NEW LACTALDEHYDE REDUCTASES FOR THE PRODUCTION OF 1,2-PROPANEDIOL

Abstract

The present invention relates to new lactaldehyde reductase (LAR) enzymes useful for the production of 1,2-propane-diol and to microorganisms overexpressing said enzymes. The invention also relates to a method for producing 1,2-propanediol by converting lactaldehyde into 1,2-propanediol with said enzymes.

Claims

1. A method for producing 1,2-propanediol, comprising the step of converting (R), (S) and/or (R,S) lactaldehyde into 1,2-propanediol with at least one enzyme having one or more of the following properties: a) a specific lactaldehyde reductase activity of at least 1850 mU/mg towards (R)-lactaldehyde and NADH in anaerobic conditions, b) a specific lactaldehyde reductase activity of at least 600 mU/mg towards (R)-lactaldehyde and NADH in aerobic conditions, c) a specific lactaldehyde reductase activity of at least 4350 mU/mg towards (S)-lactaldehyde and NADH in aerobic conditions, d) a specific lactaldehyde reductase activity of at least 200 mU/mg towards (R)-lactaldehyde and NADPH, and e) a specific lactaldehyde reductase activity of at least 150 mU/mg towards (S)-lactaldehyde and NADPH.

2. The method according to claim 1, wherein said enzyme is selected from the group consisting of YiaY (SEQ ID NO:3), GldA (SEQ ID NO:5), YqhD (SEQ ID NO:7), Yarn (SEQ ID NO:9), YeaE (SEQ ID NO:11), YqhE (SEQ ID NO:13), YdhF (SEQ ID NO:15), GOX1615 (SEQ ID NO:17), YhdN (SEQ ID NO:19), Gld2 (SEQ ID NO:21), Alr (SEQ ID NO:23), functional fragments and functional mutants thereof, and combinations thereof.

3. The method according to claim 1, wherein said enzyme has a specific lactaldehyde reductase activity of at least 1850 mU/mg towards (R)-lactaldehyde and NADH in anaerobic conditions, and a specific lactaldehyde reductase activity of at least 600 mU/mg towards (R)-lactaldehyde and NADH in aerobic conditions; and is selected from the group consisting of GldA, YiaY, functional fragments and functional mutants thereof, and combinations thereof.

4. The method according to claim 1, wherein said enzyme has a specific lactaldehyde reductase activity of at least 4350 mU/mg towards (S)-lactaldehyde and NADH in aerobic conditions, and is selected from the group consisting of GldA, functional fragments and functional mutants thereof, and combinations thereof.

5. The method according to claim 1, wherein said enzyme has a specific lactaldehyde reductase activity of at least 200 mU/mg towards (R)-lactaldehyde and NADPH, and is selected from the group consisting of YafB, YqhE, GOX1615, YhdN, functional fragments and functional mutants thereof, and combinations thereof.

6. The method according to claim 1, wherein said enzyme has a specific lactaldehyde reductase activity of at least 150 mU/mg towards (S)-lactaldehyde and NADPH in aerobic conditions, and is selected from the group consisting of YafB, YqhE, YhdN, Gld2, functional fragments and functional mutants thereof, and combinations thereof.

7. The method according to claim 2, wherein said functional mutant of YqhD (YqhD*) comprises at least one amino acid mutation selected from the group consisting of T142G, T142S, S144A, G149A, G149E, G149H, G149K, G149T, G149N, G149R, G149S, V151M, V151L, A162K, A162D, A162L, A162N, and combinations thereof, the amino acids numbers being made by reference to the YqhD E. coli amino acid sequence (SEQ ID NO:7).

8. The method according to claim 1, wherein the conversion of (R), (S) and/or (R,S) lactaldehyde into 1,2-propanediol is made by contacting in vitro a solution comprising said lactaldehyde with said enzyme having a lactaldehyde reductase activity.

9. The method according to claim 1, wherein the conversion of (R), (S) and/or (R,S) lactaldehyde into 1,2-propanediol is made by culturing a recombinant microorganism in a culture medium comprising a source of carbon, said microorganism being genetically modified to comprise at least one pathway for the production of (R), (S) and/or (R,S) lactaldehyde and the conversion thereof into 1,2-propanediol.

10. The method according to claim 9, wherein the genetic modification for the conversion of lactaldehyde into 1,2-propanediol is an overexpression of said at least one enzyme.

11. The method according to claim 9, wherein the genetic modification for the production of (R), (S) and/or (R,S) lactaldehyde is an overexpression of at least one of the following enzymes: methylglyoxal synthase; methylglyoxal reductase; glyoxalase; lactate dehydrogenase; lactate coA-transferase; lactoyl coA reductase; and any combination thereof.

12. A lactaldehyde reductase consisting of a functional mutant of YqhD as defined in claim 7.

13. (canceled)

14. (canceled)

15. A recombinant microorganism overexpressing a lactaldehyde reductase as defined in claim 12.

16. The recombinant microorganism according to claim 15, wherein said microorganism is selected from the group consisting of Enterobacteriaceae, Bacillaceae, Clostridiaceae, Streptomycetaceae and yeasts.

17. The recombinant microorganism according to claim 16, wherein said Enterobacteriaceae is Escherichia coli.

18. The method according to claim 1, wherein said enzyme has a specific lactaldehyde reductase activity of at least 5000 mU/mg towards (R)-lactaldehyde and NADPH, and is selected from the group consisting of YafB, YqhE, GOX1615, YhdN, functional fragments and functional mutants thereof, and combinations thereof.

19. The method according to claim 1, wherein said enzyme has a specific lactaldehyde reductase activity of at least 10000 mU/mg towards (R)-lactaldehyde and NADPH, and is selected from the group consisting of YafB, YqhE, GOX1615, YhdN, functional fragments and functional mutants thereof, and combinations thereof.

20. The method according to claim 1, wherein said enzyme has a specific lactaldehyde reductase activity of at least 2500 mU/mg towards (S)-lactaldehyde and NADPH in aerobic conditions, and is selected from the group consisting of YafB, YqhE, YhdN, Gld2, functional fragments and functional mutants thereof, and combinations thereof.

21. The method according to claim 1, wherein said enzyme has a specific lactaldehyde reductase activity of at least 5000 mU/mg towards (S)-lactaldehyde and NADPH in aerobic conditions, and is selected from the group consisting of YafB, YqhE, YhdN, Gld2, functional fragments and functional mutants thereof, and combinations thereof.

Description

DRAWINGS

[0197] FIG. 1. Different pathways for the production of (R) or (S)-1,2-propanediol utilizing the intermediate Methylglyoxal or Lactate by a genetically modified microorganism for the production of 1,2-propanediol from different sources of carbon.

[0198] FIG. 2. Different pathways to produce (R) or (S)-1,2-propanediol by using the lactate as intermediate and an enzyme with a racemase activity to convert (R)-lactate into (S)-lactate and vice versa.

EXAMPLES

[0199] The present invention is further defined in the following examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From above disclosure and these examples, the man skilled in the art can make various changes of the invention to adapt it to various uses and conditions without modifying the essentials means of the invention.

[0200] Exemplary genes and enzymes required for constructing microorganisms with these capabilities are described as well as methods for cloning and transformation, monitoring product formation and using the engineered microorganisms for production.

[0201] In particular, examples show modified Escherichia coli (E. coli) strains, but these modifications can easily be performed in other microorganisms of the same family or other microorganisms.

[0202] Escherichia coli belongs to the Enterobacteriaceae family, which comprises members that are Gram-negative, rod-shaped, non-spore forming and are typically 1-5 m in length. Most members have flagella used to move about, but a few genera are non-motile. Many members of this family are a normal part of the gut flora found in the intestines of humans and other animals, while others are found in water or soil, or are parasites on a variety of different animals and plants. E. coli is one of the most important model organism, but other important members of the Enterobacteriaceae family include Klebsiella, in particular Klebsiella terrigena, Klebsiella planticola or Klebsiella oxytoca, Pantoea and Salmonella.

[0203] In the examples given below, methods well known in the art were used to construct Escherichia coli strains containing replicating vectors and/or various chromosomal deletions, and substitutions using homologous recombination well described by Datsenko & Wanner, (2000) for E. coli. In the same manner, the use of plasmids or vectors to express or overexpress one or several genes in a recombinant microorganisms are well known by the man skilled in the art. Examples of suitable E. coli expression vectors include pTrc, pACYC184n pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, etc. . . . (Studier et al., 1990 and Pouwels et al., 1985).

[0204] 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.

[0205] Protocol 4: Construction of Recombinant Plasmids

[0206] 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 (that the person skilled in the art will be able to define), then ligated and transformed into competent cells. Transformants were analysed and recombinant plasmids of interest were verified by DNA sequencing.

[0207] Protocol 5: Flask Cultures for the Production of Recombinant Proteins

[0208] Flask cultures for the production of recombinant proteins were carried out as described in patent application WO 2010/076324 except that LB broth was supplemented with 5 g/L glucose.

[0209] Protocol 6: Evaluation of 1,2-Propanediol Production Strains

[0210] 1,2-propanediol production strains were cultivated in flask cultures as described in patent application EP 2532751, except that 20 g/L glucose or sucrose and 40 g/L MOPS were used. 1,2-propanediol (MPG) was quantified by HPLC-RID with Biorad HPX-87H column. MPG enantiomeric form (S or R) was identified by GC-FID with Varian Chirasil-DEX column.

Example 1: Identification of New LAR Enzymes

[0211] Construction of Strain 1

[0212] To characterize the L-1,2-propanediol oxidoreductase from Escherichia coli, the gene fucO (SEQ ID No 2) was cloned into the expression plasmid pPAL7 (Biorad). This plasmid was named pPG0249 and transformed into strain BL21(DE3)star, giving rise to strain 1.

[0213] Construction of Strain 2

[0214] To inactivate the aldehyde reductase encoded by the yqhD gene (SEQ ID No 8), the DyqhD::Km deletion described in patent application WO 2008/116853 was transferred by P1 phage transduction (according to Protocol 2) into strain BL21(DE3)star. To characterize the aldehyde reductase from Escherichia coli, the gene yqhD (SEQ ID No 8) was cloned into the expression plasmid pPAL7 (Biorad). This plasmid was named pPG0332 and transformed into strain BL21(DE3)star DyqhD::Km, giving rise to strain 2.

[0215] Construction of Strain 3

[0216] To characterize the mutated yqhD*(G149E), site-directed mutagenesis on pPG0332 was used. This plasmid was named pPG0329 and transformed into strain BL21(DE3)star DyqhD::Km, giving rise to strain 3.

[0217] Construction of Strain 4

[0218] To inactivate the glyoxal reductase encoded by the yafB gene (SEQ ID No 10), the homologous recombination strategy was used (according to Protocols 1). Oligonucleotides for DyafB: SEQ ID No 29 and 30, were used to PCR amplify the resistance cassette. The strain retained was designated MG1655 DyafB::Km. Finally, the DyafB::Km deletion was transferred by P1 phage transduction (according to Protocol 2) into strain BL21(DE3)star. To characterize the glyoxal reductase from Escherichia coli, the gene yafB was cloned into the expression plasmid pPAL7 (Biorad). This plasmid was named pPG0319 and transformed into strain BL21(DE3)star DyafB::Km, giving rise to strain 4.

[0219] Construction of Strain 5

[0220] To characterize the aldo-keto reductase from Escherichia coli, the gene yeaE (SEQ ID No 12) was cloned into the expression plasmid pPAL7 (Biorad). This plasmid was named pPG0371 and transformed into strain BL21(DE3), giving rise to strain 5.

[0221] Construction of Strain 6

[0222] To characterize the predicted aldo/keto NAD(P) oxidoreductase from Escherichia coli, the gene ydhF (SEQ ID No 16) was cloned into the expression plasmid pPAL7 (Biorad). This plasmid was named pPG0372 and transformed into strain BL21(DE3), giving rise to strain 6.

[0223] Construction of Strain 7

[0224] To characterize the predicted iron-containing alcohol dehydrogenase from Escherichia coli, the gene yiaY (SEQ ID No 4) was cloned into the expression plasmid pPAL7 (Biorad). This plasmid was named pPG0270 and transformed into strain BL21(DE3), giving rise to strain 7.

[0225] Construction of Strain 8

[0226] To characterize the glycerol dehydrogenase from Gluconobacter oxydans (SEQ ID No 17), the synthetic gene gld optimized for Escherichia coli (SEQ ID No 31) was cloned into the expression plasmid pPAL7 (Biorad). This plasmid was named pPG0381 and transformed into strain BL21(DE3), giving rise to strain 8.

[0227] Construction of Strain 9

[0228] To characterize the aldo keto reductase from Bacillus subtilis, the gene yhdN from Bacillus subtilis (SEQ ID No 20) was cloned into the expression plasmid pPAL7 (Biorad). This plasmid was named pPG0357 and transformed into strain BL21(DE3), giving rise to strain 9.

[0229] Construction of Strain 10

[0230] To inactivate the gldA gene, the homologous recombination strategy was used (according to Protocols 1 and 3). Oligonucleotides for DgldA: SEQ ID No 32 and 33, were used to PCR amplify the resistance cassette. The strain retained was designated MG1655 DgldA::Km. Finally, the DgldA::Km deletion was transferred by P1 phage transduction (according to Protocol 2) into the strain BL21(DE3)star. To characterize the glycerol dehydrogenase from Hypocrea jecorina (SEQ ID No 21), the synthetic gene gld2 optimized for Escherichia coli (SEQ ID No 34) was cloned into the expression plasmid pPAL7 (Biorad). This plasmid was named pPG0418 and transformed into strain BL21(DE3)star DgldA::Km previously described giving rise to strain 10.

[0231] Construction of Strain 11

[0232] To characterize the alcohol reductase from Leishmania donovani (SEQ ID No 23), the synthetic gene air optimized for Escherichia coli (SEQ ID No 35) was cloned into the expression plasmid pPAL7 (Biorad). This plasmid was named pPG0259 and transformed into strain BL21(DE3), giving rise to strain 11.

[0233] Construction of Strain 12

[0234] To characterize the 1,2-propanediol:NAD+ oxidoreductase from Escherichia coli, the gene gldA (SEQ ID No 6) was cloned into the expression plasmid pET101/D-TOPO (Lifetechnologies). This plasmid was named pPG0029 and transformed into strain BL21(DE3)star, giving rise to the strain 12.

[0235] Construction of Strain 13

[0236] To characterize the beta-keto ester reductase from Escherichia coli, the gene yqhE (SEQ ID No 14) was cloned into the expression plasmid pET TOPO (Lifetechnologies). This plasmid was named pPG0153 and transformed into strain BL21(DE3)star, giving rise to the strain 13.

[0237] Construction of strains 14 to 33

[0238] To characterize the mutated aldehyde reductase from Escherichia coli, first the native gene yqhD (SEQ ID No 8) was cloned into the plasmid pTRC99A (Amersham Pharmacia), this plasmid was named pME0103. Site-directed mutagenesis was used on pME0103 to introduce diverse mutations:

[0239] yqhD*(V151L); yqhD*(G149E); yqhD*(T142S); yqhD*(T142G); yqhD*(S144A); yqhD*(G149A); yqhD*(G149H); yqhD*(G149K); yqhD*(G149M); yqhD*(G149T); yqhD*(G149N); yqhD*(G149R); yqhD*(G149S); yqhD*(G149V); yqhD*(A1621); yqhD*(A162K); yqhD*(A162D); yqhD*(A162L); yqhD*(A162N); yqhD*(V151M).

[0240] The plasmids obtained were transformed into strain BL21(DE3), giving rise to strains 14 to 33.

[0241] Preparation of Cell-Free Extract

[0242] The cells (400-600 mg dry weight) were resuspended in extraction buffer (60-100 ml). The suspended cells were disrupted by 8 sonication cycles of 30 sec on ice (Branson sonifier, 70 W). Cells were incubated for 45 minutes at room temperature with 1-5 mM MgCl2 and 2 Ul/ml of DNasel. Cells debris were removed by centrifugation at 12000 g for 30 min at 4 C. The supernatant was kept as the crude extract.

[0243] For FucO, the extraction was also realized in anaerobia.

TABLE-US-00003 TABLE 3 Buffers used for preparation of cell-free extract Enzyme Extraction buffer FucO 10 mM Tris-HCl pH 7.5 2.5 mM NAD+ and protease inhibitor YqhD wild-type 100 mM Potassium phosphate pH 7.6 and protease and mutants inhibitor YafB 1M Tris-HCl pH 7 and protease inhibitor YeaE 100 mM Potassium phosphate pH 7.6 and protease inhibitor YdhF 100 mM Potassium phosphate pH 7.6 and protease inhibitor YiaY 100 mM Potassium phosphate pH 7.6 and protease inhibitor GOX1615 100 mM Potassium phosphate pH 7.6 and protease inhibitor YhdN 100 mM Potassium phosphate pH 7.6 and protease inhibitor Gld2 5 mM Sodium phosphate pH 7 and protease inhibitor Alr 100 mM Potassium phosphate pH 7.6 and protease inhibitor GldA 100 mM Potassium phosphate pH 7.6 + 20 mM Imidazole and protease inhibitor YqhE 20 mM Tris-HCl pH 8.3 and protease inhibitor

[0244] Purification

[0245] Subtilisine Affinity Purification

[0246] The enzymes were purified from the crude extract by using subtilisine affinity chromatography (Profinity 5 ml, BIORAD) according to the manufacturer's instructions. The crude extract was loaded on the column equilibrated with wash buffer. The Tag was removed from the protein by fluoride cleavage (incubation on the column with 100 mM fluoride at room temperature for 30 minutes). The protein was eluted with the elution buffer. The fractions which contain the protein were pooled, concentrated and loaded on a gel filtration column (Superdex 200 10/300 GL column, GE Healthcare) equilibrated with analysis buffer except for YafB, YeaE, YhdN and Gld2, the buffer was exchanged against analysis buffer by dialysis over night. Protein concentration was determined using Bradford assay.

[0247] For FucO, the purification was also realized in anaerobia condition.

TABLE-US-00004 TABLE 4 Buffers used for subtilisine affinity purification Enzyme Wash buffer Elution buffer Analysis buffer FucO 50 mM Tris-HCl 50 mM Tris-HCl pH 7.5 50 mM Tris-HCl pH 7.5 pH 7.5 100 mM Sodium fluoride YqhD and 100 mM Potassium 100 mM Potassium 50 mM Hepes pH 7.5 YqhD*(G14 Phosphate pH 7.6 Phosphate, 100 mM Sodium 9E) Fluoride pH 7.6 YafB 1M Tris-HCl pH 7 1M Tris-HCl pH 7 1M Tris-HCl pH 7 100 mM Sodium Fluoride 150 mM NaCl YeaE 100 mM Potassium 100 mM Potassium 50 mM Hepes pH 7.5 Phosphate pH 7.6 Phosphate, 100 mM Sodium Fluoride pH 7.6 YdhF 100 mM Potassium 100 mM Potassium 50 mM Hepes pH 7.5 Phosphate pH 7.6 Phosphate, 100 mM Sodium Fluoride pH 7.6 YiaY 100 mM Potassium 100 mM Potassium 50 mM Potassium Phosphate pH 7.6 Phosphate, 100 mM Sodium Phosphate Fluoride pH 7.6 150 mM NaCl pH 7.6 GOX1615 100 mM Potassium 100 mM Potassium 50 mM HEPES pH 7.5 Phosphate pH 7.6 Phosphate, 100 mM Sodium Fluoride pH 7.6 YhdN 100 mM Potassium 100 mM Potassium 100 mM Potassium Phosphate pH 7.6 Phosphate, 100 mM Sodium Phosphate pH 7.6 Fluoride pH 7.6 Gld2 5 mM Sodium 5 mM Sodium Phosphate 100 mM MES pH 6.5 Phosphate pH 7 100 mM Sodium Fluoride pH 7 Alr 100 mM Potassium 100 mM Potassium 100 mM Potassium Phosphate pH 7.6 Phosphate, 100 mM Sodium Phosphate pH 7.6 Fluoride pH 7.6

[0248] Nickel Affinity Purification (GIdA)

[0249] The enzyme was purified from the crude extract by using Nickel affinity chromatography (HisTrapFF 1 mL, GE Healthcare) according to the manufacturer's instructions. The enzyme was eluted by using a linear gradient of imidazole (20 to 500 mM) in 100 mM potassium phosphate (pH 7.6). The fractions containing the protein were pooled, concentrated and the buffer was exchanged against 100 mM MES (pH6.5) by dialysis over night. Protein concentration was determined using Bradford assay.

[0250] Native Purification (YqhE)

[0251] The purification of YqhE was realized in 2 steps.

[0252] Step 1: Ion Exchange Chromatography

[0253] Using an Akta Purifier (GE Healthcare), the crude extract was loaded onto a 5 ml HiTrapQ FF column (GE Healthcare) equilibrated with the wash buffer. Proteins were eluted with a gradient of 20 column volumes from 0% to 100% of elution buffer. The fractions which contain the protein were pooled and the buffer was exchanged against analysis buffer by dialysis for 2 hours.

[0254] Step 2: Affinity Chromatography

[0255] Using an Akta Purifier (GE Healthcare), the protein from the first step was loaded onto a 1 ml HiTrapBlueHP1 column (GE Healthcare) equilibrated with the wash buffer. Proteins were eluted with a gradient of 20 column volumes from 0% to 100% of NaCl elution buffer. The fractions which contain the protein were pooled, concentrated and loaded on a gel filtration column (Superdex 200 10/300 GL column, GE Healthcare) equilibrated with analysis buffer. The fractions which contain the protein were pooled and concentrated. Protein concentrations were determined using Bradford assay.

TABLE-US-00005 TABLE 5 Buffers used for affinity chromatography Step Column Wash buffer Elution buffer Analysis buffer 1 HiTrapQ 20 mM Tris- 20 mM Tris- 20 mM Tris- HCl pH 8.3 HCl pH 8.3 1M HCl pH 7 NaCl 2 HiTrapBlue 20 mM Tris- 20 mM Tris- 20 mM Tris- HCl pH 7 HCl pH 7 1M HCl 150 mM NaCl NaCl

[0256] Demonstration of the NAD(P)H Dependent Lactaldehyde Reductase Activity of Purified Proteins

[0257] NAD(P)H dependent lactaldehyde reductase assay (R-LAR with R-lactaldehyde or S-LAR with S-lactaldehyde)

[0258] The R-LAR and S-LAR activity was determined by measuring the consumption of NAD(P)H at 340 nm on a spectrophotometer (.sub.340=6290 M.sup.1 cm.sup.1) and at 30 C. The reaction mixture (1 mL) containing assay buffer, 0.2 mM to 0.4 mM NAD(P)H and protein was incubated for 5 min at 30 C. Then, 5-10 mM of lactaldehyde 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.

[0259] For FucO, the assay was also realized in anaerobia condition.

[0260] The results are presented in Table 6 and 7

TABLE-US-00006 TABLE 6 Activity of purified enzymes R-LAR S-LAR Enzyme Assay buffer Cofactor (mUI/mg) (mUI/mg) FucO 100 mM MES-KOH NADH 1835 19362 anaerobia (pH 6.5) 0.1 mM FeSO4 30 mM ammonium sulfate FucO 100 mM MES-KOH NADH 593 4313 (pH 6.5) 0.1 mM FeSO4 30 mM ammonium sulfate YiaY 20 mM Hepes (pH 7.5) NADH 4175 4199 0.1 mM FeSO4 GldA 100 mM MES-KOH NADH 46891 13827 (pH 6.5) 0.1 mM FeSO4 30 mM ammonium sulfate YqhD 20 mM Hepes (pH 7.5) NADPH 234 627 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 238 1654 (G149E) 0.1 mM ZnSO4 YafB 20 mM Hepes (pH 7.5) NADPH 6698 2959 YeaE 20 mM Hepes (pH 7.5) NADPH 241 206 YqhE 20 mM Hepes (pH 7.5) NADPH 6768 4527 0.1 mM ZnSO4 YdhF 20 mM Hepes (pH 7.5) NADPH 537 196 GOX1615 20 mM Hepes (pH 7.5) NADPH 23594 1555 YhdN 20 mM Hepes (pH 7.5) NADPH 6880 5751 0.1 mM ZnSO4 Gld2 10 mM sodium NADPH 4875 2638 phosphate (pH 7) Alr 20 mM Hepes (pH 7.5) NADPH 1512 1453

TABLE-US-00007 TABLE 7 Activity of cell extracts R-LAR S-LAR Enzyme Assay buffer cofactor (mUI/mg) (mUI/mg) YqhD* 20 mM Hepes (pH 7.5) NADPH 42 301 (G149E) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 52 117 (T142S) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 35 315 (T142G) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 22 356 (S144A) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 53 356 (G149A) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 57 151 (G149H) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 84 222 (G149K) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 45 124 (G149M) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 79 265 (G149T) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 58 245 (G149N) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 57 208 (G149R) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 62 208 (G149S) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 47 148 (G149V) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 45 120 (A162I) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 54 338 (A162K) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 20 337 (A162D) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 62 176 (A162L) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 35 320 (A162N) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 20 724 (V151L) 0.1 mM ZnSO4 YqhD* 20 mM Hepes (pH 7.5) NADPH 19 360 (V151M) 0.1 mM ZnSO4

Example II: Production of 1,2-Propanediol with New LAR Enzymes

[0261] Construction of Strain 34

[0262] Escherichia coli strain MG1655 is modified to produce lactate. To inactivate the acetate kinase and phosphotransacetylase encoded by the ackA and pta genes respectively, the pyruvate oxydase encoded by the poxB gene, the alcohol dehydrogenase encoded by the adhE gene, the pyruvate formate lyase activating enzyme and the pyruvate formate lyase encoded by the pflA and pflB genes respectively, the aldehyde dehydrogenases encoded by the aldA and aldB genes, the DackA-pta, DpoxB, DadhE, DpflAB, DaldA and DaldB deletions described in patent application WO 2008/116852 are transferred by P1 phage (according to Protocol 2) into strain MG1655 and the resistance genes are removed according to protocol 3. To inactivate the lactate dehydrogenase encoded by the dld gene and the methylglyoxal synthase encoded by the gene mgsA, Ddld and DmgsA deletions described in patent application WO 2011/012693 are transferred by P1 phage (according to Protocol 2) into the previous strain and the resistance genes are removed according to protocol 3 giving rise to strain MG1655 DackA-pta DpoxB DadhE DpflAB DaldA DaldB Ddld DmgsA. To inactivate the fumarate reductase flavoprotein complex encoded by the frdABCD operon and the phosphoenol pyruvate synthase encoded by the ppsA gene, the homologous recombination strategy is used (according to Protocols 1). Oligonucleotides for DfrdABCD: SEQ ID No 36 and 37, and DppsA: SEQ ID No 38 and 39, are used to PCR amplify the resistance cassettes. The strains retained are designated MG1655 DfrdABCD::Cm and MG1655 DppsA::Km. Finally, the DfrdABCD::Cm and the DppsA::Km deletions are transferred by P1 phage transduction (according to Protocol 2) into the previous strain and the resistance genes are removed according to protocol 3 giving rise to strain 34.

[0263] Construction of Strains 35 to 66

[0264] First, to overproduce 1,2-propanediol, all the lactaldehyde reductase candidates (from E. coli: fucO, yafB, yeaE, ydhF, yiaY, gldA, yqhE, wild-type and mutated yqhD; from Gluconobacter oxydans: GOX1615; from Bacillus subtilis: yhdN; from Hypocrea jecorina: gld2; from Leishmania donovani: alr) are each cloned into the pME101VB06 plasmid described in patent application EP 2532751 giving rise to 33 pME101VB06-lactaldehyde reductase plasmids. Then, to overproduce racemic (R)-1,2-propanediol, the lactoyl-coA transferase from Megasphaera elsdenii encoded by the pct gene, and the lactoyl-coA reductase from Salmonella enterica, encoded by the pduP gene are heterologously expressed on plasmid. The synthetic gene pct optimized for Escherichia coli (SEQ ID No 40) and the pduP gene (SEQ ID No 41) are heterologously and separately expressed under a Ptrc artificial promoter and an artificial ribosome binding site (sequence given in patent WO 2007/0770441) on a pBBR1MCS5 plasmid (Kovach et al., 1995) giving rise to plasmid pBBR1MCS5-Ptrc01/RBS01-pctmeO1ec-Ptrc01/RBS01-pduPse. Finally each pME101VB06-lactaldehyde reductase plasmid and the pBBR1MCS5-Ptrc01/RBS01-pctmeO1ec-Ptrc01/RBS01-pduPse plasmid are transformed into strain 34 giving rise to strains 35 to 66.

[0265] Construction of Strains 67 to 98

[0266] To overproduce racemic (S)-1,2-propanediol, the lactate racemase from Lactobacillus sakei encoded by the larA gene (SEQ ID No 26) is heterologously and separately expressed under a Ptrc artificial promoter and an artificial ribosome binding site on the pBBR1MCS5-Ptrc01/RBS01-pctmeO1ec-Ptrc01/RBS01-pduPse plasmid giving rise to plasmid pBBR1MCS5-Ptrc01/RBS01-pctmeO1ec-Ptrc01/RBS01-pduPse-Ptrc01/RBS01-larAls. Finally each pME101VB06-lactaldehyde reductase plasmid and the pBBR1MCS5-Ptrc01/RBS01-pctmeO1ec-Ptrc01/RBS01-pduPse-Ptrc01/RBS01-larAls plasmid are transformed into the strain 34 giving rise to strains 67 to 98.

[0267] Construction of Strains 99 to 131

[0268] To overproduce (S)-lactate and (S)-1,2-propanediol, the lactate dehydrogenase encoded by the ldhA gene is deleted as described in patent application WO 2008/116852 and transferred by P1 phage (according to Protocol 2) into strain 34 giving rise to strain 99. Then the L-lactate dehydrogenase from Bacillus coagulans encoded by the ldh gene (SEQ ID No 42) is heterologously and separately expressed under a Ptrc artificial promoter and an artificial ribosome binding site on the pBBR1MCS5-Ptrc01/RBS01-pctmeO1ec-Ptrc01/RBS01-pduPse plasmid giving rise to plasmid pBBR1MCS5-Ptrc01/RBS01-pctmeO1ec-Ptrc01/RBS01-pduPse-Ptrc01/RBS01-ldhbc. Finally each pME101VB06-lactaldehyde reductase plasmid and the pBBR1MCS5-Ptrc01/RBS01-pctmeO1ec-Ptrc01/RBS01-pduPse-Ptrc01/RBS01-ldhbc plasmid are transformed into strain 99 giving rise to strains 100 to 131.

[0269] Construction of Strains 132 to 163

[0270] To overproduce (R)-lactate and (R)-1,2-propanediol, the lactate racemase from Lactobacillus plantarum encoded by the larA gene (SEQ ID No 28) is heterologously and separately expressed under a Ptrc artificial promoter and an artificial ribosome binding site on the pBBR1MCS5-Ptrc01/RBS01-pctmeO1ec-Ptrc01/RBS01-pduPse-Ptrc01/RBS01-ldhbc plasmid giving rise to plasmid pBBR1MCS5-Ptrc01/RBS01-pctmeO1ec-Ptrc01/RBS01-pduPse-Ptrc01/RBS01-ldhbc-Ptrc01/RBS01-larAlp. Finally each pME101VB06-lactaldehyde reductase plasmid and the pBBR1MCS5-Ptrc01/RBS01-pctmeO1ec-Ptrc01/RBS01-pduPse-Ptrc01/RBS01-ldhbc-Ptrc01/RBS01-larAlp plasmid are transformed into strain 99 giving rise to strains 132 to 163.

[0271] Construction of strains 164 to 196 To re-introduce the gloA gene, the homologous recombination strategy is used (according to Protocols 1 and 3). Oligonucleotides for gloA reconstruction: SEQ ID No 43 and 4, are used to PCR amplify the resistance cassette. The strain retained is designated MG1655 gloArc::Km and the gloArc::Km modification is transferred by P1 phage transduction (according to Protocol 2) into the evolved strain MG1655 lpd* DtpiA DpflAB DadhE DldhA DgloA DaldA DaldB Dedd DarcA Dndh described in patent application WO2008/116852. Then, to inactivate the lactate dehydrogenase from Escherichia coli encoded by the dld gene, Ddld::Cm deletion described in patent application WO 2011/012693 is transferred by P1 phage (according to Protocol 2) into the previous strain giving rise to strain 164. Finally, each pME101VB06-lactaldehyde reductase plasmid and the pBBR1MCS5-Ptrc01/RBS01-pctmeO1ec-Ptrc01/RBS01-pduPse plasmid are transformed into strain 164 giving rise to strains 165 to 196.

[0272] Construction of Strains 202 to 212

[0273] To inactivate the gldA and yqhD genes, the homologous recombination strategy is used as described for construction of strain 10 and strain 2 respectively (according to Protocols 1 and 3) the DgldA::Km and DyqhD::Km are transferred by P1 phage transduction (according to Protocol 2) into strain 164 giving rise to strain 197. Finally each pME101VB06-lactaldehyde reductase plasmid, except yqhD (wild-type and mutated) and gldA plasmids, and the pBBR1MCS5-Ptrc01/RBS01-pctmeO1ec-Ptrc01/RBS01-pduPse-Ptrc01/RBS01-larAls plasmid are transformed into strain 197 giving rise to strains 198 to 207.

[0274] Production of 1,2-Propanediol in Shake Flasks

[0275] 1,2-propanediol (MPG) producing strains were cultivated and MPG was quantified as described in protocol 6.

[0276] Compared to strain 35, considered as the control strain and described in patent WO 2012/172050, all strains produced more MPG.

TABLE-US-00008 TABLE 8 1,2-propanediol produced by strains of the present invention Enantiomeric Strain LAR enzyme MPG produced form of MPG 35 FucO Control (WO 2012/172050) R 36 YafB ++ R 37 YeaE + R 38 YdhF + R 39 YiaY ++ R 40 GldA ++ R 41 YqhE ++ R 42 YqhD + R 43 GOX1615 ++ R 44 YhdN ++ R 45 Gld2 ++ R 46 Alr ++ R 47 YqhD*(G149E) ++ R 48 YqhD*(T142S) ++ R 49 YqhD*(T142G) ++ R 50 YqhD*(S144A) ++ R 51 YqhD*(G149A) ++ R 52 YqhD*(G149H) ++ R 53 YqhD*(G149K) ++ R 54 YqhD*(G149M) + R 55 YqhD*(G149T) ++ R 56 YqhD*(G149N) ++ R 57 YqhD*(G149R) ++ R 58 YqhD*(G149S) ++ R 59 YqhD*(G149V) + R 60 YqhD*(A162I) + R 61 YqhD*(A162K) ++ R 62 YqhD*(A162D) ++ R 63 YqhD*(A162L) ++ R 64 YqhD*(A162N) ++ R 65 YqhD*(V151L) ++ R 66 YqhD*(V151M) ++ R 67 FucO ++ S 68 YafB ++ S 69 YeaE + S 70 YdhF + S 71 YiaY + S 72 GldA ++ S 73 YqhE ++ S 74 YqhD + S 75 GOX1615 + S 76 YhdN ++ S 77 Gld2 ++ S 78 Alr + S 79 YqhD*(G149E) ++ S 80 YqhD*(T142S) ++ S 81 YqhD*(T142G) ++ S 82 YqhD*(S144A) ++ S 83 YqhD*(G149A) ++ S 84 YqhD*(G149H) ++ S 85 YqhD*(G149K) ++ S 86 YqhD*(G149M) + S 87 YqhD*(G149T) ++ S 88 YqhD*(G149N) ++ S 89 YqhD*(G149R) ++ S 90 YqhD*(G149S) ++ S 91 YqhD*(G149V) + S 92 YqhD*(A162I) + S 93 YqhD*(A162K) ++ S 94 YqhD*(A162D) ++ S 95 YqhD*(A162L) ++ S 96 YqhD*(A162N) ++ S 97 YqhD*(V151L) ++ S 98 YqhD*(V151M) ++ S 100 FucO ++ S 101 YafB ++ S 102 YeaE + S 103 YdhF + S 104 YiaY + S 105 GldA ++ S 106 YqhE ++ S 107 YqhD + S 108 GOX1615 + S 109 YhdN ++ S 110 Gld2 ++ S 111 Alr + S 112 YqhD*(G149E) ++ S 113 YqhD*(T142S) ++ S 114 YqhD*(T142G) ++ S 115 YqhD*(S144A) ++ S 116 YqhD*(G149A) ++ S 117 YqhD*(G149H) ++ S 118 YqhD*(G149K) ++ S 119 YqhD*(G149M) + S 120 YqhD*(G149T) ++ S 121 YqhD*(G149N) ++ S 122 YqhD*(G149R) ++ S 123 YqhD*(G149S) ++ S 124 YqhD*(G149V) + S 125 YqhD*(A162I) + S 126 YqhD*(A162K) ++ S 127 YqhD*(A162D) ++ S 128 YqhD*(A162L) ++ S 129 YqhD*(A162N) ++ S 130 YqhD*(V151L) ++ S 131 YqhD*(V151M) ++ S 132 FucO + R 133 YafB ++ R 134 YeaE + R 135 YdhF + R 136 YiaY ++ R 137 GldA ++ R 138 YqhE ++ R 139 YqhD + R 140 GOX1615 ++ R 141 YhdN ++ R 142 Gld2 + R 143 Alr + R 144 YqhD*(G149E) ++ R 145 YqhD*(T142S) ++ R 146 YqhD*(T142G) ++ R 147 YqhD*(S144A) ++ R 148 YqhD*(G149A) ++ R 149 YqhD*(G149H) ++ R 150 YqhD*(G149K) ++ R 151 YqhD*(G149M) + R 152 YqhD*(G149T) ++ R 153 YqhD*(G149N) ++ R 154 YqhD*(G149R) ++ R 155 YqhD*(G149S) ++ R 156 YqhD*(G149V) + R 157 YqhD*(A162I) + R 158 YqhD*(A162K) ++ R 159 YqhD*(A162D) ++ R 160 YqhD*(A162L) ++ R 161 YqhD*(A162N) ++ R 162 YqhD*(V151L) ++ R 163 YqhD*(V151M) ++ R 165 FucO + R/S 166 YafB ++ R/S 167 YeaE + R/S 168 YdhF + R/S 169 YiaY ++ R/S 170 GldA ++ R/S 171 YqhE ++ R/S 172 YqhD + R/S 173 GOX1615 ++ R/S 174 YhdN ++ R/S 175 Gld2 + R/S 176 Alr + R/S 177 YqhD*(G149E) ++ R/S 178 YqhD*(T142S) ++ R/S 179 YqhD*(T142G) ++ R/S 180 YqhD*(S144A) ++ R/S 181 YqhD*(G149A) ++ R/S 182 YqhD*(G149H) ++ R/S 183 YqhD*(G149K) ++ R/S 184 YqhD*(G149M) + R/S 185 YqhD*(G149T) ++ R/S 186 YqhD*(G149N) ++ R/S 187 YqhD*(G149R) ++ R/S 188 YqhD*(G149S) ++ R/S 189 YqhD*(G149V) + R/S 190 YqhD*(A162I) + R/S 191 YqhD*(A162K) ++ R/S 192 YqhD*(A162D) ++ R/S 193 YqhD*(A162L) ++ R/S 194 YqhD*(A162N) ++ R/S 195 YqhD*(V151L) ++ R/S 196 YqhD*(V151M) ++ R/S 198 FucO + R 199 YafB ++ R 200 YeaE + R 201 YdhF + R 202 YiaY ++ R 203 YqhE ++ R 204 GOX1615 ++ R 205 YhdN ++ R 206 Gld2 + R 207 Alr + R (the symbol + indicates an increase of more than 10% compared to the control strain, and the symbol ++ indicates an increase of more than 50% compared to the control strain)

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