherein its production of NADPH is increased. The increase of NADPH production is achieved by increasing the PntAB transhydrogenase activity as a sole modification, or in combination with the attenuation of the activity of UdhA transhydrogenase. In a particular embodiment of the invention, the increase of NADPH production is coupled with an increase of the one carbon metabolism.

The invention is also related to a method for the production of methionine, in a fermentative process comprising the steps of:

culturing a modified methionine-producing microorganism in an appropriate culture medium comprising a source of carbon, a source of sulphur and a source of nitrogen, and

recovering methionine and/or its derivatives from the culture medium, wherein in said microorganism, the transhydrogenase activity of PntAB is enhanced in a way that the production of reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) is increased.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to a microorganism producing methionine, wherein said microorganism is modified to enhance the transhydrogenase activity of PntAB.

The invention is also related to a method for the production of methionine in a fermentative process.

■ Definitions

The present invention relates to microorganisms that contain genetics modifications to enhance gene expression or protein production or activity.

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

PFAM (protein families database of alignments and hidden Markov models; http://www.sanger.ac.uk/Software/Pfam/) represents a large collection of protein sequence alignments. Each PFAM makes it possible to visualize multiple alignments, see protein domains, evaluate distribution among organisms, gain access to other databases, and visualize known protein structures. COGs (clusters of orthologous groups of proteins; http://www.ncbi.nlm.nih.gov/COG/ are obtained by comparing protein sequences from 66 fully sequenced genomes representing 38 major phylogenic lines. Each COG is defined from at least three lines, which permits the identification of former conserved domains.

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

Using the references given on GenBank for known genes, those skilled in the art are able to determine the equivalent genes in other organisms, bacterial strains, yeasts, fungi, mammals, plants, etc. This routine work is advantageously done using consensus sequences that can be determined by carrying out sequence alignments with genes derived from other microorganisms, and designing degenerate probes to clone the corresponding gene in another organism. These routine methods of molecular biology are well known to those skilled in the art, and are claimed, for example, in Sambrook et al. (1989 Molecular Cloning: a Laboratory Manual. 2 ^nd ed. Cold Spring Harbor Lab., Cold Spring Harbor, New York.).

The term "attenuation of activity" according to the invention could be employed for an enzyme or a gene and denotes, in each case, the partial or complete suppression of the expression of the corresponding gene, which is then said to be 'attenuated' . 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 the gene expression, a deletion in the coding region of the gene, or the exchange of the wildtype promoter by a weaker natural or synthetic promoter. Preferentially, the attenuation of a gene is essentially the complete deletion of that gene, which can be replaced by a selection marker gene that facilitates the identification, isolation and purification of the strains according to the invention. A gene is inactivated preferentially by the technique of homologous recombination (Datsenko, K.A. & Wanner, B.L. (2000) "One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products". Proc. Natl. Acad. Sci. USA 97: 6640-6645).

The term "enhanced transhydrogenase PntAB activity" designates an enzymatic activity that is superior to the enzymatic activity of the non modified microorganism. The man skilled in the art knows how to measure the enzymatic activity of said enzyme. In a preferred embodiment of the invention, the PntAB activity is increased of 10% in comparison with the activity observed in a non-modified microorganism; preferentially, said activity is increased of 20%, more preferentially of 30%, even more preferentially of 50%), or is superior to 60%>.

To enhance an enzymatic activity, the man skilled in the art knows different means : modifiying the catalytic site of the protein, increasing the stability of the protein, increasing the stability of the messenger RNA, increasing the expression of the gene encoding the protein.

Elements stabilizing the proteins are known in the art (for example the GST tags, Amersham Biosciences), as well as elements stabilizing the messenger RNA (Carrier and Keasling (1998) Biotechnol. Prog. 15, 58-64).

The terms "increased expression of the gene" "enhanced expression of the gene" or

"overexpression of the gene" are used interchangeably in the text and have similar meaning.

To increase the expression of a gene, the man skilled in the art knows different techniques: increasing the copy-number of the gene in the microorganism, using a promoter inducing a high level of expression of the gene, attenuating the activity and/or the expression of a direct or indirect transcription repressor of the gene.

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 to the expert in the field (including gene replacement). When the gene is located extra-chromosomally, the gene is 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).

In a specific embodiment of the invention, the gene is expressed using promoters with different strength. In one embodiment of the invention, the promoters are inducible. These promoters are homologous or heterologous. The man skilled in the art knows which promoters are the most convenient, for example promoters Ptrc, Ptoc, Viae or the lambda promoter cl are widely used.

^■ Derivatives of methionine:

According to the invention, methionine is recovered from the culture medium. However, it is also possible to recover from the culture medium some derivatives of methionine, that can be converted into methionine in a simple reaction. Derivatives of methionine stem from methionine transforming and/or degrading pathways such as S-acyl methionine and N-acyl methionine pathways. In particular these products are S-adenosyl- methionine (SAM) and N-acetyl-methionine (NAM). Especially NAM is an easily recoverable methionine derivative that may be isolated and transformed into methionine by deacylation.

^■ Microorganisms

The term "microorganism for the production of methionine" or "methionine- producing microorganism" designates a microorganism producing higher levels of methionine than non-producing microorganisms, which produce methionine only for their endogenous needs. Microorganisms "optimized" for methionine production are well known in the art, and have been disclosed in particular in patent applications WO2005/111202, WO2007/077041 and WO2009/043803.

The term "modified microorganism" is related to a microorganism modified for an improved methionine production. According to the invention, the amount of methionine produced by the microorganism, and particularly the methionine yield (ratio of gram/mol methionine produced per gram/mol carbon source), is higher in the modified microorganism compared to the corresponding unmodified microorganism. Usual modifications include deletions of genes by transformation and recombination, gene replacements, and introduction of vectors for the expression of heterologous genes.

The microorganism used in the invention is a bacterium, a yeast or a fungus. Preferentially, the microorganism is selected among Enter obacteriaceae, Bacillaceae, Streptomycetaceae and Corynebacteriaceae . More preferentially, the microorganism is of the genus Escherichia, Klebsiella, Pantoea, Salmonella or Corynebacterium. Even more preferentially, the microorganism is either the species Escherichia coli or Corynebacterium glutamicum.

^■ Increase of NADPH

According to the invention, the modified microorganism comprises, on the one hand, the modifications for an improved methionine production and, on the other hand, modifications for an improved production of available NADPH, by increasing transhydrogenase activity of PntAB.

PntAB is a transmembrane pyridine nucleotide transhydrogenase which catalyses the reduction of NADP ⁺ into NADPH via oxidation of NADH into NAD ⁺ , and which is composed of two subunits alpha and beta encoded respectively by the pntA and pntB genes.

The increased activity of PntAB enhances the production of available NADPH in the cell. This does not lead automatically to an increased concentration in the cell, since available NADPH may be directly consumed by NADPH dependent enzymes. The term "increased activity of PntAB" in this context describes the increase in the intracellular activity of the transhydrogenase which is encoded by the corresponding pntA and pntB genes, for example by increasing the number of copies of the gene, using a stronger promoter or using an allele with increased activity and possibly combining these means. In a specific embodiment of the invention, the genes encoding pntAB are overexpressed.

In a particular embodiment of the invention, the pntAB genes are overexpressed using promoters exhibiting high strength. Such promoters, for example, are those belonging to the Ptrc family which each exhibits a specific strength. The Ptrc promoter is an artificial hybrid promoter exhibiting consensus sequences of typical E. coli promoter Plac and Ptrp, in particularly the -35 box of Plac and -10 box of Ptrp (Amann et al, 1983, Gene and Amann et al, 1988, Gene). The inventors modified this artificial promoter by modifying consensus sequences of -10 or -35 boxes in accordance with promoter comparison in Hawley study (Hawley & McClure, 1983, Nucleic Acids Research) to obtain series of promoters with different strengths. The more the sequences are different from the consensus -10 and -35 boxes; the lower is the strength of the promoter. The Ptrc promoters used in the invention are named Ptrc weighted by a number: the higher is the number, the larger is the difference between the artificial promoter sequence and the consensus sequence.

In a specific embodiment of the invention, the increase of the available NADPH is obtained by increasing the activity of PntAB in combination with an attenuation of UdhA activity. UdhA is a soluble pyridine nucleotide transhydrogenase which catalyses essentially the oxidation of NADPH into NADP ⁺ via the reduction of NAD ⁺ into NADH.

As described above, the attenuation of UdhA activity may be achieved by suppressing partially or completely the corresponding udhA gene, by inhibiting the expression of the udhA gene, deleting all or part of the promoter region necessary for the gene expression, by a deletion in the coding region of the gene or by the exchange of the wildtype promoter by a weaker, natural or synthetic, promoter.

In a preferred embodiment of the invention, the attenuation of UdhA activity is achieved by a complete deletion of the udhA gene by homologous recombination.

■ CI metabolism

In a preferred embodiment of the invention, the increased production of NADPH is coupled with the increase of the one carbon metabolism (said CI metabolism) in the methionine-producing microorganism. The CI metabolism is well known by the man skilled in the art and is based on the folate metabolisation. The folate is reduced into dihydrofolate by folate reductase and then into tetrahydrofolate (said THF) by dihydrofolate reductase. THF is a compound involved in DNA base and amino acid biosynthesis.

THF accepts a methylene group originating from glycine or serine and forms methylene-THF. In the case of serine the transfer of the methylene group to THF is catalysed by serine hydroxymethyltransferase (GlyA) or in the case of glycine by the glycine cleavage complex (GcvTHP). 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 H3 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.

In the methionine biosynthesis, methylene-THF can be reduced by methylene tetrahydrofolate reductase (MetF) to form methyl-THF. Methyl-THF is the donor of methyl during the methylation of homocysteine by the methyltransferases MetE or MetH to form methionine.

Increasing CI metabolism leads to an improved methionine production.

According to the invention, "increasing CI metabolism" relates to the increase of the activity of at least one enzyme involved in the CI metabolism chosen among MetF, GcvTHP, Lpd, GlyA,; MetE or MetH. For increasing enzyme activity, the corresponding genes of these different enzymes may be overexpressed or modified in their nucleic sequence to expressed enzyme with improved activity or their sensitivity to feed-back regulation may be decreased.

In a preferred embodiment of the invention, the one carbon metabolism is increased by enhancing the activity of methylenetetrahydrofolate reductase MetF and/or the activity of glycine cleavage complex GcvTHP and/or the activity of serine hydroxymethyltransferase GlyA.

In a specific embodiment of the invention, the activity of MetF is enhanced by overexpressing the gene metF and/or by optimizing the translation.

In a specific embodiment of the invention, overexpression of metF gene is achieved by expressing the gene under the control of a strong promoter belonging to the Ptrc family promoters, or under the control of an inducible promoter, like a temperature inducible promoter P _R as described in application PCT/FR2009/052520.

According to another embodiment of the invention, optimisation of the translation of the protein MetF is achieved by using a RNA stabiliser. Other means for the overexpression of a gene are known to the expert in the field and may be used for the overexpression of the metF gene. ^■ Optimization of methionine biosynthesis pathway:

As said above, the modified microorganism used in this invention may comprise further modifications in available NADPH production and CI metabolism, modifications for an improved methionine production.

Genes involved in methionine production in a microorganism 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 been described in patent applications WO2005/111202, WO2007/077041 and WO2009/043803. These applications are incorporated as reference into this application.

The patent application WO2005/111202 describes a methionine producing strain that overexpresses homoserine succinyltransferase alleles with reduced feed-back sensitivity to its inhibitors SAM and methionine. This application describes also the combination of theses alleles with a deletion of the methionine repressor MetJ responsible for the down-regulation of the methionine regulon. In addition, application describes combinations of the two modifications with the overexpression of aspartokinase/homoserine dehydrogenase.

For improving the production of methionine, the microorganism may exhibit:

- an increased expression of at least one gene selected in the group consisting of:

• cysP which encodes a periplasmic sulphate binding protein, as described in WO2007/077041 and in WO2009/043803,

• cysU which encodes a component of sulphate ABC transporter, as described in WO2007/077041 and in WO2009/043803,

• cysW which encodes a membrane bound sulphate transport protein, as described in WO2007/077041 and in WO2009/043803,

• cysA which encodes a sulphate permease, as described in WO2007/077041 and in WO2009/043803,

• cysM which encodes an O-acetyl serine sulfhydralase, as described in WO2007/077041 and in WO2009/043803,

• cysl and cysJ encoded respectively the alpha and beta subunits of a sulfite reductase as described in WO2007/077041 and in WO2009/043803. Preferably cysl and cysJ are overexpressed together,

• cysH which encodes an adenylyl sulfate reductase, as described in WO2007/077041 and in WO2009/043803,

• cysE which encodes a serine acyltransferase, as described in WO2007/077041, • serA which encodes a phosphoglycerate dehydrogenase, as described in WO2007/077041 and in WO2009/043803,

• serB which encodes a phosphoserine phosphatase, as described in WO2007/077041 and in WO2009/043803,

• serC which encodes a phosphoserine aminotransferase, as described in WO2007/077041 and in WO2009/043803,

• metA alleles which encode an homoserine succinyltransferases with reduced feed-back sensitivity to S-adenosylmethionine and/or methionine as described in WO2005/111202,

• thrA or thrA alleles which encode aspartokinases/homoserine dehydrogenase with reduced feed-back inhibition to threonine, as described in WO2009/043803 and WO2005/111202,

- or an inhibition of the expression of at least one of the following genes:

• pykA which encodes a pyruvate kinase, as described in WO2007/077041 and in WO2009/043803,

• pykF which encodes a pyruvate kinase, as described in WO2007/077041 and in WO2009/043803,

• purU which encodes a formyltetrahydrofolate deformylase, as described in WO2007/077041 and in WO2009/043803.

• yncA which encodes a N-acetyltransferase, as described in WO 2010/020681

• metJ which encodes for a repressor of the methionine biosynthesis pathway, as described in WO2005/111202.

In a specific embodiment of the invention, genes may be under control of an inducible promoter. Patent application PCT/FR2009/052520 describes a methionine producing strain that expressed thrA allele with reduced feed-back inhibition to threonine and cysE under control of an inducible promoter. This application is incorporated as reference into this application.

In a preferred embodiment of the invention, thrA gene or allele is under control of a temperature inducible promoter. In a most preferred embodiment, the temperature inducible promoter used is belonging to the family of P _R promoters.

In another aspect of the invention, the activity of the pyruvate carboxylase is enhanced. Increasing activity of pyruvate carboxylase is obtained by overexpressing the corresponding gene or modifying the nucleic sequence of this gene to expresse an enzyme with improved activity. In another embodiment of the invention, the pyc gene is introduced on the chromosome in one or several copies by recombination or carried by a plasmid present at least at one copy in the modified microorganism. The pyc gene originates from Rhizobium etli, Bacillus subtilis, Pseudomonas fluorescens, Lactococcus lactis or Corynebacterium species.

In a particular embodiment of the invention, the overexpressed genes are at their native position on the chromosome, or are integrated at a non-native position. For an optimal methionine production, several copies of the gene may be required, and these multiple copies are integrated into specific loci, whose modification does not have a negative impact on methionine production.

Examples for locus into which a gene may be integrated, without disturbing the metabolism of the cell, are:

accession

Locus function

number

Pseudogene, phage terminase protein A homolog, N-terminal aaaD 87081759 fragment

Pseudogene, phage terminase protein A homolog, C-terminal aaaE 1787395 fragment

Pseudogene, ferric ABC family transporter permease; C- afuB 1786458 terminal fragment

predicted ferric ABC transporter subunit (ATP -binding afuC 87081709 component)

agaA 48994927 Pseudogene, C-terminal fragment, GalNAc-6-P deacetylase agaW 1789522 Pseudogene, N-terminal fragment, PTS system EIICGalNAc alpA 1788977 protease

appY 1786776 DNA-binding transcriptional activator

argF 1786469 ornithine carbamoyltransferase

argU none arginine tRNA

argW none Arginine tRNA(CCU) 5

arpB 87081959 Pseudogene reconstruction, ankyrin repeats

arrD 1786768 lysozyme

arrQ 1787836 Phage lambda lysozyme R protein homolog

arsB 87082277 arsenite transporter

arsC 1789918 arsenate reductase

arsR 1789916 DNA-binding transcriptional repressor

beeE 1787397 Pseudogene, N-terminal fragment, portal protein

borD 1786770 bacteriophage lambda Bor protein homolog

cohE 1787391 Cl-like repressor

croE 87081841 Cro-like repressor

cspB 1787839 Cold shock protein

cspF 1787840 Cold shock protein homolog

cspl 1787834 Cold shock protein

cybC 1790684 Pseudogene, N-terminal fragment, cytochrome b562

dicA 1787853 Regulatory for dicB

dicB 1787857 Control of cell division dicC 1787852 Regulatory for dicB

dicF none DicF anti sense sRNA

eaeH 1786488 Pseudogene, intimin homolog

efeU 87081821 Pseudogene reconstruction, ferrous iron permease emrE 1786755 multidrug resistance pump

essD 1786767 predicted phage lysis protein

essQ 87081934 Phage lambda S lysis protein homolog

exoD 1786750 Pseudogene, C-terminal exonuclease fragment

eyeA none novel sRNA, unknown function

flu 48994897 Antigen 43

flxA 1787849 Unknown

gapC 87081902 Pseudogene reconstruction, GAP dehydrogenase gatR 87082039 Pseudogene reconstruction, repressor for gat operon glvC 1790116 Pseudogene reconstruction

glvG 1790115 Pseudogene reconstruction, 6-phospho-beta-glucosidase gnsB 87081932 Multicopy suppressor of secG(Cs) and fabA6(Ts) gtrA 1788691 Bactoprenol-linked glucose translocase

gtrB 1788692 Bactoprenol glucosyl transferase

gtrS 1788693 glucosyl transferase

hokD 1787845 Small toxic membrane polypeptide

icd 1787381 Isocitrate dehydrogenase

icdC 87081844 Pseudogene

ilvG 87082328 Pseudogene reconstruction, acetohydroxy acid synthase II insA 1786204 IS 1 gene, transposition function

insA 1786204 IS 1 gene, transposition function

insB 1786203 IS 1 insertion sequence transposase

insB 1786203 IS 1 insertion sequence transposase

insC 1786557 IS2 gene, transposition function

insD 1786558 IS2 gene, transposition function

insD 1786558 IS2 gene, transposition function

insE 1786489 IS3 gene, transposition function

insF 1786490 IS3 gene, transposition function

insH 1786453 IS5 gene, transposition function

insH 1786453 IS5 gene, transposition function

insH 1786453 IS5 gene, transposition function

insl 1786450 IS30 gene, transposition function

insl(-l) 1786450 IS30 gene, transposition function

insM 87082409 Pseudogene, truncated IS600 transposase

insN 1786449 Pseudogene reconstruction, IS911 transposase ORFAB insO none Pseudogene reconstruction, IS911 transposase ORFAB insX 87081710 Pseudogene, IS3 family transposase, N-terminal fragment insZ 1787491 Pseudogene reconstruction, IS4 transposase family, in ISZ' intA 1788974 Integrase gene intB 1790722 Pseudogene reconstruction, P4-like integrase

intD 1786748 predicted integrase

intE 1787386 el4 integrase

intF 2367104 predicted phage integrase

intG 1788246 Pseudogene, integrase homolog

intK 1787850 Pseudogene, integrase fragment

intQ 1787861 Pseudogene, integrase fragment

intR 1787607 Integrase gene

intS 1788690 Integrase

intZ 1788783 Putative integrase gene

isrC none Novel sRNA, function unknown

jayE 87081842 Pseudogene, C-terminal fragment, baseplate

kilR 87081884 Killing function of the Rac prophage

lafU none Pseudogene, lateral flagellar motor protein fragment lfhA 87081703 Pseudogene, lateral flagellar assembly protein fragment lit 1787385 Cell death peptidase

Pseudogene reconstruction, lorn homolog; outer membrane lomR 1787632 protein interrupted by IS5Y, missing N-terminus

malS 1789995 a-amylase

mcrA 1787406 5-methylcytosine-specific DNA binding protein

Pseudogene reconstruction, lipoprotein drug pump OMF mdtQ 87082057 family

melB 1790561 melibiose permease

mmuM 1786456 homocysteine methyltransferase

mmuP 870811708 S-methylmethionine permease

mokA none Pseudogene, overlapping regulatory peptide, enables hokB ninE 1786760 unknown

nmpC 1786765 Pseudogene reconstruction, OM porin, interrupted by IS5B nohD 1786773 DNA packaging protein

Pseudogene, phage lambda Nul homolog, terminase small nohQ 1787830 subunit family, putative DNA packaging protein

ogrK 1788398 Positive regulator of P2 growth

ompT 1786777 outer membrane protease VII

oweE none Pseudogene, lambda replication protein O homolog oweS 1788700 Pseudogene, lambda replication protein O homolog pauD none argU pseudogene, DLP12 prophage attachment site pawZ none CPS-53 prophage attachment site attR, argW pseudogene pbl 87082169 Pseudogene reconstruction, pilT homolog

Pseudogene, phage lambda replication protein P family; C- peaD 87081754 terminal fragment

perR 1786448 predicted DNA-binding transcriptional regulator

outer membrane porin of poly-P-l,6-N-acetyl-D-glucosamine pgaA 1787261 (PGA) biosynthesis pathway

pgaB 1787260 PGA N-deacetylase UDP-N-acetyl-D-glucosamine β-Ι,ό-Ν-acetyl-D- pgaC 1787259 glucosaminyl transferase

pgaD 1787258 predicted inner membrane protein

phnE 87082370 Pseudogene reconstruction, phosphonate permease pinE 1787404 DNA invertase

pinH 1789002 Pseudogene, DNA invertase, site-specific recombination pinQ 1787827 DNA invertase

pinR 1787638 DNA invertase

prfH 1786431 Pseudogene, protein release factor homolog

psaA none ssrA pseudogene, CP4-57 attachment site duplication ptwF none thrW pseudogene, CP4-6 prophage attachment site quuD 1786763 predicted antitermination protein

quuQ 87081935 Lambda Q antitermination protein homolog

racC 1787614 unknown

racR 1787619 Rac prophage repressor, cl-like

ralR 1787610 Restriction alleviation gene

rbsA 1790190 D-ribose ABC transporter subunit (ATP -binding component) rbsD 87082327 D-ribose pyranase

recE 1787612 RecET recombinase

recT 1787611 RecET recombinase

relB 1787847 Antitoxin for RelE

relE 1787846 Sequence-specific mRNA endoribonuclease

rem 1787844 unknown

Pseudogene reconstruction, lambda ren homolog, interrupted renD 87081755 by IS3C; putative activator of lit transcription

rhsE 1787728 Pseudogene, rhs family, encoded within RhsE repeat rnlA 1788983 RNase LS, endoribonuclease

rph 1790074 Pseudogene reconstruction, RNase PH

rusA 1786762 Endonuclease

rzoD 87081757 Probable Rzl-like lipoprotein

rzoQ none Probable Rzl-like lipoprotein

rzoR 87081890 Probable Rzl-like lipoprotein

rzpD 1786769 predicted murein endopeptidase

rzpQ 1787835 Rz-like equivalent

rzpR 87081889 Pseudogene, Rz homolog

sieB 87081885 Superinfection exclusion protein

Pseudogene, antisense sRNA blocking mokA/hokA sokA none translation

C-terminal Stf variable cassette, alternate virion-host stfE 87081843 specificity protein; Tail Collar domain, pseudogene stfP 1787400 Predicted tail fiber protein

stfR 87081892 Side-tail fiber protein

tfaD 87081759 Pseudogene, tail fiber assembly gene, C-terminal fragment tfaE 1787402 Predicted tail fiber assembly gene tfaP 1787401 Predicted tail fiber assembly gene

tfaQ 2367120 Phage lambda tail fiber assembly gene homolog tfaR 1787637 Phage lambda tail fiber assembly gene homolog tfaS 87082088 Pseudogene, tail fiber assembly gene, C-terminal fragment

Pseudogene reconstruction, tail fiber assembly gene, C- tfaX 2367110 terminal fragment

thrW none threonine tRNA (attachment site of the CP4-6 prophage) tori 87082092 CPS-53/KpLEl exisionase

treB 2367362 subunit of trehalose PTS permease (IIB/IIC domains) treC 1790687 trehalose-6-phosphate hydrolase

trkG 1787626 Major constitutive K+ uptake permease

ttcA 1787607 Integrase gene

ttcC none Pseudogene, prophage Rac integration site ttcA duplication uidB 1787902 Glucuronide permease, inactive point mutant

uxaA 1789475 altronate hydrolase

uxaC 2367192 uronate isomerase

wbbL 1788343 Pseudogene reconstruction, rhamnosyl transferase wcaM 1788356 predicted colanic acid biosynthesis protein

Pseudogene, exisionase fragment in defective prophage xisD none DLP12

xisE 1787387 el4 excisionase

yabP 1786242 Pseudogene reconstruction

Pseudogene, C-terminal fragment, H repeat-associated yafF 87081701 protein

yafU 1786411 Pseudogene, C-terminal fragment

yafW 1786440 antitoxin of the Ykfl-YafW toxin-antitoxin system yafX 1786442 unknown

predicted DNA-binding transcriptional regulator; inner yafY 1786445 membrane lipoprotein

yafZ 87081705 unknown

yagA 1786462 predicted DNA-binding transcriptional regulator yagB 87081711 Pseudogene, antitoxin-related, N-terminal fragment yagE 1786463 predicted lyase/synthase

yagF 1786464 predicted dehydratase

yagG 1786466 putative sugar symporter

yagH 1786467 putative β-xylosidase

yagi 1786468 predicted DNA-binding transcriptional regulator yagJ 1786472 unknown

yagK 1786473 unknown

yagL 1786474 DNA-binding protein

yagM 2367101 unknown

yagN 2367102 unknown

yagP 1786476 Pseudogene, LysR family, fragment

yaiT 1786569 Pseudogene reconstruction, autotransporter family yaiX 87082443 Pseudogene reconstruction, interrupted by IS2A ybbD 1786709 Pseudogene reconstruction, novel conserved family ybcK 1786756 predicted recombinase

ybcL 1786757 predicted kinase inhibitor

ybcM 1786758 predicted DNA-binding transcriptional regulator ybcN 1786759 DNA base-flipping protein

ybcO 1786761 unknown

ybcV 87081758 unknown

ybcW 1786772 unknown

ybcY 48994878 Pseudogene reconstruction, methyltransferase family ybeM 1786843 Pseudogene reconstruction, putative CN hydrolase ybfG 87081771 Pseudogene reconstruction, novel conserved family ybfl none Pseudogene reconstruction, KdpE homolog

ybfL 87081775 Pseudogene reconstruction, H repeat-associated protein ybfO 1786921 Pseudogene, copy of Rhs core with unique extension ycgH 87081847 Pseudogene reconstruction

ycgl 1787421 Pseudogene reconstruction, autotransporter homolog ycjV 1787577 Pseudogene reconstruction, malK paralog

ydaC 1787609 unknown

ydaE 87081883 Metallothionein

ydaF 87081886 unknown

ydaG 87081887 unknown

ydaQ 87081882 Putative exisionase

ydaS 1787620 unknown

ydaT 1787621 unknown

ydaU 1787622 unknown

ydaV 1787623 unknown

ydaW 87081888 Pseudogene, N-terminal fragment

ydaY 1787629 pseudogene

ydbA 87081898 Pseudogene reconstruction, autotransporter homolog yddK 1787745 Pseudogene, C-terminal fragment, leucine-rich yddL 1787746 Pseudogene, OmpCFN porin family, N-terminal fragment ydeT 1787782 Pseudogene, FimD family, C-terminal fragment ydfA 1787854 unknown

ydffl 87081937 unknown

ydfC 1787856 unknown

ydfD 1787858 unknown

ydffi 1787859 Pseudogene, N-terminal fragment

ydfJ 1787824 Pseudogene reconstruction, MFS family

ydfK 1787826 Cold shock gene

ydfO 87081931 unknown

ydfR 1787837 unknown

ydfU 87081936 unknown ydfV 1787848 unknown

ydfX 1787851 pseudogene

yedN 87082002 Pseudogene reconstruction, IpaH/YopM family

yedS 87082009 Pseudogene reconstruction, outer membrane protein homolog yeeH none Pseudogene, internal fragment

yeeL 87082016 Pseudogene reconstruction, glycosyltransferase family yeeP 87082019 Pseudogene, putative GTP -binding protein

yeeR 87082020 unknown

yeeS 1788312 unknown

yeeT 1788313 unknown

Antitoxin component of toxin-antitoxin protein pair YeeV- yeeU 1788314 YeeU

yeeV 1788315 Toxin component of toxin-antitoxin protein pair YeeV- YeeU yeeW 1788316 pseudogene

Pseudogene, gpD phage P2-like protein D; C-terminal yegZ none fragment

yehH 87082046 Pseudogene reconstruction

yehQ 87082050 Pseudogene reconstruction

yejO 1788516 Pseudogene reconstruction, autotransporter homolog

Pseudogene reconstruction, C-terminal fragment, LysR yfaH 1788571 homolog

yfaS 87082066 Pseudogene reconstruction

yfcU 1788678 Pseudogene reconstruction, FimD family

yfdK 1788696 unknown

yfdL 1788697 Pseudogene, tail fiber protein

Pseudogene, intact gene encodes a predicted DNA adenine yfdM 87082089 methyltransferase

yfdN 1788699 unknown

yfdP 1788701 unknown

yfdQ 1788702 unknown

yfdR 87082090 unknown

yfdS 1788704 unknown

yfdT 1788705 unknown

yfEL 1788784 unknown

yffM 1788785 unknown

yffN 1788786 unknown

yffO 1788787 unknown

yffP 1788788 unknown

yffQ 1788790 unknown

yffR 1788791 unknown

yffS 1788792 unknown

y 1788976 unknown

yfji 1788978 unknown

1788979 unknown yfjK 1788980 unknown

yfjL 1788981 unknown

yfjM 1788982 unknown

yfjO 87082140 unknown

yfjP 48994902 unknown

yfjQ 1788987 unknown

yfjR 1788988 unknown

yfjS 87082142 unknown

yfjT 1788990 unknown

yfjU 1788991 pseudogene

yfjV 1788992 Pseudogene reconstruction, arsB-like C-terminal fragment yfjW 2367146 unknown

yfjX 1788996 unknown

yfjY 1788997 unknown

yfjZ 1788998 Antitoxin component of putative toxin-antitoxin YpjF-YfjZ

Pseudogene reconstruction, has alpha-amylase-related ygaQ 1789007 domain

ygaY 1789035 Pseudogene reconstruction, MFS family

ygeF 2367169 Pseudogene reconstruction, part of T3SS PAI ETT2 remnant ygeK 87082170 Pseudogene reconstruction, part of T3SS PAI ETT2 remnant ygeN 1789221 Pseudogene reconstruction, orgB homolog

ygeO 1789223 Pseudogene, orgA homolog, part of T3SS PAI ETT2 remnant ygeQ 1789226 Pseudogene reconstruction, part of T3SS PAI ETT2 remnant yghE 1789340 Pseudogene reconstruction, general secretion protein family yghF 1789341 Pseudogene, general secretion protein

yghO 1789354 Pseudogene, C-terminal fragment

yghX 1789373 Pseudogene reconstruction, S9 peptidase family

yhcE 1789611 Pseudogene reconstruction, interrupted by IS5R

yhdW 1789668 Pseudogene reconstruction

yhiL 87082275 Pseudogene reconstruction, FliA regulated

yhiS 1789920 Pseudogene reconstruction, interrupted by IS5T

yhjQ 1789955 Pseudogene reconstruction

yibJ 48994952 Pseudogene reconstruction, Rhs family

yibS none Pseudogene reconstruction, Rhs family, C-terminal fragment yibU none Pseudogene reconstruction, H repeat-associated protein yibW none Pseudogene reconstruction, rhsA-linked

yicT none Pseudogene, N-terminal fragment

yifN 2367279 Pseudogene reconstruction

yjbl 1790471 Pseudogene reconstruction

yjdQ none Pseudogene reconstruction, P4-like integrase remnant yjgX 1790726 Pseudogene reconstruction, EptAB family

yjhD 87082406 Pseudogene, C-terminal fragment

yjhE 87082407 Pseudogene, putative transporter remnant Pseudogene reconstruction, helicase family, C-terminal yjhR 1790762 fragment

yjhV 1790738 Pseudogene, C-terminal fragment

yjhY none Pseudogene reconstruction, novel zinc finger family

Pseudogene reconstruction, rimK paralog, C-terminal yjhZ none fragment

yjiP 1790795 Pseudogene reconstruction, transposase family yjiT 87082428 Pseudogene, N-terminal fragment

Pseudogene reconstruction, helicase-like, C-terminal yjiv none fragment

yjjN 87082432 predicted oxidoreductase

ykfA 87081706 putative GTP -binding protein

ykffl 1786444 unknown

Pseudogene, retron-type reverse transcriptase family, N ykfC 87081707 terminal fragment

ykfF 1786443 unknown

ykfG 2367100 unknown

ykfH 87081704 unknown

ykfl 1786439 toxin of the Ykfl-YafW toxin-antitoxin system ykfj 1786430 Pseudogene, N-terminal fragment

ykfK 1786445 Pseudogene, N-terminal fragment

ykfL none Pseudogene, C-terminal fragment

ykfN none Pseudogene, N-terminal remnant, YdiA family ykgA 87081714 Pseudogene, N-terminal fragment, AraC family ykgP none Pseudogene, oxidoreductase fragment

Pseudogene, C-terminal fragment of a putative ykgQ none dehydrogenase

ykgS none Pseudogene internal fragment

ykiA 1786591 Pseudogene reconstruction, C-terminal fragment ylbE 1786730 Pseudogene reconstruction, yahG paralog

Pseudogene reconstruction, discontinuous N-terminal ylbG 87081748 fragment

ylbH 1786708 Pseudogene, copy of Rhs core with unique extension ylbl none Pseudogene, internal fragment, Rhs family

ylcG 87081756 unknown

ylcH none unknown

ylcl none unknown

ymdE 87081823 Pseudogene, C-terminal fragment

ymfD 1787383 Putative SAM-dependent methyltransferase ymfE 1787384 unknown

ymfl 87081839 unknown

ymfj 87081840 unknown

ymfL 1787393 unknown

ymfM 1787394 unknown

ymfQ 1787399 Putative baseplate or tail fiber proteintt ymfR 1787396 unknown

ymjC none Pseudogene, N-terminal fragment

ymjD none Expressed deletion pseudogene fusion remnant protein ynaA 1787631 Pseudogene, N-terminal fragment

ynaE 1787639 Cold shock gene

ynaK 1787628 unknown

ynci 1787731 Pseudogene reconstruction, H repeat-associated, RhsE-linked yncK none Pseudogene reconstruction, transposase homolog

Pseudogene reconstruction, C-terminal fragment, AraC yneL 1787784 family

Pseudogene reconstruction, putative OM autotransporter yneO 1787788 adhesi

ynfN 87081933 Cold shock gene

ynfO none unknown

yoeA 87082018 Pseudogene reconstruction, interrupted by IS2F

yoeD none Pseudogene, C-terminal fragment of a putative transposase yoeF 87082021 Pseudogene, C-terminal fragment

yoeG none pseudogene, N-terminal fragment

yoeH none pseudogene, C-terminal fragment

ypdJ 87082091 Pseudogene, exisonase fragment

ypjc 1789003 Pseudogene reconstruction

ypjF 1788999 Toxin component of putative toxin-antitoxin pair YpjF-YfjZ ypji none Pseudogene reconstruction

ypjJ 87082144 unknown

ypjK 87082141 unknown

Pseudogene reconstruction, C-terminal fragment, LysR yqffi 1789281 family

yqiG 48994919 Pseudogene reconstruction, FimD family, interrupted by IS2I

Pseudogene reconstruction, C-terminal fragment, yedZ yrdE none paralog

yrdF none Pseudogene, N-terminal fragment

yrhA 87082266 Pseudogene reconstruction, interrupted by IS IE

yrhC 87082273 Pseudogene reconstruction, N-terminal fragment

ysaC none Pseudogene, C-terminal remnant

ysaD none Pseudogene, internal sequence remnant

ytfA 1790650 Pseudogene, C-terminal fragment

yzgL 87082264 Pseudogene, putative periplasmic solute binding protein

The present invention is also related to a method for the production of methionine, comprising the steps of:

culturing a modified methionine-producing microorganism in an appropriate culture medium comprising a source of carbon, a source of sulphur and a source of nitrogen, and - recovering the methionine or one of its derivatives from the culture medium, wherein said microorganism is modified to present an enhanced transhydrogenase activity ofPntAB.

^■ Culture conditions

The terms "fermentative process', 'culture' or 'fermentation" are used interchangeably to denote the growth of bacteria on an appropriate growth medium containing a simple carbon source, a source of sulphur and a source of nitrogen.

An appropriate culture medium is a medium appropriate for the culture and growth of the microorganism. Such media are well known in the art of fermentation of microorganisms, depending upon the microorganism to be cultured.

The term "source of carbon" according to the invention denotes any source of carbon that can be used by those skilled in the art to support the normal growth of a microorganism, which can be hexoses such as glucose, galactose or lactose; pentoses; monosaccharides; disaccharides such as sucrose, cellobiose or maltose; oligosaccharides; molasses; starch or its derivatives; hemicelluloses; glycerol and combinations thereof. An especially preferred carbon source is glucose. Another preferred carbon source is sucrose.

In a particular 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, 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.

In a specific aspect of the invention, the culture is performed in such conditions that the microorganism is limited or starved for an inorganic substrate, in particular phosphate and/or potassium. Subjecting an organism to a limitation of an inorganic substrate defines a condition under which growth of the microorganisms is governed by the quantity of an inorganic chemical supplied that still permits weak growth. Starving a microorganism for an inorganic substrate defines the condition under which growth of the microorganism stops completely due, to the absence of the inorganic substrate. The fermentation is generally conducted in fermenters with an appropriate culture medium adapted to the microorganism being used, containing at least one simple carbon source, and if necessary co-substrates for the production of metabolites.

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.

As an example of known culture medium for E. coli, the culture medium can be of identical or similar composition to an M9 medium (Anderson, 1946, Proc. Natl. Acad. Sci. USA 32: 120-128), an M63 medium (Miller, 1992; A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) or a medium such as defined by Schaefer et al. (1999, Anal. Biochem. 270: 88-96).

As an example of known culture medium for C. glutamicum, the culture medium can be of identical or similar composition to BMCG medium (Liebl et al., 1989, Appl. Microbiol. Biotechnol. 32: 205-210) or to a medium such as described by Riedel et al. (2001, J. Mol. Microbiol. Biotechnol. 3: 573-583).

■ Recovering of methionine

The action of "recovering methionine from the culture medium" designates the action of recovering methionine or one of its derivatives, in particular SAM and NAM and all other derivatives that may be useful.

The present invention is also related to a method for the production of methionine, comprising the step of isolation of methionine, its precursors or derivatives, of the fermentation broth and/or the biomass, optionally remaining in portions or in the total amount (0-100%) in the end product.

After fermentation, L-methionine, its precursors or compounds derived thereof, is/are recovered and purified if necessary. The methods for the recovery and purification of the produced compound such as methionine, S-adenosyl-methionine and N-acetyl- methionine in the culture media are well known to those skilled in the art (WO 2005/007862, WO 2005/059155).

Optionally, from 0 to 100%, preferentially at least 90 %, more preferentially 95 %, even more preferentially at least 99% of the biomass may be retained during the purification of the fermentation product.

Optionally, the methionine derivative N-acetyl-methionine is transformed into methionine by deacylation, before methionine is recovered. EXEMPLES PROTOCOLES

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

Protocol 1: Chromosomal modifications by homologous recombination and selection of recombinants (Datsenko, K.A. & Wanner, B.L. (2000)

Allelic replacement or gene disruption in specified chromosomal loci was carried out by homologous recombination as described by Datsenko. & Wanner (2000). The chloramphenicol (Cm) resistance cat, the kanamycin (Km) resistance kan, or the gentamycin (Gt) resistance gm genes, flanked by Flp recognition sites, were amplified by PCR by using pKD3 or pKD4 or p34S-Gm (Dennis et Zyltra, AEM july 1998, p 2710- 2715) plasmids as template respectively. The resulting PCR products were used to transform the recipient E. coli strain harbouring plasmid pKD46 that expresses the λ Red (γ, β,.εχο) recombinase. Antibiotic-resistant transformants were then selected and the chromosomal structure of the mutated loci was verified by PCR analysis with the appropriate primers listed in Table 2.

The cat, kan and gm -resistance genes were removed by using plasmid pCP20 as described by Datsenko. & Wanner (2000), except that clones harbouring the pCP20 plasmid were cultivated at 37°C on LB and then tested for loss of antibiotic resistance at 30°C. Antibiotic sensitive clones were then verified by PCR using primers listed in Table 2

Protocol 2: Transduction of phage PI

Chromosomal modifications were transferred to a given E. coli recipient strain by PI transduction. The protocol is composed of 2 steps: (i) preparation of the phage lysate on a donor strain containing the resistance associated chromosomal modification and (ii) infection of the recipient strain by this phage lysate.

Preparation of the phage lysate

Inoculate 100 μΐ of an overnight culture of the strain MG1655 with the chromosomal modification of interest in 10 ml of LB + Cm 30μg/ml or Km 50μg/ml or Gt ^g/mL + glucose 0.2% + CaCl ₂ 5 mM.

Incubate 30 min at 37°C with shaking.

Add 100 μΐ of PI phage lysate prepared on the donor strain MG1655 (approx. 1 x 10 ⁹ phage/ml).

Shake at 37°C for 3 hours until the complete lysis of cells.

- Add 200 μΐ of chloroform, and vortex.

Centrifuge 10 min at 4500 g to eliminate cell debris.

Transfer of supernatant to a sterile tube.

- Store the lysate at 4°C. Transduction

Centrifuge 10 min at 1500 g 5 ml of an overnight culture of the E. coli recipient strain cultivated in LB medium.

- Suspend the cell pellet in 2.5 ml of MgS0 ₄ 10 mM, CaCl ₂ 5 mM.

Infect 100 μΐ cells with 100 μΐ PI phage of strain MG1655 with the modification on the chromosome (test tube) and as a control tubes 100 μΐ cells without PI phage and 100 μΐ PI phage without cells.

Incubate 30 min at 30°C without shaking.

Add 100 μΐ sodium citrate 1 M in each tube, and vortex.

- Add 1 ml of LB.

Incubate 1 hour at 37°C with shaking.

Centrifuge 3 min at 7000 rpm.

Plate on LB + Cm 30 μg/ml or Km 50 μg/ml or Gt 10μg/mL

Incubate at 37°C overnight.

The antibiotic-resistant transductants were then selected and the chromosomal structure of the mutated locus was verified by PCR analysis with the appropriate primers listed in Table 2.

Table 1 : Genotypes and corresponding numbers of intermediate strains and producer strains that appear in the following examples.

Strain Genotype

number

1 MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl 7-metF Ptrc07-serB ArnetJ ApykF ApykA ApurU AyncA

2 MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl 7-metF Ptrc07-serB ArnetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE

3 MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl 7-metF Ptrc07-serB ArnetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *1- cysE-PgapA-metA *11 : : Cm

4 MG1655 metA *ll Ptrc-metH VtrcF-cysPUWAM FtrcF-cysJIH Ftrc09-gcvTHP Vtrc36- ARNmstl 7-metF Ptrc07-serB ArnetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- VlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *1- cysE-PgapA-metA *11 AuxaCA::TT07-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA- metA *11

5 MG1655 metA *ll Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Ftrc09-gcvTHP Vtrc36- ARNmstl 7-metF Ptrc07-serB ArnetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l- cysE-PgapA-metA *11 AuxaCA ::TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 ACP4-6::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11

6 MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl 7-metF Ptrc07-serB ArnetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *1- cysE-PgapA-metA *11 AuxaCA ::TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 ACP4-6::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC::TT02-serA-serC ::Gt MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- metFr.Km Ptrc07-serB AmetJ ApykF ApykA ApurlJ AyncA AmalS::TTadc-CI857-PlambdaR*(- 35)-thrA *l-cysE ApgaABCD: :TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 AuxaCA ::TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 ACP4-6::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC ::TT02- serA-serC ::Gt

MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36-metF Ptrc07-serB AmetJ ApykF ApykA ApurlJ AyncA AmalS::TTadc-CI857-PlambdaR*(-35)- thrA *l-cysE ApgaABCD: :TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AuxaCA ::TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 ACP4-6::TT02- TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *ll AtreBC :: TT02-serA-serC :: Gt AudhA ::Km

MG1655 metA *ll Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36-metF Ptrc07-serB AmetJ ApykF ApykA ApurlJ AyncA AmalS::TTadc-CI857-?lambdaR*(-35)- thrA *l-cysE ApgaABCD: :TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 AuxaCA ::TT07-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 ACP4-6::TT02- TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *ll DtreBC :: TT02-serA-serC :: Gt PtrcOl-pntAB ::Cm AudhA ::Km

MG1655 metA *ll Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Ftrc09-gcvTHP Vtrc36- ARNmstl 7-metF Ptrc07-serB AmetJ ApykF ApykA ApurlJ AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE ApgaABCD: :TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *1- cysE-PgapA-metA *11 AuxaCA ::TT07-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA- metA *11 ACP4-6::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 DtreBC: :TT02- serA-serC

MG1655 metA *ll Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Ftrc09-gcvTHP Vtrc36- ARNmstl 7-metF Ptrc07-serB AmetJ ApykF ApykA ApurlJ AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE ApgaABCD: :TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *1- cysE-PgapA-metA *11 AuxaCA ::TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 DCP4-6: :TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC ::TT02- serA-serC PtrcOl-pntAB ::Cm

MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl 7-metF Ptrc07-serB AmetJ ApykF ApykA ApurlJ AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE ApgaABCD: :TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *1- cysE-PgapA-metA *11 AuxaCA: :TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 ACP4-6::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC ::TT02- serA-serC PtrcOl-pntAB ::Cm pCL1920-PgapA-pycRe-TT07

MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl 7-metF Ptrc07-serB AmetJ ApykF ApykA ApurlJ AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE DpgaABCD: :TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *1- cysE-PgapA-metA *11 AuxaCA ::TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 ACP4-6::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC ::TT02- serA-serC PtrcOl-pntAB ::Cm AudhA ::Km

MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36-metF Ptrc07-serB AmetJ ApykF ApykA ApurlJ AyncA AmalS::TTadc-CI857-PlambdaR*(-35)- thrA *l-cysE ApgaABCD: :TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AuxaCA ::TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 ACP4-6::TT02- TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AwcaM: : TT02-TTadc- PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC ::TT02-serA-serC PtrcOl- pntAB :: Cm AudhA ::Km MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl 7-metF Ptrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *1- cysE-PgapA-metA *11 AuxaCA::TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 ACP4-6::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC ::TT02- serA-serC PtrcOl-pntAB r. Cm AudhA ::Km pCL1920-PgapA-pycRe-TT07

MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl 7-metF Ptrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *1- cysE-PgapA-metA *11 AuxaCA::TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 ACP4-6::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC ::TT02- serA-serC PtrcOl-pntAB :: AudhA

MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl 7-metF Ptrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *1- cysE-PgapA-metA *11 AuxaCA::TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 ACP4-6::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC ::TT02- serA-serC Ptrc30-pntAB ::Cm AudhA

MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl 7-metF Ptrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *1- cysE-PgapA-metA *11 AuxaCA::TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 ACP4-6::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC ::TT02- serA-serC Ptrc30-pntAB r. Cm AudhA pCL1920-PgapA-pycRe-TT07

MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl 7-metF Ptrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *1- cysE-PgapA-metA *11 AuxaCA::TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 ACP4-6::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC ::TT02- serA-serC Ptrc97-pntAB ::Cm AudhA

MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl 7-metF Ptrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *1- cysE-PgapA-metA *11 AuxaCA::TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 ACP4-6::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC ::TT02- serA-serC Ptrc97-pntAB r. Cm AudhA pCL1920-PgapA-pycRe-TT07

MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl 7-metF Ptrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *1- cysE-PgapA-metA *11 AuxaCA::TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 ACP4-6::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC ::TT02- serA-serC Ptrc55-pntAB rCm AudhA MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl 7-metF Ptrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *1- cysE-PgapA-metA *11 AuxaCA::TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 ACP4-6::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 AwcaM::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC ::TT02- serA-serC ¥trc55-pntAB r. Cm AudhA pCL1920-?gapA-pycRe-TT07

MG1655 metA *ll Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Ftrc09-gcvTHP Vtrc36- ARNmstl 7-metF Ptrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *1- cysE-PgapA-metA *11 AuxaCA::TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 ACP4-6::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC ::TT02- serA-serC AudhA ::Km

MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl 7-metF Ptrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857- PlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *1- cysE-PgapA-metA *11 AuxaCA::TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 ACP4-6::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC ::TT02- serA-serC AudhA ::Km pCL1920-PgapA-pycRe-TT07

Table 2: Primers used for PCR verifications of chromosomal modifications described above

Location of the

homology with

Genes SEQ

Primers name the Sequences name ID °

chromosomal

region

3734778-

Ome0826-malS-F 1 GGTATTCCACGGGATTTTTCGCG

3734800

malS

3738298- CGTCAGTAATCACATTGCCTGTT

Ome0827-malS-R 2

3738322 GG

Ome 1691- 1093002-

11 GGGCTGATGCTGATTGAAC DpgaABCD verif F 1093020

pgaABCD

Ome-1692- 1084333-

12 GTGTTACCGACGCCGTAAACGG DpgaABCD verif R 1084354

3238676-

Ome 1612-uxaCA_R3 15 GGTGTGGTGGAAAATTCGTCG

3238696

uxaCA

3243726-

Ome 1774-DuxaCA F 16 GCATTACGATTGCCCATACC

3243707

259527-

Ome 1775-DCP4-

22 GCTCGCAGATGGTTGGCAAC 6_verif_F

259546

CP4-6

297672-

Ome 1776-DCP4-

23 TGGAGATGATGGGCTCAGGC 6_verif_R

297691

Ome 1595-DtreB- 4464951-

27 GTTGCCGAACATTTGGGAGTGC verif F 4464930

treBC

Omel596- 4462115-

28 GGAGATCAGATTCACCACATCC DtreB verif R 4462136

4130309- GCCCGGTACTCATGTTTTCGGGT

Ome0726-PtrcmetF F 29

4130337 TTATGG

metF

4130967- CCGTTATTCCAGTAGTCGCGTGC

Ome0727 PtrcmetF R 30

4130940 AATGG

4159070-

Oag0055-udhAF2 33 GTGAATGAACGGTAACGC

4159053

udhA

4157088-

Oag0056-udhAR2 34 GATGCTGGAAGATGGTCACT

4157107

1676137-

Omel l51-pntAF 37 CCACTATCACGGCTGAATCG

1676118

pntAB

1675668-

Omel l52-pntAR 38 GTCCCAGGATTCAGTAACGC

1675687

Omel707- 2115741-

43 GCCGTTCAACACTGGCTGGACG DwcaM verif F 2115762

wcaM

Omel708- 2110888-

44 TGCCATTGCAGGTGCATCGC DwcaM verif R 2110907 I. EXAMPLE 1: Construction of strain 7, MG1655 metA *ll Ytrc-metH YtrcF- cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36-metF::Km Vtrc07-serB AmetJ ApykF ApykA Apurll AyncA AmalS::TTadc-CI857-YlambdaR*(-35)-thrA *l-cysE ApgaABCD:: TT02-TTadc-YlambdaR *(-35)-RBS01-thrA *l-cysE-YgapA-metA *11

AuxaCA ::TT07-TTadc-YlambdaR*(-35)-RBS01-thrA *l-cysE-YgapA-metA *11 ACP4-6::TT02-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *ll AtreBC ::TT02-serA-serC ::Gt LI. Construction of strain 1

Methionine producing strain 1 (Table 1) has been described in patent application WO2007/077041 which is incorporated as reference into this application.

1.2. Construction of strain 2

The TTadc-CI857-PlambdaR*(-35)-thrA *l-cysE fragment was inserted at the malS locus in two steps. First the plasmid y\JC\%-DmalS:.TTadc-CI857-PlambdaR*(-35)-thrA *l- cysEr.Km was constructed and second the TTadc-CI857-PlambdaR*(-35)-thrA *l- cysE::Km fragment containing upstream and downstream sequences homologous to malS locus was introduced into the chromosome. Subsequently, the resistance cassette was removed according to Protocol 1.

All descriptions of integrations at different loci on the chromosome presented below are following the same method; 1) construction of duplication vector containing upstream and downstream homologous sequence of the locus of interest, the DNA fragment and a resistance cassette 2) construction of minimal strain (MG1655) containing the chromosomal modification of interest and 3) transduction into the complex strain (MG1655 containing already several modifications).

1.2.1. Construction of pUC18-AmalS::TTadc-CI857-PlambdaR*(-35)-thrA *l- cysEr.Km plasmid

The plasmid p\JCl8-AmalS: :TTadc-CI857-PlambdaR*(-35)-thrA *l-cysE is derived from plasmids pSCB-TTadc-cI857-VlambdaR*(-35)-thrA *l-cysE and pUC18-Awa/S: :MCS: :Km that have been described in patent application PCT/FR2009/052520. Briefly, the ApallBamHI digested TTadc-cI857-PlambdaR*(-35)-thrA *l-cysE fragment was cloned between the Apal and BamHI sites of the pUC18-Awa/S: :MCS-Km. The resulting plasmid was verified by DNA sequencing and called p\JCl8-AmalS: :TTadc-CI857-PlambdaR*(- 35)-thrA *l-cysE::Km.

1.2.2. Construction of strain MG1655 metA *ll AmalS::TTadc-CI857-

VlambdaR*(-35)-thrA *l-cysE::Km (pKD46)

To replace the malS gene by the TTadc-CI857-PlambdaR*(-35)-thrA * 1-cysE: :Km DNA fragment, pUCl&-DmalS: :TTadc-CI857-PlambdaR*(-35)-thrA *l-cysE::Km was digested by Seal and EcoR V and the remaining digested fragment TTadc-CI857-PlambdaR*(-35)- thrA *l-cysE::Km containing upstream and downstream sequence homologous to the malS locus was introduced into the strain MG1655 metA*ll pKD46 according to Protocol 1. Kanamycin resistant recombinants were selected and the presence of the AmalS.. TTadc- CI857-PlambdaR*(-35)-thrA * 1-cysE: :Km chromosomal modification was verified by PCR with primers Ome 0826-malS-F (SEQ ID N°l) and Ome 0827-malS-R (SEQ ID N°2) (Table 2) and by DNA sequencing. The verified and selected strain was called MG1655 metA *11 pKD46 AmalS: : TTadc-CI857-?lambdaR*(-35)-thrA *l-cysE::Km.

1.2.3. Transduction of AmalS: :TTadc-CI857-PlambdaR*(-35)-thrA *l-cysE::Km into strain 1 and removal of resistance cassette

The AmalS :.TTadc-CI857-PlambdaR*(-35)-thrA *l-cysE::Km chromosomal modification was transduced into the strain MG1655 metA * 11 Vtrc-metH VtrcF-cysPUWAM VtrcF- cysJIH Vtrc09-gcvTHP Vtrc36-ARNmstl7-metF Vtrc07-serB AmetJ ApykF ApykA Apurll AyncA with a PI phage lysate from strain MG1655 metA * 11 pKD46 AmalS: :TTadc- CI857-PlambdaR*(-35)-thrA *l-cysE::Km described above, according to Protocol 2.

Kanamycin resistant transductants were selected and the presence of AmalS::TTadc- CI857-PlambdaR*(-35)-thrA * 1-cysE: :Km chromosomal modification was verified by PCR with primers Ome 0826-malS-F (SEQ ID N°l) and Ome 0827-malS-R (SEQ ID N°2) (Table 2). The resulting strain has the genotype MG1655 metA * 11 Vtrc-metH VtrcF- cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36-ARNmstl7-metF Vtrc07-serB AmetJ ApykF ApykA Apurll AyncA AmalS: :TTadc-CI857-PlambdaR*(-35)-thrA * 1-cysE: :Km Finally, the kanamycin resistance of the above strain was removed according to Protocol 1. The loss of the kanamycin resistant cassette was verified by PCR by using the primers Ome 0826-malS-F (SEQ ID N°l) and Ome 0827-malS-R (SEQ ID N°2) (Table 2). The resulting strain MG1655 metA * 11 Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Vtrc09- gcvTHP Vtrc36-ARNmstl7-metF Ftrc07-serB AmetJ ApykF ApykA Apurll AyncA AmalS: :TTadc-CI857-PlambdaR*(-35)-thrA *l-cysE was called strain 2.

1.3. Construction of strain 3

1.3.1. Construction of the plasmid pUC18-ApgaABCD: :TT02-TTadc- PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *ll::Cm

Plasmid pVC\8-ApgaABCD: :TT02-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *ll::Cm is derived from plasmids yCL\920-TTadc-VlambdaR*(-35)-RBS01-thrA *l- cysE-PgapA-metA *ll and pUC\8-ApgaABCD: :TT02-MCS::Cm described below.

Construction of plasmid ^ L\920-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-VsapA- metA * 11

Plasmid pCL1920-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *ll is derived from plasmid yCL\920-TTadc-CI857-VlambdaR*(-35)-thrA *l-cysE-VgapA-metA *11 described in patent application PCT/FR2009/052520. To construct plasmid pCL1920-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *11, plasmid yCL\920-TTadc-CI857-VlambdaR*(-35)-thrA *l-cysE-VgapA-metA *11 was amplified with primers PR-RBS01 F (SEQ ID N°3) and TTadc-pCL1920_R (SEQ ID N°4). The PCR product was digested by Dpnl in order to eliminate the parental DNA template. The remaining cohesive overhang was phosphorylated and introduced in an E. coli strain to form plasmid pCL1920-TTadc-PlambdaR*(-35)-RBS0I-thrA *]-cysE-PgapA- metA *ll. The TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *l 1 insert and especially the presence of the RBS01 sequence upstream of the thrh gene were verified by DNA sequencing, using the following primers.

PR-RBS01 F (SEQ ID N°3)

ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACA

ATTTTACCTCTGGCGGTGATAATGGTTGCATGTACTAAGGAGGTTATAAATGA

GAGTGTTGAAGTTCGG

with

- upper case sequence homologous to lambda bacteriophage P _R promoter (PlambdaR*{- 35), (Mermet-Bouvier & Chauvat, 1994, Current Microbiology, vol. 28, pp 145-148 _; Tsurimoto T, Hase T, Matsubara H, Matsubara K, Mol Gen Genet. 1982; 187(l):79-86).

- underlined upper case sequence corresponding to RBS01 sequence with a Psil restriction site

- bold upper case sequence homologous to 5' end of thrA (1-20)

TTadc-pCL1920_R (SEQ ID N°4)

TAAAAAAAATAAGAGTTACCATTTAAGGTAACTCTTATTTTTAGGGCCCGGTA CCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGC

with

- upper case sequence homologous to TTadc transcriptional terminator sequence (transcription terminator of the adc gene from Clostridium acetobutylicum, homologous from 179847 to 179807 of the pSLOl megaplasmid),.

- bold upper case sequence homologous to pCL\920-TTadc-CI857-PlambdaR*(-35)- thrA *l-cysE-VgapA-metA *11 plasmid

Construction of pUC18-Apga^£C/J: :TT02-MCS: :Cm plasmid

To construct the ApgaABCD: :TT02-MCS: :Cm fragment, the upstream region of pgaA (uppgaA), the TT02 transcriptional terminator, the multiple cloning site (MCS) and the downstream region of pgaD (downpgaD) were joined by PCR. The chloramphenicol cassette {Cm) was subsequently amplified and added to the previous construct.

First, the uppgaA-TT02 was amplified from E. coli MG1655 genomic DNA using primers Ome 1687-ApgaABCD_amont_EcoRI_F (SEQ ID N°5) and Ome 1688- ApgaABCD_amont_TT02_BstZ 17I R (SEQ ID N°6) by PCR. Then, the TT02-MCS- down-pgaD fragment was amplified from E. coli MG1655 genomic DNA using primers Ome 1689-ApgaABCD_aval_MCS_BstZ17I_F (SEQ ID N°7) and Ome 1690- Apga AB CD aval EcoRI R (SEQ ID N°8) by PCR. Primers Ome 1688- ApgaABCD_amont_TT02_BstZ17I_R (SEQ ID N°6) and Ome 1689- ApgaABCD_aval_MCS_BstZ17I_F (SEQ ID N°7) have been designed to overlap through a 36 nucleotides-long region. Finally, the uppgaA-TT02-MCS-downpgaD fragment was amplified by mixing the uppgaA-TT02 and the TT02-MCS-down-pgaD amplification products using primers Ome 1687-ApgaABCD_amont_EcoRI_F (SEQ ID N°5) and Ome 1690-ApgaABCD_aval_EcoRI_R (SEQ ID N°8). The resulting fusion PCR product was digested by Hpal and cloned between the Hindlll and EcoRI of pUC18 plasmid that had been treated by Large (Klenow) Fragment of E. coli DNA Polymerase I. The resulting plasmid was verified by DNA sequencing and called p\JCl8-ApgaABCD: :TT02-MCS. Ome 1687-ApgaABCD_amont_EcoRI_F (SEQ ID N°5)

CGTAGTTAACGAATTCGACTAGAAAGTATGTGAGCAACTATCGGCCCCCC

with

- bold upper case sequence for Hpal and EcoRI restriction site and extrabases,

- upper case sequence homologous to sequence upstream pgaA gene (1092609- 1092576, reference sequence on the website http://ecouene.oru/)

Ome 1688-ApgaABCD_amont_TT02_BstZ17I_R (SEQ ID N°6)

GCTTGTATACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCG TTTTATTTGATGAGGCTGCGCTACGGCACTCACGG

with

- bold upper case sequence for BstZlll restriction site and extrabases,

- underlined upper case sequence corresponding to the transcriptional terminator 7) of E. coli rrnB (Orosz A, Boros I and Venetianer P. Eur. J. Biochem. 1991 Nov l;201(3):653-9)

- upper case sequence homologous to sequence upstream pgaA gene (1091829-1091851, reference sequence on the website http://ecogene.org/)

Ome 1689-ApgaABCD_aval_MCS_BstZ17I_F (SEQ ID N°7)

AGACTGGGCCTTTCGTTTTATCTGTTGTATACAAGCTTAATTAAGGGCCCGG GCGGATCCCCCAAAACAAAGCCCGGTTCGC

with

- bold upper case sequence complementary to the 3' end sequence of the transcriptional terminator 7) of E. coli rrnB (Orosz A, Boros I and Venetianer P. Eur. J. Biochem. 1991 Nov l;201(3):653-9),

- underlined upper case sequence containing a MCS multiple cloning site: BstZlll,

Hindlll, Pad, Apal, BamHl,

- upper case sequence homologous to sequence upstream of the pgaD gene ( 1085325- 1085304, reference sequence on the website http: //ecouen e . or u/ ) Ome 1690-ApgaABCD_aval_EcoRI_R (SEQ ID N°8)

CGTAGTTAACGAATTCAGCTGATATTCGCCACGGGC

with

- bold upper case sequence for Hpal and EcoBl restriction sites and extrabases, - upper case sequence homologous to sequence upstream of the pgaD gene

( 1084714- 1084733, reference sequence on the website http://ecogene.org/)

Finally, primers Ome 1603- K7_Cm_ampl_SmaI_BstZ17I_F (SEQ ID N°9) and Ome 1604-K7_Cm_ampl_HindIII_R (SEQ ID N°10) were used to amplify the chloramphenicol cassette and the FRT sequences from plasmid pKD3 and the fragment was cloned between the BstZlTl and Hindlll sites of plasmid pUC18-Ap o^5CD: :TT02-MCS. The resulting plasmid was verified by DNA sequencing and called p\JCl8-ApgaABCD: :TT02- MCS: :Cm.

Ome 1603- K7_Cm_ampl_SmaI_BstZ17I_F (SEQ ID N°9)

TCCCCCGGGGTATACTGTAGGCTGGAGCTGCTTCG

With

- underlined upper case sequence for BstZ17I and Smal restriction site and extrabases,

- upper case sequence corresponding to site primer 1 of plasmid pKD3 (Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645)

Ome 1604-K7_Cm_ampl_HindIII_R (SEQ ID N° 10)

GCCCAAGCTTCATATGAATATCCTCCTTAG

with

- underlined upper case sequence Hindlll restriction site and extrabases,

- upper case sequence corresponding to site primer 2 of pKD3 plasmid (Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645).

Construction of the pVC\8-Ap£aABCD: :TT02-TTadc-VlambdaR*(-35)-RBS01-thrA *l- cysE-PgapA-metA *ll::Cm

To construct pUC 1 -ApgaABCD: : TT02-TTadc-?lambdaR *(-35)-RBS01-thrA *l-cysE- VgapA-metA *ll::Cm, the ApallBamHl fragment TTadc-VlambdaR*(-35)-RBS01-thrA *l- cysE-?gapA-metA *ll isolated from pCL\920-TTadc-VlambdaR*(-35)-RBS01-thrA *l- cysE-PgapA-metA * 11 described above was cloned between the Apal and BamHI sites of plasmid :TT02-MCS: :Cm described above. The resulting plasmid was verified by DNA sequencing and called y\JC\%-ApgaABCD:.TT02-TTadc-VlambdaR*(- 35)-RBS01-thrA *l-cysE-VgapA-metA *ll::Cm.

1.3.2. Construction of strain MG1655 metA *ll ApgaABCD: : TT02-TTadc-

PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *ll::Cm (pKD46)

To replace the pgaABCD operon by the TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l- cysE-VgapA-metA *ll::Cm region, pUCl -ApgaABCD: : TT02-TTadc-?lambdaR*(-35)- RBS01-thrA *l-cysE-PgapA-metA *l l::Cm was digested by Sapl and Aatll restriction enzymes and the remaining digested fragment ApgaABCD: : TT02-TTadc-PlambdaR*(-35)- RBS01-thrA *l-cysE-PgapA-metA *l 1 : :Cm was introduced into strain MG1655 met A* 11 pKD46 according to Protocol 1.

Chloramphenicol resistant recombinants were selected and the presence of the ApgaABCD: :TT02-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *ll::Cm chromosomal modification was verified by PCR with primers Ome 1691- DpgaABCD verif F (SEQ ID N°l l) and Ome 1692-DpgaABCD verif R (SEQ ID N°12) (Table 2) and by DNA sequencing. The verified and selected strain was called MG1655 metA *11 ApgaABCD: : TT02-TTadc-?lambdaR *(-35)-RBS01-thrA *l-cysE-?gapA- metA *ll::Cm (pKD46).

1.3.3. Transduction of ApgaABCD: : TT02-TTadc-PlambdaR *(-35)-RBS01-thrA *1- cysE-PgapA-metA * 11 : :Cm into strain 2

The ApgaABCD: : TT02-TTadc-?lambdaR *(-35)-RBS01-thrA *l-cysE-?gapA-metA *ll::Cm chromosomal modification was transduced into strain 2 (Table 1), described above, with a PI phage lysate from strain MG1655 metA * 11 pKD46 ApgaABCD: :TT02-TTadc- PlambdaR*(-35)-RBS01-thrA * 1-cysE-PgapA-metA * 11 : :Cm described above, according to Protocol 2.

Chloramphenicol resistant transductants were selected and the presence of the ApgaABCD: :TT02-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *ll::Cm chromosomal modification was verified by PCR with Ome 1691-DpgaABCD verif F (SEQ ID N°l l) and Ome 1692-DpgaABCD verif R (SEQ ID N°12) (Table 2). The resulting strain MG1655 metA * 11 Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Vtrc09- gcvTHP Vtrc36-ARNmstl7-metF Ftrc07-serB AmetJ ApykF ApykA Apurll AyncA AmalS: : TTadc-CI857-VlambdaR *(-35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-

VlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *ll::Cm was called strain 3.

1.4. Construction of strain 4

1.4.1. Construction of pUC18-AuxaCA::TT07-TTadc-PlambdaR*(-35)-RBS01- thrA * 1-cysE-PgapA-metA *ll::Km plasmid

To construct the plasmid pUC18-AwxaCA::rr07-MCS-Km, the AuxaCA:.TT07-MCS-Km fragment was obtained by PCR using the E.coli MG1655 AwxaCA: :TT07-MCS-Km genomic DNA as template and cloned into pUC18 (Norrander et al., 1983, Gene 26,101- 106).

Construction of strain MG1655 A¾xaCA: :TT07-MCS-Km pKD46

To replace the uxaCA region by the TT07-MCS-Km fragment, Protocol 1 was used except that primers Ome 1506-DuxaCA-MCS-F (SEQ ID N°13) and Ome 1507-DuxaCA-MCS-R (SEQ ID N°14) were used to amplify the kanamycin resistance cassette from pKD4 plasmid. Ome 1506-DuxaCA-MCS-F (SEQ ID N°13)

GCAAGCTAGCTCACTCGTTGAGAGGAAGACGAAAATGACTCCGTTTATGACTGAAGA TTTCCTGTTAGA Z4CCGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCTGTA GGCTGGAGCTGCTTCG

with

- italic upper case sequence homologous to sequence upstream uxaCA locus (3242797-3242724, reference sequence on the website http://ecogene.org/),

- underlined upper case sequence for T7Te transcriptional terminator sequence from phage T7 (Harrington K.J., Laughlin R.B. and Liang S. Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):5019-24.),

- upper case sequence corresponding to primer site 2 of plasmid pKD4 (Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645).

Ome 1507-DuxaCA-MCS-R (SEQ ID N°14)

TTAACAACTCATTTCGACTTTATAGCGTTACGCCGCTTTTGAAGATCGCCGAATTCG AGCTCGGTACCCGGGGATCCATCTCGAGATCCGCGGATGTATACATGGGCCCC ATATGAATATCCTCCTTAG

with

- italic upper case sequence homologous to sequence downstream of the uxaCA locus (3239830-3239879, reference sequence on the website http://ecogene.org/),

- a region (underlined upper case) for the MCS containing the sequence of the restriction sites Apal, BstZlTl, SacH, Xhol, Aval, BamHI, Smal, Kpnl, Sad, EcoRI,

- upper case corresponding to primer site 1 of plasmid pKD4 (Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645),

Kanamycin resistant recombinants were selected and the insertion of the resistance cassette was verified by PCR with primers Ome 1612-uxaCA_R3 (SEQ ID N°15) and Ome 1774- DuxaCA F (SEQ ID N°16) (Table 2) and by DNA sequencing. The verified and selected strain was called MG1655 AuxaCA: :TT07-MCS-Km pKD46.

Construction of plasmid pUC18-A¾xaCA::rr07-MCS: :Km

The AwxaCA: :TT07-MCS-Km region was amplified by PCR from E. coli MG1655 AwxaCA: :TT07-MCS-Km genomic DNA described above as template with primers Ome 1515-uxaCA-R2 (SEQ ID N°17) and Ome 1516-uxaCA-F2 (SEQ ID N°18).

Ome 1515-uxaCA R2 (SEQ ID N ⁰ 17)

CCCACTGGCCTGTAATATGTTCGG, homologous to sequence downstream uxaCA locus (3239021-3239044)

Ome 1516-uxaC A F2 (SEQ ID N° 18)

ATGCGAT ATCGACCGTATAAGCAGCAGAATAGGC

with

- underlined upper case sequence for the restriction site EcoR V and extra-bases - italic upper case sequence homologous to the sequence upstream of the uxaCA locus (3243425-3243402)

Then, the resulting PCR product (obtained with a blunt-end DNA polymerase) was digested by the restriction enzyme EcoKV and cloned into the Smal site of pUC18. The resulting plasmid was verified by DNA sequencing and called pUC18-AwxaCA: :TT07- MCS-Km.

Construction of pVC\8-AuxaCA-TT07-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE- VgapA-metA *ll::Km

To construct pVC\8-AuxaCA-TT07-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA- metA *ll::Km, the TTadc-?lambdaR*(-35)-RBS01-thrA * 1-cysE-VgapA-metA * 11 ApallBamm digested fragment from pCL\920-TTadc-VlambdaR*(-35)-RBS01-thrA *l- cysE-PgapA-metA *l 1 described above was cloned into the Apal and BamHI sites of pUC18-AuxaCA: :TT07-MCS-Km described above. The resulting plasmid was verified by DNA sequencing and called pVC\8-AuxaCA-TT07-TTadc-VlambdaR*(-35)-RBS01- thrA *l-cysE-VgapA-metA *ll::Km

1.4.2. Construction of strain MG1655 metA *ll AuxaCA: :TT07-TTadc- PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 (pKD46) To replace the uxaCA operon by the TT07-TTadc-PlambdaR*(-35)-RBS01-thrA * 1-cysE- VgapA-metA *11: :Km region, pUC 1 - AuxaCA : : TTO 7-TTadc-VlambdaR *(-35)-RBS01- thrA * 1-cysE-PgapA-metA * 11 : :Km was digested by BamHI and Ahdl and the remaining digested fragment AuxaCA: :TT07-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *ll::Km was introduced into strain MG1655 metA *ll pKD46 according to Protocol 1.

Kanamycin resistant recombinants were selected and the presence of the AuxaCA: :TT07- TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *ll::Km chromosomal modification was verified by PCR with primers Ome 1612-uxaCA_R3 (SEQ ID N°15) and Ome 1774-DuxaCA F (SEQ ID N°16) (Table 2) and by DNA sequencing. The resulting strain was designated MG1655 metA *ll AuxaCA: :TT07-TTadc-VlambdaR*(-35)-RBS01- thrA *l-cysE-VgapA-metA *ll::Km (pKD46)

1.4.3. Transduction of AuxaCA: :TT07-TTadc-PlambdaR*(-35)-RBS01-thrA * 1- cysE-PgapA-metA * 11 : :Km into strain 3

The AuxaCA : : TT07-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *ll::Km chromosomal modification was transduced into strain 3 (Table 1), described above, with a PI phage lysate from strain MG1655 metA *ll pKD46 AuxaCA: :TT07-TTadc- VlambdaR*(-35)-RBS01-thrA * 1-cysE-VgapA-metA * 11 : :Km described above, according to Protocol 2.

The AuxaCA: :TT07-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *ll::Km chromosomal modification was verified by PCR with Ome 1612-uxaCA_R3 (SEQ ID N°15) and Ome 1774-DuxaCA_F (SEQ ID N°16) (Table 2). The resulting strain was called MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Vtrc36-ARNmstJ7-metF Ftrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc- CI857-VlambdaR *(-35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-?lambdaR *(-35)-RBS01- thrA *l-cysE-?gapA-metA *ll::Cm AuxaCA::TT07-TTadc-VlambdaR*(-35)-RBS01- thrA *l-cysE-VgapA-metA *ll::Km

Finally, the chloramphenicol and kanamycin resistance cassettes of the above strain were removed according to Protocol 1. The loss of the chloramphenicol and kanamycin resistance cassettes was verified by PCR with primers Ome 1691-DpgaABCD_verif_F (SEQ ID N°l l), Ome 1692-DpgaABCD verif R (SEQ ID N°12), Ome 1612-uxaCA_R3 (SEQ ID N°15) and Ome 1774-DuxaCA F (SEQ ID N°16) (Table 2). The resulting strain MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl7-metF Ftrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS: :TTadc-CI857- VlambdaR *(-35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-?lambdaR *(-35)-RBS01-thrA *1- cysE-VgapA-metA *11 AuxaCA::TT07-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-

VgapA-metA *l 1 was called strain 4.

1.5. Construction of strain 5

1.5.1. Construction of plasmid pMA-DCP4-6::TT02-TTadc-PlambdaR*(-35)- thrA *l-cysE-PgapA-metA *ll::Km

Plasmid pMA-ACP4-6::TT02-TTadc-VlambdaR*(-35)-thrA *l-cysE-?gapA-metA *ll::Km is derived from pCL\920-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *l 1 described above and pMA-ACP4-6: :TT02-MCS:.Km described below.

Construction of plasmid ρΜΑ-ΔΟ^-fr :Jr02-MCS: :Km

First pMA-ACP4-6: :TT02 -MCS was synthesized by GeneArt (http://www.geneart.com/). The ACP4-6: :TT02-MCS fragment was cloned into the Ascl and Pad sites of plasmid pMA from GeneArt and contains the following sequence identified as SEQ ID N° 19: ggcgcgccaggcctagggCCGACGATGTACGTCAGGTGTGCAACCTCGCCGATCCGGTG GGGCAGGTAATCGATGGCGGCGTACTGGACAGCGGCCTGCGTCTTGAGCGTCG TCGCGTACCGCTGGGGGTTATTGGCGTGATTTATGAAGCGCGCCCGAACGTGA CGGTTGATGTCGCTTCGCTGTGCCTGAAAACCGGTAATGCGGTGATCCTGCGC GGTGGCAAAGAAACGTGTCGCACTAACGCTGCAACGGTGGCGGTGATTCAGG ACGCCCTGAAATCCTGCGGCTTACCGGCGGGTGCCGTGCAGGCGATTGATAAT CCTGACCGTGCGCTGGTCAGTGAAATGCTGCGTATGGATAAATACATCGACAT GCTGATCCCGCGTGGTGGCGCTGGTTTGCATAAACTGTGCCGTGAACAGTCGA CAATCCCGGTGATCACAGGTGGTATAGGCGTATGCCATATTTACGTTGATGAA AGTGTAGAGATCGCTGAAGCATTAAAAGTGATCGTCAACGCGAAAACTCAGCG

TCCGAGCACATGTAATACGGTTGAAACGTTGCTGGTGAATAAAAACATCGCCG

ATAGCTTCCTGCCCGCATTAAGCAAACAAATGGCGGAAAGCGGCGTGACATTA CACGCAGATGCAGCTGCACTGGCGCAGTTGCAGGCAGGCCCTGCGAAGGTGGT TGCTGTTAAAGCCGAAGAGTATGACGATGAGTTTCTGTCATTAGATTTGAACGT CAAAATCGTCAGCGATCTTGACGATGCCATCGCCCATATTCGTGAACACGGCA CACAACACTCCGATGCGATCCTGACCCGCGATATGCGCAACGCCCAGCGTTTT GTTAACGAAGTGGATTCGTCCGCTGTTTACGTTAACGCCTCTACGCGTTTTACC GACGGCGGCCAGTTTGGTCTGGGTGCGGAAGTGGCGGTAAGCACACAAAAAC TCCACGCGCGTGGCCCAATGGGGCTGGAAGCACTGACCACTTACAAGTGGATC GGCATTGGTGATTACACCATTCGTGCGTAACATCAAATAAAACGAAAGGCTC AGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTG7¾ TACAAGCTTAATAAGGG CCCGGGCGGA TCCC ACC ACGTTTCCTCCTGTGCCGT ATTTGTGCC ATTGT AACC TTGGCAATTCATCAAAATACTGTTCTGACATCAGGCAGTGCAGGTGCAGACAT TTAAGCCAATTGCTGCCGCCATTCTTTGACGTAGTCAATCAGGGCGCGGAGCTT TGGTGCAATATTGCGACGCTGTGGGAAATACAGATAGAAGCCCGGAAATTGTG GAAGAAAGTCATCAAGCAGCGATACAAGTTTACCGCTTTCAATATATGGCCTG AAAGTTTCCTGAGTGGCAATTGTTATTCCTCCGCCGGC AAGAGCCAGCCTC AA CATCAGACGCAGATCATTAGTCGTAATCTGCGGTTCAATCGCAAGGTCGAAAG TTCTCCCGTTTTCTTCAAATGGCCAGCGATAAGGCGCAACCTCCGGGGACTGAC GCCAGCCGATACACTTATGGGTATTTCCCCCGGAGGCGAGAAAGCACTCTCCA CGCCCGGCCGCAAGGATCAGGACGACGGGGGCAGGCATGAATCCTCCTCCTG ATGGAGACGTACAGAGGCGACTTCTGCCAGCACGGAGAGTGCCAGAGTATGC GCATCCCGGGCTTTGGGGAATATCCCGACGGGTGCCCGGATTTGCGTTGTTTCC TCCCTGGACCATCCCAGCTCGTGGAGCTTTTGCAGACGTAACGTGTGGGTTCGA TAGCTGCCCAATGCGCCGAGATAAAAGGGTTTTGCTTCTCGCGCGGCCTGCAA CACTGGCAGCTCCCGGTTGAGATCATGGCACAGCAAAATGACCGCCGTATCGG TATCGATCTGAGCGCTGGCTGAGGCCGGAAAAAGATCGAAGATATGGCTGTCA TAGCCTGTGGCTGCTGCAAGACTCGCGGTTGCCTGCGCCTCAAGAGAACGTCC GTAAATCATCAGCCTGACGCATGGCCTGAACCCCACCTCAAAGCCATTGAGAT TCCAGCCCGTCCGGGTTTGCGTGGGCAGGCACACCAGCGATTGTGCTTGCGGA TCGTAGCGCAGCCCCACCGGTTTTCTCTGTTCCAGGCGGTTCAGCACGGCGAG CAGAGGCTGTGCCGAGCGTAGggtacctcttaattaa

- lower case corresponding to Ascl, Stul and Avrll restriction sites

- underlined lower cases homologous to sequence upstream of the CP4-6 locus (260955- 261980, http://ecouene.oru/).

- bold upper case corresponding to a TT02 transcriptional terminator sequence from the T] of E. coli rrnB gene (Orosz A, Boros I and Venetianer P. Eur. J. Biochem. 1991 Nov l;201(3):653-9)

- italic upper case sequence corresponding to a MCS containing BstZlll, Hindlll, Apal, Smal and BamHl restriction sites - upper case homologous to sequence downstream of the CP4-6 locus (296511-297581, http://ecogene.org/)

- underlined lower case corresponding to Kpnl and Pad restriction sites.

Second, primers Ome 1605-K7_Km_ampl_SmaI_BstZ17I_F (SEQ ID N°20) and Ome 1606-K7_Km_ampl_HindIII_R (SEQ ID N°21), were used to amplify the kanamycin cassette from plasmid pKD4 that was subsequently cloned between the BstZlll and Hindlll sites of the pMA-ACP4-6: :TT02-MCS. The resulting plasmid was verified by DNA sequencing and called pMA-ACP4-6::TT02-MCS-Km.

Ome 1605-K7_Km_ampl_SmaI_BstZ17I_F (SEQ ID N°20)

TCCCCCGGGGTATACCATATGAATATCCTCCTTAG

with

- underlined upper case sequence for BstZlll and Smal restriction sites and extrabases,

- upper case sequence corresponding to site primer 1 of plasmid pKD4 (Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645)

Ome 1606-K7_Km_ampl_HindIII_R (SEQ ID N°21)

GCCCAAGCTTTGTAGGCTGGAGCTGCTTCG

with

- underlined upper case sequence for Hindlll restriction site and extrabases, - upper case sequence corresponding to site primer 2 of plasmid pKD4 (Datsenko,

K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645)

To construct pMA-ACP4-6::TT02-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA- metA *ll::Km the TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 ApallBamHl digested fragment from pCL\920-TTadc-VlambdaR*(-35)-RBS01-thrA *l- cysE-PgapA-metA *11 described above was cloned into the Apal and BamHl sites of pMA- ACP4-(5:. r02-MCS-Km described above. The resulting plasmid was verified by DNA sequencing and called pMA-ACP4-6::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE- VgapA-metA *ll::Km

1.5.2. Construction of strain MG1655 metA *ll DCP4-6::TT02-TTadc- PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *ll::Km (pKD46)

To replace the CP 4-6 operon by the TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE- VgapA-metA *ll::Km fragment plasmid pMA-ACP4-6::TT02-TTadc-VlambdaR*(-35)- RBS01-thrA *l-cysE-PgapA-metA *ll::Km was digested by Kpnl and Avrll and the remaining digested fragment ACP4-6: :TT02-TTadc-PlambdaR*(-35)-RBS01-thrA * 1-cysE- VgapA-metA * 11 : :Km was introduced into the strain MG1655 met A* 11 pKD46 according to Protocol 1.

Kanamycin resistant recombinants were selected and the presence of the ACP4-6::TT02- TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *ll::Km chromosomal modification was verified by PCR with primers Omel775-DCP4-6_verif_F (SEQ ID N°22) and Omel776-DCP4-6_verif_R (SEQ ID N°23) (Table 2) and by DNA sequencing. The resulting strain was called MG1655 metA *ll ACP4-6::TT02-TTadc-?lambdaR*(-35)- RBSOl-thrA *l-cysE-?gapA-metA *ll::Km (pKD46)

1.5.3. Transduction of ACP4-6::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA * 1- cysE-PgapA-metA *ll::Km into strain 4 and removal of the resistance cassette.

The ACP4-6::TT02-TTadc-?\ambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *ll::Km chromosomal modification was transduced into strain 4 (Table 1), described above, with a PI phage lysate from strain MG1655 metA *11 pKD46 ACP4-6::TT02-TTadc-?lambdaR*(- 35)-RBS01-thrA * 1-cysE-PgapA-metA * 11 : :Km described above, according to Protocol 2. Kanamycin resistant transductants were selected and the presence of the ACP4-6::TT02- TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *ll::Km chromosomal modification was verified by PCR with Omel775-DCP4-6_verif_F (SEQ ID N°22) and Omel776-DCP4-6_verif_R (SEQ ID N°23) (Table 2). The resulting strain was called MG1655 metA * 11 Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl7-metF Ftrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS: :TTadc-CI857- VlambdaR *(-35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-?lambdaR *(-35)-RBS01-thrA *1- cysE-VgapA-metA *11 AuxaCA::TT07-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE- VgapA-metA *11 ACP4-6::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA- metA *ll::Km..

Finally, the kanamycin resistance cassette of the above strain was removed by using Protocol 1. The loss of the kanamycin resistance cassette was verified by PCR by using primers Omel775-DCP4-6_verif_F (SEQ ID N°22) and Omel776-DCP4-6_verif_R (SEQ ID N°23) (Table 2). The resulting strain MG1655 metA * 11 Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36-ARNmstl7-metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS: :TTadc-CI857-?lambdaR*(-35)-thrA *l-cysE ApgaABCD: :TT02- TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AuxaCA::TT07-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 ACP4-6::TT02-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 was called strain 5.

1.6. Construction of strain 6

To insert the TT02-serA-serC region, which encodes an E. coli phosphoglycerate dehydrogenase and a phosphoserine aminotransferase, respectively, at the treBC locus, a two steps methods was used. First plasmid pMA-AtreBC::TT02-serA-serC::Gt was constructed and second the AtreBC : :TT02-serA-serC: :Gt fragment that contains upstream and downstream regions homologous to the treBC locus was recombined into the chromosome according to Protocol 1. 1.6.1. Construction of plasmid pMA -AtreBC: : TT02-serA -serC: : Gt

Plasmid pMA-AtreBC::TT02-serA-serC::Gt is derived from pMA-AtreBC::TT02-MCS::Gt described below, p34S-Gm (Dennis et Zyltra, AEM july 1998, p 2710-2715), and pUC18- serA-serC, which has been described in patent application PCT FR2009/052520.

First plasmid pMA-AtreBC::TT02-MCS was been synthesized by GeneArt (http://www.geneart.com/). The AtreBC: :TT02 -MCS fragment was cloned into the AscI and Pad sites of plasmid pMA from GeneArt and which contains the following sequence, identified as SEQ ID N° 24:

ggcgcgccgGCAATCAAAATCCTGATGCAACGGCTGTATGACCAGGGGCATCGTAA TATCAGTTATCTCGGCGTGCCGCACAGTGACGTGACAACCGGTAAGCGACGTC ACGAAGCCTACCTGGCGTTCTGCAAAGCGCATAAACTGCATCCCGTTGCCGCC CTGCCAGGGCTTGCTATGAAGCAAGGCTATGAGAACGTTGCAAAAGTGATTAC GCCTGAAACTACCGCCTTACTGTGCGCAACCGACACGCTGGCACTTGGCgcaagta aatacctgcaagagcaacgcatcgacaccttgcaactggcgagcgtcggtaatacgccgt taatgaaattcctccatccggagatc gtaaccgtagatcccggttacgccgaagctggacgccaggcggcttgccagttgatcgcg caggtaaccgggcgcagcgaacc gcaacaaatcatcatccccgccaccctgtcctgatcgtttcctgaacgataaattgtgat ctCATCAAATAAAACGA AAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTG7¾ TACAAGCTT ^J 4GGGCCCGGGCGG^JCCGTCTGTCAGTATGTTGTTTTTGTTGATTTTTCAA CCAGCAAATTCATTAAAAAATTTACATATCGCTGTAGCGCCCGTCATCCGTACG CTCTGCTTTTTACTTTGAGCTACATCAAAAAAAGCTCAAACATCCTTGATGCAA AGCACTATATATAGACTTTAAAATGCGTCCCAACCCAATATGTTGTATTAATCG ACTATAATTGCTACTACAGCTCCCCACGAAAAAGGTGCGGCGTTGTGGATAAG CGGATGGCGATTGCGGAAAGCACCGGAAAACGAAACGAAAAAACCGGAAAAC GCCTTTCCCAATTTCTGTGGATAACCTGTTCTTAAAAATATGGAGCGATCATGA CACCGCATGTGATGAAACGAGACGGCTGCAAAGTGCCGTTTAAATCAGAGCGC ATCAAAGAAGCGATTCTGCGTGCAGCTAAAGCAGCGGAAGTCGATGATGCCG ATTATTGCGCCACTGTTGCCGCGGTTGTCAGCGAGCAGATGCAGGGCCGCAAC CAGGTGGATATCAATGAGATCCAGACCGCAGTTGAAAATCAGCTGttoattoa

- lower cases corresponding to AscI restriction site

- underlined upper cases homologous sequence upstream of the treBC locus (4460477- 4461034).

- bold upper cases corresponding to a TT02 transcriptional terminator sequence from the 7 _/ terminator of the E. coli rrn gene (Orosz A, Boros I and Venetianer P. Eur. J. Biochem. 1991 Nov l;201(3):653-9)

- italic upper cases corresponding to a multiple cloning sites containing BstZIlI,

Hindlll, Apal, Smal and BamH\ restriction sites

- upper cases homologous to sequence downstream of the treBC locus (4464294-4464787) -italic lower cases corresponding to Pad restriction site To construct plasmid pMA-AtreBC::TT02-MCS : :Gt, the FRT-Gt-FRT resistance cassette was amplified by PCR with primers BstZ17I-FRT-Gt-F (SEQ ID N°25) and Hindlll-FRT- Gt-R (SEQ ID N°26) using p34S-Gm as template.

BstZ17I-FRT-Gt-F (SEQ ID N°25)

TCCCCCGGGGTATACTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTC TAGAGAATAGGAACTTCGGAATAGGAACTTCATTTAGATGGGTACCGAGCT CGAATTG

with

- underlined upper case sequence for Smal and BstZlll restriction sites and extrabases,

- bold upper case sequence corresponding to the FRT sequence (Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645)

- upper case sequence homologous to sequence of the gentamycin gene located on p34S- Gm (Dennis et Zyltra, AEM July 1998, p 2710-2715).

Hindlll-FRT-Gt-R (SEQ ID N°26)

CCCAAGCTTCATATGAATATCCTCCTTAGTTCCTATTCCGAAGTTCCTATTC

TCTAGAAAGTATAGGAACTTCGGCGCGGATGGGTACCGAGCTCGAATTG

with

- underlined upper case sequence for the Hindlll restriction site and extrabases, - bold upper case sequence corresponding to the FRT sequence (Datsenko, K.A. &

Wanner, B.L., 2000, PNAS, 97: 6640-6645)

- upper case sequence homologous to the sequence of the gentamycin gene located on p34S-Gm (Dennis et Zyltra, AEM July 1998, p 2710-2715).

The FRT-Gt-FRT PCR product was then cloned between the BstZlll and Hindlll sites of pMA-AtreB::TT02-MCS. The resulting plasmid was verified by DNA sequencing and called pMA-AtreBC::TT02-MCS-Gt.

To construct the final plasmid pMA-AtreBC: :TT02-serA-serC: :Gt, pUCl&serA-serC was digested by Hindlll and the serA-serC digested fragment was cloned into the pMA- AtreBC ::TT02-MCS-Gt that had been linearized by Hindlll. The resulting plasmid was verified by digestion and by DNA sequencing and was called pMA-AtreBC::TT02-serA- serC :.Gt

1.6.2. Construction of strain MG1655 metA *ll pKD46 AtreBC::TT02-serA- serCr. Gt

To replace the treBC gene by the TT02-serA-serC::Gt fragment, pMA-AtreBC::TT02- serA-serCr. Gt was digested by Apal and Sail and the remaining digested fragment AtreBC: :TT02-serA-serC: :Gt was introduced into the strain MG1655 metA *ll pKD46 according to Protocol 1. Gentamycin resistant recombinants were selected and the presence of the AtreBC ::TT02- serA-serC::Gt fragment was verified by PCR with primers Ome 1595-DtreB verif F (SEQ ID N°27) and Ome 1596- DtreB_verif_R (SEQ ID N°28) (Table 2) and by DNA sequencing. The resulting strain was called MG1655 metA *ll pKD46 AtreBC ::TT02 -serA- serC::Gt.

1.6.3. Transduction of AtreBC: : TT02-serA -serC: : Gt into strain 5

The AtreBC ::TT02-serA-serC::Gt chromosomal modification was then transduced into strain 5 (Table 1) with a PI phage lysate from strain MG1655 metA *ll pKD46 AtreBC :: TTO 2 -serA-serC: :Gt described above, according to Protocol 2.

Gentamycin resistant transductants were selected and the presence of the DtreBC: :TT02- serA-serC: :Gt chromosomal modification was verified by PCR with Ome 1595- DtreB verif F (SEQ ID N°27) and Ome 1596-DtreB verif R (SEQ ID N°28) (Table 2). The resulting strain MG1655 metA *ll Vtrc-metH VtrcF-cysPlJWAMV\rcF-cysJIH Vtrc09- gcvTHP Vtrc36-ARNmstJ7-metF Ftrc07-serB AmetJ ApykF ApykA Apurll AyncA AmalS: : TTadc-CI857-VlambdaR *(-35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-

VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 AuxaCA ::TT07-TTadc-

VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 ACP4-6::TT02-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *ll AtreBC ::TT02-serA-serC ::Gt was called strain 6.

1.7. Construction of strain 7 by transduction of Pfrc?6-metF::Km into strain 6

Overexpression of the metF gene from artificial promoter integrated at the metF locus into the chromosome, strain MG1655 metA *ll AmetJ Vtrc36-metF::Km, has been described in patent application WO 2007/077041.

The ^' Ptrc36-metF::Km promoter construct was transduced into strain 6 (Table 1) with a PI phage lysate from strain MG1655 metA *ll AmetJ Vtrc36-metF r.Km according to Protocol 2.

Kanamycin resistant transductants were selected and the presence of the 7trc36-metF: :Km promoter construct was verified by PCR with primers Ome0726-PtrcmetF-F (SEQ ID N°29) and Ome0727-PtrcmetF-R (SEQ ID N°30) (Table 2). The resulting strain MG1655 metA *ll Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc07-serB AmetJ ApykF ApykA Apurll AyncA AmalS: :TTadc-CI857-PlambdaR*(-35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-?lambdaR *(-35)-RBS01-thrA *l-cysE-?gapA-metA *11

AuxaCA: :TT07-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 ACP4- 6::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 AtreBC ::TT02- serA-serCr. Gt Vtrc36-metF ::Km was called strain 7. II. EXAMPLE 2: Construction of strain 9, MG1655 metA Hl Ytrc-metH YtrcF- cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36-metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857-VlambdaR*(-35)-thrA *l-cysE ApgaABCD::TT02-TTadc-YlambdaR*(-35)-RBS01-thrA *l-cysE-YgapA-metA *ll AuxaCA::TT07-TTadc-YlambdaR*(-35)-RBS01-thrA *l-cysE-YgapA-metA *11

A CP4-6:: TT02-TTadc-VlambdaR *(-35)-RBS01-thrA Π-cysE-VgapA-metA *11 DtreBC ::TT02-serA-serC::Gt Ytrc01-pntAB::Cm AudhAr.Km

II.1. Construction of strain 8

II.1.1. Construction of MG1655 metA *ll AmetJ Vtrc36-metF by removal of the kanamycine resistance cassette

The kanamycin resistance of the strain MG1655 metA *ll AmetJ Vtrc36-metF::Km, described in patent application WO2007/077041, was removed according to Protocol 1. Loss of the kanamycin resistant cassette was verified by PCR by using the primers Ome0726-PtrcmetF-F (SEQ ID N°29) and Ome0727-PtrcmetF-R(SEQ ID N°30) (Table 2). The resulting strain was called MG1655 metA *ll AmetJVtrc36-metF.

11.1.2. Construction o ^" MG1655 metA *ll AmetJ Vtrc36-metF AudhA::Km pKD46 To delete the udhA gene in strain MG1655 metA *ll AmetJ Vtrc36-metF, Protocol 1 has been used except that primers Ome0032-DUdhAF (SEQ ID N°31) and Ome0034- DUdhAR (SEQ ID N°32) were used to amplify the kanamycin resistance cassette from plasmid pKD4.

Kanamycin resistant recombinants were selected. The insertion of the resistance cassette was verified by PCR with primers Oag0055-udhAF2 (SEQ ID N°33) and Oag0056- udhAR2 (SEQ ID N°34) (Table 2) and by DNA sequencing. The resulting strain was called MG1655 metA *11 AmetJ Vtrc36-metF AudhAr.Km pKD46.

Ome0032-DUdhAF (SEQ ID N°31)

GGTGCGCGCGTCGCAGTTATCGAGCGTTATCAAAATGTTGGCGGCGGTTGCAC

CCACTGGGGCACCATCCCGTCGAAAGCCATATGAATATCCTCCTTAG

with:

- upper case sequence homologous to sequence upstream of the udhA gene

(4158729-4158650, reference sequence on the website http://ecogene.org/)

- underlined upper case sequence corresponding to the primer site 1 of plasmid pKD4 (Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645)

Ome0034-DUdhAR (SEQ ID N°32)

CCC AGAATCTCTTTTGTTTCCCGATGGAAC AAAATTTTC AGCGTGCCC ACGTTC ATGCCGACGATTTGTGCGCGTGCCAGTGTAGGCTGGAGCTGCTTCG

with - upper case sequence homologous to sequence downstream of the udhA gene (4157588-4157667, reference sequence on the website http://ecogene.org/)

- underlined upper case sequence corresponding to the primer site 2 of plasmid pKD4 (Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645)

II.1.3. Co-transduction of Ptrc36-metF AudhAr.Km into strain 6

The genes udhA and metF are close on the E. coli chromosome, 89.61 min and 89.03 min respectively and since phage PI can package 2 min of the E. coli chromosome, the genes udhA and metF are co-transducibles. Thereby, the Vtrc36-metF promoter modification and AudhA::Km deletion described above, were co-transduced into strain 6 (Table 1) by using a PI phage lysate from the strain MG1655 metA *11 AmetJVtrc36-metF AudhA::Km pKD46, described above, according to Protocol 2.

Kanamycin resistant transductants were selected. The presence of the Ptrc36-wet promoter modification was verified by PCR with primers Ome0726-PtrcmetF-F (SEQ ID N°29) and Ome0727-PtrcmetF-R (SEQ ID N°30) followed by DNA sequencing. The presence of the OudhA: :Km deletion was verified by PCR with primers Oag0055-udhAF2 (SEQ ID N°33) and Oag0056-udhAR2 (SEQ ID N°34) (Table 2). The resulting strain MG1655 metA * 11 Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS: :TTadc-CI857-PlambdaR*(- 35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-?lambdaR *(-35)-RBS01-thrA *l-cysE-?gapA- metA *11 AuxaCA::TT07-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 ACP4-6::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *ll

AtreBC : : TT02-serA-serC: :Gt AudhA ::Km was called strain 8.

Π.2. Construction of strain 9

11.2.1. Construction of strain MG1655 metA *11 pKD46 PtrcOl -pntAB:: Cm

To increase the expression level of pyridine nucleotide transhydrogenase alpha and beta subunits, PntA and PntB, a constitutive artificial trcOl promoter was added upstream of the pntAB operon into the strain MG1655 metk *ll pKD46 according to Protocol 1, except that primers Omel 149-Ptrc-pntAF (SEQ ID N°35) and Ome 1150-Ptrc-pntAR (SEQ ID N°36) were used to amplify the chloramphenicol resistance cassette from plasmid pKD3. Chloramphenicol resistant recombinants were selected. The presence of the artificial promoter VtrcOl and the insertion of the resistance cassette were verified by PCR with primers Omel 151-pntAF (SEQ ID N°37) and Omel l52-pntAR (SEQ ID N°38) (Table 2) and by DNA sequencing. The resulting strain was called MG1655 metA * 11 pKD46 PtrcOl-pntABr. Cm.

Ome 1149-Ptrc-pntAF (SEQ ID N°35)

GCTCGTACATGAGCAGCTTGTGTGGCTCCTGACACAGGCAAACCATCAT CAATAAAACCGATTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCC4 TA TGA

ATATCCTCCTTAG with

- upper case sequence homologous to sequence upstream of the pntA gene (1676002-1675941 reference sequence on the website http://ecogene.org/)

- underlined upper case sequence for T7Te transcriptional terminator sequence from phage T7 (Harrington K.J., Laughlin R.B. and Liang S. Proc Natl Acad Sci U S A.

2001 Apr 24;98(9):5019-24.),

- italic upper case sequence corresponding to the primer site 1 of plasmid pKD3 (Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645)

Ome 1150-Ptrc-pntAR (SEQ ID N°36)

GCTGC AAC ACGGGTTTC ATTGGTT AACCGTTCTCTTGGT ATGCC AATTCG

CATGATATTCCCTTCCTTCCACACATTATACGAGCCGGATGATTAATTGTCAAC AG ICTGTAGGCTGGAGCTGCTTCG

with

- upper case sequence homologous to sequence upstream of the pntA gene ( 1675875- 1675940, reference sequence on the website http://ecogene.org/)

- underlined upper case sequence for the trc promoter sequence

- Italic upper case sequence corresponding to the primer site 2 of plasmid pKD3 (Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645)

11.2.2. Transduction of PtrcO 1-pntAB : :Cm into strain 8

The ^' Ptrc01-pntAB::Cm promoter modification was transduced into strain 8 (Table 1) according to Protocol 2 by using a PI phage lysate from strain MG1655 metA * 11 pKD46 VtrcOl-pntAB: :Cm described above.

Chloramphenicol resistant transductants were selected and the presence of the VtrcOl- pntABr. Cm chromosomal modification was verified by PCR with Omel 151-pntAF (SEQ ID N°37) and Omel 152-pntAR (SEQ ID N°38) (Table 2). The resulting strain MG1655 metA *11 Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36-metF Vtrc07- serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857-VlambdaR*(-35)-thrA *l- cysE ApgaABCD::TT02-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 AuxaCA::TT07-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 ACP4- 6::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 AtreBC::TT02- serA-serCr. Gt VtrcO 1-pntAB: :Cm AudhAr.Km was called strain 9. III. EXAMPLE 3: Construction of strain 10, MG1655 metA Hl Ytrc-metH YtrcF- cysPUWAM YtrcF-cysJIH Ytrc09-gcvTHP Vtrc36-A RNmstl 7-metF Ytrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857-VlambdaR*(-35)-thrA *l- cysE ApgaABCD:: TT02- TTadc-YlambdaR *(-35)-RBS01-thrA *l-cysE-YgapA- metA *11 AuxaCA::TT07-TTadc-YlambdaR*(-35)-RBS01-thrA *l-cysE-YgapA- metA *11 ACP4-6::TT02-TTadc-VlambdaR*(-35)-RBS01-thrA Π-cysE-VgapA- metA ni AwcaM::TT02-TTadc-YlambdaR*(-35)-RBS01-thrA *l-cysE-YgapA- metA Hl AtreBC::TT02-serA-serC IIL1. Construction of plasmid pUC18-AwcaM::TT02-TTadc-YlambdaR*(-35)-

RBSOl-thrA *l-cysE-PgapA-metA *ll::Cm

Plasmid pUC 1 -AwcaM: : TT02-TTadc-?lambdaR *(-35)-RBS01-thrA *l-cysE-?gapA- metA *ll::Cm is derived from plasmid pCL1920-TTadc-PlambdaR*(-35)-RBS01-thrA *l- cysE-VgapA-metA *ll described above and plasmid pUC18-A vca ::rr02-MCS: :Cm described below.

III.1.1. Construction of plasmid pUC18-AwcaM: :TT02-MCS: :Cm

To construct the DwcaM::TT02-MCS::Cm fragment, overlapping PCR between the upstream region of wcaM (upwcaM), the TT02 transcriptional terminator, the multiple cloning site (MCS) and the downstream region of wcaM (downwcaM) was done and the chloramphenicol cassette (Cm) was amplified and cloned subsequently.

First, the fragment upwcaM-TT02 was amplified from E. coli MG1655 genomic DNA using primers Ome 1703-DwcaM-HpaI-EcoRI-F (SEQ ID N°39) and Ome 1704-DwcaM- TT02-BstZ17I-R (SEQ ID N°40) by PCR. Then, the TT02-MCS-downwcaM fragment was amplified from E. coli MG1655 genomic DNA using primers Ome 1705-DwcaM- MCS-BstZ17I-F (SEQ ID N°41) and Ome 1706-DwcaM-EcoRI-R (SEQ ID N°42) by PCR. Primers Ome 1704-DwcaM-TT02-BstZ17I-R (SEQ ID N°40) and Ome 1705- DwcaM-MCS-BstZ17I-F (SEQ ID N°41) have been designed to overlap through a 36 nucleotides-long region. Finally, the upwcaM-TT02-MCS-downwcaM fragment was amplified by mixing the upwcaM-TT02 and the TT02-MCS-downwcaM amplicons and using primers Ome 1703-DwcaM-HpaI-EcoRI-F (SEQ ID N°39) and Ome 1706-DwcaM- EcoRI-R (SEQ ID N°42). The resulting fusion PCR product was digested by Hpal and cloned between the Hindlll and EcoRl sites of plasmid pUC18 that had been treated by Large (Klenow) Fragment of E. coli DNA Polymerase I. The resulting plasmid was verified by DNA sequencing and called pUC18-A vcaM:. r02-MCS.

Ome 1703-DwcaM-HpaI-EcoRI-F (SEQ ID N°39)

CGTAGTTAACGAATTCCTGCGCCACCGAGCCAGCCAGACCTTGCGC

with - underlined upper case sequence for Hpal and EcoRI restriction sites and extrabases,

- upper case sequence homologous to sequence downstream of the wcaM gene (2114909-2114938, reference sequence on the website http://ecogene.org/)

Ome 1704-DwcaM-TT02-BstZ17I-R (SEQ ID N°40)

GCTTGTATACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCG

TTTTATTTGATGGCGTTCTCCTCTATAAAGCCTGCAGCAAGC

with

- bold upper case sequence for the BstZlll restriction site and extrabases,

- underlined upper case sequence corresponding to the transcriptional terminator Ti of E. coli rrnB (Orosz A, Boros I and Venetianer P. Eur. J. Biochem. 1991 Nov l;201(3):653-9)

- upper case sequence homologous to sequence downstream of the wcaM gene (2113921- 2113950, reference sequence on the website http://ecogene.org/).

Ome 1705 (DwcaM-MCS-BstZ17I-F) (SEQ ID N°41)

AGACTGGGCCTTTCGTTTTATCTGTTGTATACAAGCTTAATTAAGGGCCCGG

GCGGATCCATTTGCGACCATTCCTGGAAAAATGGAGTC

with

- bold upper case sequence complementary to the 3' end sequence of the transcriptional terminator Ti of E. coli rrnB (Orosz A, Boros I and Venetianer P. Eur. J.

Biochem. 1991 Nov l;201(3):653-9),

- underlined upper case sequence containing a multiple cloning site: BstZlll, Hindlll, Pad, Apal, BamHl,

- upper case sequence homologous to sequence upstream of the wcaM gene (2 1 12496-2 1 12525, reference sequence on the website http://ecogene.org/)

Ome 1706 (DwcaM-EcoRI-R) (SEQ ID N°42)

CGTAGTTAACGAATTCCGCCCCTTCTTTCAGGTTGCGTAGGCCATAC

with

- bold upper case sequence for Hpal and EcoRI restriction sites and extrabases, - upper case sequence homologous to sequence upstream of the wcaM gene

(2 1 1 1497-2 1 1 1527, reference sequence on the website http://ecogene.org/)

Finally, primers Ome 1603- K7_Cm_ampl_SmaI_BstZ17I_F (SEQ ID N°9) and Ome 1604-K7_Cm_ampl_HindIII_R (SEQ ID N°10), described above, were used to amplify the chloramphenicol cassette from plasmid pKD3. The cassette was then cloned between the BstZlll and Hindlll sites of plasmid p\JCl8-AwcaM: :TT02-MCS. The resulting plasmid was verified by DNA sequencing and called pUCl8-AwcaM: :TT02-MCS-Cm.

III.1.2. Construction of plasmid pUC18-AwcaM::TT02-TTadc-PlambdaR*(-35)- RBSOl-thrA *l-cysE-PgapA-metA *ll::Cm To construct pOC18-AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA- metA *ll::Cm, the TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 ApallBamRl digested fragment from pCL\920-TTadc-VlambdaR*(-35)-RBS01-thrA *l- cysE-PgapA-metA * 11 described above was cloned between the Apal and BamHI sites of pUC18-A vcaM:. r02-MCS-Cm described above. The obtained plasmid was verified by DNA sequencing and called pVC\8-AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01- thrA *l-cysE-VgapA-metA *ll::Cm

IIL2. Construction of strain MG1655 metA Hl AwcaM:: TT02- TTadc- YlambdaR*(-35)-RBS01-thrA *l-cysE-YgapA-metA *ll::Cm pKD46

To replace the wcaM gene by the TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE- VgapA-metA *ll::Cm region plasmid pOC18-AwcaM::TT02-TTadc-PlambdaR*(-35)- RBS01-thrA *l-cysE-PgapA-metA *ll::Cm was digested by BspHI and the remaining digested fragment AwcaM: :TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA- metA *ll::Cm was introduced into strain MG1655 metA *ll pKD46 according to Protocol 1.

Chloramphenicol resistant recombinants were selected and the presence of the AwcaM: :TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *ll::Cm chromosomal modification was verified by PCR with primers Omel707-DwcaM_verif_F (SEQ ID N°43) and Omel708-DwcaM_verif_R (SEQ ID N°44) (Table 2) and by DNA sequencing. The verified and selected strain was called MG1655 metA *ll AwcaM: :TT02- TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *ll::Cm (pKD46)

IIL3. Transduction of DwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01- thrA*l-cysE-PgapA-metA*ll::Cm into strain 6 and removal of the resistance cassette.

The AwcaM::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *ll::Cm chromosomal modification was transduced into the strain 6 (Table 1) described above with a PI phage lysate from strain MG1655 metA *ll pKD46 AwcaM: :TT02-TTadc- VlambdaR*(-35)-RBS01-thrA * 1-cysE-PgapA-metA * 11 : :Cm described above, according to Protocol 2.

Chloramphenicol resistant transductants were selected and the presence of the AwcaM: :TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *ll::Cm chromosomal modification was verified by PCR with Omel707-DwcaM_verif_F (SEQ ID N°43) and Omel708-DwcaM_verif_R (SEQ ID N°44) (Table 2). The resulting strain was called MG1655 metA *ll Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36-ARNmstl 7-metF Ftrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc- CI857-PlambdaR *(-35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-?lambdaR *(-35)-RBS01- thrA *l-cysE-?gapA-metA *11 AuxaCA::TT07-TTadc-?lambdaR*(-35)-RBS01-thrA *1- cysE-VgapA-metA *11 ACP4-6::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE- PgapA-metA *Π AtreBC::TT02-serA-serC::Gt AwcaM::TT02-TTadc-PlambdaR*(-35)- RBSOl-thrA *l-cysE-PgapA-metA *ll::Cm.

The gentamycin and chloramphenicol cassettes of the above strain were then removed according to Protocol 1. The loss of the gentamycin and chloramphenicol resistance cassettes was verified by PCR by using primers Ome 1595-DtreB_verif_F (SEQ ID N°27) and Ome 1596-DtreB verif R (SEQ ID N°28) and primers Omel707-DwcaM_verif_F (SEQ ID N°43) and Omel708-DwcaM_verif_R (SEQ ID N°44) (Table 2) respectively. The resulting strain MG1655 metA *ll Vtrc-metH PtrcF-cysPUWAMVtrcF-cysJIH Vtrc09- gcvTHP Vtrc36-ARNmstl 7-metF Ftrc07-serB AmetJ ApykF ApykA Apurll AyncA AmalS: : TTadc-CI857-PlambdaR *(-35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-

VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 AuxaCA::TT07-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 ACP4-6::TT02-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 AwcaM::TT02-TTadc-?lambdaR*(- 35)-RBS01-thrA *l-cysE-VgapA-metA *ll AtreBC ::TT02 -serA-serC was called strain 10.

IV. EXAMPLE 4 : Construction of strain 12, MG1655 metA *ll Vtrc-metH VtrcF- cysPUWAM YtrcF-cysJIH Ytrc09-gcvTHP Ptrc36-A RNmstl 7-metF Ytrc07-serB AmetJ ApykF ApykA Apurll AyncA AmalS: :TTadc-CI857-PlambdaR*(-35)-thrA *1- cysE ApgaABCD:: TT02- TTadc-PlambdaR *(-35)-RBS01-thrA H-cysE-PgapA- metA Hl AuxaCA ::TT07-TTadc-YlambdaR*(-35)-RBS01-thrA *l-cysE-YgapA- metA HI ACP4-6::TT02-TTadc-¥lambdaR*(-35)-RBS01-thrA Π-cysE-VgapA- metA HI AwcaM::TT02-TTadc-YlambdaR*(-35)-RBS01-thrA *l-cysE-YgapA- metA Hl DtreBC::TT02-serA-serC YtrcOl-pntAB ::Cm pCL1920-PgapA-pycre- TT07

IV.1. Construction of strain 11 by transduction of VtrcOl-pntAB: :Cm into strain 10

The ^' Ptrc01-pntAB::Cm promoter modification described above was transduced into strain 10 (Table 1) according to Protocol 2 by using a PI phage lysate from strain MG1655 metA *ll pKD46 Vtrc01-pntAB::Cm described above.

Chloramphenicol resistant transductants were selected and the presence of the VtrcOl- pntABr. Cm chromosomal modification was verified by PCR with primers Omel 151-pntAF (SEQ ID N°37) and Omel 152-pntAR (SEQ ID N°38) (Table 2). The resulting strain MG1655 metA *ll Vtrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl 7-metF Ftrc07-serB AmetJ ApykF ApykA Apurll AyncA AmalS: :TTadc-CI857- VlambdaR *(-35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-?lambdaR *(-35)-RBS01-thrA *1- cysE-VgapA-metA *11 AuxaCA: :TT07-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-

VgapA-metA *11 ACP4-6::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA- metA *Π AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC ': :TT02-serA-serC VtrcO 1-pntAB ': :Cm was called strain 11.

IV.2. Construction of pCL1920-PgapA-pycRe-TT07 and introduction into strain 11

In order to overexpress the pyruvate carboxylase gene of Rhizobium etli, plasmid pCL\920-PgapA-pycRe-TT07 was constructed, which is derived from plasmid pCL1920 (Lerner & Inouye, 1990, NAR 18, 15 p 4631).

To construct the VgapA-pycRe-TTO? insert, overlapping PCR between the gapA promoter, its Ribosome Binding Site (RBS) and the pycRe gene of Rhizobium etli has been used.

First, the gapA promoter and RBS region (corresponding to the -156 to -1 region upstream of the gapA start codon) was amplified from E. coli MG1655 genomic DNA by PCR using primers PgapA-Sall-F (SEQ ID N°45) and PgapA-pycRe-R (SEQ ID N°46). Then, the pycRe gene was amplified from Rhizobium etli CFN 42 genomic DNA using primers PgapA-pycRe F (SEQ ID N°47) and pycRe-TT07-SmaI-R (SEQ ID N°48). Primers PgapA-pycRe-R (SEQ ID N°46) and PgapA-pycRe F (SEQ ID N°47) have been designed to overlap through a 42 nucleotides-long region. Finally, the ¥gapA-RBSgapA-pycRe-TT07 fragment was amplified by mixing the gap A promoter-RBS and the pycRe amplicons using primers PgapA-Sall-F (SEQ ID N°45) and pycRe-TT07-SmaI-R (SEQ ID N°48).The resulting fusion PCR product was cloned between the Sail and Smal sites of plasmid pCL1920. The resulting plasmid was verified by DNA sequencing and called pCL1920- PgapA-pycRe-TT07.

PgapA-Sall-F (SEQ ID N°45)

ACGCGTCGACGGTATCGATAAGCTTCGTTTAAACAAGCCCAAAGGAAGAGTG

A

- underlined upper case sequence for Sail, Clal, Hindlll and Pmel restriction site and extrabases,

- bold upper case sequence homologous to the gapA promoter sequence (1860640- 1860658, reference sequence on the website http://ecouene.oru/)

PgapA-pycRe-R (SEQ ID N°46)

GTATCTTGGATATGGGCATATGTTCCACCAGCTATTTGTTAG

with

- bold upper case sequence homologous to the promoter of gapA gene (1860772-1860791, reference sequence on the website http://ecogene.org/)

- upper case sequence homologous to pycRe gene, except that the GTG start codon of pycRe gene was replaced by ATG (4236889-4236906, reference sequence on the website http://www.ncbi.nlm.nih.gov/).

PgapA-pycRe F (SEQ ID N°47) CTAACAAATAGCTGGTGGAACATATGCCCATATCCAAGATAC with

- bold upper case sequence homologous to the promoter of gapA gene (1860772-1860791, reference sequence on the website http://ecogene.org/)

- upper case sequence homologous to the pycRe gene, except that the GTG start codon of pycRe gene was replaced by an ATG (4236889-4236906, reference sequence on the website http://www.ncbi.nlm.nih.gov/).

pycRe-TT07-SmaI-R (SEQ ID N°48)

TCCCCCCGGGGATCCGAATTCGCAGAAAGGCCCACCCGAAGGTGAGCCAGC7U G^GGGC AAGGACGGGCGAACGAAACCTTTCGTGCCGTTCGCTCATCCGCC GTAAACCGCCAG

with

- underlined upper case sequence for a Smal, BamHl and EcoRl restriction sites and extrabases,

- upper case sequence corresponding to T7Te transcriptional terminator sequence from T7 phage (Harrington K.J., Laughlin R.B. and Liang S. Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):5019-24.),

- italic upper case sequence for Xhol restriction site and extrabases,

- bold upper case sequence homologous to sequence downstream of the pycRe gene (4240332-4240388, reference sequence on the website http://www.ncbi.nlm.nih.gov/).

Then, the p L\920-PgapA-pycRe-TT07 was introduced by electroporation into strain 1 1 (Table 1). The presence of plasmid p L\920-PgapA-pycRe-TT07 was verified and the resulting strain MG1655 metA *ll Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Vtrc09- gcvTHP Vtrc36-ARNmstl7-metF Ftrc07-serB AmetJ ApykF ApykA Apurll AyncA AmalS: : TTadc-CI857-VlambdaR *(-35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-

VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 AuxaCA ::TT07-TTadc-

VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 ACP4-6::TT02-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 AwcaM::TT02-TTadc-?lambdaR*(- 35)-RBS01-thrA *l-cysE-VgapA-metA *ll AtreBC ::TT02-serA-serC Vtrc01-pntAB::Cm pCL1920-Pg^-#ycRe-TT07 was called strain 12.

V. EXAMPLE 5 : Construction of strain 13, MG1655 metA *ll Vtrc-metH VtrcF- cysPUWAM YtrcF-cysJIH Ytrc09-gcvTHP Ptrc36-A RNmstl 7-metF Ytrc07-serB AmetJ ApykF ApykA Apurll AyncA AmalS: :TTadc-CI857-PlambdaR*(-35)-thrA *1- cysE ApgaABCD:: TT02- TTadc-PlambdaR *(-35)-RBS01-thrA H-cysE-PgapA- metA HI AuxaCA ::TT07-TTadc-YlambdaR*(-35)-RBS01-thrA *l-cysE-YgapA- metA HI ACP4-6::TT02-TTadc-¥lambdaR*(-35)-RBS01-thrA Π-cysE-VgapA- metA *11 AwcaM::TT02-TTadc-YlambdaR*(-35)-RBS01-thrA *l-cysE-YgapA- metA Hl DtreBC::TT02-serA-serC Vtrc01-pntAB ::Cm AudhA::Km

The AudhA::Km deletion described previously was transduced into strain 11 (Table 1) by using a PI phage lysate from the strain MG1655 metA * 11 AmetJ ^r Vtrc36-metF AudhAr.Km pKD46 described above according to Protocol 2

Kanamycin resistant transductants were selected. The presence of the T ^> trc36-ARNmstl 7- metF promoter modification was verified by PCR with primers Ome0726-PtrcmetF-F (SEQ ID N°29) and Ome0727-PtrcmetF-R (SEQ ID N°30) followed by DNA sequencing and the presence of the AudhA. Km deletion was verified by PCR with primers Oag0055- udhAF2 (SEQ ID N°33) and Oag0056-udhAR2 (SEQ ID N°34) (Table 2). The resulting strain MG1655 metA * 11 Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36-ARNmstl 7-metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalSrTTadc- CI857-PlambdaR *(-35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-VlambdaR *(-35)-RBS01- thrA *l-cysE-VgapA-metA *11 AuxaCA ::TT07-TTadc-VlambdaR*(-35)-RBS01-thrA *1- cysE-VgapA-metA *11 ACP4-6: : TT02-TTadc-VlambdaR *(-35)-RBS01-thrA *l-cysE-

VgapA-metA *11 AwcaM::TT02-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA- metA *11 AtreBC ::TT02-serA-serC VtrcOl-pntABrCm AudhAr.Km was called strain 13.

VI. EXAMPLE 6 : Construction of strain 14 MG1655 metA *ll Vtrc-metH VtrcF- cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36-metF Vtrc07-serB AmetJ ApykF

ApykA ApurU AyncA AmalS::TTadc-CI857-YlambdaR*(-35)-thrA *l-cysE ApgaABCD:: TT02-TTadc-YlambdaR *(-35)-RBS01-thrA *l-cysE-YgapA-metA *11 AuxaCA ::TT07-TTadc-YlambdaR*(-35)-RBS01-thrA *l-cysE-YgapA-metA *11 ACP4-6::TT02-TTadc-¥lambdaR*(-35)-RBS01-thrA *l-cysE-¥gapA-metA *ll AwcaM:: TT02-TTadc-YlambdaR *(-35)-RBS01-thrA *l-cysE-YgapA-metA *11

AtreBC ::TT02-serA-serC Vtrc01-pntAB ::Cm AudhA ::Km

The genes udhA and metF are close on the E. coli chromosome, 89.61 min and 89.03 min respectively and since phage PI can package 2 min of the E. coli chromosome, the genes udhA and metF are co-transducibles. Thereby, the Vtrc36-metF promoter modification and AudhAr.Km deletion described above, were co-transduced into strain 11 (Table 1) by using a PI phage lysate from the strain MG1655 metA * 11 AmetJ Vtrc36-metF AudhAr.Km pKD46, described above, according to Protocol 2.

Kanamycin resistant transductants were selected. The presence of the 7trc36-metF promoter modification was verified by PCR with primers Ome0726-PtrcmetF-F (SEQ ID N°29) and Ome0727-PtrcmetF-R (SEQ ID N°30) followed by DNA sequencing. The presence of the AudhArKm deletion was verified by PCR with primers Oag0055-udhAF2 (SEQ ID N°33) and Oag0056-udhAR2 (SEQ ID N°34) (Table 2). The resulting strain MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM VtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS: :TTadc-CI857-VlambdaR*(- 35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-?lambdaR *(-35)-RBS01-thrA *l-cysE-?gapA- metA *11 AuxaCA ::TT07-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 ACP4-6::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11

AwcaM::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *ll

AtreBC::TT02-serA-serC ¥trc01-pntAB::Cm AudhAr.Km was called strain 14.

VII. EXAMPLE 7 : Construction of strain 15, MG1655 metA Hl Ytrc-metH YtrcF- cysPUWAM YtrcF-cysJIH Ytrc09-gcvTHP Vtrc36-A RNmstl 7-metF Ytrc07-serB

AmetJ ApykF ApykA ApurU AyncA AmalS: :TTadc-CI857-VlambdaR*(-35)-thrA *1- cysE ApgaABCD:: TT02- TTadc-PlambdaR *(-35)-RBS01-thrA *l-cysE-PgapA- metA HI AuxaCA: :TT07-TTadc-YlambdaR*(-35)-RBS01-thrA *l-cysE-YgapA- metA *11 ACP4-6::TT02-TTadc-¥lambdaR*(-35)-RBS01-thrA Π-cysE-VgapA- metA *11 AwcaM::TT02-TTadc-YlambdaR*(-35)-RBS01-thrA *l-cysE-YgapA- metA Hl DtreBC ::TT02-serA-serC YtrcOl-pntABrCm AudhAr.Km pCL1920- PgapA-pycre- TTO 7

The pCL\920-PgapA-pycRe-TW7 , described previously, was introduced by electroporation into strain 13 (Table 1). The presence of plasmid p L\920-PgapA-pycRe- TT07 was verified and the resulting strain MG1655 metA *ll Vtrc-metHVtrcF-cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36-ARNmstl 7-metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS: :TTadc-CI857-?lambdaR*(-35)-thrA *l-cysE ApgaABCD: :TT02- TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 AuxaCA ::TT07-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 ACP4-6::TT02-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 AwcaM::TT02-TTadc-?lambdaR*(- 35)-RBS01-thrA *l-cysE-VgapA-metA *ll AtreBC ::TT02-serA-serC VtrcOl-pntAB Cm AudhAr.Km p L\920-PgapA-pycre-TT07 was called strain 15. VIII. EXAMPLE 8 : Construction of strain 18, MG1655 metA *ll Vtrc-metH VtrcF- cysPUWAM YtrcF-cysJIH Ytrc09-gcvTHP Ytrc36-ARNmstl 7-metF Ytrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857-VlambdaR*(-35)- thrA *l-cysE ApgaABCD:: TT02-TTadc-PlambdaR *(-35)-RBS01-thrA *l-cysE- PgapA-metA *11 AuxaCA: :TT07-TTadc-YlambdaR*(-35)-RBS01-thrA *l-cysE- VgapA-metA *11 ACP4-6::TT02-TTadc-¥lambdaR*(-35)-RBS01-thrA Π-cysE-

YgapA-metA *11 AwcaM:: TT02-TTadc-YlambdaR *(-35)-RBS01-thrA *l-cysE- YgapA-metA *ll DtreBC ::TT02-serA-serC Ytrc30-pntAB::Cm AudhA pCL1920- PgapA-pycRe-TT07 VIII.1. Construction of strain 16 by removal of the resistance cassettes of strain 13

The kanamycin and chloramphenicol resistance cassettes of the strain 13, described above, were removed according to Protocol 1. The loss of the kanamycin and chloramphenicol resistant cassettes were verified by PCR by using the primers with primers Oag0055- udhAF2 (SEQ ID N°33), Oag0056-udhAR2 (SEQ ID N°34), Omel 151-pntAF (SEQ ID N°37) and Omel 152-pntAR (SEQ ID N°38) (Table 2). The resulting strain MG1655 metA * 11 Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36-ARNmstl7- metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS: :TTadc-CI857-PlambdaR*(- 35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-PlambdaR *(-35)-RBS01-thrA *l-cysE-PgapA- metA *11 AuxaCA::TT07-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 ACP4-6: : TT02-TTadc-?lambdaR *(-35)-RBS01-thrA *l-cysE-?gapA-metA *11

AwcaM::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *ll

DtreBC ::TT02-serA-serC VtrcOl-pntAB AudhA pCL1920-PgapA-pycRe-TT07 was called strain 16.

VIII.2. Construction of strain MG1655 metA *11 pKD46 Ptrc30-pntAB::Cm

To increase the expression level of pyridine nucleotide transhydrogenase alpha and beta subunits, PntA and PntB, a constitutive artificial trc30 promoter was added upstream of pntA-pntB operon into the strain MG1655 metA *ll pKD46 according to Protocol 1, except that primers Ome2104 Ptrc30-pntAR (SEQ ID N°49) and Ome 1150-Ptrc-pntAF (SEQ ID N°36) were used to amplify the chloramphenicol resistance cassette from pKD3 plasmid.

Chloramphenicol resistant recombinants were selected. The presence of the artificial promoter trc30 and the insertion of the resistance cassette were verified by PCR with primers Omel 151-pntAF (SEQ ID N°37) and Omel 152-pntAR (SEQ ID N°38) (Table 2) and by DNA sequencing. The verified and selected strain was called MG1655 metA * 11 pKD46 Vtr30-pntAB::Cm.

Ome2104 Ptrc30-pntA R (SEQ ID N°49)

GCTGCAACACGGGTTTCATTGGTTAACCGTTCTCTTGGTATGCCAATTCGCATG ATATTCCCTTCCTTCCACACAGTATACGAGCCGGATGATTAATCGTCAACAGCT

CTGTAGGCTGGAGCTGCTTCG

with

- upper case sequence homologous to sequence upstream the pntA gene ( 1675875- 1675940, reference sequence on the website http://ecogene.org/)

- underlined upper case sequence for the trc30 promoter sequence

- italic upper case sequence corresponding to the primer site 2 of pKD3 plasmid (Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645) VIII.3. Construction of strain 17 by transduction of Ptrc30-pntAB: :Cm into strain 16

The 7trc30-pntAB: :Cm promoter modification was transduced into the strain 16 (Table 1) by using a PI phage lysate from the strain MG1655 metA *ll pKD46 Vtrc30-pntAB: :Cm described above according to Protocol 2.

Chloramphenicol resistant transductants were selected and the presence of the 7trc30- pntABr. Cm chromosomal modification was verified by PCR with Omel 151-pntAF (SEQ ID N°37) and Omel 152-pntAR (SEQ ID N°38) (Table 2). The verified and selected strain MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl7-metF VtrcOV-serB AmetJ ApykF ApykA ApurU AyncA AmalS: :TTadc-CI857- VlambdaR *(-35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-?lambdaR *(-35)-RBS01-thrA *1- cysE-VgapA-metA *11 AuxaCA::TT07-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-

VgapA-metA *11 ACP4-6::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA- metA *11 AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 AtreBC: :TT02-serA-serC Vtrc30-pntAB: :Cm AudhA was called strain 17.

VIII.4. Construction of strain 18 by introduction of pCL1920-PgapA-pycre-TT07 into strain 17

The pCL\920-PgapA-pycRe-TT07 , described previously, was introduced by electroporation into strain 17 (Table 1). The presence of plasmid p L\920-PgapA-pycRe- TT07 was verified and the resulting strain MG1655 metA *11 Vtrc-metHVtrcF-cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36-ARNmstl7-metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS: :TTadc-CI857-?lambdaR*(-35)-thrA *l-cysE ApgaABCD: :TT02- TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 AuxaCA ::TT07-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 ACP4-6::TT02-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 AwcaM::TT02-TTadc-?lambdaR*(- 35)-RBS01-thrA *l-cysE-VgapA-metA *ll AtreBC ::TT02-serA-serC Vtrc30-pntAB::Cm AudhA pCL1920-PgapA-pycre-TT07 was called strain 18.

IX. EXAMPLE 9 : Construction of strain 20, MG1655 metA Hl Ytrc-metH YtrcF- cysPUWAM YtrcF-cysJIH Ytrc09-gcvTHP Vtrc36-A RNmstl 7-metF Ytrc07-serB

AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857-VlambdaR*(-35)- thrA *l-cysE ApgaABCD:: TT02-TTadc-PlambdaR *(-35)-RBS01-thrA *l-cysE- PgapA-metA *11 AuxaCA: :TT07-TTadc-YlambdaR*(-35)-RBS01-thrA *l-cysE- VgapA-metA *11 ACP4-6::TT02-TTadc-¥lambdaR*(-35)-RBS01-thrA Π-cysE- YgapA-metA *11 AwcaM:: TT02-TTadc-YlambdaR *(-35)-RBS01-thrA *l-cysE-

YgapA-metA *ll DtreBC ::TT02-serA-serC Ytrc97-pntAB::Cm AudhA pCL1920- PgapA-pycRe-TT07 IX.l. Construction of strain MG1655 metA *11 pKD46 Ptrc97-pntAB::Cm

To increase the expression level of pyridine nucleotide transhydrogenase alpha and beta subunits, PntA and PntB, a constitutive artificial trc97 promoter was added upstream of pntA-pntB operon into the strain MG1655 metk *ll pKD46 according to Protocol 1, except that primers Ome2105 Ptrc97-pntAR (SEQ ID N°50) and Ome 1150-Ptrc-pntAF (SEQ ID N°36) were used to amplify the chloramphenicol resistance cassette from pKD3 plasmid.

Chloramphenicol resistant recombinants were selected. The presence of the artificial promoter trc97 and the insertion of the resistance cassette were verified by PCR with primers Omel 151-pntAF (SEQ ID N°37) and Omel l52-pntAR (SEQ ID N°38) (Table 2) and by DNA sequencing. The verified and selected strain was called MG1655 metA * 11 pKD46 Vtr97-pntAB::Cm.

Ome 2105 Ptrc97-pntAR (SEQ ID N°50)

GCTGCAACACGGGTTTCATTGGTTAACCGTTCTCTTGGTATGCCAATTCGCATG ATATTCCCTTCCTTCCACACATTTTACGAGCCGGATGATTAATAGCCAACAGCT CTGTAGGC TGGAGC TGCTTCG

with

- upper case sequence homologous to sequence upstream the pntA gene (1675875-1675940, reference sequence on the website http://ecogene.org/)

- underlined upper case sequence for the trc97 promoter sequence

- Italic upper case sequence corresponding to the primer site 2 of pKD3 plasmid

(Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645)

IX.2. Constrcution of strain 19 by transduction of Ptrc97-pntA Ii::Cm into strain 16

The ^' Ptrc97-pntAB: :Cm promoter modification was transduced into the strain 16 (Table 1) by using a PI phage lysate from the strain MG1655 metA * 11 pKD46 Vtrc97-pntAB::Cm described above according to Protocol 2.

Chloramphenicol resistant transductants were selected and the presence of the T*trc97- pntABr. Cm chromosomal modification was verified by PCR with Omel 151-pntAF (SEQ ID N°37) and Omel 152-pntAR (SEQ ID N°38) (Table 2). The verified and selected strain MG1655 metA * 11 Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl7-metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS: :TTadc-CI857- VlambdaR *(-35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-?lambdaR *(-35)-RBS01-thrA *1- cysE-VgapA-metA *11 AuxaCA::TT07-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-

VgapA-metA *11 ACP4-6::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA- metA *11 AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-PgapA-metA *11 AtreBC: :TT02-serA-serC: :Gt Vtrc97-pntAB: :Cm AudhA was called strain 19. IX.3. Construction of strain 20 by introduction of pCL1920-PgapA-pycre-TT07 into strain 19

The CL\920-PgapA-pycRe-TT01 ', described previously, was introduced by electroporation into strain 19 (Table 1). The presence of plasmid p L\920-PgapA-pycRe- TT07 was verified and the resulting strain MG1655 metA *11 Vtrc-metHVtrcF-cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36-ARNmstl7-metF VtrcOV-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857-?lambdaR*(-35)-thrA *l-cysE ApgaABCD: : TT02- TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 AuxaCA ::TT07-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 ACP4-6::TT02-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 AwcaM::TT02-TTadc-?lambdaR*(- 35)-RBS01-thrA *l-cysE-VgapA-metA *ll AtreBC ::TT02-serA-serC Vtrc97-pntAB::Cm AudhA pCL\920-PgapA-pycre-TT07 was called strain 20.

X. EXAMPLE 10 : Construction of strain 22, MG1655 metA *11 Vtrc-metH VtrcF- cysPUWAM YtrcF-cysJIH Ytrc09-gcvTHP Ptrc36-A RNmstl 7-metF Ytrc07-serB

AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857-VlambdaR*(-35)- thrA *l-cysE ApgaABCD:: TT02-TTadc-PlambdaR *(-35)-RBS01-thrA *l-cysE- PgapA-metA *11 AuxaCA: :TT07-TTadc-YlambdaR*(-35)-RBS01-thrA *l-cysE- VgapA-metA *11 ACP4-6::TT02-TTadc-¥lambdaR*(-35)-RBS01-thrA Π-cysE- YgapA-metA *11 AwcaM:: TT02-TTadc-YlambdaR *(-35)-RBS01-thrA *l-cysE-

YgapA-metA *ll DtreBC ::TT02-serA-serC Ytrc55-pntAB::Cm AudhA pCL1920- PgapA-pycRe-TT07

X. l. Construction of strain MG1655 metA *ll pKD46 Ptrc55-pntAB::Cm

To increase the expression level of pyridine nucleotide transhydrogenase alpha and beta subunits, PntA and PntB, a constitutive artificial trc55 promoter was added upstream of pntA-pntB operon into the strain MG1655 metA*ll pKD46 according to Protocol 1, except that primers Oag 0699-Ptrc55-pntAB R (SEQ ID N°51) and Ome 1150-Ptrc-pntAF (SEQ ID N°36) were used to amplify the chloramphenicol resistance cassette from pKD3 plasmid.

Chloramphenicol resistant recombinants were selected. The presence of the artificial promoter trc55 and the insertion of the resistance cassette were verified by PCR with primers Omel 151-pntAF (SEQ ID N°37) and Omel l52-pntAR (SEQ ID N°38) (Table 2) and by DNA sequencing. The verified and selected strain was called MG1655 metA * 11 pKD46 Vtr55-pntAB::Cm.

Oag 0699-Ptrc55-pntAB R (SEQ ID N°51) GCTGCAACACGGGTTTCATTGGTTAACCGTTCTCTTGGTATGCCAATTCGCATG ATATTCCCTTCCTTCCACACACTATACGAGCCGGATGATTAATGGTCAACAGCT

CTGTAGGCTGGAGCTGCTTCG

with

- upper case sequence homologous to sequence upstream the pntA gene

( 1675875- 1675940, reference sequence on the website http://ecogene.org/)

- underlined upper case sequence for the trc55 promoter sequence

- italic upper case sequence corresponding to the primer site 2 of pKD3 plasmid (Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645)

X.2. Transduction of Ptrc55-pntAB: :Cm into strain 16

The 7trc55-pntAB: :Cm promoter modification was transduced into the strain 16 (Table 1) by using a PI phage lysate from the strain MG1655 metA *ll pKD46 Vtrc55-pntAB: :Cm described above according to Protocol 2.

Chloramphenicol resistant transductants were selected and the presence of the T*trc55- pntABr. Cm chromosomal modification was verified by PCR with Omel 151-pntAF (SEQ ID N°37) and Omel 152-pntAR (SEQ ID N°38) (Table 2). The verified and selected strain MG1655 metA *ll Ptrc-metH PtrcF-cysPUWAM PtrcF-cysJIH Ptrc09-gcvTHP Ptrc36- ARNmstl7-metF VtrcOV-serB AmetJ ApykF ApykA ApurU AyncA AmalS: :TTadc-CI857- VlambdaR *(-35)-thrA *l-cysE ApgaABCD: : TT02-TTadc-?lambdaR *(-35)-RBS01-thrA *1- cysE-VgapA-metA *11 AuxaCA::TT07-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-

VgapA-metA *11 ACP4-6::TT02-TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-?gapA- metA *11 AwcaM::TT02-TTadc-PlambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 AtreBC : : TT02-serA-serC: :Gt Vtrc55-pntAB : :Cm AudhA was called strain 21.

X.3. Construction of strain 22 by introduction of pCL1920-PgapA-pycre-TT07 into strain 21

The pCL\920-PgapA-pycRe-TT07 , described previously, was introduced by electroporation into strain 21 (Table 1). The presence of plasmid p L\920-PgapA-pycRe- TT07 was verified and the resulting strain MG1655 metA *ll Vtrc-metHVtrcF-cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36-ARNmstl7-metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS: :TTadc-CI857-?lambdaR*(-35)-thrA *l-cysE ApgaABCD: :TT02- TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 AuxaCA ::TT07-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 ACP4-6::TT02-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 AwcaM::TT02-TTadc-?lambdaR*(- 35)-RBS01-thrA *l-cysE-VgapA-metA *ll AtreBC ::TT02-serA-serC Vtrc55-pntAB::Cm AudhA pCL1920-PgapA-pycre-TT07 was called strain 22. XL EXAMPLE 11 : Construction of strain 24, MG1655 metA *11 Ytrc-metH YtrcF- cysPUWAM YtrcF-cysJIH Ytrc09-gcvTHP Ptrc36-A RNmstl 7-metF Ytrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857-VlambdaR*(-35)- thrA *l-cysE ApgaABCD:: TT02-TTadc-PlambdaR *(-35)-RBS01-thrA *l-cysE- PgapA-metA *11 AuxaCA: :TT07-TTadc-VlambdaR*(-35)-RBS01-thrA *l-cysE-

VgapA-metA *11 ACP4-6::TT02-TTadc-¥lambdaR*(-35)-RBS01-thrA Π-cysE- YgapA-metA *11 AwcaM:: TT02-TTadc-YlambdaR *(-35)-RBS01-thrA *l-cysE- YgapA-metA *ll DtreBC ::TT02-serA-serC AudhAr.Km pCL1920-PgapA-pycRe- TT07

XI.1. Construction of strain 23 by transduction of AudhAr.Km into strain 10

The AudhAr.Km deletion described previously was transduced into strain 10 (Table 1) by using a PI phage lysate from the strain MG1655 metA * 11 AmetJ Vtrc36-metF AudhArKm pKD46 described above according to Protocol 2.

Kanamycin resistant transductants were selected. The presence of the T ^> trc36-ARNmstl 7- metF promoter modification was verified by PCR (reasons explained in example 6) with primers Ome0726-PtrcmetF-F (SEQ ID N°29) and Ome0727-PtrcmetF-R (SEQ ID N°30) followed by DNA sequencing and the presence of the AudhA : :Km deletion was verified by PCR with primers Oag0055-udhAF2 (SEQ ID N°33) and Oag0056-udhAR2 (SEQ ID N°34) (Table 2). The resulting strain MG1655 metA * 11 Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36-ARNmstl7-metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857-PlambdaR*(-35)-thrA *l-cysE ApgaABCD: :TT02- TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 AuxaCA ::TT07-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 ACP4-6::TT02-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 AwcaM: :TT02-TTadc-?lambdaR*(- 35)-RBS01-thrA *l-cysE-VgapA-metA *ll AtreBC ::TT02-serA-serC AudhAr.Km was called strain 23.

XI.2. Construction of strain 24: introduction of pCL1920-PgapA-pycre-TT07 into strain 23

The pCL\920-PgapA-pycRe-TT07 , described previously, was introduced by electroporation into strain 23 (Table 1). The presence of plasmid p L\920-PgapA-pycRe- TT07 was verified and the resulting strain MG1655 metA *ll Vtrc-metH VtrcF-cysPUWAM VtrcF-cysJIH Vtrc09-gcvTHP Vtrc36-ARNmstl 7-metF Vtrc07-serB AmetJ ApykF ApykA ApurU AyncA AmalS::TTadc-CI857-?lambdaR*(-35)-thrA *l-cysE ApgaABCD: :TT02- TTadc-?lambdaR*(-35)-RBS01-thrA *l-cysE-VgapA-metA *11 AuxaCA ::TT07-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 ACP4-6::TT02-TTadc- VlambdaR*(-35)-RBS01-thrA *l-cysE-?gapA-metA *11 AwcaM: :TT02-TTadc-?lambdaR*(- 35)-RBS01-thrA *l-cysE-VgapA-metA *ll AtreBC ::TT02-serA-serC AudhAr.Km p L\920-PgapA-pycre-TT07 was called strain 24. XII. EXAMPLE 12: Production of L-methionine by fermentation in shake flasks

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 (LB Broth Miller 25 g/L) with 2.5 g.L ^"1 glucose and 90 % minimal medium PCI). It was used to inoculate a 50 mL culture of PCI medium to an OD ₆ oo of 0.2. When necessary, antibiotics were added at concentrations of 50 mg.L ^"1 for kanamycin and spectinomycin, 30 mg.L ^"1 for chloramphenicol and 10 mg.L ^"1 for gentamycin. The temperature of the culture was maintained at 37°C for two hours, 42°C for two hours and then 37°C until the end of the culture. 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. For each strain, several repetitions were made.

Table 3 : Minimal medium composition (PCI).

Table 4: Methionine yield (Yme _t ), in % g of methionine per g of glucose produced in batch culture by the different strains. For the definition of methionine/glucose yield see below. SD denotes the standard deviation for the yields that was calculated on the basis of several repetitions (N = number of repetitions).

The extracellular methionine concentration was quantified by HPLC after OPA FMOC derivatization. The residual glucose concentration was analyzed using HPLC with refractometric detection. Methionine yield was expressed as follows:

methionine (g) .

Y _met = — * 100

consummed glu cose (g)

As can be seen in table 4 methionine/glucose yield (Y _met ) is increased upon pntAB overexpression and/or udhA deletion (strains 9 and 14 compared to strain 7 and strains 12, 13, 15 and 24 compared to strain 10) .

The improvement of the yield is even better upon strong overexpression of the metF gene. Strain 13 that contains both an mRNA stabilizing sequence in front of the metF gene and overexpression of pntAB and udhA deletion shows a higher yield than strain 14 without a strong overexpression of the CI pathway.

XIII. EXAMPLE 13: Transhydrogenase and 5,10-methylenetetrahydrofolate reductase activities

Results described in example 12, table 4 are confirmed by the analysis of transhydrogenase and 5,10-methylenetetrahydrofolate reductase activities carried out by PntAB and MetF, respectively (Table 5). In small Erlenmeyer flask cultures, both activities increase upon overexpression.

Table 5: Transhydrogenase (TH, PntAB) and 5,10-methylenetetrahydrofolate reductase (MTHFR, MetF) activities were determined in the above described strains and are given in mUI/mg of proteins (N = number of independent Erlenmeyer flask cultures).

For the in vitro determination of enzyme activities, E. coli strains were cultured in minimal medium as described above. Preceding extraction method, cells were harvested from culture broth by centrifugation. The pellet was resuspended in cold 20mM potassium phosphate buffer (pH 7.2) and cells were lysed by bead beating with a Precellys (Bertin Technologies; 2x10s at 5000rpm, 2 minutes on ice between the two steps) followed by centrifugation at 12000g (4°C) for 30 minutes. The supernatant was desalted and used for analysis. Protein concentrations were determined using Bradford assay reagent.

Transhydrogenase activities (TH) were assayed as previously described (Yamagushi and Stout, 2003). Cyclic transhydrogenase activity was assayed spectrophotometrically for 30 minutes at 375nm in a 37°C reaction mixture containing 50mM MES-KOH buffer (pH 6.0), 0.2 mM NADH, 0.2 mM AcPyAD with or without 10μΜ NADPH and 6μg of crude cell extract. All results are the average of at least three measurements.

5, 10-methylenetetrahydrofolate reductase activities (MTHFR) were assayed as previously described (Matthews, 1986) with some modifications. The method is shown in equation (1). (1) Methyl-THF + Menadione→ Methylene-THF + Menadiol

Methyl-THF is used as the substrate and the formation of methylene-THF is measured by acid decomposition of methylene-THF which yields formaldehyde. The produced formaldehyde reacts with the indicator BDH to form a highly fluorescent hydrazone derivative at 530nm. 5,10-methylenetetrahydrofolate reductase activity was assayed for 30 minutes at 37°C in a reaction mixture containing 50mM potassium phosphate buffer (pH 6.7), 0.3 mM EDTA, 0.2 % (w/v) bovine serum albumin, saturated solution of menadione in 20% methanol/80%) H ₂ 0, and 20μg of crude cell extract. After incubation, the reaction is terminated by addition of sodium acetate buffer (pH 4.7) and by placing the tube in a heater block at 100°C for 2 minutes. After centrifugation for 5 minutes at lOOOOg, the produced formaldehyde is measured by addition of 0.001%> NBDH in 95% ethanol/6%> phosphoric acid with a FLx800 Fluorescence Microplate Reader (Biotek).

XIV. EXAMPLE 14: Production of L-methionine by fermentation in bio-reactor Strains that produced substantial amounts of metabolites of interest were subsequently tested under production conditions in 2.5 L fermentors (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 (B la) 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 , increased exponentially for 24 hours with a growth rate of 0.13 h ^"1 in order to obtain a final cellular concentration of about 18 g.L ^"1 . Table 6: Preculture batch mineral medium composition (Bla and Bib).

Table 7: Preculture fedbatch mineral medium composition (Fl)

Table 8: Culture batch mineral medium composition (B2).

Table 9: Culture fedbatch medium composition (F2)

Subsequently, 2.5 L fermentors (Pierre Guerin) were filled with 600 mL of minimal medium (B2) and were inoculated to a biomass concentration of 2.1 g.L ^"1 with a preculture volume ranging between 55 to 70 mL.

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 (ML OH 10 % for 9 hours and ML OH 28 % until the culture end). 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.

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 according to a sigmoid profile with an increasing flow rate that reached 24 mL.h ^"1 after 26 hours of growth. The precise feeding conditions were calculated by the equation: Q{t) = p\ + -pl(t-p4)

\ + e

where Q(t) is the feeding flow rate in mL.h ^"1 for a batch volume of 600 mL with pi = 1.80, p2 = 22.40, p3 = 0.270, p4 = 6.5.

After 26 hours, the 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.

Table 10: Maximal methionine yield (Y _me t max) in % g of methionine per g of glucose produced in fedbatch culture by the different strains. For the definition of methionine/glucose yield see below. SD denotes the standard deviation for the yields which was calculated on the basis of several repetitions (N = number of repetitions).

* Strains 10 and 13 were cultivated with respectively 49.1 and 55.5 g.L ^"1 of ammonium thiosulfate and with 5 g.L ^"1 of magnesium sulphate instead of magnesium thiosulfate in fedbatch medium. The batch medium contained 1 g.L ^"1 of magnesium sulphate instead of magnesium thiosulfate for the two strains. As observed previously in flask experiments, in the same way, in bioreactor, methionine/glucose yield (Y _met max) is increased upon pntAB overexpression and/or udhA deletion (compare strains 10 and 13 of table 10).

Moreover we show that a fine tuned regulation of pntAB overexpression improved methionine production as can be seen with strains 12, 15, 18, 20 and 22 of table 10 above. Methionine production is modulated by the expression level of the pntAB genes and we show that it is better with a moderate overexpression of pntAB.

The fermentor 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, - Methionine^ * VQ X 100

m ^et Consumed glu cos e _t

The maximal yield obtained during the culture was presented here for each strain.

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 volume, = fed weight, - fed weight,

density fed solution

Injected Glucose _t = fed volume _t * [Glucose]

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

EXAMPLE 15: Transhydrogenase activity

Results described in example 14, table 10 are confirmed by the analysis of transhydrogenase activities carried out by PntAB (Table 1 1). As observed previously in flask experiments, transhydrogenase activities are increased upon pntAB overexpression (see strains 10 and 13 of table 1 1). Moreover, a fine tuned regulation of pntAB overexpression decreased about 10 times the transhydrogenase activities as can be seen with strain 18 of table 1 1. Table 11 : Transhydrogenase (TH, PntAB) activities were determined in the above described strains and are given in mUI/mg of proteins (N = minimum two points of independent fermentor cultures).

Transhydrogenase activities (TH) were assayed as previously described in Example 13. All results are the average of at least three measurements.

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