CONVERSION OF METHYLGLYOXAL INTO HYDROXYACETONE USING NOVEL ENZYMES AND APPLICATIONS THEREOF

Abstract

The present invention relates to new methylglyoxal reductase (MGR) enzymes which are useful for efficiently converting methylglyoxal into hydroxyacetone. The invention more particularly relates to a method for efficiently converting methylglyoxal into hydroxyacetone using said enzymes, to a method for producing 1,2-propanediol using a microorganism overexpressing said enzymes, and to said microorganism.

Claims

1.-15. (canceled)

16. A method for the fermentative conversion of methylglyoxal into hydroxyacetone, comprising the step of expressing, in a microorganism, at least one methylglyoxal reductase having a catalytic efficiency k.sub.cat/Km equal or superior to 5 mM.sup.1s.sup.1 wherein said methylglyoxal reductase is selected from the group consisting of the YjgB enzyme of sequence SEQ ID NO: 1 and its mutants.

17. The method according to claim 16, wherein said methylglyoxal reductase is the YjgB enzyme of sequence SEQ ID NO: 1.

18. The method according to claim 17, wherein the YjgB enzyme is expressed in combination with the YahK enzyme of sequence SEQ ID NO: 3, the YhdN enzyme of sequence SEQ ID NO: 5, the Gld enzyme of sequence SEQ ID NO: 7, the YafB enzyme of sequence SEQ ID NO: 11 or the YqhD enzyme of sequence SEQ ID NO: 13.

19. A method for the fermentative production of 1,2-propanediol, comprising the steps of: a) culturing, under fermentative conditions, a microorganism genetically modified for the production of 1,2-propanediol, in a culture medium comprising a carbohydrate as a source of carbon; and b) recovering 1,2-propanediol from said culture medium, wherein said microorganism overexpresses at least one gene coding for a methylglyoxal reductase as defined in claim 16 and converts methylglyoxal into hydroxyacetone.

20. The method according to claim 19, further comprising the step c) of purifying the 1,2-propanediol recovered from step b).

21. The method according to claim 19, wherein said microorganism further comprises the deletion of the yqhD or yqhD* gene coding for the methylglyoxal reductase of sequence SEQ ID NO: 13 or SEQ ID NO: 15.

22. The method according to claim 19, wherein said microorganism further overexpresses the gldA gene coding for the NADH dependent glycerol dehydrogenase of sequence SEQ ID NO: 21, or a mutant thereof coding for a NADH dependent glycerol dehydrogenase of sequence SEQ ID NO: 23.

23. The method according to claim 19, wherein said microorganism further overexpresses at least one gene coding for a NADPH dependent acetol reductase, said NADPH dependent acetol reductase having at least 60% amino acid identity with a sequence selected from the group consisting of SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, and SEQ ID NO: 5.

24. The method according to claim 23, wherein said microorganism further comprises the deletion of the gldA or gldA* gene coding for the NADH dependent glycerol dehydrogenase of sequence SEQ ID NO: 21 or SEQ ID NO: 23, and/or overexpresses a mutant thereof coding for a NADPH dependent glycerol dehydrogenase.

25. The method according to claim 19, wherein said microorganism further comprises at least one of the following genetic modifications: the overexpression of the pntAB gene operon coding for the nicotinamide nucleotide transhydrogenase of sequences SEQ ID NO: 89 and SEQ ID NO: 91, the attenuation of the pgi gene coding for the phosphoglucose isomerase of sequence SEQ ID NO: 93, the attenuation of the pJkA gene coding for the phosphofructokinase of sequence SEQ ID NO: 95, the overexpression of the zwf gene coding for the glucose-6-phosphate dehydrogenase of sequence SEQ ID NO: 97, the overexpression of the yjeF gene coding for the ADP-dependent dehydratase of sequence SEQ ID NO: 99, the overexpression of the gapN gene coding for the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase of sequence SEQ ID NO: 101, the overexpression of a mutant lpd* gene coding for the NADP-dependent lipoamide dehydrogenase of sequence SEQ ID NO: 103, and combinations thereof.

26. The method according to claim 19, wherein said microorganism further comprises the deletion of the gloA gene coding for the glyoxalase I of sequence SEQ ID NO: 31.

27. The method according to claim 19, wherein said microorganism is selected from the group consisting of Enterobacteriaceae, Clostridiaceae, Bacillaceae, Streptomycetaceae, Corynebacteriaceae, and Saccharomycetaceae.

28. The method of claim 27, wherein said microorganism is selected from the group consisting of Escherichia coli, Klebsiella pneumoniae, Thermoanaerobacterium thermosaccharolyticum, Clostridium sphenoides, Corynebacterium glutamicum and Saccharomyces cerevisiae.

29. The method according to claim 28, wherein the microorganism is Escherichia coli.

30. A microorganism genetically modified for the production of 1,2-propanediol, wherein said microorganism is as defined in claim 19.

31. The method of claim 16, wherein the YjgB mutant is selected from the group consisting of YjgB* (N240Y) of sequence SEQ ID NO: 9, YjgB*(I165V) of SEQ ID NO: 125 and YjgB*(Q39R/I165V/A296V) of sequence SEQ ID NO: 127.

32. The method according to claim 19, wherein said microorganism further overexpresses the mgsA gene coding for the methylglyoxal synthase of sequence SEQ ID NO: 17 or of sequence SEQ ID NO: 19.

33. The method according to claim 19, wherein said microorganism overexpresses a mutant gldA gene coding for a NADPH dependent glycerol dehydrogenase comprising at least a replacement of the aspartic acid amino acid residue at position 37 of SEQ ID NO: 21 or 23 with a glycine, an alanine, or a valine.

34. The method according to claim 19, wherein said microorganism overexpresses a mutant gldA gene coding for a NADPH dependent glycerol dehydrogenase of sequence SEQ ID NO: 87.

35. The method according to claim 19, wherein said carbohydrate is selected from the group consisting of arabinose, fructose, galactose, glucose, lactose, maltose, sucrose, and xylose, and any mixture thereof.

Description

DRAWINGS

[0126] FIG. 1. Methylglyoxal reductase (MGR) specific activity of strains harbouring different MGR enzymes and percentages of expression of the related MGR enzymes in the related strains.

EXAMPLES

[0127] In currently available 1,2-propanediol production E. coli strains, methylglyoxal is transformed into hydroxyacetone by the methylglyoxal reductase (MGR) enzyme YqhD*(G149E). However, YqhD*(G149E) exhibits a low catalytic efficiency and must be highly expressed so as to allow the production of 1,2-propanediol (it represents up to 40% of the total proteins expressed in the strain). This high level of expression results in a metabolic burden for the microorganism, and therefore generates a stress to the cell due to deprivation of carbon and energy.

[0128] Furthermore, even if the expression level of YqhD*(G149E) was pushed at a higher expression level, its catalytic efficiency would not be sufficient to reach a maximal 1,2-propanediol production performance. Thus, in order to increase 1,2-propanediol production, it is necessary to use a methylglyoxal reductase enzyme with a higher catalytic efficiency than YqhD*(G149E). To do so, several candidate enzymes, not known for reducing methylglyoxal, were evaluated by measuring their catalytic efficiencies in vitro. The best performing enzymes were then screened for their capacity to detoxify methylglyoxal (MG) in vivo. The enzymes exhibiting the highest resistance to methylglyoxal were then introduced into a 1,2-propanediol production E. coli strain.

Material and Methods:

[0129] In the examples given below, methods well-known in the art were used to construct E. coli strains containing replicating vectors and/or various chromosomal deletions, and substitutions using homologous recombination well described by Datsenko & Wanner, (2000) for Escherichia coli. In the same manner, the use of plasmids or vectors to express or overexpress one or several genes in a recombinant microorganism are well-known by the man skilled in the art. Examples of suitable E. coli expression vectors include pTrc, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, etc. . . . (Studier et al., 1990; and Pouwels et al., 1985)

[0130] Several protocols have been used in the following examples. Protocol 1 (chromosomal modifications by homologous recombination, selection of recombinants), protocol 2 (transduction of phage P1) and protocol 3 (antibiotic cassette excision, the resistance genes were removed when necessary) used in this invention have been fully described in patent application EP 2532751, incorporated herein by reference. Chromosomal modifications were verified by a PCR analysis with appropriate oligonucleotides that the person skilled in the art is able to design.

Protocol 4: Construction of Recombinant Plasmids

[0131] Recombinant DNA technology is described in Molecular Cloning: Sambrook and Russell (2001). Briefly, the DNA fragments were PCR amplified using oligonucleotides and appropriate genomic DNA as 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, then ligated and transformed into competent cells. Transformants were analysed and recombinant plasmids of interest were verified by DNA sequencing.

Example 1: Identification of New Methylglyoxal Reductase (MGR) Enzymes

1.1 Determination of the Methylglyoxal Reductase Activity of Various Candidate Enzymes

1.1.1. Construction of Strains 1 to 16

[0132] To determine the kinetic parameters of various aldehyde reductase enzymes candidates, the following strains were constructed:

TABLE-US-00002 TABLE 2 strains constructed and used for the determination of the kinetic parameters of 16 aldehyde reductase enzyme candidates Strain Methylglyoxal reductase enzyme Number Name Uniprot Ref Microorganism of origin gene sequence 1 YqhD Q46856 E. coli SEQ ID No14 2 YqhD*(G149E) E. coli SEQ ID No16 3 YafB P30863 E. coli SEQ ID No12 4 YhdN P80874 Bacillus subtilis SEQ ID No6 5 YahK P75691 E. coli SEQ ID No4 6 Gld Q5FQJ0 Gluconobacter oxydans SEQ ID No8 7 YdjG*(D232E) E. coli SEQ ID No106 8 Adh3.2 R4Z7U3 Dickeya zeae SEQ ID No108 9 YdhF P76187 E. coli SEQ ID No110 10 YeaE P76234 E. coli SEQ ID No112 11 Gld2 Q0GYU74 Hypocrea jecorina SEQ ID No114 12 YiaY P37686 E. coli SEQ ID No116 13 BudC Q48436 Klebsiella pneumoniae SEQ ID No118 14 GldA*(A160T) E. coli SEQ ID No24 15 YjgB P27250 E. coli SEQ ID No2 16 YjgB*(N240Y) E. coli SEQ ID No10 17 YjgB*(I165V) E. coli SEQ ID No126 18 YjgB* (Q39R/I165V/A296V) E. coli SEQ ID No128

[0133] The genes coding for the different putative methylglyoxal reductase enzymes were cloned into the expression plasmid pPAL7 (Biorad) and the plasmids obtained were transformed into strain BL21(DE3)star, except for strains 2 and 7.

[0134] For strain 2, the plasmid was cloned into a BL21(DE3)star strain deleted for yqhD obtained as following. The yqhD gene was inactivated in strain MG1655 using the homologous recombination strategy (according to Protocol 1). Oligonucleotides for DyqhD: SEQ ID No 119 and 120, were used to PCR amplify the resistance cassette. The strain retained was designated MG1655 DyqhD::Cm. Finally, the DyqhD::Cm deletion was transferred by P1 phage transduction (according to Protocol 2) into the strain BL21(DE3)star and the pPAL7-yqhD*(G149E) plasmid was introduced resulting in strain 2.

[0135] For strain 7, the plasmid was cloned into a BL21(DE3)star strain deleted for ydjG obtained as following. The ydjG gene was inactivated in strain MG1655 using the homologous recombination strategy (according to Protocol 1). Oligonucleotides for DydjG: SEQ ID No 121 and 122, were used to PCR amplify the resistance cassette. The strain retained was designated MG1655 DydjG::Km. Finally, the DydjG::Km deletion was transferred by P1 phage transduction (according to Protocol 2) into the strain BL21 (DE3)star and the pPAL7-ydjG*(D232E) plasmid was introduced resulting in strain 7.

[0136] Strain 14, bearing the GldA*(A160T) enzyme, was the same as strain number 20 described in patent application EP14305691, incorporated herein by reference.

1.1.2. Overproduction of Proteins

[0137] Cultures for the overproduction of proteins were realized in a 2 L Erlenmeyer flask, using LB broth (Bertani, 1951) that was supplemented with 2.5 g/l glucose and 100 mg/L of ampicillin. An overnight preculture was used to inoculate a 500 mL culture to an OD.sub.600nm of about 0.15. This preculture was carried out in a 500 mL Erlenmeyer flask filled with 50 mL of LB broth that was supplemented with 2.5 g/L glucose and 100 mg/L of ampicillin. The culture was first kept on a shaker at 37 C. and 200 rpm until OD.sub.600 nm was about 0.5 and then the culture was moved on a second shaker at 25 C. and 200 rpm until OD.sub.600 nm was 0.6-0.8 (about one hour), before induction with 500 M IPTG. The culture was kept at 25 C. and 200 rpm until OD.sub.600 nm was around 4, and then it was stopped. Cells were centrifuged at 7000 rpm, 5 minutes at 4 C., and then stored at 20 C.

1.1.3. Protein Purification

Step 1: Preparation of Cell-Free Extracts.

[0138] About 400 mg of E. coli biomass was suspended in 60 ml of 100 mM potassium phosphate pH 7.6, and a protease inhibitor cocktail. The cell suspension (15 ml per conical tube) was sonicated on ice (Bandelin sonoplus, 70 W) in a 50 ml conical tube during 8 cycles of 30 sec with 30 sec intervals. After sonication, cells were incubated for 30 min at room temperature with 5 mM MgCl2 and 1 UI/ml of DNasel. Cells debris were removed by centrifugation at 12000 g for 30 min at 4 C.

Step 2: Affinity Purification

[0139] Except for the strain 14, the proteins were purified from the crude cell-extract by affinity on a Profinity column (BIORAD, Bio-Scale Mini Profinity exact cartridge 5 ml) according to the protocol recommended by the manufacturer. The crude extract was loaded on a 5 ml Profinity exact cartridge equilibrated with 100 mM potassium phosphate pH 7.6. The column was washed with 10 column volumes of the same buffer and incubated 30 min with 100 mM potassium phosphate pH 7.6, 100 mM fluoride at room temperature. The protein was eluted from the column with 2 column volumes of 100 mM potassium phosphate pH 7.6. The tag remained tightly bound to the resin and the purified protein was released. The fractions containing the protein were pooled, concentrated and loaded on a gel filtration column (Superdex 200 10/300 GL column, GE Healthcare) equilibrated with different storage buffers (Table 3). Protein concentration was measured using the Bradford protein assay.

[0140] For strain 14, the purification protocol was previously described in patent application WO 2015/173247, incorporated herein by reference.

TABLE-US-00003 TABLE 3 Protein storage buffer Enzyme Storage buffer YqhD 50 mM Hepes pH 7.5 YqhD*(G149E) 50 mM Hepes pH 7.5 YafB 1M Tris-HCl pH 7 150 mM NaCl YeaE 50 mM Hepes pH 7.5 YdhF 50 mM Hepes pH 7.5 YhdN 100 mM Potassium Phosphate pH 7.6 Gld2 100 mM MES pH 6.5 YahK 100 mM Potassium Phosphate pH 7.6 150 mM NaCl Gld 50 mM Hepes pH 7.5 YdjG*(D232E) 100 mM Potassium Phosphate pH 7.6 Adh3.2 100 mM Potassium Phosphate pH 7.6 150 mM NaCl YiaY 100 mM Potassium Phosphate pH 7.6 150 mM NaCl BudC 100 mM Potassium Phosphate pH 7.6 150 mM NaCl GldA*(A160T) 100 mM MES pH 6.5 YjgB 100 mM Potassium Phosphate pH 7.6 150 mM NaCl YjgB*(N240Y) 100 mM Potassium Phosphate pH 7.6 150 mM NaCl YjgB*(I165V) 100 mM Potassium Phosphate pH 7.6 150 mM NaCl YjgB* 100 mM Potassium Phosphate pH 7.6 150 mM NaCl (Q39R/I165V/ A296V)

1.1.4. Determination of Kinetic Parameters of Purified Putative Methylalyoxal Reductase Enzymes

[0141] Methylglyoxal reductase activity (MGR) was determined by measuring the consumption of NAD(P)H at 340 nm on a spectrophotometer at 30 C. (.sub.340=6290 M.sup.1 cm.sup.1). The reaction mixture (1 mL) containing assay buffer and purified enzyme was incubated for 5 min at 30 C. Then, 0.1-40 mM methylglyoxal was added to start the reaction. One unit of enzyme activity was defined as the amount of enzyme catalyzing the decrease of 1 mol of NAD(P)H per min. Kinetic parameters were determined with Sigmaplot. The kinetic parameters of the purified enzymes are provided in Table 4.

1.1.5. Determination of Reaction Product

[0142] The reaction product by the different putative enzymes from methylglyoxal (MG) was measured by GC-MS (Agilent Technologies) for the Hydroxyacetone (HA) and by UHPLC-MS/MS for the Lactaldehyde (LA) after reaction with methylbenzothiazolinone-2-hydrazone (MBTH) and FeCl.sub.3. The reaction mixture (1 mL) containing assay buffer, 10 mM methylglyoxal, 5 mM NADPH and 5-10 g of purified enzyme was incubated for 30 min at 30 C. 1 l of the reaction product was injected. A reaction mixture without MG was prepared as a control. The reaction product of the purified enzymes is provided in Table 4.

TABLE-US-00004 TABLE 4 Kinetic parameters and reaction product of purified enzymes Km kcat/Km Reaction Enzyme Assay buffer Cofactor mM mM.sup.1s.sup.1 product YqhD 20 mM Hepes (pH 7.5) NADPH 2.09 0.40 HA 0.1 mM ZnSO4 YqhD* (G149E) 20 mM Hepes (pH 7.5) NADPH 2.92 0.80 HA 0.1 mM ZnSO4 YafB 20 mM Hepes (pH 7.5) NADPH 8.20 2.06 HA YeaE 20 mM Hepes (pH 7.5) NADPH 1.59 0.91 ND YdhF 20 mM Hepes (pH 7.5) NADPH 21.8 0.35 HA YhdN 20 mM Hepes (pH 7.5) NADPH 0.64 5.92 HA 0.1 mM ZnSO4 Gld2 10 mM sodium phosphate NADPH 7.7 11.1 LA (pH 7) YahK 20 mM Hepes (pH 7.5) NADPH 1.41 8.3 HA Gld 20 mM Hepes (pH 7.5) NADPH 1.10 8.74 HA YdjG* (D232E) 20 mM Hepes (pH 7.5) NADPH 3.25 0.01 HA Adh3.2 20 mM Hepes (pH 7.5) NADPH 6.7 0.17 HA YiaY 20 mM Hepes (pH 7.5) NADH 2.84 0.34 HA 0.1 mM FeSO4 BudC 50 mM Imidazole (pH 7) NADH 74.8 4.2 ND GldA* (A160T) 100 mM MES-KOH (pH 6.5) NADH 3.17 7.8 LA 0.1 mM FeSO4 30 mM ammonium sulfate YjgB 40 mM Hepes (pH 7.5) NADPH 10.6 51.6 HA YjgB* (N240Y) 40 mM Hepes (pH 7.5) NADPH 1.6 40.4 ND YjgB*(I165V) 40 mM Hepes (pH 7.5) NADPH 5.5 57.02 ND YjgB* (Q39R/I165V/A296V) 40 mM Hepes (pH 7.5) NADPH 0.88 39.9 ND HA: Hydroxyacetone, LA: Lactaldehyde, ND: Not determined

[0143] Five enzymes producing Hydroxyacetone and having a catalytic efficiency at least two times higher than that of YqhD*(G149E) (mutated enzyme which itself has a catalytic efficiency two times higher than the native YqhD enzyme) were selected for further characterization and screening: Gld, YhdN, YafB, YahK and YjgB.

1.2. Selection of the Best Methylglyoxal Reductase Enzymes

1.2.1. Construction of Strains 17 to 23

[0144] The selected MGR enzymes were subsequently screened by cloning the corresponding genes into the modified E. coli strain 15: MG1655 DgloA Dedd DpflAB DldhA DadhE DgldA DyqhD constructed as following. To inactivate the glyoxalase I encoded by gloA, the phosphogluconate dehydratase encoded by edd, the pyruvate formate lyase activating enzyme and the pyruvate formate lyase encoded by pflA and pflB respectively, the lactate dehydrogenase encoded by IdhA and the alcohol dehydrogenase encoded by adhE, the DgloA, Dedd, DpflAB, DdhA and DadhE deletions described in patent application WO 2008/116852 (incorporated herein by reference) were transferred by P1 phage (according to Protocol 2) into strain MG1655 and the resistance genes were removed according to protocol 3. To inactivate the glycerol dehydrogenase encoded by gldA, the DgldA deletion described in patent application patent application WO 2015/173247 (incorporated herein by reference) was transferred by P1 phage (according to Protocol 2) into the previous strain. Finally, to inactivate the aldehyde reductase encoded by yqhD, the DyqhD::Cm deletion described above was transferred by P1 phage transduction (according to Protocol 2) into the previous strain, resulting in strain 17.

[0145] Then, the genes described in Table 5 below were expressed under defined RBS on pME101VB06 plasmid described in patent application EP 2532751 (incorporated herein by reference), and each plasmid was introduced into strain 17 resulting in strains 18 to 23.

TABLE-US-00005 TABLE 5 description of the methylglyoxal reductase strains 18 to 23 Strain Enzyme 18 Gld 19 YhdN 20 YafB 21 YqhD*(G149E) 22 YahK 23 YjgB

1.2.2. Methylalyoxal Reductase Assay on Crude Extract

[0146] Methylglyoxal reductase activity (MGR) was determined by measuring the consumption of NAD(P)H at 340 nm on a spectrophotometer at 30 C. (.sub.340=6290 M.sup.1 cm.sup.1). The reaction mixture (1 mL) containing assay buffer and crude extract was incubated for 5 min at 30 C. Then, 10 mM methylglyoxal was added to start the reaction. One unit of enzyme activity was defined as the amount of enzyme catalyzing the decrease of 1 mol of NAD(P)H per min. Specific enzyme activity was expressed as units of enzyme activity per mg of protein. The activity value determined without substrate in the assay was subtracted.

1.2.3. Determination of Expression Level of the Methylalyoxal Reductase Enzymes

[0147] In parallel to the specific activity in all strains, the expression level of the different MGR was quantified by SDS-PAGE analysis. A same quantity of crude extract was loaded on SDS-PAGE and the expression level was determined as the ratio of the band volume of the MGR relative to the total lane volume, using BioRad Image Lab Software.

[0148] The specific activities of these different enzymes were very different and not directed related to the expression level. For example, strain 21 shows a high expression with a low specific activity while strain 22 shows a 5 times lower expression level but a 4 times higher specific activity (FIG. 1).

1.2.4. Screening on Methylglyoxal (MG)

[0149] Since in vitro activity may not reflect real in vivo activity, strains 17 to 23 were screened for their resistance to MG on LB agar plates. Strains were cultivated at 37 C. in LB rich medium supplemented with 50 g/mL spectinomycin, up to an OD600 nm of about 1. Then 100 L of 0, 10.sup.1 or 10.sup.2 dilutions were plated on LB agar plates supplemented with 50 g/mL spectinomycin, and 0, 2, 3 or 4 mM MG. Plates were incubated at 37 C. for 48 h.

[0150] Table 6 below indicates the least dilution and the highest MG concentration at which some clones grew, which gave an indication of the resistance level of the strain (the higher the MG concentration and the lower dilution for a given concentration, the higher resistance).

TABLE-US-00006 TABLE 6 screening on methylglyoxal of strains 17 to 23 Strain 17 18 19 20 21 22 23 MGR x Gld YhdN YafB YqhD* YahK YjgB mM MG 0 2 2 3 2 3 3 Dilution 10.sup.2 10.sup.2 10.sup.2 10.sup.1 10.sup.2 10.sup.2 10.sup.2 Resistance level + + ++ + +++ +++ : no resistance; +: medium resistance; ++: high resistance; +++: very high resistance

[0151] The YahK and YjgB enzymes allowing the better MG resistance, these candidate MGR enzymes were retained to replace YqhD*(G149E) in the MPG producing strains. The skilled practitioner would nevertheless readily understand that the enzymes Gld, YafB and YhdN, would also be suitable to replace YqhD*(G149E) in said MPG producing strains.

Example 2: Production of 1.2-Propanediol with the New Methylglyoxal Reductase (MGR) Enzymes According to the Invention

2.1. Construction of Strains 24 to 26

[0152] To inactivate the ptsG gene, the homologous recombination strategy was used (according to Protocol 1). Oligonucleotides for DptsG: SEQ ID No 123 and 124, were used to PCR amplify the resistance cassette. The strain retained was designated MG1655 DptsG::Km. The DptsG::Km deletion was transferred by P1 phage transduction (according to Protocol 2) into E. coli MPG production strain 5 described in patent application WO 2015/173247 (incorporated herein by reference), giving rise to strain 24. Thereafter, yahK and yjgB were chromosomally overexpressed under the Ptrc promoter and under defined RBS and either construction was transferred by P1 phage (according to Protocol 2) into strain 24 further modified by deleting yqhD as described in Example 1, giving rise to strain 25 for YahK and strain 26 for YjgB.

2.2. Evaluation of MPG Production Strains

[0153] 1,2-propanediol production strains were cultivated in shake flasks (as described in patent application EP 2532751, incorporated herein by reference, except that glucose and xylose were used as carbon sole and 40 g.Math.L-1 of MOPS were added in the media in order to maintain a pH above 6.0 throughout fermentation course) and in 2 L fermenters as follows:

[0154] Inocula were obtained after 24 hour precultures realised in baffled flasks containing 50 mL of minimal media (M1) completed with 10% of LB media (w:w) at 37 C.

[0155] Subsequently, 2.5 L fermentors (Pierre Guerin) were filled with 700 mL of minimal medium (M2) and were inoculated to a biomass concentration of 0.2 g.Math.L.sup.1 with a preculture volume ranging between 55 to 80 mL. For the strain 26, zinc was added in the batch medium at a final concentration of 4 mg.Math.L.sup.1.

[0156] The culture temperature was maintained constant at 37 C. and pH was maintained to the working value (6.8) by automatic addition of NH.sub.4OH solution (10%) The initial agitation rate was set at 200 RPM and the initial airflow rate was set at 40 NL.Math.h.sup.1. The dissolved oxygen concentration was maintained at values between 20 and 40%, preferentially 30% saturation by increasing the agitation and then if necessary by increasing the aeration. When it was needed, antibiotics were added at a concentration of 50 mg.Math.L.sup.1 for spectinomycin.

[0157] Fedbatch media was composed of sugars (glucose/xylose; ratio 2:1) and alimentation rate was adjusted automatically to maintained a sugar concentration of 30 g.Math.L.sup.1 in the fermentation broth by measuring CO.sub.2 production.

[0158] Cultures were stopped after 54 hours.

TABLE-US-00007 TABLE 7 Composition of M1 and M2 media M1 M2 Component Concentration (g/L) Concentration (g/L) Glucose 20.00 21.0 Xylose 0.00 9.0 (NH.sub.4).sub.2SO.sub.4 4.88 4.88 Citric acidH.sub.2O 1.70 0.00 KH.sub.2PO.sub.4 1.65 6.76 MgSO.sub.47H.sub.2O 1.00 1.80 K.sub.2HPO.sub.43H.sub.2O 0.92 0.00 (NH.sub.4).sub.2HPO.sub.4 0.40 0.00 Fe(III) citrateH.sub.2O 0.1064 0.0000 FeSO.sub.47H.sub.2O 0.0000 0.1000 CaCl.sub.22H.sub.2O 0.08 0.08 MnCl.sub.24H.sub.2O 0.0150 0.0000 Zn(CH.sub.3COO).sub.22H.sub.2O 0.0130 0.0000 ThiamineHCl 0.0100 0.0140 EDTA, 2Na2H.sub.2O 0.0084 0.0000 H.sub.3BO.sub.3 0.0030 0.0000 CoCl.sub.26H.sub.2O 0.0025 0.0036 Na.sub.2MoO.sub.42H.sub.2O 0.0025 0.0000 CuCl.sub.22H.sub.2O 0.0015 0.0000

[0159] 1.2-propanediol (PG) and its precursor hydroxyacetone (HA) were quantified by HPLC-RID with Biorad HPX-87H column.

[0160] In shake flasks, production strains with yahKor yjgB overexpression produced more PG+HA in gram by gram of biomass than the strain with yqhD*(G149E) overexpression. In 2 L fermenters, only the strain with yjgB overexpression was better.

TABLE-US-00008 TABLE 8 PG + HA Yield for PG producing strains in gram PG + HA per gram of biomass. Strain 24 Strain 25 Strain 26 Shake flasks 1.88 2.15 2.14 2 L fermenters 3.25 3.63 5.42

[0161] The behaviour of strain 25 in 2 L fermenters was attributed to inhibition of YahK by HA.

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