MODIFIED MICROORGANISM AND METHOD FOR THE IMPROVED PRODUCTION OF ECTOINE

Abstract

The present invention relates to a microorganism genetically modified for production of ectoine, wherein said microorganism comprises the following modifications: expression of a heterologous gene ectA encoding a diaminobutyric acid acetyltransferase having at least 90% similarity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, a heterologous gene ectB encoding a diaminobutyric acid aminotransferase having at least 90% similarity with SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, a heterologous gene ectC encoding an ectoine synthase having at least 90% similarity with SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 and deletion of pykA and pykF genes. The present invention also relates to a method for the production of ectoine using said microorganism.

Claims

1. Microorganism genetically modified for the production of ectoine, wherein the microorganism comprises the following modifications: expression of heterologous gene ectA encoding a diaminobutyric acid acetyltransferase having at least 80% identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, and heterologous gene ectB encoding a diaminobutyric acid aminotransferase having at least 80% identity with SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, and heterologous gene ectC encoding an ectoine synthase having at least 80% identity with SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, deletion of pykA and pykF genes, and at least a 50% reduction in citrate synthase enzyme activity as compared to an unmodified microorganism.

2. Microorganism of claim 1 further comprising at least a 75% reduction in citrate synthase enzyme activity.

3. Microorganism of claim 1, wherein the citrate synthase enzyme has at least 80% identity with the citrate synthase enzyme of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, encoded by the gene gltA.

4. Microorganism of claim 1, wherein citrate synthase enzyme activity is reduced by placing the gltA gene encoding the citrate synthase under the control of promoter PgltA or a heterologous inducible promoter.

5. Microorganism of claim 3, wherein expression of the gltA gene, encoding the citrate synthase enzyme having at least 80% identity with the citrate synthase enzyme of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, is under the control of promoter PgltA or under the control of a heterologous inducible promoter.

6. Microorganism of claim 1 further comprising a deletion of the gene ppc and an overexpression of the gene pck encoding a phosphoenolpyruvate carboxykinase having at least 80% identity with SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33.

7. Microorganism of claim 1 further comprising an overexpression of an aspartate transaminase having at least 80% identity with SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and a glutamate dehydrogenase having at least 80% identity with SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44.

8. Microorganism of claim 1 further comprising a deletion of at least one gene selected from the group consisting of ackA-pta, adhE, frdABCD, ldhA, mgsA, pflAB, and mdh.

9. Microorganism of claim 1, wherein the microorganism has been genetically modified to be able to utilize sucrose as a carbon source, and wherein said microorganism further comprises the overexpression of: the heterologous cscBKAR genes of E. coli EC3132, or the heterologous scrKYABR genes of Salmonella sp.

10. Microorganism of claim 1, wherein the microorganism belongs to the family of the bacteria Enterobacteriaceae, Clostridiaceae, Bacillaceae, Streptomycetaceae, or Corynebacteriaceae, or to the family of yeasts Saccharomycetaceae.

11. Microorganism of claim 10, wherein the Enterobacteriaceae bacterium is Escherichia coli or Klebsiella pneumoniae, the Clostridiaceae bacterium is Clostridium acetobutylicum, the Corynebacteriaceae bacterium is Corynebacterium glutamicum, or the Saccharomycetaceae yeast is Saccharomyces cerevisiae.

12. Microorganism of claim 11, wherein the Enterobacteriaceae bacterium is Escherichia coli.

13. Method for the production of ectoine comprising the steps of: a) culturing a microorganism genetically modified for the production of ectoine in an appropriate culture medium comprising a source of carbon and a source of nitrogen, and b) recovering ectoine from the culture medium, wherein the microorganism genetically modified for the production of ectoine comprises the following modifications: expression of heterologous gene ectA encoding a diaminobutyric acid acetyltransferase having at least 80% identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, and heterologous gene ectB encoding a diaminobutyric acid aminotransferase having at least 80% identity with SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, and heterologous gene ectC encoding an ectoine synthase having at least 80% identity with SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, deletion of pykA and pykF genes, and at least a 50% reduction in citrate synthase enzyme activity as compared to an unmodified microorganism.

14. Method of claim 13, wherein the source of carbon is glycerol and/or glucose and/or sucrose.

15. Method of claim 13, wherein step b) comprises a step of filtration, desalination, cation exchange, liquid extraction, or distillation.

16. Microorganism of claim 5, wherein expression of the gltA gene is under the control of a promoter selected from the group consisting of a trc promoter, a tac promoter, a lac promoter, a tet promoter, a lambda P.sub.L promoter, and a lambda P.sub.R promoter.

17. Microorganism of claim 7 further comprising a deletion of at least one gene selected from the group consisting of ackA-pta, adhE, frdABCD, ldhA, mgsA, pflAB, and mdh.

18. Microorganism of claim 17, wherein the microorganism belongs to the family of the bacteria Enterobacteriaceae, Clostridiaceae, Bacillaceae, Streptomycetaceae, or Corynebacteriaceae, or to the family of yeasts Saccharomycetaceae.

19. Method for the production of ectoine of claim 13 wherein the microorganism genetically modified for the production of ectoine is a microorganism further comprising an overexpression of an aspartate transaminase having at least 80% identity with SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and a glutamate dehydrogenase having at least 80% identity with SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and a deletion of at least one gene selected from the group consisting of ackA-pta, adhE, frdABCD, ldhA, mgsA, pflAB, and mdh.

20. Method for the production of ectoine of claim 13 wherein the microorganism genetically modified for the production of ectoine is a microorganism further comprising an overexpression of an aspartate transaminase having at least 80% identity with SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and a glutamate dehydrogenase having at least 80% identity with SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and a deletion of at least one gene selected from the group consisting of ackA-pta, adhE, frdABCD, ldhA, mgsA, pflAB, and mdh, and wherein the microorganism belongs to the family of the bacteria Enterobacteriaceae, Clostridiaceae, Bacillaceae, Streptomycetaceae, or Corynebacteriaceae, or to the family of yeasts Saccharomycetaceae.

Description

FIGURES

[0129] FIG. 1: Map of the recombinant vector pEC1.

p15A: plasmid origin of replication; aadA1: aminoglycoside 3′-adenylyltransferase conferring spectinomycin/streptomycin resistance; Placl Q: laclQ promoter; laclQ: mutant lac repressor gene binding the lac operator more tightly than the wild-type Lacl; Ptrc01: artificial promoter with lac operator binding site (Brosius et al., 1985); operateur lac: lac operator binding site; ectA-ectB-ectC: H. elongata ectoine operon.

[0130] FIG. 2: Map of the recombinant vector pEC3.

p15A: plasmid origin of replication; aadA1: aminoglycoside 3′-adenylyltransferase conferring spectinomycin/streptomycin resistance; Placl Q: laclQ promoter; laclQ: mutant lac repressor gene binding the lac operator more tightly than the wild-type Lacl; Ptrc01: artificial promoter with lac operator binding site (Brosius et al., 1985); operateur lac: lac operator binding site; ectA-ectB-ectC: H. elongata ectoine operon; rocG: B. subtilis glutamate dehydrogenase gene; PaspC: aspC promoter; aspC: E. coli aspartate transaminase gene

[0131] FIG. 3: Metabolic pathway for the production of ectoine from glucose. The first step (1) is the phosphotransferase system (PTS) responsible for both glucose uptake and phosphorylation on the carbon 6. In this schematic representation G6P, PEP, and OAA mean respectively glucose-6-phosphate, phosphoenolpyruvate and oxaloacetic acid. Enzymatic activities are (2) phosphoenolpyruvate carboxykinase (EC: 4.1.1.49), (3) pyruvate decarboxylase (EC: 1.2.4.1), (4) aspartate aminotransferase (EC: 2.6.1.1), (5) glutamate dehydrogenase (EC: 1.4.1.2), (6) aspartokinase (EC: 2.7.2.4), (7) aspartate-semialdehyde dehydrogenase (EC: 1.2.1.11), (8) diaminobutyrate-2-oxoglutarate transaminase (EC: 2.6.1.76), (9) L-2,4-diaminobutyric acid acetyltransferase (EC: 2.3.1.178), (10) L-ectoine synthase (EC: 4.2.1.108). Citrate synthase activity (11) (EC: 2.3.3.1) is strongly regulated by environmental conditions (12).

EXAMPLES

[0132] The present invention is further defined in the following examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only. The person skilled in the art will readily understand that these examples are not limitative and that various modifications, substitutions, omissions, and changes may be made without departing from the scope of the invention.

Methods

[0133] In the examples given below, methods well-known in the art were used to construct E. coli strains containing replicating vectors and/or various chromosomal deletions, and substitutions using homologous recombination, as is well-described in Datsenko & Wanner, (2000) for E. coli. In the same manner, the use of plasmids or vectors to express or overexpress one or more genes in a recombinant microorganism are well-known by the person skilled in the art. Examples of suitable E. coli expression vectors include pTrc, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, etc.

[0134] Chromosomal modifications. Several protocols have been used in the following examples. Protocol 1 (chromosomal modifications by PCR amplification using oligonucleotides and appropriate genomic DNA as a matrix (that the person skilled in the art will be able to define), homologous recombination and selection of recombinants), protocol 2 (transduction of phage P1) and protocol 3 (antibiotic cassette excision, the resistance genes were removed when necessary) used in this invention have been fully described in patent application EP 2532751 (see in particular example 1 and example 3, points 1.2 and 1.3, incorporated herein by reference). Chromosomal modifications were verified by PCR analysis with appropriate oligonucleotides that the person skilled in the art is able to design.

[0135] Construction of recombinant plasmids. Recombinant DNA technology is well described in the literature and routinely used by the person skilled in the art. Briefly, DNA fragments were PCR amplified using oligonucleotides and appropriate genomic DNA as a matrix (that the person skilled in the art will be able to define). The DNA fragments and chosen plasmid were digested with compatible restriction enzymes (that the person skilled in the art will be able to define), then ligated and transformed into competent cells. Transformants were analyzed and recombinant plasmids of interest were verified by DNA sequencing.

[0136] Overlap extension PCR method (OE-PCR). The overlap extension PCR method consists of two or more primary PCR reactions which generate DNA fragments with overlapping ends and a secondary reaction which joins the two or more fragments into a single fragment (Horton et al., 1990). The person skilled in the art will be able to define appropriate oligonucleotides for this purpose.

[0137] Strain selection based on the expression of genes responsible for antibiotic resistance. Strains construction requires the selection of cells harboring a DNA fragment responsible for a specific antibiotic resistance. To achieve this selection, bacteria are spread on petri dishes containing LB solid medium (10 g/L bactopeptone, 5 g/L yeast extract, 5 g/L NaCl and 20 g/L agar). Three antibiotics could be added according to the selection marker: [0138] Chloramphenicol (30 mg/L) [0139] Kanamycin (50 mg/L) [0140] Gentamycin (10 mg/L) [0141] Spectinomycin (50 mg/L) [0142] Streptomycin (100 mg/L)

[0143] Determination of ectoine and acetic acid production. Ectoine and acetic acid concentrations are determined using an ultra high-pressure liquid chromatography system (UPLC ACQUITY, Waters®). Samples collected at different time points are centrifuged for 2 minutes at 5,000 g and at 4° C. to remove the insoluble part. The upper phase of each sample is diluted 1000-fold in distilled water. Ectoine and acetic acid are then separated on an Acquity UPLC-HSS-T3-C18/2.1 mm×150 mm×1.8 μm column (Waters®) using a water/acetonitrile gradient as the mobile phase. The gradient table is presented in Table 1 below.

TABLE-US-00001 TABLE 1 Gradient table for acetic acid and ectoine separation. The total flow rate is constant at 400 μL/minute. Time course - Percent of Solvant A Percent of Solvant B min (water/acetonitrile 99/1 v/v) (acetonitrile) 0 100 0 4.5 100 0 5.5 50 50 6 10 90 8 10 90 9 100 0 11 100 0

[0144] Ectoine and acetic acid are quantified using the mass spectrometer API3200 (Sciex®).

[0145] Biomass estimation. Biomass quantity variation is monitored using a spectrophotometer (Nicolet Evolution 100 UV-Vis, THERMO®). Biomass production increases the turbidity of the culture medium. It is assayed by measuring the absorbance at 600 nm. Each unit of absorbance represents 2.2×10.sup.9+/−2×10.sup.8 cells/mL.

[0146] Determination of citrate synthase activity. Citrate synthase activity is defined as the ability of a protein mixture to condense the acetyl group from acetyl-CoA and an oxaloacetic Acid (OAA) into citric acid. Such a reaction also releases a co-enzyme A (CoA) harboring a free thiol group. The principle of the present assay is to monitor the release of the free thiol group. To do this, the method described by Georges Ellman in 1959 (Ellman GL, 1959) was adapted. Ellman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid)), also called DTNB, which reacts with free thiol groups, was used. This reagent cleaves the disulfide bond to release 2-nitro-5-thiobenzoate (TNB.sup.−), which ionizes to the TNB.sup.2− dianion in water at neutral and alkaline pH. This TNB.sup.2− ion has a yellow color. This reaction is stoichiometric and the molar extinction coefficient of TNB.sup.2− at 412 nm is 13,600 mol.Math.L.sup.−1.Math.cm.sup.−1. A bacterial crude extract is prepared through mechanical grinding (i.e., using glass beads or a french press) or bacterial cell membrane permeabilization (i.e., using guanidine-HCl and Triton X100 treatment). Proteins of interest are separated from cell debris by centrifugation (1,500 g at 4° C.).

[0147] The initial assay mix contained from 2 to 6 μg/mL of protein extract, 20 mM HEPES (pH=7.5), 0.15 mM Acetyl-CoA, 0.2 mM DTNB, 0.3 mM OAA. The apparition of the yellow color as a function of time is linearly correlated with specific enzymatic activity and is monitored by analyzing the absorbance at 420 nm with a spectrophotometer (Nicolet Evolution 100 UV-Vis, THERMO®).

Example 1: Strain Construction

[0148] To overexpress the ectoine operon, the ectA, ectB and ectC genes from Halomonas elongata (SEQ ID NO: 1) were cloned under the IPTG inducible trc promoter of SEQ ID NO: 58 into the pACYC184 plasmid (Chang and Cohen, 1978).

[0149] The resulting plasmid pEC0001 was then transformed into strain MG1655 giving rise to strain 1.

[0150] To optimize the strain for ectoine production, combinations of mutations were introduced into the E. coli strain resulting in strains 2a: MG1655 DackA+pta DadhE DldhA DfrdABCD DmgsA DpflAB Dmdh DaspA DdcuA and 2b: strain 2a with DpykA DpykF, constructed as described below.

[0151] To inactivate the ackA+pta, frdABCD, pflAB operons and the adhE, IdhA, mgsA, mdh, aspA, dcuA, pykA, and pykF genes, the homologous recombination strategy was used (according to Protocol 1). The strains retained were designated MG1655 DackA+pta::Gt, MG1655 DadhE::Cm, MG1655 DldhA::Km, MG1655 DfrdABCD::Gt, MG1655 DmgsA::Km, MG1655 DpflAB::Cm, MG1655 Dmdh::Gt, MG1655 DaspA::Km, MG1655 DdcuA::Cm, MG1655 DpykA::Cm and MG1655 DpykF::Km were Km, Cm and Gt designate respectively DNA sequence conferring resistance to kanamycin, chloramphenicol and gentamycin. All these deletions were transferred by P1 phage transduction (according to Protocol 2) into E. coli MG1655 and resistance genes were removed according to protocol 3 when necessary. The resulting strains were named strain 2a and 2b, as indicated above.

[0152] The aspC gene from E. coli and the glutamate dehydrogenase gene rocG from B. subtilis (SEQ ID NO: 59) were cloned under the native promoter and the inducible trc promoter SEQ ID NO: 23, respectively, into the previously described pEC0001 plasmid. The resulting plasmid pEC0003 was then transformed into strains 2a and 2b giving rise respectively to strains 3a and 3b.

[0153] To increase ectoine production, the phosphoenolpyruvate carboxykinase pck gene from A. succiniciproducens (SEQ ID NO: 60) was chromosomally overexpressed under the inducible trc promoter into the ppc locus, thereby deleting the ppc gene. Specifically, a fragment carrying the native pck gene and a resistance marker flanked by homologous DNA sequences to the targeted integration locus ppc was PCR amplified by OE-PCR. The PCR product obtained was then introduced by electroporation into E. coli strain MG1655 (pKD46). pck overexpression was transferred by P1 phage transduction (according to Protocol 2) into strain 3b and the resistance gene was removed according to protocol 3, giving rise to strain 4.

[0154] Results

[0155] As can be seen in Table 2, strains 1, 3a, 3b and 4 showed an increase in ectoine production. Ectoine production was better in strain 3b as compared to strain 3a (data not shown), therefore strain 3b was used in the examples below.

TABLE-US-00002 TABLE 2 Ectoine production, in batch cultures for the different strains. Ectoine production Control Strain − Strain 1 + Strain 3a + Strain 3b + Strain 4 + The symbol “+” indicates detectable ectoine in the medium. The “−” represents undetectable ectoine.

Example 2: CitS Reduction

[0156] Biological regulation of citrate synthase activity. In all cases, the sequence of the citrate synthase (gltA) open reading frame (SEQ ID NO: 61) is that present in the wild-type strain (MG1655) or an equivalent nucleotide sequence coding a protein with at least 90% sequence similarity based on Blosum62 homology matrix as compared to native E. coli citrate synthase.

[0157] The attenuation of expression can notably be due to either the exchange of the wild-type promoter for a weaker natural or synthetic promoter or the use of an agent reducing gene expression, such as an antisense RNA or interfering RNA (RNAi), and more particularly a small interfering RNA (siRNA) or short hairpin RNA (shRNA).

[0158] Regulation systems are controlled by environmental conditions responsible for repression of citrate synthase gene expression. Environmental conditions are listed in Table 3 below. The growth medium “nitrogen base” is composed of 10 g/L bactopeptone and 5 g/L yeast extract supplemented with 0.5 g/L sodium chloride.

TABLE-US-00003 TABLE 3 List of environmental culture conditions used. Medium name 1 2 3 4 5 Nitrogen Base 1x 1x 1x 1x 1x Glucose (6 g/L) + + + + + Temperature (in ° C.)  37 37  30  37  37 Oxygen pO.sub.2 en mm Hg 160 45 160 160 160 IPTG 100 μM − − − + − Anhydrotetracycline 0.4 μM − − − − +

[0159] We also used strains in which the genomic sequence of the citrate synthase gene is under the control of a different promoter associated with a specific transcriptional factor, if needed. Strain names and regulator systems used are listed in Table 4 below.

TABLE-US-00004 TABLE 4 List of strains used for analysis of citrate synthase regulation. Strain A B C D E F G Regulator PgltA Ptrc Ptac Plac Ptet PL PR controlling gltA SEQ ID SEQ SEQ SEQ SEQ SEQ SEQ SEQ NO: ID ID ID ID ID ID ID NO: NO: NO: NO: NO: NO: NO: 22 23 24 25 26 27 28

[0160] Initial biomass is obtained after an overnight culture (16 hours) in condition 4 (see Table 3). All bacteria present in said biomass are in physiological stationary phase due to carbon starvation. The biomass is diluted 100-fold in new medium. After 4 hours in the indicated growth conditions, citrate synthase activity is monitored as previously described. Table 5 provides the obtained results.

TABLE-US-00005 TABLE 5 Citrate synthase activity. The “+” indicates a strain/condition association leading to an activity between 0.8 and 1.2 fold of the reference measured in strain A and condition 1. The represents a strain/condition association with an activity below 0.8-fold of the reference. The “++” means that citrate synthase activity is increased by more than 1.2-fold of the reference when the said strain is grown in the indicated condition. Strains A B C D E F G Culture 1 reference − − − + + + conditions 2 − − − − + + + 3 + − − − + − − 4 + ++ ++ + + + + 5 + − − − − + +

[0161] Results presented in Table 5 shown that citrate synthase activity is strongly reduced when PL λ or PR λ controls its expression in association with environmental conditions in which the growing temperature is 30° C. Similar results can be observed when pTet controls citrate synthase gene expression and in presence of tetracycline in the medium. When the gltA gene (coding the citrate synthase) is under the control of its native promoter, citrate synthase activity is also strongly reduced if bacteria are growing in oxygen starvation.

[0162] For the pTrc, pTac and pLac regulation systems, expression of the citrate synthase is reduced when IPTG is absent from the medium. Such results indicate that the absence of a specific molecule is controlling the gltA expression. In the present context, the PgltA, Ptet, PL and PR systems are particularly preferred for limiting the citrate synthase activity. Strains constructed using these systems are discussed in further detail below.

Example 3: Optimisation of Carbon Utilisation for Ectoine Production

[0163] Strains constructed for ectoine production test. Regulation systems A, E, F and G described in example 2 have been introduced in strains 1, 3b and 4 presented in the example 1 above. These new strains (Table 6) exhibit the same regulation of the citrate synthase activity.

TABLE-US-00006 TABLE 6 List of strains used for analysis of ectoine production. Genetic Background Strain 1 Strain H I J K Regulator controlling gltA PgltA Ptet PL PR Seq ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 22 NO: 26 NO: 27 NO: 28 Genetic Background Strain 3b Strain L M N O Regulator controlling gltA PgltA Ptet PL PR Seq ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 22 NO: 26 NO: 27 NO: 28 Genetic Background Strain 4 Strain P Q R S Regulator controlling gltA PgltA Ptet PL PR Seq ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 22 NO: 26 NO: 27 NO: 28

[0164] The production conditions tested are those presented in example 2 above. In all conditions, the medium is supplemented with 100 μM IPTG to induce ectA/B/C gene expression. Results obtained are provided in Table 7 below.

TABLE-US-00007 TABLE 7 Ectoine, acetic acid and biomass production. Conditions Strains 1 2 3 5 H Ectoine: reference Ectoine: + Ectoine: + Ectoine: + Acetic acid: reference Acetic acid: ++ Acetic acid: + Acetic acid: + Biomass: reference Biomass: − − Biomass: + Biomass: + I Ectoine: + Ectoine: + Ectoine: + Ectoine: + Acetic acid: + Acetic acid: + Acetic acid: + Acetic acid: ++ Biomass: + Biomass: − Biomass: + Biomass: − − J Ectoine: + Ectoine: + Ectoine: + Ectoine: + Acetic acid: + Acetic acid: + Acetic acid: ++ Acetic acid: + Biomass: + Biomass: − Biomass: − − Biomass: + K Ectoine: + Ectoine: + Ectoine: + Ectoine: + Acetic acid: + Acetic acid: + Acetic acid: ++ Acetic acid: + Biomass: + Biomass: − Biomass: − − Biomass: + L Ectoine: + Ectoine: +++ Ectoine: + Ectoine: + Acetic acid: − Acetic acid: − Acetic acid: − Acetic acid: − Biomass: + Biomass: − − Biomass: + Biomass: + M Ectoine: + Ectoine: + Ectoine: + Ectoine: +++ Acetic acid: − Acetic acid: − Acetic acid: − Acetic acid: − Biomass: + Biomass: − Biomass: + Biomass: − − N Ectoine: + Ectoine: + Ectoine: +++ Ectoine: + Acetic acid: − Acetic acid: − Acetic acid: − Acetic acid: − Biomass: + Biomass: − Biomass: − − Biomass: + O Ectoine: + Ectoine: + Ectoine: +++ Ectoine: + Acetic acid: − Acetic acid: − Acetic acid: − Acetic acid: − Biomass: + Biomass: − Biomass: − − Biomass: + P Ectoine: + Ectoine: +++ Ectoine: + Ectoine: + Acetic acid: − Acetic acid: − Acetic acid: − Acetic acid: − Biomass: + Biomass: − − Biomass: + Biomass: + Q Ectoine: + Ectoine: + Ectoine: + Ectoine: +++ Acetic acid: − Acetic acid: − Acetic acid: − Acetic acid: − Biomass: + Biomass: − Biomass: + Biomass: − − R Ectoine: + Ectoine: + Ectoine: +++ Ectoine: + Acetic acid: − Acetic acid: − Acetic acid: − Acetic acid: − Biomass: + Biomass: − Biomass: − − Biomass: + S Ectoine: + Ectoine: + Ectoine: +++ Ectoine: + Acetic acid: − Acetic acid: − Acetic acid: − Acetic acid: − Biomass: + Biomass: − Biomass: − − Biomass: + The “+” indicates a strain/condition association leading to a production level between 0.8 and 1.2 fold of the reference measured in strain H and condition 1. The “−” indicates a strain/condition association leading to a production level between 0.6 and 0.8 fold of the reference. The “− −” indicates a strain/condition association leading to a production level below 0.6 fold of the reference. The “++” indicates a strain/condition association leading to a production level between 1.2 and 2 fold of the reference. The “+++” means that the production level is equal or superior to 2-fold of the reference when said strain is grown in the indicated condition.

[0165] As illustrated in Table 7, the deletion of pykA and pykF in particular, coupled with a reduction of citrate synthase activity (here by limiting gltA gene expression), surprisingly and advantageously improves ectoine production while also reducing acetic acid production in all conditions (compare e.g., results obtained for strains L to O with strains H to K). provides a relatively simple and robust microorganism that may be used to cost-effectively produce ectoine on an industrial scale.

[0166] Results presented in Table 7 also show that while a reduction of citrate synthase activity alone advantageously reduce the conversion of the carbon source into biomass, this is not sufficient to improve ectoine production (see strain H/condition 2; strain I/condition 5; strains J and K/condition 3). Indeed, although incorporation of a carbon source into biomass is a limiting factor for ectoine production, a significant part of the acetyl-CoA present in the microorganism is used for acetic acid production due to a disequilibrium between the oxaloacetic acid and acetyl-CoA production.

[0167] However, when weak citrate synthase activity is associated with an optimization of the metabolic pathway as described herein, ectoine production is advantageously improved (see Table 7, strain L or strain P/condition 2; strain M or strain Q/condition 5; strains N and O or strain R and S/condition 3).

REFERENCES

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