Patent Publication Number: US-8969053-B2

Title: Mutant YqhD enzyme for the production of a biochemical by fermentation

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a §371 National Stage Application of PCT/EP2010/061106, filed Jul. 30, 2010, which claims priority to European Application No. 09166856.6, filed Jul. 30, 2009 and U.S. Provisional Application No. 61/229,866, filed Jul. 30, 2009. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention concerns a method for the production of a biochemical selected among acetol and 1,2-propanediol, 1,3-propanediol, ethylene glycol and 1,4-butanediol comprising culturing a microorganism modified for an improved production of the biochemical selected among acetol and 1,2-propanediol, 1,3-propanediol, ethylene glycol and 1,4-butanediol in an appropriate culture medium and recovery of the desired biochemical which may be further purified wherein the microorganism expresses a YqhD enzyme which catalytic efficiency toward NADPH is increased. 
     The present invention also relates to a mutant YqhD enzyme comprising at least one amino acid residue in the protein sequence of the parent enzyme replaced by a different amino acid residue at the same position wherein
         the mutant enzyme has retained more than 50% of the YqhD activity of the parent enzyme and   the catalytic efficiency toward NADPH of the mutant YqhD is increased as compared with the catalytic efficiency toward NADPH of the parent enzyme.       

     1. Description of Related Art 
     YqhD was first identified as an 1,3-propanediol oxidoreductase catalysing the NADPH-dependent reduction of 3-hydroxypropionaldehyde (3-HPA) into 1,3-propanediol (PDO). This activity, endogenous to  E. coli , was found to be more efficient that the recombinant NADH-dependent activity (encoded by dhaT gene from  Klebsiella pneumoniae ) used to built a PDO biosynthetic pathway in  E. coli  (Nakamura and Whited, 2003). The Du Pont de Nemours—Genencor project for the production of PDO in  E. coli  resulted in an industrial process with high titers and high yields (WO2001/012833 and WO2004/033646). 
     Expression of the yqhD gene was later used for purpose of production of PDO in different organisms,  E. coli  or  Saccharomyces cerevisiae  (Wang et al, 2007, Rao et al, 2008, Wang et al, 2009). 
     We showed that YqhD can act as a methylglyoxal reductase in the biosynthesis of 1,2-propanediol in  E. coli  (WO2008/116853). 
     The crystal structure of YqhD was determined with bound NADP co-factor. YqhD was shown to be a tetramer containing 1 atom of Zn by monomer. Alcohol dehydrogenase activity was recorded on several alcohols but with a weak affinity. However, the discovery of the presence of a modified (hydroxylated) co-factor in the enzyme cast doubt about the activity data provided (Sulzenbacher et al, 2004). 
     A more recent study characterized YqhD as an NADPH-dependent aldehyde reductase working on different substrates: acetaldehyde, propanaldehyde, butanaldehyde, acrolein and malondialdehyde (Perez et al, 2008). In this study, no activity was detected on short or medium chain alcohols. These authors proposed that YqhD was of relevance for the detoxification of toxic aldehydes inside the cell. 
     Miller et al (2009) showed that purified YqhD exhibited a furfural reductase activity. In this study, YqhD was found to have a very strong affinity for its NADPH co-factor with Km value for NADPH of 8 μM. With such an affinity, an active YqhD could scavenge NADPH normally used for the biosynthetic reactions of the cell and would thus inhibit growth. 
     Mutants of YqhD were produced by error-prone PCR in the aim of improving the enzyme activity towards 3-HPA for the production of 1,3-propanediol (Li et al, 2008). Two mutants, a double mutant (D99Q N147H) and a single mutant ((Q202A) were found to have an improved affinity (lower Km) for 3-HPA and an improved catalytic efficiency (Kcat/Km) for 3-HPA. 
     PDO is a monomer used in the production of polyester fibers, especially poly(trimethylene terephthalate) (PTT) and with potential applications in the manufacture of polyurethanes and cyclic compounds. 
     PDO can be produced by different chemical routes from i) acrolein, water and hydrogen, ii) ethylene oxide, carbon monoxide and water in the presence of phosphine and iii) glycerol and hydrogen in the presence of carbon monoxide. All of these methods have in common to be expensive and to generate waste streams containing toxic substances potentially harmful to the environment. 
     PDO is a typical product of glycerol fermentation and has not been found in anaerobic conversions of other organic substrates. Only very few organisms, all of them bacteria, are able to form it. They include enterobacteria of the genera  Klebsiella  ( K. pneumoniae ),  Enterobacter  ( E. agglomerans ) and  Citrobacter  ( C. freundii ), lactobacilli ( L. brevis  and  L. buchneri ) and clostridia of the  C. butyricum  and the  C. pasteurianum  group. 
     New bioprocesses for the production of PDO in recombinant  E. coli  have been disclosed in WO2001/012833 and WO2004/033646. These processes rely on the utilization of the yqhD gene encoding the YqhD enzyme for the last step of synthesis of PDO. Production of PDO from glycerol in a recombinant  Klebsiella  expressing the yqhD gene was described in CN1011260379. The same system applied to the production of PDO from glucose in a recombinant  Saccharomyces cerevisiae  was disclosed in CN101130782. 
     An alternative solution to produce PDO in recombinant organisms using a different biosynthetic pathway without glycerol as an intermediate has been proposed in patent application EP08173129.1. Production of other diols, such as ethylene glycol and 1,4-butanediol, according to the same scheme was also incorporated in the same patent application. All these processes are using the enzyme YqhD for the last step of synthesis. 
     1,2-propanediol or propylene glycol, a C3 dialcohol, is a widely-used chemical. It is a component of unsaturated polyester resins, liquid detergents, coolants, anti-freeze and de-icing fluids for aircraft. Propylene glycol has been increasingly used since 1993-1994 as a replacement for ethylene derivatives, which are recognised as being more toxic than propylene derivatives. 
     1,2-propanediol is currently produced by chemical means using a propylene oxide hydration process that consumes large amounts of water. Propylene oxide can be produced by either of two processes, one using epichlorhydrin, and the other hydroperoxide. Both routes use highly toxic substances. In addition, the hydroperoxide route generates by-products such as tert-butanol and 1-phenyl ethanol. For the production of propylene to be profitable, a use must be found for these by-products. The chemical route generally produces racemic 1,2-propanediol, whereas each of the two stereoisomers (R)1,2-propanediol and (S)1,2-propanediol are of interest for certain applications (e.g. chiral starting materials for specialty chemicals and pharmaceutical products). 
     Acetol or hydroxyacetone (1-hydroxy- 2-propanone) is a C3 keto alcohol. This product is used in vat dyeing process in the textile industry as a reducing agent. It can advantageously replace traditional sulphur containing reducing agents in order to reduce the sulphur content in wastewater, harmful for the environment. Acetol is also a starting material for the chemical industry, used for example to make polyols or heterocyclic molecules. It possesses also interesting chelating and solvent properties. 
     Acetol is currently produced mainly by catalytic oxidation or dehydration of 1,2-propanediol. New processes starting from renewable feedstocks like glycerol are now proposed (see DE4128692 and WO 2005/095536). Currently, the production cost of acetol by chemical processes reduces its industrial applications and markets. 
     The disadvantages of the chemical processes for the production of 1,2-propanediol and acetol make biological synthesis an attractive alternative. 1,2-propanediol can be derived from central metabolism in three steps whereas acetol can be obtained in two steps. Methylglyoxal synthase converting dihydroxyacetone phosphate into methylglyoxal (MG) is the mandatory first step for the production of these two compounds. MG can then be converted to either acetol or D- or L-lactaldehyde by methylglyoxal reductases (Cameron et al, 1998, Bennett and San, 2001, Ko et al, 2005). As mentioned earlier, YqhD was shown to be very efficient in the production of acetol from methylglyoxal (WO2008/116853). Acetol or lactaldehyde can be converted to 1,2-propanediol by several enzymatic activities, especially glycerol dehydrogenase (encoded by gldA gene) or 1,2-propanediol oxidoreductase (encoded by fucO gene) in  E. coli  (Altaras and Cameron, 2000). 
     Processes for the production of 1,2-propanediol or acetol using different microorganisms,  Clostridium sphenoides  (DE3336051),  Klebsiella pneumoniae  (WO 2004/087936), recombinant yeast (WO 99/28481) or recombinant  E. coli  (WO 98/37204) have been disclosed. We recently proposed alternative approaches for the production of 1,2-propanediol or acetol (WO 2005/073364, WO 2008/116852, WO 2008/116848, WO 2008/116849, WO 2008/116851)), some of them relying on the use of the methylglyoxal reductase YqhD. 
     During their investigations on 1,2-propanediol production, the inventors identified new mutant YqhD enzymes with increased catalytic efficiency (increased Kcat/Km) toward NADPH, while keeping most of their specific activity for the conversion of methylglyoxal into acetol, as demonstrated in Example 2 by the characterization of purified enzymes. Use of these mutants is a key element in the design of more efficient processes for the production of all the products using a metabolic pathway based on YqhD activity. 
     SUMMARY 
     The present invention concerns a method for the production of a biochemical selected among acetol and 1,2-propanediol, 1,3-propanediol, ethylene glycol and 1,4-butanediol comprising culturing a microorganism modified for an improved production of the biochemical selected among acetol and 1,2-propanediol, 1,3-propanediol, ethylene glycol and 1,4-butanediol in an appropriate culture medium and recovery of the desired biochemical which may be further purified wherein the microorganism expresses a YqhD enzyme which catalytic efficiency toward NADPH is increased. 
     The present invention concerns a mutant YqhD enzyme comprising at least one amino acid residue in the protein sequence of the parent enzyme replaced by a different amino acid residue at the same position wherein
         the mutant enzyme has retained more than 50% of the YqhD activity of the parent enzyme and   the catalytic efficiency toward NADPH of the mutant YqhD is increased as compared with the catalytic efficiency toward NADPH of the parent enzyme.       

     The invention also concerns a DNA sequence comprising a sequence coding for the mutant YqhD enzyme of the invention and a microorganism expressing such YqhD, which catalytic efficiency toward NADPH is increased, particularly a microorganism comprising a gene coding for the mutant YqhD enzyme of the invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1-3  depict embodiments of the present invention as described herein. 
     
    
    
     DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT 
     In the present application, terms are employed with their usual meaning, except when precised otherwise. 
     Microorganisms 
     A “microorganism” means all kind of unicellular organisms, including procaryotic organisms like bacteria, and eucaryotic organisms like yeasts. Preferentially, the microorganism is selected among the group consisting of bacteria, yeasts and fungi, more preferentially selected among Enterobacteriaceae, Bacillaceae, Streptomycetaceae, Clostridiaceae and Corynebacteriaceae. More preferentially, the microorganism is a species of  Escherichia, Klebsiella, Pantoea, Salmonella, Bacillus, Streptomyces, Clostridium  or  Corynebacterium . Even more preferentially, the microorganism is selected among the group consisting of  Escherichia coli, Klebsiella pneumoniae, Thermoanaerobacterium thermosaccharolyticum, Clostridium sphenoides  or  Saccharomyces cerevisiae.    
     As used herein, the term “modified microorganism” or “modified” or “recombinant” refer to a host cell that has a modification of its genome, e.g., as by addition of nucleic acid not naturally occurring in the organism or by a modification of nucleic acid naturally occurring in the host cell. 
     A “microorganism modified for an improved production of acetol and/or 1,2-propanediol and/or 1,3-propanediol and/or ethylene glycol and/or 1,4-butanediol” is a microorganism in which pathways to favour the production of the desired biochemical by conversion of a simple source of carbon have been modified. The microorganism modified for such improved production produces more of the desired biochemical than a native, unmodified microorganism. 
     Such microorganisms modified for an improved production of acetol and/or 1,2-propanediol and/or 1,3-propanediol and/or ethylene glycol and/or 1,4-butanediol are described in the scientific publications and patent applications cited in the “background of the invention” section, the content of said publications and patent applications being incorporated herein by reference. 
     In a preferred embodiment, the desired biochemical is 1,2-propanediol. The preferred biosynthetic pathways for the production of 1,2-propanediol with the microorganism of the invention are represented on  FIG. 2 . The person skilled in the art shall identify the enzymatic activities related to the pathway to be promoted and the other enzymatic activities to be attenuated. 
     Microorganisms modified for the improved production of 1,2-propanediol by conversion of methyl glyoxal are also disclosed in Cameron et al, 1998, Bennett and San, 2001, Ko et al, 2005 and WO 99/28481, WO 98/37204, WO 2005/073364, WO 2008/116852, WO 2008/116848, PCT/EP2009/053093 which content is incorporated herein by reference. 
     In the case of yeasts, the following modifications of the host organism are preferred:
         attenuation of expression of at least one of the following genes: TPI1, NDE1, NDE2, GUT2, GPD1, GPD2, PDC1, PDC2, PDC5, PDC6, GLO1   enhancement of expression of GRE3 gene.       

     In the microorganisms of the invention, the DNA sequence coding for a mutant YqhD enzyme of the invention may be introduced in a vector for the expression and translation of the mutant YqhD enzyme. It can also be integrated in the chromosome of the said microorganism. 
     Integration of the DNA sequence can be done either entirely, or simply by introducing in the native gene of the microorganism, the mutation in the coding sequence by replacing the nucleotide(s) coding for the amino acid to be changed by the nucleotide(s) coding for the amino acid of the mutated protein. 
     Total, partial or specific nucleotides replacement in a gene of a microorganism is well known in the art of genetic engineering, including Sambrook J et al., Molecular cloning: a laboratory manual, Cold Spring Harbour Press, New York (2001), Ausubel F M et al., Current protocols in molecular biology, John Wiley and sons, New York (1999), Adams A et al., Methods in yeast genetics, Cold Spring Harbour Press, New York (1997). 
     The microorganism of the invention may additionally comprise a gene coding for a glycerol dehydrogenase (GlyDH) enzyme with reduced inhibition of its activity by NAD+ and/or its substrate and/or its product. 
     “The inhibition of which activity by NAD+ and/or its substrate and/or its product is reduced” means that the inhibition of the activity of the GlyDH enzyme expressed in the microorganism is less inhibited that the activity of the native GlyDH enzyme of the same microorganism. The inhibition of the activity of the GlyDH enzyme can be defined by the Inhibition Concentration 50 (IC50) or the Inhibition Constant (Ki) or any other techniques known by the skilled person. The reduced inhibition of the activity of the GlyDH enzyme means that the IC50 or the Ki of the GlyDH enzyme of the invention is higher than the IC50 or the Ki of the native GlyDH enzyme. The skilled person knows the relation between IC50 and Ki, and their meaning on the activity of enzyme according to the classic Michaelis-Menten kinetics. 
     Preferably, the activity of the GlyDH enzyme is less inhibited than the native GlyDH enzyme of  E. coli . In a preferred embodiment, the enzyme activity is less inhibited for at least two members of the group consisting of NAD+, the enzyme&#39;s substrate and the enzyme&#39;s product. More preferably the enzyme activity is less inhibited by the three of NAD+, its substrate and its product. 
     The enzyme “substrate” is dihydroxyacetone, hydroxyacetone, methylglyoxal, lactaldehyde, glyceraldehyde, glycolaldehyde and derivatives thereof. 
     The enzyme “product” is the molecule obtained from the selected substrate by reduction of the carbonyl function. 
     For the production of 1,2-propanediol, the substrate is hydroxyacetone and the product is 1,2-propanediol. 
     Particularly the GlyDH enzyme is a mutant enzyme, wherein
         the mutant enzyme has retained more than 50% of the activity of the parent enzyme and   the glycerol dehydrogenase activity of the mutant GlyDH is less inhibited by NAD+and/or by its substrate as compared to the parent enzyme and/or by its product as compared to the parent enzyme.       

     Preferably, the mutant GlyDH preferably comprises at least a mutation selected among the group consisting of A160T and T120N, and combinations thereof. The aminoacids positions are given by reference to the GlyDH sequence of  E. coli , encoded by the gldA gene. The person skilled in the art shall find the corresponding aminoacids in sequences from other organisms by standard techniques of sequence alignment. 
     The microorganism of the invention may also comprise, additionally, a gene coding for a methylglyoxal synthase (MGS) enzyme which activity is not inhibited by orthophosphate, 
     “Not inhibited by orthophosphate” or “lacking inhibition by orthophosphate” means that no inhibition by orthophosphate is identified in an activity assay, when activity of the enzyme is studied in presence of orthophosphate. 
     In addition, kinetics of the MGS enzyme of the invention follows Michaelis-Menten kinetics regardless of the presence or absence of orthophosphate. Kinetics of the native enzyme follow a Michaelis-Menten model only in the absence of orthophosphate. The presence of orthophosphate makes the kinetic profile (specific activity over substrate concentration) of the native enzyme to become sigmoidal, which denotes the allosteric inhibition by orthophosphate. 
     Such enzyme has preferably a methylglyoxal synthase activity of at least 50% of the activity of the methylglyoxal synthase of  E. coli.    
     Particularly, the MGS enzyme is a mutant MGS enzyme wherein
         the mutant enzyme has retained more than 50% of the activity of the parent enzyme and   the methylglyoxal synthase activity of the mutant MGS is not inhibited by orthophosphate as compared to the parent enzyme.       

     Preferably the mutant MGS comprises at least a mutation selected among the group consisting of H21Q, A95V and V116L, and combinations thereof. The aminoacids positions are given by reference to the MgsA sequence of  E. coli . The person skilled in the art shall find the corresponding aminoacids in sequences from other organisms by standard techniques of sequence alignment. 
     YqhD Enzyme 
     The invention concerns an YqhD enzyme which catalytic efficiency toward NADPH is increased, a microorganism comprising the same and a method for the production of a desired biochemical by fermentation of said microorganism on a culture medium comprising a simple source of carbon. 
     An YqhD enzyme “which catalytic efficiency toward NADPH is increased” means that the catalytic efficiency towards NADPH of the YqhD enzyme expressed in the microorganism is higher than the catalytic efficiency towards NADPH of the native YqhD enzyme of the same microorganism. The catalytic efficiency is defined as the ratio between the catalytic constant (Kcat) and the Michaelis constant (Km). Increase of catalytic efficiency of YqhD enzyme means that the Kcat of the enzyme is increased or that the Km of the enzyme is decreased. In a preferred embodiment the Kcat of the YqhD enzyme is increased and the Km of the YqhD enzyme is decreased. 
     Preferably, the catalytic efficiency towards NADPH of the YqhD enzyme is higher than the efficiency of the native YqhD enzyme of  E. coli.    
     In another aspect of the invention, the YqhD enzyme is more sensitive to inhibition by NADP + . Inhibition of catalytic activity of YqhD by NADP +  is higher than the inhibition of catalytic activity of the native YqhD enzyme of  E. coli.    
     In a preferred embodiment of the invention, the catalytic efficiency towards NADPH of the YqhD enzyme and its sensitivity to NADP +  are higher than the efficiency and sensitivity of the native YqhD of  E. coli.    
     Catalytic efficiency of the enzyme towards NADPH and its sensitivity to NADP +  can be measured by methods known to the person skilled in the art, and particularly as disclosed in example 3. 
     Such enzyme has preferably an enzymatic activity of at least 50% of the activity of the YqhD of  E. coli , more preferably at least 60% of the activity of the YqhD of  E. coli.    
     YqhD is reducing methylglyoxal with NADPH as a cofactor and the methylglyoxal reductase activity of the enzyme can be measured by means known to the person skilled in the art. Such an activity assay is well known in the art and can be carried out as disclosed in Example 2. 
     The enzymes may be obtained by various ways known to the person skilled in the art. 
     A first approach consists in screening native enzymes of various organisms for their increased catalytic efficiency towards NADPH. 
     A second approach consists in inducing mutation(s) in enzymes of known organisms and selecting the enzymes for increased catalytic efficiency towards NADPH. Mutations may be induced by methods known in the art such as subjecting the microorganism to mutagenic agents. Another method to induce mutations is to growth the microorganism under selection pressure and identify the microorganism growing under such conditions and select the enzymes obtained for their increased catalytic efficiency towards NADPH. 
     Other methods are also known in the art to obtain mutations by shuffling DNA from various origins and select the proteins encoded by the shuffled DNA so obtained based on their methylglyoxal reductase activity and their increased catalytic efficiency towards NADPH. 
     In a particular embodiment of the invention, the inventors obtained several mutants YqhD retaining their methylglyoxal reductase activity and having an increased catalytic efficiency towards NADPH by selecting strains modified for an improved production of 1,2-propanediol cultured under selection pressure as disclosed in WO 2005/073364 and as shown in Example 1. 
     The present invention concerns a mutant YqhD enzyme comprising at least one amino acid residue in the protein sequence of the parent enzyme replaced by a different amino acid residue at the same position wherein
         the mutant enzyme has retained more than 50% of the YqhD activity of the parent enzyme and   the catalytic efficiency toward NADPH of the mutant YqhD is increased as compared with the catalytic efficiency toward NADPH of the parent enzyme.       

     “Mutant” means that a mutation was introduced in a protein sequence by a person. According to the invention, a mutant enzyme is an enzyme comprising at least one amino acid difference with a parent enzyme. In the mutant enzyme of the invention, any change in amino acids may be introduced, either by directed mutagenesis or random mutagenesis, but also chimerical enzymes can be constructed, comprising parts of a second enzyme replacing corresponding parts of the parent enzyme. 
     The “parent enzyme” is the enzyme prior mutation. The parent enzyme may be of any origin, natural, isolated from another organism or synthetic. 
     The method for determining that the mutated YqhD has retained “more than 50%” of the activity of the parent enzyme is well known in the art and disclosed in Example 2. 
     Indeed, the skilled person shall choose the level of desired activity according to the final use of mutant YqhD. Indeed, when a high activity is necessary, the skilled person will choose a mutant having more than 80% of activity, compared to the non mutated parent enzyme, more preferably more than 90% of activity. In other cases, selecting a mutant YqhD with an activity around and above 50% compared to the parent enzyme may prevent additional modifications in a microorganism, like modifying the promoter to lower the level of expression of the enzyme. 
     According to the invention, “increased catalytic efficiency towards NADPH” means that the catalytic efficiency towards NADPH is at least 80 000 M−1 s−1, more preferably at least 100 000 M−1 s−1. 
     Methods used to calculate the kinetic parameters of the enzymes are given in Examples 2 and 3 but can also be found e.g. in Segel, I H, Enzyme Kinetics, John Wiley &amp; Sons (1993). 
     In a preferred embodiment, the mutant YqhD enzyme of the invention comprises at least one amino acid residue of an identified region in the native parent YqhD enzyme replaced by a different amino acid residue at the same position. 
     The inventors have identified mutants YqhD comprising at least one amino acid residue of one of the following Conserved Regions in the native parent YqhD which has been replaced by a different amino acid residue at the same position: 
                            -Glu-Xa1-Xa2-Xa3-Xa4-Xa5-Xa6-Xa7-Xa8-(CR1)            
wherein
     Xa1 represents Met, Ser, Thr and Val, preferably Met and Val,   Xa2 represents Asn, Asp, Ser and Thr, preferably Thr,   Xa3 represents Ala, Arg, Asn, Cys, Gln, Ile, Pro, Ser, Thr and Trp,   Xa4 represents Ala, Asn, Asp, Gly, H is, Ile, Leu, Phe, Trp and Val,   Xa5 represents Ala, Cys, Gly, Phe, Ser, Thr, Trp and Tyr,   Xa6 represents Gly, Ile and Val, preferably Val,   Xa7 represents Ala, Ile, Leu and Val, preferably Ile, and   Xa8 represents Glu, Gly, Ile, Ser and Thr, preferably Thr,   

                            -His-Xa9-X10-X11-X12-X13-X14-X15-X16-X17-(CR2)            
wherein
     Xa9 represents Ala and Gly, preferably Gly,   X10 represents Arg, Cys, Gln, Glu, Gly, Ile, Leu, Pro and Val,   X11 represents Ala, Asn, Cys, Gly, Met and Thr,   X12 represents Asn, H is, Leu and Val, preferably Asn,   X13 represents Ala, Ser, Thr and Tyr, preferably Ala,   X14 represents Ala, Ile, Leu, Met and Val, preferably Ile and Val,   X15 represents Ala, Asn, Leu, Met, Phe, Thr, Tyr and Val, preferably Leu,   X16 represents Ile, Leu, Met, Phe and Thr, preferably Leu, and   X17 represents Ala, Cys, Gly, Pro and Thr, preferably Pro.   

     These conserved regions can be identified in different YqhD enzymes by simple sequence alignment using standard sequence alignment tools such as ClustalW2, Kalign, MAFFT, MUSCLE or T-coffee, all available on the European Bioinformatics Institute website. A sequence alignment of several YqhD enzymes of different species is given in  FIG. 1 . 
     Amino acids numbers in the present application are given by reference to the proteins of  E. coli.    
     It can be found in  FIG. 1  that CR1 correspond to amino acids 145 to 153 of  E. coli  YqhD and CR2 correspond to amino acids 281 to 290 of  E. coli  YqhD. 
     According to the invention, the mutant YqhD can have at least one mutation in one of CR1 or CR2, and can have at least two mutations in CR1 and CR2. 
     “At least” in such context means that the mutated enzyme may have other mutations, but nor related to the identified Conserved Regions CR1 and CR2. These other non identified mutations have no substantial impact on the mutated enzyme of the invention, provided that:
         the mutant enzyme has retained more than 50% of the YqhD activity of the parent enzyme and   the catalytic efficiency toward NADPH of the mutant YqhD is increased as compared with the catalytic efficiency toward NADPH of the parent enzyme.       

     In preferred embodiments, the amino acid residue in the conserved regions CR1 and CR2 in the native parent YqhD replaced by a different amino acid residue at the same position in the mutant YqhD is selected among the group consisting of amino acid Xa4 in CR1 and amino acid X13 in CR2 and combinations thereof. 
     Xa4 correspond to amino acid 149 in the YqhD sequence of  E. coli . X13 corresponds to amino acid 286 in the YqhD sequence of  E. coli.    
     Particularly, the mutated YqhD of the invention comprises at least one of the mutations selected among the group consisting in G149E, G149S, A286T and combinations thereof, the aminoacid positions being given by reference to the YqhD sequence of  E. coli.    
     More preferably, the mutated YqhD of the invention comprises at least one of the following amino acid sequence in conserved regions CR1 and CR2: 
     
       
         
           
               
               
            
               
                   
                 CR1: Glu Ser Asn Ala    Ser    Ala Val Ile Ser or 
               
               
                   
                   
               
               
                   
                 Glu Ser Asn Ala    Glu    Ala Val Ile Ser 
               
               
                   
                   
               
               
                   
                 CR2: His Ala Gln Thr Leu    Thr    Ile Val Leu Pro 
               
            
           
         
       
     
     the amino acid residue marked in bold and underlined corresponding to the amino acid in the mutant YqhD different from the amino acid in the parent YqhD. 
     Particularly, the mutant YqhD of the invention has at least 25% sequence identity compared to the YqhD sequence of  E. coli , provided that it comprises at least one of the following mutations in CR1 and/or CR2: 
     
       
         
           
               
               
            
               
                   
                 CR1: Glu Ser Asn Ala    Ser    Ala Val Ile Ser or 
               
               
                   
                   
               
               
                   
                 Glu Ser Asn Ala    Glu    Ala Val Ile Ser 
               
               
                   
                   
               
               
                   
                 CR2: His Ala Gln Thr Leu    Thr    Ile Val Leu Pro. 
               
            
           
         
       
     
     Sequence identity is defined after sequence alignment of the YqhD sequence of  E. coli  with the protein sequence to be compared using CLUSTALW2 available on the EBI website (see above) with default parameters. The sequence identity is then calculated with the sequence alignment by the ratio of the number of identical amino acids at the same position with the total number of amino acids in the reference sequence ( E. coli ). 
     Preferably, the mutant YqhD has at least 35% sequence identity. 
     In most preferred embodiments, the mutant YqhD of the invention comprises the sequence selected among the group consisting of YqhD identified in SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3. 
     DNA, Vectors, Genes 
     The present invention also concerns a DNA sequence comprising a sequence coding for the mutant YqhD of the invention. The sequence sequence coding for the mutant YqhD of the invention is not a limiting factor by itself. The skilled person can easily obtain the sequence of a native YqhD from a microorganism and introduce in the coding sequence the mutation(s) to be introduced in the protein by changing one or more appropriate nucleotide. 
     The skilled person can also perform a mutagenesis in the sequence of a microorganism, and isolate the mutated DNA sequence by standard methods. 
     Mutations can be introduced by site-directed mutagenesis by usual methods like Polymerase Chain Reaction (PCR, see Sambrook J et al., Molecular cloning: a laboratory manual, Cold Spring Harbour Press, New York (2001), Ausubel F M et al., Current protocols in molecular biology, John Wiley and sons, New York (1999), Adams A et al., Methods in yeast genetics, Cold Spring Harbour Press, New York (1997)), or by random mutagenesis techniques, such as use of mutagenic agents (Ultra-Violet rays or chemical agents like nitrosoguanidine (NTG) or ethylmethanesulfonate (EMS)) or use of PCR techniques (DNA shuffling or error-prone PCR). 
     The person skilled in the art can also prepare synthetic genes with preferred codons selected for an improved expression in a specific organism. Codons usages by various organisms are well known in the art and several companies are proposing the manufacture of synthetic genes with codon optimization. 
     The sequence of the invention can be isolated, consisting in the coding sequence as defined above, or within a gene comprising regulatory elements upstream and downstream the coding sequence for its expression in a specific organism. 
     The sequence can also be present in a vector, for its replication (replication vector) or for the expression and translation of the mutated protein of the invention in a microorganism (expression vector). Such vectors are known in the art and not a limiting factor for the definition of the invention. 
     Said genes and vectors are also part of the invention. 
     Preferably, the DNA sequence of the invention is in a microorganism with regulatory elements allowing expression and translation of the mutated YqhD of the invention. 
     Production of the Desired Biochemical 
     The invention also concerns a method for the production of acetol and/or 1,2-propanediol and/or 1,3-propanediol and/or ethylene glycol and/or 1,4-butanediol by fermentation comprising culturing a microorganism of the invention, modified for an improved production of acetol and/or 1,2-propanediol and/or 1,3-propanediol and/or ethylene glycol and/or 1,4-butanediol and recovery of the acetol and/or 1,2-propanediol and/or 1,3-propanediol and/or ethylene glycol and/or 1,4-butanediol. 
     In a particular embodiment, the recovered acetol and/or 1,2-propanediol and/or 1,3-propanediol and/or ethylene glycol and/or 1,4-butanediol is purified is purified. 
     Methods for the purification acetol and/or 1,2-propanediol and/or 1,3-propanediol and/or ethylene glycol and/or 1,4-butanediol are known in the art. 
     In a preferred embodiment, the desired biochemical is 1,2-propanediol. Purification of 1,2-propanediol are known in the art and described in U.S. Pat. No. 5,076,896 and WO 2007/074066. 
     Advantageously, the production is done by fermentation in a batch, fed-batch or continuous process, according to processes known to the person skilled in the art of microorganisms fermentation. Preferably, the production is done by fermentation in a fed-batch process. 
     Culture Medium and Carbon Source 
     In the production method of the invention, the microorganism is cultured on an appropriate culture medium. 
     An “appropriate culture medium” means a medium of known molecular composition adapted to the growth of the micro-organism. In particular, said medium contains at least a source of phosphorus and a source of nitrogen. Said appropriate medium is for example a mineral culture medium of known set composition adapted to the bacteria used, containing at least one carbon source. Said appropriate medium may also designate any liquid comprising a source of nitrogen and/or a source of phosphorus, said liquid being added and/or mixed to the source of sucrose. In particular, the mineral growth medium for Enterobacteriaceae can thus be of identical or similar composition to M9 medium (Anderson, 1946), M63 medium (Miller, 1992) or a medium such as defined by Schaefer et al. (1999). 
     The carbon source ‘glucose’ can be replaced in this medium by any other carbon source, in particular by sucrose or any sucrose-containing carbon source such as sugarcane juice or sugar beet juice. 
     A “carbon source” or “carbon substrate” means any carbon source capable of being metabolized by a microorganism wherein the substrate contains at least one carbon atom. 
     Preferably, the carbon source is selected among the group consisting of glucose, sucrose, mono- or oligosaccharides, starch or its derivatives or glycerol and mixtures thereof. 
     Indeed the microorganisms used in the method of the present invention can be modified to be able to grow on specific carbon sources when the non modified microorganism cannot grow on the same source of carbon, or grow at to low rates. These modifications may be necessary when the source of carbon is a byproduct of biomass degradation such as by-products of sugarcane including; filter cake from clarification of raw juice and different kind of molasses. 
       FIG. 1  represents the alignment of 23 protein sequences of YqhD or homologous enzymes of various sources. The sequences 4HDB_CLOKL (SEQ ID NO:55); YQHD_ECOLI (SEQ ID NO: 56); ADH3_ENTHI (SEQ ID NO: 57); ADHA_BACSU (SEQ ID NO: 58); ADHB_BACSU (SEQ ID NO: 59); ADHB_CLOAB (SEQ ID NO: 60); ADHA_CLOAB (SEQ ID NO: 61); MACA1_RHOOP (SEQ ID NO: 62); TFDF2_RALEJ (SEQ ID NO: 63); TFDF1_RALEJ (SEQ ID NO: 64); CLCE_PSESB (SEQ ID NO: 65); ADH1_CLOSA (SEQ ID NO: 66); ADH2_ENTHI (SEQ ID NO: 67); ADHE_ECO57 (SEQ ID NO: 68); MEDH_RHOER (SEQ ID NO: 69); EUTG_SALTY (SEQ ID NO: 70); EUTG_ECOLI (SEQ ID NO: 71); GBSB_BACSU (SEQ ID NO: 72); FUCO_ECO57 (SEQ ID NO: 73); MEDH_BACMT (SEQ ID NO: 74); DHAT_CITFR (SEQ ID NO: 75); DHAT_KLEPN (SEQ ID NO: 76); ADH4_YEAST (SEQ ID NO: 77); ADH4_SCHPO (SEQ ID NO: 78); ADH2_ZYMMO (SEQ ID NO: 79); ADH2_ECOLI (SEQ ID NO: 80) were obtained from the UniProt Knowledge Base (The UniProt consortium (2008)) and the alignment made using MUSCLE with default parameters. 
       FIG. 2  represents the metabolic pathways for the production of lactic acid, acetol and 1,2-propanediol in the microorganisms of the invention. 
       FIG. 3  represents an analysis on SDS 4-15% gradient polyacrylamide gel the different purification steps of the protein yqhD. Lane 1: Molecular weight marker, Lane 2: Crude extract, Lane 3, Ammonium sulphate precipitate dissolved, Lane 4, HiTrap PhenylHP column pool, Lane 5, Superdex 200 pool. 
     EXAMPLES 
     Example 1 
     Evolution of 2 Modified Strains of  E. coli  MG1655 in Chemostat Culture and Identification of 2 Mutant YqhD Enzymes in the Evolved Clones 
     The construction of the strains  E. coli  MG1655 lpd* ΔtpiA, ΔpflAB, ΔadhE, ΔldhA::Cm, ΔgloA, ΔaldA, ΔaldB, Δedd (strain 1) and  E. coli  MG1655 lpd* ΔtpiA, ΔpflAB, ΔadhE, ΔldhA, ΔgloA, ΔaldA, ΔaldB, Δedd, ΔarcA, Δndh::Km (strain 2) were previously described in patent application WO 2008/116852. 
     To evolve them toward improved 1,2 propanediol production, the 2 strains were cultivated in continuous culture, either under anaerobic conditions, or under microaerobic conditions (1% oxygen) in the culture medium MPG (given in patent application WO 2008/116852) with 0.42 or 0.84 g/l sodium nitrate, with excess glucose (from 20 g/l initially with addition if the glucose becomes exhausted). The temperature was set at 37° C., the pH was regulated at 6.5 by addition of base and the dilution rate of the chemostat was set between 0.04 h −1  and 0.08 h −1 . The evolution of the strain in the chemostats was followed by the increase of the biomass concentration coupled with the increase of the concentrations of the product, 1,2-propanediol and the co-product acetate, over several weeks. This denoted the improvement of the performances of the strains. When the cultures reached a steady state with no further increase of the concentrations under these conditions, the evolution was done. 
     The characteristics of the strains before and after evolution were assessed. Single colonies representing individual clones were isolated on Petri dishes. These clones were assessed using the initial strain as control in an Erlenmeyer flask assay, using the same medium MPG used in the chemostat culture, but buffered with MOPS. Among these clones, several presented better 1,2-propanediol specific production rates as compared to the control. The results obtained on the best clone for each condition of evolution are reported in Table 1 and 2 below. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Comparison of the best evolved clone obtained after 66 days of 
               
               
                 evolution under anaerobic conditions with the initial strain 
               
            
           
           
               
               
               
            
               
                   
                 Initial strain 
                 Best evolved 
               
               
                 Strain  E. coli  MG1655 lpd* 
                 before evolution 
                 clone 
               
               
                 ΔtpiA ΔpflAB ΔadhE ΔldhA::Cm 
                 (performances 
                 (performances 
               
               
                 ΔgloA Δald, ΔaldB Δedd 
                 measured after 
                 measured after 
               
               
                 (Strain 1) 
                 2 days of culture) 
                 2 days of culture) 
               
               
                   
               
               
                 Glucose specific consumption rate 
                 0.12 
                 0.21 (+75%) 
               
               
                 (g glucose/g biomass/h) 
               
               
                 1,2-propanediol specific 
                 0.02 
                 0.07 (+250%) 
               
               
                 production rate (g 1,2-propane- 
               
               
                 diol/g biomass/h) 
               
               
                 1,2-propanediol + hydroxyacetone 
                 0.04 
                 0.08 (+100%) 
               
               
                 specific production rate 
               
               
                 (g 1,2-propanediol + hydroxy- 
               
               
                 acetone/g biomass/h) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Comparison of the best evolved clone obtained after 132 days of 
               
               
                 evolution under microaerobic conditions with the initial strain 
               
            
           
           
               
               
               
            
               
                   
                 Initial strain 
                 Best evolved 
               
               
                 Strain  E. coli  MG1655 lpd* 
                 before evolution 
                 clone 
               
               
                 ΔtpiA ΔpflAB ΔadhE ΔldhA::Cm 
                 (performances 
                 (performances 
               
               
                 ΔgloA Δald, ΔaldB, Δedd, 
                 measured after 
                 measured after 
               
               
                 ΔarcA Δndh (Strain 2) 
                 2 days of culture) 
                 2 days of culture) 
               
               
                   
               
               
                 Glucose specific consumption rate 
                 0.15 
                 0.28 (+87%) 
               
               
                 (g glucose/g biomass/h) 
               
               
                 1,2-propanediol specific 
                 0.00 
                 0.10 
               
               
                 production rate (g 1,2-propane- 
               
               
                 diol/g biomass/h) 
               
               
                 1,2-propanediol + hydroxyacetone 
                 0.04 
                 0.10 (+150%) 
               
               
                 specific production rate 
               
               
                 (g 1,2-propanediol + hydroxy- 
               
               
                 acetone/g biomass/h) 
               
               
                   
               
            
           
         
       
     
     Specific genes involved in the terminal 1,2-propanediol biosynthetic pathway were sequenced in the 2 best evolved clones of strain 1 and strain 2. For each clone, one mutated yqhD gene was identified resulting in expression of mutated YqhD protein: YqhD*(G149E) for evolved clone of strain 1 and YqhD*(G149S)(A286T) for evolved clone of strain 2. 
     Example 2 
     Production and Purification of Native YqhD and 3 Mutant YqhD (G149E, G149S and G149S A286T) 
     1. Construction of the Strains for Production of YqhD Proteins 
     1.1. Construction of the Plasmid for the Overexpression of Native yqhD: pTRC99A-yqhD 
     The gene yqhD (sequence 3153377 to 3154540) was PCR amplified from genomic DNA of  E. coli  MG1655 using the following oligonucleotides: 
                    yqhD F ptrc99A F, consisting of 43 pb        (SEQ ID NO 4):       cgatgcacg tc     atg aacaactttaatctgcacaccccaacccg ,            
with:—a region (underlined letters) homologous to the sequence (3153377 to 3153408) of the gene yqhD.
         a BspHI restriction site (bold)       

                        yqhD F ptrc99A R, consisting of 43 pb (SEQ ID NO 5):           ggcgtaaa aagc     tt agcgggcggctcgtatatacggcggctgacatccaacgtaatgtcgtgattttcg              
with:—a region (underlined letters) homologous to the sequence (3154540 to 3154483) of the gene yqhD.
         a HindIII restriction site (bold)       

     The PCR fragment amplified was digested by BspHI-HindIII and ligated in the pTRC99A digestion by NcoI-HindIII. The plasmid obtained was named pTRC99A-yqhD. 
     1.2. Construction of the Plasmids for the Overexpression of yqhD* 
     The three mutant YqhD bear the mutation (G149E), (G149S) or (G149S)(A286T). The plasmids for the overexpression of the three mutant proteins were built by site-directed mutagenesis using Quickchange site-directed mutagenesis kit from Stratagene® and the oligonucleotides described in table 3. The double mutant YqhD was built by two successive site-directed mutagenesis, G149S and then A286T. 
     The plasmids obtained were named pTRC99A-yqhD*(G149E), pTRC99A-yqhD*(G149S), pTRC99A-yqhD*(G149S)(A286T). 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Oligonucleotides used for the site-directed mutagenesis of yqhD 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Name of 
                   
                   
                 Homologous 
               
               
                   
                 oligonuc- 
                 Sequence of the 
                 Modification to create 
                 to the 
               
               
                 Mutation 
                 leotides 
                 oliognucleotide 
                 mutation 
                 sequence 
               
               
                   
               
               
                 G149E 
                 yqhD* 
                 ggttcagaatccaacgcagAAgcgg 
                 Red capital letter: AA instead 
                 3153803 to 
               
               
                   
                 (G149E) 
                 tgatAtcccgtaaaacc 
                 of GC to create mutation 
                 3153844 
               
               
                   
                  EcoRVF 
                 (SEQ ID NO 6) 
                 G149E. 
                   
               
               
                   
                   
                   
                 Green capital letter: A instead 
                   
               
               
                   
                   
                   
                 of C to create EcoRV 
                   
               
               
                   
                   
                   
                 restriction site without change 
                   
               
               
                   
                   
                   
                 in protein sequence 
                   
               
               
                   
                 yqhD* 
                 ggttttacgggaTatcaccgcTTctg 
                 Red capital letter: TT instead 
                   
               
               
                   
                 (G149E)  
                 cgttggattctgaacc 
                 of CG to create mutation 
                   
               
               
                   
                 EcoRVR 
                 (SEQ ID NO 7) 
                 G149E. 
                   
               
               
                   
                   
                   
                 Green capital letter: T instead 
                   
               
               
                   
                   
                   
                 of G to create EcoRV 
                   
               
               
                   
                   
                   
                 restriction site without change 
                   
               
               
                   
                   
                   
                 in protein sequence 
                   
               
               
                   
               
               
                 G149S 
                 yqhD 
                 ggttcagaatccaacgcaAgcgcggt 
                 Red capital letter: A instead of 
                 3153803 to 
               
               
                   
                 G149S MD 
                 gatAtcccgtaaaaccacaggc 
                 G to create mutation G149S. 
                 3153850 
               
               
                   
                 F 
                 (SEQ ID NO 8) 
                 Green capital letter: A instead 
                   
               
               
                   
                   
                   
                 of C to create EcoRV 
                   
               
               
                   
                   
                   
                 restriction site without change 
                   
               
               
                   
                   
                   
                 in protein sequence 
                   
               
               
                   
                 yqhD 
                 gcctgtggttttacgggaTatcaccgc 
                 Red capital letter: T instead of 
                   
               
               
                   
                 G149S MD 
                 gcTtgcgttggattctgaacc 
                 C to create mutation G149S. 
                   
               
               
                   
                 R 
                 (SEQ ID NO 9) 
                 Green capital letter: T instead 
                   
               
               
                   
                   
                   
                 of G to create EcoRV 
                   
               
               
                   
                   
                   
                 restriction site without change 
                   
               
               
                   
                   
                   
                 in protein sequence 
                   
               
               
                   
               
               
                 A286T 
                 yqhD 
                 gatcacgcgcaaacactgActatAgt 
                 Red capital letter: A instead of 
                 3154214 to 
               
               
                   
                 A286T MD 
                 Actgcctgcactgtggaatgaaaaac 
                 G to create mutation A286T. 
                 3154268 
               
               
                   
                 R 
                 gcg 
                 Green capital letter : A instead 
                   
               
               
                   
                   
                 (SEQ ID NO 10) 
                 of C to create ScaI restriction 
                   
               
               
                   
                   
                   
                 site without change in protein 
                   
               
               
                   
                   
                   
                 sequence 
                   
               
               
                   
                 yqhD 
                 cgcgtttttcattccacagtgcaggcag 
                 Red capital letter: T instead of 
                   
               
               
                   
                 A286T MD 
                 TacTatagTcagtgtttgcgcgtgatc 
                 C to create mutation A286T. 
                   
               
               
                   
                 scaI F 
                 (SEQ ID NO 11) 
                 Green capital letter : T instead 
                   
               
               
                   
                   
                   
                 of G to create ScaI restriction 
                   
               
               
                   
                   
                   
                 site without change in protein 
                   
               
               
                   
                   
                   
                 sequence 
               
               
                   
               
            
           
         
       
     
     1.3. Construction of the Strain MG1655 ΔyqhD::Km 
     The gene yqhD was inactivated in strain  E. coli  MG1655 by inserting a Kanamycin resistance cassette and deleting most of the gene concerned using the technique described in Protocol 1. 
     Protocol 1: Introduction of a PCR Product for Recombination and Selection of the Recombinants (FRT System). 
     The oligonucleotides chosen and given in Table 4 for replacement of a gene or an intergenic region were used to amplify either the chloramphenicol resistance cassette from the plasmid pKD3 or the kanamycin resistance cassette from the plasmid pKD4 (Datsenko, K. A. &amp; Wanner, B. L. (2000)). The PCR product obtained was then introduced by electroporation into the recipient strain bearing the plasmid pKD46 in which the system λ Red (γ, β, exo) expressed greatly favours homologous recombination. The antibiotic-resistant transformants were then selected and the insertion of the resistance cassette was checked by PCR analysis with the appropriate oligonucleotides given in Table 5. 
     If they are other modifications in the strain, they were checked with the oligonucleotides given in Table 5. 
     The resulting strain was named  E. coli  MG1655 ΔyqhD::Km. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Oligonucleotides used for replacement of a chromosomal 
               
               
                 region by recombination with a PCR product 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Homology with 
               
               
                   
                 Region name 
                 Names of oligos 
                 chromosomal region 
               
               
                   
                   
               
               
                   
                 gldA 
                 DgldA F 
                 4137058 to 4136979 
               
               
                   
                   
                 DgldA R 
                 4135955 to 4136034 
               
               
                   
                 edd-eda 
                 DedaR 
                 1930144 to 1930223 
               
               
                   
                   
                 DeddF 
                 1932501 to 1932582 
               
               
                   
                 aldA 
                 DaldAR 
                 1487615 to 1487695 
               
               
                   
                   
                 DaldAF 
                 1486256 to 1486336 
               
               
                   
                 aldB 
                 DaldBR 
                 3754534 to 3754455 
               
               
                   
                   
                 DaldBF 
                 3752996 to 3753075 
               
               
                   
                 arcA 
                 DarcAF 
                 4637868 to 4637791 
               
               
                   
                   
                 DarcAR 
                 4637167 to 4637245 
               
               
                   
                 ndh 
                 DndhF 
                 1165071 to 1165149 
               
               
                   
                   
                 DndhR 
                 1166607 to 1166528 
               
               
                   
                 Ptrc01-gapA 
                 Ptrc-gapAF 
                 1860800 to 1860762 
               
               
                   
                   
                 Ptrc-gapAR 
                 1860478 to 1860536 
               
               
                   
                 gloA 
                 DgloA F 
                 1725861 to 1725940 
               
               
                   
                   
                 DgloA R 
                 1726268 to 1726189 
               
               
                   
                 yqhD 
                 DyqhDF 
                 3153377 to 3153456 
               
               
                   
                   
                 DyqhDR 
                 3154540 to 3154460 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Oligonucleotides used for checking the insertion of resistance 
               
               
                 cassette or the loss of resistance cassette 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Homology with 
               
               
                   
                 Region name 
                 Names of oligos 
                 chromosomal region 
               
               
                   
                   
               
               
                   
                 gldA gene 
                 talC F 
                 4137144 to 4137121 
               
               
                   
                   
                 yijF R 
                 4135136 to 4135159 
               
               
                   
                 edd-eda genes 
                 edaR 
                 1929754 to 1929777 
               
               
                   
                   
                 eddF 
                 1932996 to 1932968 
               
               
                   
                 aldA gene 
                 aldAF 
                 1485877 to 1485901 
               
               
                   
                   
                 aldAR 
                 1487714 to 1487689 
               
               
                   
                 aldB gene 
                 aldBF 
                 3752056 to 3752095 
               
               
                   
                   
                 aldBR 
                 3754644 to 3754674 
               
               
                   
                 arcA gene 
                 arcAF 
                 4638292 to 4638273 
               
               
                   
                   
                 arcAR 
                 4636854 to 4636874 
               
               
                   
                 ndh gene 
                 ndhF 
                 1164722 to 164742 
               
               
                   
                   
                 ndhR 
                 1167197 to 1167177 
               
               
                   
                 Ptrc01-gapA 
                 yeaAF 
                 1860259 to 1860287 
               
               
                   
                   
                 gapAR 
                 1861068 to 1861040 
               
               
                   
                 gloA 
                 gloAF2 
                 1725641 to 1725660 
               
               
                   
                   
                 gloAR2 
                 1726450 to 1726431 
               
               
                   
                 yqhD 
                 yqhDF 
                 3153068 to 3153100 
               
               
                   
                   
                 yqhDR 
                 3154825 to 3154797 
               
               
                   
                 mgsA::Km 
                 helDF 
                 1025242 to 1025260 
               
               
                   
                   
                 mgsA R3 
                 1026734 to 1026715 
               
               
                   
                   
               
            
           
         
       
     
     1.4. Introduction of the Plasmids Built in the Strain  E. coli  MG1655 ΔyqhD::Km. 
     The plasmids pTRC99A-yqhD, pTRC99A-yqhD*(G149E), pTRC99A-yqhD*(G149S), pTRC99A-yqhD*(G149S)(A286T) were transformed by electroporation in the strain  E. coli  MG1655 AyqhD::Km. The strains obtained were named respectively:
           E. coli  MG1655 ΔyqhD::Km pTRC99A-yqhD     E. coli  MG1655 ΔyqhD::Km pTRC99A-yqhD*(G149E)     E. coli  MG1655 ΔyqhD::Km pTRC99A-yqhD*(G149S)     E. coli  MG1655 ΔyqhD::Km pTRC99A-yqhD*(G149S)(A286T).
 
2. Production of YqhD Proteins
       

     The four strains previously described were cultivated at 37° C. under aerobic conditions in 2 l baffled Erlenmeyer flasks with 500 ml LB medium with 2.5 g/l glucose. The flasks were agitated at 200 rpm on an orbital shaker. When the optical density measured at 550 nm reached 0.5 units, the flasks were incubated at 25° C. When the optical density reached 1.2 units, the production of YqhD proteins was induced by adding 500 μM IPTG in the cultures. The biomass was harvested by centrifugation when the cultures reached an optical density above 3.5 units. The supernatant was discarded and the pellet was stored at −20° C. before use. 
     3. Methylglyoxal Reductase Activity Assay 
     Methylglyoxal reductase activity was assayed by measuring the initial rate of NADPH oxidation with a spectrophotometer at a wavelength of 340 nm and at a constant temperature of 37° C. The reaction mixture using methylglyoxal as substrate was carried out in 20 mM Hepes pH 7.5, 0.1 mM Zinc sulphate, 0.2 mM NADPH, 0.6 μg of purified enzyme in a final volume of 1 ml. The reaction mixture was incubated for 5 min at 37° C. and then the reaction was initiated by the addition of the substrate methylglyoxal at a final concentration of 10 mM. Control assay (blank), lacking the substrate was run in parallel and the value measured for the control was subtracted to the value measured for the assay to take into account non-specific oxidation of NADPH. 
     One unit of enzyme activity was defined as the amount of enzyme that consumed 1 μmol substrate per minute under the conditions of the assay. Specific enzyme activity was expressed as units per mg of protein. 
     4. Purification of the YqhD Enzymes 
     4.1. Step 1: Preparation of Cell-Free Extracts 
     Between 350-400 mg of  E. coli  biomass were resuspended in 70 ml of 50 mM Hepes pH 7.5, and a protease inhibitor cocktail. Cells were sonicated on ice (Branson sonifier, 70 W) in a Rosett cell RZ3 during six cycles of 30 sec with 30 sec intervals. After sonication, cells were incubated for 1 hour at room temperature with 1 mM MgCl2 and 1 UI/ml of DNaseI. Cells debris were removed by centrifugation at 12000 g for 30 min at 4° C. Supernatant was kept as the crude extract. 
     4.2. Step 2: Ammonium Sulphate Precipitation 
     The crude extract was precipitated at a concentration of 50% ammonium sulphate: solid ammonium sulphate (300 g/l) was added to the crude extract on ice. After 15 min of incubation at 4° C., the mix was centrifuged at 12000 g for 15 min at 4° C. The supernatant was discarded and the precipitate dissolved in 40 ml of 50 mM Hepes pH 7.5, 1 M ammonium sulphate. 
     4.3. Step 3: Hydrophobic Chromatography. 
     Using an Akta Purifier (GE Healthcare), the protein extract from the previous step was loaded onto a 5 ml HiTrap PhenylHP column (GE Healthcare) equilibrated with the same buffer. The column was washed with 10 column volumes of the same buffer. Proteins were eluted with two steps gradient, a gradient of 10 column volumes from 1 M to 0.5 M ammonium sulphate and a gradient of 20 column volumes from 0.5 M to 0 M ammonium sulphate. After elution, the column was washed with 10 column volumes of 50 mM Hepes pH 7.5. The flow rate of the column was 2.5 ml/min and 2.5 ml fractions were collected. The fractions which contain the protein were pooled and concentrated to be loaded on a gel filtration column. 
     4.4. Step 4: Gel Filtration 
     The concentrated fractions from the HiTrap PhenylHP column were loaded onto a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with 50 mM Hepes pH 7.5. The flow rate of the column was 0.5 ml/min and 0.5 ml fractions were collected. The protein was eluted with about 13.8 ml of buffer. The expression and purification of the three mutant yqhD was remarkably similar to that of the wild type enzyme. There were no differences in the oligomerisation state between the native yqhD and the mutated yqhD*. All proteins were stored at −20° C. 
     The pool of each purification step was analysed on a SDS 4-15% gradient polyacrylamide gel ( FIG. 3 ). This gel showed that the purity increased along the purification step. After the superdex 200 column, the protein was almost 90% pure. The final pool showed one major band at about 42 kDa corresponding to the protein yqhD. 
     Example 3 
     Characterization of Native YqhD and 3 Mutant YqhD (G149E, G149S and G149S A286T) with the Substrate Methylglyoxal and with the Co-Factor NADPH 
     1. Characterization of the YqhD Enzymes with the Substrate Methylglyoxal 
     Kinetic constants (Km, kcat and kcat/Km) for the four purified enzymes (yqhD, yqhD*(G149E), yqhD*(G149S) and yqhD*(G149S A286T)) toward methylglyoxal were determined using the activity assay previously described. In this case the reaction was started by addition of the purified enzyme. Seven methylglyoxal concentrations between 1.3 mM and 30 mM were analysed for each enzyme. For all kinetics, initial velocity was determined in triplicate for all substrate concentrations. 
     Kinetic constants (Km and Vmax) of each protein were determined with the module enzyme kinetics from the software Sigma Plot (Systat Software Inc, San Jose, Calif.). The data sets exhibiting Michaelis-Menten kinetics were fitted to a Michaelis-Menten equation. The different kinetic parameters of four YqhD enzymes were compiled in Table 6. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 kinetics parameters for the YqhD enzymes toward methylglyoxal 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 YqhD*(G149S 
                 YqhD* 
                 YqhD* 
               
               
                   
                 YqhD 
                 A286T) 
                 (G149S) 
                 (G149E) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Km (mM) 
                 4.81 ± 0.41 
                 3.28 ± 0.38 
                 3.37 ± 0.34 
                 7.80 ± 0.79 
               
               
                 Specific 
                 3.91 ± 0.11 
                 2.69 ± 0.10 
                 3.80 ± 0.12 
                 7.65 ± 0.31 
               
               
                 activity 
               
               
                 (μmol/min/ 
               
               
                 mg) 
               
               
                 kcat (s−1) 
                 2.74 ± 0.08 
                 1.89 ± 0.07 
                 2.67 ± 0.09 
                 5.38 ± 0.21 
               
               
                 kcat/Km 
                 570 ± 65  
                 577 ± 88  
                 792 ± 105 
                 689 ± 98  
               
               
                 (M−1s−1) 
               
               
                   
               
            
           
         
       
     
     The kinetics parameters of the four enzymes were very similar. The kinetic parameters, substrate affinity (Km), catalytic (kcat), and catalytic efficiency (kcat/Km) of the proteins yqhD*(G149S A286T)), yqhD*(G149S), were identical to the native protein. 
     Specific activity for each YqhD is directly calculated from the kcat value. Specific activity of YqhD and YqhD*(G149S) were similar. Specific activity of YqhD*(G149S)(A286T) represented 69% of the specific activity of YqhD. Specific activity of YqhD*(G149E) was almost doubled compared to that of YqhD. The mutations were not detrimental for the activity of the enzyme. 
     2. Characterization of the YqhD Enzymes with the co-Factor NADPH 
     Kinetic constants (Km, kcat and kcat/Km) for the four purified enzymes (yqhD, yqhD*(G149E), yqhD*(G149S) and yqhD*(G149S A286T)) toward methylglyoxal were determined using the activity assay previously described. In this case the reaction was started by addition of the purified enzyme. Nine NADPH concentrations between 6 μM and 300 μM were analysed for each enzyme. For all kinetics, initial velocity was determined in triplicate for all co-factor concentrations. 
     Kinetic constants (Km and Vmax) of each protein were determined with the module enzyme kinetics from the software Sigma Plot (Systat Software Inc, San Jose, Calif.). The data sets exhibiting Michaelis-Menten kinetics were fitted to a Michaelis-Menten equation. The different kinetic parameters of four YqhD enzymes were compiled in Table 7. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 kinetics parameters for the YqhD enzymes toward NADPH 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 YqhD*(G149S 
                   
               
               
                   
                 YqhD 
                 YqhD*G149E 
                 A286T) 
                 YqhD*G149S 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Km (μM) 
                  32 ± 4.2 
                  28 ± 1.8 
                  20 ± 2.2 
                  22 ± 3.7 
               
               
                 Specific activity 
                 2.99 ± 0.1  
                 4.86 ± 0.08 
                 3.17 ± 0.02 
                 3.78 ± 0.15 
               
               
                 (μmol/min/mg) 
               
               
                 kcat (s−1) 
                 2.1 ± 0.1 
                 3.4 ± 0.1 
                 2.2 ± 0.1 
                 2.7 ± 0.1 
               
               
                 kcat/Km 
                 6.6E+04 ± 1.1E+04 
                 1.22E+05 ± 9.8E+03  
                 1.13E+05 ± 1.3E+04  
                 1.22E+05 ± 2.5E+04  
               
               
                 (M−1s−1) 
               
               
                   
               
            
           
         
       
     
     The kinetic parameters, substrate affinity (Km) and catalytic constant (kcat) were similar for the native enzyme ant the three mutants. The catalytic efficiency (kcat/Km) of the three mutant proteins was around 1.22 E+05 M −1  s −1 , but two times higher than the native enzyme (kcat/Km 6.6 E+04 M −1  s −1 ). 
     To summarize, the properties of the 3 mutant YqhD were very similar: the mutant YqhD have a better catalytic efficiency toward NADPH, allowing a more efficient use of this co-factor. 
     3. Characterization of the Inhibition of YqhD Enzymes by NADP+ 
     The NADP+ inhibition constant for three purified enzymes (yqhD, yqhD*(G149E) and yqhD*(G149S A286T)) were determined in the conditions described for the activity assay, excepted for the concentration of methylglyoxal that was increased to 20 mM. Enzyme kinetics with regards to NADPH were realised with increasing concentration of NADPH between 50 and 200 μM. Five concentrations of the inhibitor NADP+ were used (0-0.05-0.1-0.2-0.5 mM) and the kinetics were recorded for each condition of inhibition. The NADP+ inhibition constant (Ki) of each protein was determined with the module enzyme kinetic from the software Sigma Plot (Systat Software Inc, San Jose, Calif.). A model of competitive inhibition was fitted by the software. The kinetic parameters calculated for these three proteins were summarized in Table 8. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Inhibition of the YqhD enzymes by NADP+ 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 YqhD*(G149S 
               
               
                   
                 YqhD 
                 YqhD*G149E 
                 A286T) 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                 Km (μM) 
                 51 ± 11 
                     24 ± 5.5 
                 19 ± 3.5 
               
               
                 Specific activity 
                  3.63 ± 0.284 
                  5.53 ± 0.292 
                  5.25 ± 0.183 
               
               
                 (μmol/min/mg) 
               
               
                 kcat (s−1) 
                 2.5 ± 0.2 
                  3.9 ± 0.2 
                  3.7 ± 0.13 
               
               
                 kcat/Km 
                 4.99E+04 ± 1.5E+04  
                 1.62E+05 ± 4.5E+04 
                 1.94E+05 ± 4.25E+04  
               
               
                 (M−1s−1) 
               
               
                 Ki NADP+ (μM) 
                 68 ± 11 
                 28 ± 5 
                 16 ± 2.5 
               
               
                   
               
            
           
         
       
     
     The native YqhD enzyme was highly inhibited by the inhibitor NADP+ (Ki=68 μM). However, the two mutant YqhD enzymes were even more sensitive to the inhibition by NADP+ (Ki lowered by a factor of 2 or 3). 
     Example 4 
     Construction of Two  E. coli  1,2-Propanediol Producer Strains Expressing Wildtype or Modified YqhD and Assessment of 1,2-Propanediol Production 
     1. Construction of the Modified Strain  E. coli  MG1655 mgsA*(H21Q)::Km, Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101-VB01-yqhD-gldA*(160T). 
     1.1. Construction of a Modified Strain  E. coli  ΔgloA::Cm 
     The gene gloA was inactivated in strain  E. coli  MG1655 by inserting a chloramphenicol antibiotic cassette and deleting most of the gene concerned using the technique described in protocol 1 with the oligonucleotides given in Table 4. The deletion was checked by a PCR analysis with the appropriate oligonucleotides given in Table 5. The resulting strain was named  E. coli  MG1655 ΔgloA::Cm. 
     1.2. Construction of a Modified Strain  E. coli  ΔgloA::Cm Δedd-eda::Km 
     1.2.1. Construction of a Modified Strain  E. coli  Δedd-eda::Km 
     The genes edd-eda were inactivated in strain  E. coli  MG1655 by inserting a kanamycin antibiotic cassette and deleting most of the gene concerned using the technique described in Protocol 1 with the oligonucleotides given in Table 4. The deletion was checked by a PCR analysis with the appropriate oligonucleotides given in Table 5. 
     The resulting strain was named  E. coli  MG1655 Δedd-eda::Km. 
     1.2.2. Construction of a Modified Strain  E. coli  ΔgloA::Cm, Δedd-eda::Km. 
     The deletion of the genes edd-eda by replacement of the genes by a kanamycin resistance cassette in the strain  E. coli  Δedd-eda::Km was performed by the technique of transduction with phage P1 according to Protocol 2. 
     Protocol 2: Transduction with Phage P1 for Deletion of a Gene 
     The deletion of the chosen gene by replacement of the gene by a resistance cassette (kanamycin or chloramphenicol) in the recipient  E. coli  strain was performed by the technique of transduction with phage P1. The protocol was in two steps, (i) the preparation of the phage lysate on the strain MG1655 with a single gene deleted and (ii) the transduction of the recipient strain by this phage lysate. 
     Preparation of the Phage Lysate
         Seeding with 100 μl of an overnight culture of the strain MG1655 with a single gene deleted of 10 ml of LB+Cm 30 μg/ml+glucose 0.2%+CaCl 2  5 mM.   Incubation for 30 min at 37° C. with shaking.   Addition of 100 μl of phage lysate P1 prepared on the wild type strain MG1655 (approx. 1×10 9  phage/ml).   Shaking at 37° C. for 3 hours until all cells were lysed.   Addition of 200 μl of chloroform, and vortexing.   Centrifugation for 10 min at 4500 g to eliminate cell debris.   Transfer of supernatant in a sterile tube and addition of 200 μl of chloroform.   Storage of the lysate at 4° C.       

     Transduction
         Centrifugation for 10 min at 1500 g of 5 ml of an overnight culture of the  E. coli  recipient strain in LB medium.   Suspension of the cell pellet in 2.5 ml of MgSO 4  10 mM, CaCl 2  5 mM.   Control tubes: 100 μl cells
           100 μl phages P1 of the strain MG1655 with a single gene deleted.   
           Tube test: 100 μl of cells+100 μl phages P1 of strain MG1655 with a single gene deleted.   Incubation for 30 min at 30° C. without shaking.   Addition of 100 μl sodium citrate 1 M in each tube, and vortexing.   Addition of 1 ml of LB.   Incubation for 1 hour at 37° C. with shaking   Plating on dishes LB+Cm 30 μg/ml after centrifugation of tubes for 3 min at 7000 rpm.   Incubation at 37° C. overnight.       

     The antibiotic-resistant transformants were then selected and the insertion of the deletion was checked by a PCR analysis with the appropriate oligonucleotides given in Table 5. 
     The deletion was checked by a PCR analysis with the appropriate oligonucleotides given in Table 5 as well as the other deletion already present in the strain. 
     The resulting strain was named  E. coli  ΔgloA::Cm, Δedd-eda::Km. 
     1.3. Construction of a Modified Strain  E. coli  MG1655 Δedd-eda ΔgloA 
     The antibiotic resistance cassette were eliminated in the strain  E. coli  Δedd-eda::Km, ΔgloA::Cm according to Protocol 3. 
     Protocol 3: Elimination of Resistance Cassettes (FRT System) 
     The chloramphenicol and/or kanamycin resistance cassettes were eliminated according to the following technique. The plasmid pCP20 carrying the gene coding for FLP recombinase acting at the FRT sites of the chloramphenicol and/or kanamycin resistance cassettes was introduced into the strain by electroporation. After serial culture at 42° C., the loss of the antibiotic resistance cassettes was checked by PCR analysis with the oligonucleotides given in Table 5. 
     The presence of the modifications previously built in the strain was checked using the oligonucleotides given in Table 5. 
     The strain obtained was named  E. coli  MG1655 Δedd-eda ΔgloA. 
     1.4. Construction of the Modified Strain  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA::Cm 
     1.4.1. Construction of the Modified Strain  E. coli  MG1655 ΔaldA::Cm 
     The genes aldA was inactivated in strain  E. coli  MG1655 by inserting a chloramphenicol antibiotic cassette and deleting most of the gene concerned using the technique described in protocol 1 with the oligonucleotides given in Table 4. The deletion was checked by a PCR analysis with the appropriate oligonucleotides given in Table 5. 
     The resulting strain was named  E. coli  MG1655 ΔaldA::Cm. 
     1.4.2. Construction of the Modified Strain  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA::Cm 
     The deletion of the gene aldA by replacement of the gene by a chloramphenicol resistance cassette in the strain  E. coli  MG1655 Δedd-eda ΔgloA was performed by the technique of transduction with phage P1 (Protocol 2). 
     The deletion ΔaldA::Cm and the others modifications were checked using the oligonucleotides described in table 5. 
     The resulting strain was named  E. coli  ΔgloA::Cm, Δedd-eda::Km, ΔaldA::Cm 
     1.5. Construction of the Modified Strain  E. coli  MG1655 Δedd-eda, ΔgloA, ΔaldA::Cm, ΔaldB::Km 
     1.5.1. Construction of the Modified Strain  E. coli  MG1655 ΔaldB::Km 
     The genes aldB was inactivated in strain  E. coli  MG1655 by inserting a Kanamycin antibiotic cassette and deleting most of the gene concerned using the technique described in protocol 1 with the oligonucleotides given in Table 4. The deletion was checked by a PCR analysis with the appropriate oligonucleotides given in Table 5. 
     The resulting strain is named  E. coli  MG1655 ΔaldB::Km. 
     1.5.2. Construction of the Modified Strain  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA::Cm, ΔaldB::Km 
     The deletion of the gene aldA by replacement of the gene by a Kanamycin resistance cassette in the strain  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA::Cm, was performed by the technique of transduction with phage P1 (Protocol 2). 
     The deletion ΔaldB::Km and the others modifications were checked using the oligonucleotides described in Table 5. 
     The resulting strain was named  E. coli  ΔgloA, Δedd-eda, ΔaldA::Cm, ΔaldB::Km. 
     1.6. Construction of the Modified Strain  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB 
     The antibiotic resistance cassette was eliminated in the strain  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA::Cm, ΔaldB::Km according to Protocol 3. 
     The loss of the antibiotic resistance cassettes was checked by PCR analysis with the oligonucleotides given in Table 5. The presence of the modifications previously built in the strain was also checked using the oligonucleotides given in Table 5. 
     The strain obtained was named  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB. 
     1.7. Construction of the Modified Strain  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔarcA::Km 
     1.7.1. Construction of the Modified Strain  E. coli  MG1655 ΔarcA::Km 
     The genes arcA was inactivated in strain  E. coli  MG1655 by inserting a Kanamycin antibiotic cassette and deleting most of the gene concerned using the technique described in Protocol 1 with the oligonucleotides given in Table 4. The deletion was checked by a PCR analysis with the appropriate oligonucleotides given in Table 5. 
     The resulting strain was named  E. coli  MG1655 ΔarcA::Km. 
     1.7.2. Construction of the Modified Strain  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔarcA::Km 
     The deletion of the gene arcA by replacement of the gene by a Kanamycin resistance cassette in the strain  E. coli  Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔarcA::Km, was performed by the technique of transduction with phage P1 (Protocol 2) 
     The deletion ΔarcA::Km and the others modifications were checked using the oligonucleotides described in Table 5. 
     The resulting strain was named  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔarcA::Km. 
     1.8. Construction of the Modified Strain  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔarcA::Km, Δndh::Cm 
     1.8.1. Construction of the Modified Strain  E. coli  MG1655 Δndh::Cm 
     The gene ndh was inactivated in strain  E. coli  MG1655 by inserting a chloramphenicol resistance cassette and deleting most of the gene concerned using the technique described in Protocol 1 with the oligonucleotides given in Table 4. The deletion was checked by a PCR analysis with the appropriate oligonucleotides given in Table 5. 
     The resulting strain was named  E. coli  MG1655 Δndh::Cm. 
     1.8.2. Construction of the Modified Strain  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔarcA::Km, Δndh::Cm 
     The deletion of the gene ndh by replacement of the gene by a chloramphenicol resistance cassette in the strain  E. coli  Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔarcA::Km, was performed by the technique of transduction with phage P1 (Protocol 2). 
     The deletion Δndh::Cm and the others modifications were checked using the oligonucleotides described in Table 5. 
     The resulting strain was named  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔarcA::Km, Δndh::Cm. 
     1.9. Construction of the Modified Strain  E. coli  MG1655 Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh 
     The antibiotic resistance cassette was eliminated in the strain  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔarcA::Km, Δndh::Cm according to Protocol 3. 
     The loss of the antibiotic resistance cassettes was checked by PCR analysis with the oligonucleotides given in Table 4. The presence of the modifications previously built in the strain was also checked using the oligonucleotides given in Table 5. 
     The strain obtained was named  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh. 
     1.10. Construction of the Modified Strain  E. coli  MG1655 Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101VB01-yqhD-gldA*(A160T)) 
     1.10.1. Construction of the Plasmid pME101-VB01-yqhD-gldA*(A160T) 
     1.10.1.1. Construction of the Plasmid pME101-VB01 
     The plasmid pME101VB01 was derived from plasmid pME101 and harbors a multiple cloning site containing recognition site sequences specific for the rare restriction endonucleases NheI, SnaBI, PacI, BglII, AvrII, SacII and AgeI following by the adc transcription terminator of  Clostridium acetobutylicum  ATCC824. 
     For the expression from a low copy vector the plasmid pME101 was constructed as follows. The plasmid pCL1920 (Lerner &amp; Inouye, 1990, NAR 18, 15 p 4631-GenBank AX085428) was PCR amplified using the oligonucleotides PME101F and PME101R and the BstZ17I-XmnI fragment from the vector pTrc99A (Amersham Pharmacia Biotech, Piscataway, N.J.) harboring the lacI gene and the trc promoter was inserted into the amplified vector. 
                    PME101F (SEQ ID NO 12): ccgacagtaagacgggtaagcctg               PME101R (SEQ ID NO 13): agcttagtaaagccctcgctag            
A synthetic double-stranded nucleic acid linker comprising the multicloning site and adc transcriptional terminator was used to generate pME101VB01. Two 100 bases oligonucleotides that complement flanked by NcoI or HindIII digested restriction sites were annealed. The 100-base pair product was subcloned into NcoI/HindIII digested plasmid pME101 to generate pME101VB01.
 
     
       
         
           
               
            
               
                 pME101VB01 1, consisting of 100 bases 
               
               
                 (SEQ ID NO 14): 
               
               
                 catgg gctagctacgtattaattaaagatctcctagggagctcaccggt   
               
               
                   
               
               
                 TAAAAATAAGAGTTACCTTAAATGGTAACTCTTATTTTTTTAggcgcgc 
               
               
                   
               
               
                 ca 
               
               
                   
               
               
                 pME101VB01 2, consisting of 100 bases 
               
               
                 (SEQ ID NO 15): 
               
               
                 agcttggcgcgccTAAAAAAATAAGAGTTACCATTTAAGGTAACTCTTA 
               
               
                   
               
               
                 TTTTTA accggtgagctccctaggagatctttaattaatacgtagctag   
               
               
                   
               
               
                   c c 
               
            
           
         
       
     
     with:
         a region (underlined lower-case letters) corresponding to the multicloning site   a region (upper-case letters) corresponding to the adc transcription terminator (sequence 179847 to 179814) of  Clostridium acetobutylicum  ATCC 824 pSOL1 (NC — 001988).       

     1.10.1.2. Construction of the Plasmid pME101-VB01-yqhD-gldA*(A160T) 
     1.10.1.2.1. Construction of the Plasmid pME101-VB01-yqhD 
     The gene yqhD was PCR amplified from genomic DNA of  E. coli  MG1655 using the following oligonucleotides 
                            yqhD F, consisting of 43 pb, (SEQ ID NO 16)           cgatgcacg t     catga acaactttaatctgcacaccccaacccg              
with:
         a region (underlined letter) homologous to the sequence (3153377 to 3153408) of the gene yqhD   a restriction site BspHI (bold face letters)       

                            yqhD R, consisting of 29 pb (SEQ ID NO 17)           Cta gctagc   ttagcgggcggcttcgtata              
With:
         a region (underlined letter) homologous to the sequence (3154540 to 3154521) the gene yqhD   a restriction site NheI (bold face letters)       

     The PCR amplified fragment was cut with the restriction enzymes BspHI and NheI and cloned into the NcoI/NheI sites of the vector pME101VB01. The resulting plasmid was named pME101VB01-yqhD 
     1.10.1.2.2. Construction of the Plasmid pSCB-gldA*(A160T) 
     The gene gldA, PCR amplified from genomic DNA of  E. coli  MG1655 using the oligonucleotides gldA F and gldA R was cloned in pSCB (Strataclone®). The resulted plasmid was named pSCB-gldA. 
     A directed mutagenesis was performed on this plasmid with the following oligonucleotide: gldA*A160TmutDirF (consisting of 45 pb, gacaccaaaatcgtcgctggcacacctgcacgtctgCtagcggcg, SEQ ID NO 18) and gldA*A160TmutDirR (consisting of 45 pb cgccgctaGcagacgtgcaggtgtgccagcgacgattttggtgtc, SEQ ID NO 19). The two oligonucleotides were homologous to the region 4136602 to 4136558. In bold face letter, bases which were changed to create the mutation A160T and underlined letter, the base which were change to create EcoRV restriction site. The resulted plasmid was named pSCB-gldA*(A 160T). 
     1.10.1.3. Construction of the pME101VB01-yqhD-gldA*(A160T) 
     The pSCB-gldA*(A160T) was cut with the restriction enzymes avrII and SacI and the fragment containing gldA*(A160T) was cloned into the avrII/SacI sites of the vector pME101VB01-yqhD. The resulted plasmid was named pME101VB01-yqhD-gldA*(A160T). 
     1.10.2. Construction of the Modified Strain  E. coli  MG1655 Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101VB01-yqhD-gldA*(A160T)) 
     The plasmid pME101VB01-yqhD-gldA*(A160T) was introduced by electroporation into the strain  E. coli  MG1655 Ptrc01-gapA::cm, Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh. 
     The strain obtained was named  E. coli  MG1655 Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101VB 01-yqhD-gldA*(A160T). 
     1.11. Construction of the Modified Strain  E. coli  MG1655 mgsA*(H21Q)::Km Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101VB01-yqhD-gldA*(A160T)) 
     1.11.1. Construction of the Modified Strain  E. coli  MG1655 mgsA*(H21Q)::Km 
     1.11.1.1. Construction of the Modified Strain  E. coli  MG1655 mgsA*(H21Q) 
     A mutation was introduced in the mgsA gene in order to obtain the mutant protein MgsA*(H21Q). The technique used to build this modification was described by Heermann et al. (2008), Microbial Cell Factories. 7(14): 1-8. 
     The following oligonucleotides were used to amplify the rpsL-Neo cassette: 
                    1. mgsA*(H21Q)::rpsL-Neo F, consisting in 105 pb,       (SEQ ID NO 20)         gttaactacggatgtacattatggaactgacgactcgcactttacctgc                   gcggaaacatattgcgctggtggcacacgatca   ggcctggtgatgatgg                   cgggatc              
with,
         a region (underlined letter) homologous to the sequence of the gene mgsA.   a region (bold face letter) to amplified rpsL-Neo cassette.       

                    2. mgsA*(H21Q)::rpsL-Neo R (SEQ ID NO 21)         gggaaattaagttaccggtagtgcctgttgcatacagtacgtgttgttc                   cagtaacggttgatgccgttccacccagctcatcagcatctgtttgcat                   tcagaagaactcgtcaagaagg              
with,
         a region (underlined letter) homologous to the sequence of the gene mgsA with to mutation, the first one (in red) to create the mutation H21Q and the second one (in yellow) to create the restriction site AlwN1.   a region (bold face letter) to amplified rpsL-Neo cassette.       

     The fragment obtained was introduced into the strain MG1655 rpsL* (built as described in Heermann et al.) according to Protocol 1. The strain obtained was checked by PCR and sequence analysis. The strain obtained was named  E. coli  mgsA*(H21Q)::rpsL-Neo. 
     The deletion of the cassette rpsL-Neo was performed according to Protocol 1. The fragment transformed was obtained by the restriction with NcoI and SacI of the plasmid pETTOPO-mgsA*(H21Q). 
     The modification was checked by PCR using oligonucleotides described in Table 5. 
     The strain obtained was named strain  E. coli  MG1655 mgsA*(H21Q). 
     1.11.1.2. Construction of the Modified Strain  E. coli  MG1655 mgsA*(H21Q)::Km 
     A kanamycin resistance cassette was introduced in 3′ of mgsA*(H21Q) open reading frame using the following primers: 
                    mgsA::Km F consisting of 100 bp: (SEQ ID NO 22)         tccagtcgccgcatttcaacgacgcggtcgatattctgatccccgatta                   tcagcgttatctcgcggaccgtctgaagtaa   tgtaggctggagctgctt                   cg              
with:
         a region (underlined letters) homologous to the end of mgsA*(H21Q) ORF,   a region (Bold letter) to amplified Kanamycin cassette.       

     
       
         
           
               
            
               
                 mgsA::Km R consisting of 100 bp: (SEQ ID NO 23) 
               
               
                 
                   Tgtggaaatactgaaaaatctggatgtgccggtggcgagaaaaccgtaa 
                 
               
               
                   
               
               
                 
                   gaaacaggtggcgtttgccacctgtgcaata 
                   catatgaatatcctcctt 
                 
               
               
                   
               
               
                 
                   ag 
                 
               
            
           
         
       
         
         
           
             a region (underlined letters) homologous to the end of helD ORF, 
             a region (Bold letter) to amplified Kanamycin cassette. 
           
         
       
    
     The fragment obtained was introduced into the strain MG1655 mgsA*(H21Q) according to Protocol 1. The strain obtained was checked by PCR. The strain obtained is named  E. coli  mgsA*(H21Q)::Km 
     1.11.2. Construction of the Modified Strain  E. coli MG1655 mgsA*(H21Q)::Km Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101VB01-yqhD-gldA*(A160T)) 
     The replacement of the mgsA with the mgsA*(H21Q)::Km into the strain  E. coli  Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101VB01-yqhD-gldA*(A160T)) was performed by the technique of transduction with phage P1. IPTG was added to the culture to promote the expression of the genes borne on the plasmid. 
     The modifications mgsA*(H21Q)::Km and the others deletions were checked using the oligonucleotides described in Table 5. 
     The resulting strain was named  E. coli  MG1655 mgsA*(H21Q)::Km Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101VB01-yqhD-gldA*(A160T)) 
     2. Construction of the Modified Strain  E. coli  MG1655 mgsA*(H21Q)::Km, Ptrc01-gapA::cm, Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101-VB01-yqhD*(G149E)-gldA*(A160T)) 
     2.1. Construction of the Plasmid (pME101-VB01-yqhD*(G149E)-gldA*(A160T)) 
     2.1.1. Construction of the Plasmid pSCB-yqhD*(G149E) 
     The gene yqhD, PCR amplified from genomic DNA of  E. coli  MG1655 using the oligonucleotides yqhD F and yqhD R was cloned in pSCB (Strataclone®). The resulted plasmid was named pSCB-yqhD. A directed mutagenesis was performed on this plasmid with the following oligonucleotides: yqhD*G149EmutDirF (consisting of 45 pb, ggttcagaatccaacgcagaagcggtgatAtcccgtaaaaccacaggc, SEQ ID NO 24) and yqhD*G149EmutDirR (consisting of 45 pb gcctgtggttttacgggaTatcaccgcttctgcgttggattctgaacc, SEQ ID NO 25). The two oligonucleotides are homologous to the region 3153803 to 3153850. In bold face letter, bases which were changed to create the mutation G149E and capital letter, the base which were changed to create EcoRV restriction site. The resulted plasmid was named pSCB-yqhD*(G149E). 
     2.1.2. Construction of the Plasmid (pME101VB01-yqhD*(G149E)-gldA*(A160T) 
     The pSCB-yqhD*(G149E) was cut with the restriction enzymes BspHI and NheI and the fragment containing yqhD*(G149E) was cloned into the NcoI/NheI sites of the vector pME101VB01. The resulting plasmid was named pME101VB01-yqhD*(G149E). The pSCB-gldA*(A160T) was cut with the restriction enzymes avrII and SacI and the fragment containing gldA*(A160T) was cloned into the avrII/SacI sites of the vector pME101VB01-yqhD*(G149E). The resulted plasmid was named pME101VB01-yqhD*(G149S)-gldA*(A160T). 
     2.2. Construction of the Modified Strain  E. coli  MG1655 Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101-VB01-yqhD*(G149E)-gldA*(A160T)) 
     The plasmid pME101-VB01-yqhD*(G149E)-gldA*(A160T) was introduced by electroporation into the strain  E. coli  MG1655 Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh. 
     The strain obtained was named  E. coli  MG1655 Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101-VB01-yqhD*(G149E)-gldA*(A160T)) 
     2.3. Construction of the Modified Strain  E. coli  MG1655 mgsA*(H21Q)::Km, Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101-VB01-yqhD*(G149E)-gldA*(A160T)) 
     The replacement of the mgsA with the mgsA*(H21Q)::Km into the strain  E. coli  Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101VB01-yqhD*(G149E)-gldA*(A160T)) was performed by the technique of transduction with phage P1. IPTG was added to the culture to promote the expression of the genes borne on the plasmid. 
     The modifications mgsA*(H21Q)::Km and the others deletion were checked using the oligonucleotides described in Table 5. 
     The resulting strain was named  E. coli  MG1655 mgsA*(H21Q)::Km, Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101-VB01-yqhD*(G149E)-gldA*(A160T). 
     3. Assessment of 1,2-Propanediol Production in Two  E. coli  Isogenic Strains Differing Only in the yqhD Alleles 
     The two strains described above were cultivated in an Erlenmeyer flask assay (500 ml flasks with 50 ml of medium) under aerobic conditions in minimal medium MML11PG1 — 100 (see composition in Table 9) with 20 g/l glucose as sole carbon source. Spectinomycin was added at a concentration of 50 mg/l. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 composition of minimal medium MML11PG1_100. 
               
            
           
           
               
               
               
            
               
                   
                 Constituent 
                 Concentration (g/l) 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 EDTA 
                 0.0084 
               
               
                   
                 CoCl 2  6H 2 O 
                 0.0025 
               
               
                   
                 MnCl 2  4H 2 O 
                 0.0150 
               
               
                   
                 CuCl 2  2H 2 O 
                 0.0015 
               
               
                   
                 H 3 BO 3   
                 0.0030 
               
               
                   
                 Na 2 MoO 4  2H 2 O 
                 0.0025 
               
               
                   
                 Zn(CH 3 COO) 2  2H 2 O 
                 0.0130 
               
               
                   
                 Fe(III) citrate H 2 O 
                 0.1064 
               
               
                   
                 Citric acid 
                 1.70 
               
               
                   
                 KH 2 PO 4   
                 1.65 
               
               
                   
                 K 2 HPO 4  3H 2 O 
                 0.92 
               
               
                   
                 (NH 4 ) 2 HPO 4   
                 0.40 
               
               
                   
                 (NH 4 ) 2 SO 4   
                 4.88 
               
               
                   
                 MgSO 4  7H 2 O 
                 1.00 
               
               
                   
                 CaCl 2  2H 2 O 
                 0.08 
               
               
                   
                 Thiamine 
                 0.01 
               
               
                   
                 Glucose or Sucrose 
                 20.00 
               
               
                   
                 MOPS buffer 
                 40.00 
               
               
                   
                   
               
            
           
         
       
     
     The pH of the medium was adjusted to 6.8 with sodium hydroxide 
     The culture was carried out at 37° C. and the pH was maintained by buffering the culture medium with MOPS. 
     At the end of the culture, 1,2-propanediol and residual glucose in the fermentation broth were analysed by HPLC using a Biorad HPX 97H column for the separation and a refractometer for the detection. The biomass concentration was recorded as dry cell weight. The 1,2-propanediol specific productions were then calculated. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 production of 1,2-propanediol in minimal 
               
               
                 medium with glucose as carbon source. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 1,2- 
                 1,2-propanediol 
               
               
                   
                   
                 propanediol 
                 specific 
               
               
                   
                 Carbon 
                 titer 
                 production 
               
               
                 Strain/ 
                 source 
                 (g/l) 
                 (g/g biomass) 
               
               
                   
               
               
                   E. coli  MG1655 
                 glucose 
                 0.813 +/− 0.033 
                 0.569 +/− 0.015 
               
               
                 mgsA*(H21Q)::Km 
                   
                 (n = 3) 
                 (n = 3) 
               
               
                 ΔgloA Δedd-eda ΔaldA 
               
               
                 ΔaldB ΔarcA Δndh 
               
               
                 (pME101VB01-yqhD- 
               
               
                 gldA*(A160T)) 
               
               
                   E. coli  MG1655 
                 glucose 
                 0.904 +/− 0.049 
                 0.613 +/− 0.017 
               
               
                 mgsA*(H21Q)::Km 
                   
                 (n = 3) 
                 (n = 3) 
               
               
                 ΔgloA Δedd-eda ΔaldA 
               
               
                 ΔaldB ΔarcA Δndh 
               
               
                 (pME101VB01-yqhD* 
               
               
                 (G149E)-gldA*(A160T)) 
               
               
                   
               
               
                 n is the number of repetitions of the same experiment - The figures given are the mean and standard deviation of the figures obtained for n repetitions. 
               
            
           
         
       
     
     The titer and the specific production of 1,2-propanediol in the  E. coli  strain with a mutant YqhD were improved as compared with the isogenic strain with a native YqhD. 
     The improved properties of the mutant YqhD enzyme shown in Example 3 will lead to a more significant improvement of the performances of the strain with a mutant YqhD over the isogenic strain when compared in fed-batch culture. 
     Example 5 
     Production of 1,2-Propanediol by  E. coli  with a Mutant MGS, a Mutant YqhD and a Mutant GlyDH on Glucose and Sucrose 
     1. Construction of the Modified Strain  E. coli  MG1655 mgsA*(H21Q), Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101-VB01-yqhD*(G149E)-gldA*(A160T))) 
     The construction of this strain was described in example 4. 
     2. Construction of the Modified Strain  E. coli  MG1655 mgsA*(H21Q,) Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101-VB01-yqhD*(G149E)-gldA*(A160T)) (pBBR1MCS5-cscBKAR) 
     2.1. Construction of the Plasmid pBBR1MCS5-cscBKAR 
     The plasmid pKJL101.1 (Jahreis et al. (2002), J. Bacteriol. 184:5307-5316) was digested by EcoRI. The fragment containing the cscBKAR gene was cloned in pBBR1MCS5 (Kovach et al. (1995), Gene, 166 175-176) also digested by EcoRI. 
     The plasmid obtained was named pBBR1MCS5-cscBKAR. 
     2.2. Construction of the Modified Strain  E. coli  MG1655 mgsA*(H21Q), Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101-VB01-yqhD*(G149E)-gldA*(A160T)) (pBBR1MCS5-cscBKAR) 
     The plasmid pBBR1MCS5-cscBKAR was introduced by electroporation in the strain  E. coli  MG1655 mgsA*(H21Q), Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh. pME101-VB01-yqhD*(G149E)-gldA*(A160T)). 
     The strain obtained was named strain  E. coli  MG1655 mgsA*(H21Q), Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔarcA, Δndh (pME101-VB01-yqhD*(G149E)-gldA*(A160T)) (pBBR1MCS5-cscBKAR). 
     3. Assessment of 1,2-Propanediol Production in Two  E. coli  with a Mutant MGS, a Mutant YqhD and a Mutant GlyDH on Glucose and Sucrose 
     The two strains described above were cultivated in an Erlenmeyer flask assay (500 ml flasks with 50 ml of medium) under aerobic conditions in minimal medium MML11PG1 — 100 (see composition in Table 9) with 20 g/l glucose or sucrose as sole carbon source. Spectinomycin was added at a concentration of 50 mg/l. 
     The culture was carried out at 37° C. and the pH was maintained by buffering the culture medium with MOPS. 
     At the end of the culture, 1,2-propanediol and residual glucose or sucrose in the fermentation broth were analysed by HPLC using a Biorad HPX 97H column for the separation and a refractometer for the detection. The yields of 1,2-propanediol over glucose or sucrose were then calculated. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 production of 1,2-propanediol in minimal medium 
               
               
                 with glucose or sucrose as carbon source. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 1,2-propanediol 
               
               
                   
                   
                 1,2-propanediol 
                 yield 
               
               
                   
                 Carbon 
                 titer 
                 (g/g carbon 
               
               
                 Strain 
                 source 
                 (g/l) 
                 source) 
               
               
                   
               
               
                   E. coli  MG1655 
                 glucose 
                 0.904 +/− 0.049 
                 0.133 +/− 0.009 
               
               
                 mgsA*(H21Q)::Km 
                   
                 (n = 3) 
                 (n = 3) 
               
               
                 ΔgloA Δedd-eda ΔaldA 
               
               
                 ΔaldB ΔarcA Δndh 
               
               
                 (pME101VB01-yqhD* 
               
               
                 (G149E)-gldA*(A160T)) 
               
               
                   E. coli  MG1655 
                 sucrose 
                 1.823 +/− 0.098 
                 0.196 +/− 0.007 
               
               
                 mgsA*(H21Q)::Km 
                   
                 (n = 3) 
                 (n = 3) 
               
               
                 ΔgloA Δedd-eda ΔaldA 
               
               
                 ΔaldB ΔarcA Δndh 
               
               
                 (pME101VB01-yqhD* 
               
               
                 (G149E)-gldA*(A160T)) 
               
               
                 (pBBR1MCS5- 
               
               
                 cscBKAR) 
               
               
                   
               
               
                 n is the number of repetitions of the same experiment - The figures given are the mean and standard deviation of the figures obtained for n repetitions. 
               
            
           
         
       
     
     The production of 1,2-propanediol in a  E. coli  strain with a mutant YqhD was improved on sucrose as sole carbon source as compared with glucose. 
     Example 6 
     Construction of Two  E. coli  Acetol Producer Strains Expressing Wildtype or Modified YqhD and Assessment of Acetol Production 
     1. Construction of the Modified Strain  E. coli  MG1655 mgsA*(H21Q)::Km Ptrc01-gapA::cm, Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔgldA, (pME101-VB01-yqhD) 
     1.1. Construction of the Modified Strain  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA 
     1.1.1. Construction of the Modified Strain ΔgldA::Km 
     The gene gldA was inactivated in strain  E. coli  MG1655 by inserting a Kanamycin antibiotic cassette and deleting most of the gene concerned using the technique described in protocol 1 with the oligonucleotides given in Table 4. The deletion was checked by a PCR analysis with the appropriate oligonucleotides given in Table 5. The resulting strain was named  E. coli  MG1655 ΔgldA::Km. 
     1.1.2. Construction of the Modified Strain  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA::Km 
     The deletion of the gene gldA by replacement of the gene by a Kanamycine resistance cassette in the strain  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB, Δedd-eda, ΔgloA, ΔaldA, ΔaldB was performed by the technique of transduction with phage P1 (Protocol 2). 
     The deletion was checked by a PCR analysis with the appropriate oligonucleotides given in Table 5 as well as the others deletions already present in the strain. 
     The resulting strain was named  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA::Km 
     1.1.3. Construction of the Modified Strain  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA 
     The antibiotic resistance cassette was eliminated in the strain  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA::Km according to Protocol 3. 
     The loss of the antibiotic resistance cassette was checked by PCR analysis with the oligonucleotides given in Table 5. The presence of the modifications previously built in the strain was also checked using the oligonucleotides given in Table 5. 
     The strain obtained was named  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB, Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔgldA. 
     1.2. Construction of the Modified Strain  E. coli  MG1655 Ptrc01-gapA::cm Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA 
     1.2.1. Construction of the Modified Strain MG1655 Ptrc01-gapA::Cm 
     The replacement of the natural gapA promoter with the synthetic short Ptrc01 promoter (SEQ ID NO 26: gagctgttgactattaatcatccggctcgaataatgtgtgg) into the strain  E. coli  MG1655 was made by replacing 225 pb of upstream gapA sequence with FRT-CmR-FRT and an engineered promoter using the technique described in Protocol 2 with the oligonucleotides given in Table 4. 
     The deletion was checked by a PCR analysis with the appropriate oligonucleotides given in Table 5. The resulting strain was named  E. coli  MG1655 Ptrc01-gapA::Cm. 
     1.2.2. Construction of the Modified Strain  E. coli  MG1655 Ptrc01-gapA:: cm Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA 
     The replacement of the natural gapA promoter with the synthetic short Ptrc01 promoter into the strain  E. coli  MG1655 Δedd-eda ΔgloA, ΔaldA, ΔaldB, Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔgldA was performed by the technique of transduction with phage P1. 
     The modifications Ptrc01-gapA::cm and the others deletion were checked using the oligonucleotides described in Table 5. 
     The strain obtained was named  E. coli  MG1655 Ptrc01-gapA::cm Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA. 
     1.3. Construction of the Modified Strain  E. coli  MG1655 Ptrc01-gapA::cm Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA pME101-VB01-yqhD, 
     The plasmids pME101VB01-yqhD (see Example 4) was introduced by electroporation into the strain  E. coli  MG1655 Ptrc01-gapA:: cm Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA. 
     The strain obtained was named  E. coli  MG1655 Ptrc01-gapA::cm Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA (pME101-VB01-yqhD). 
     1.4. Construction of the Modified Strain  E. coli  MG1655 mgsA*(H21Q)::Km Ptrc01-gapA::cm Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA pME101-VB01-yqhD, 
     The replacement of the mgsA with the mgsA*(H21Q)::Km into the strain  E. coli  Δedd-eda ΔgloA, ΔaldA, ΔgldA (pME101VB01-yqhD) was performed by the technique of transduction with phage P1. IPTG was added to the culture to promote the expression of the gene borne on the plasmid. 
     The modifications mgsA*(H21Q)::Km and the others deletion were checked using the oligonucleotides described in Table 5. 
     2. Construction of the Strain  E. coli  MG1655 Ptrc01-gapA::cm Δedd-eda ΔgloA, ΔaldA, ΔaldB, Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔgldA (pME101-VB01-yqhD*(6149E) 
     2.1. Construction of the Modified Strain  E. coli  MG1655 Ptrc01-gapA::cm Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA pME101-VB01-yqhD*(G149E), 
     The plasmid pME101-VB01-yqhD*(G149E) (see Example 4) was introduced by electroporation in the strain  E. coli  MG1655 Ptrc01-gapA::cm Δedd-eda ΔgloA, ΔaldA, ΔaldB, Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔgldA. 
     The strain obtained was named  E. coli  MG1655 Ptrc01-gapA::cm Δedd-eda ΔgloA, ΔaldA, ΔaldB, Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔgldA pME101-VB01-yqhD*(G149E). 
     2.2. Construction of the Modified Strain  E. coli  MG1655 mgsA*(H21Q)::Km Ptrc01-gapA::cm Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA pME101-VB01-yqhD*(G149E), 
     The replacement of the mgsA with the mgsA*(H21Q)::Km into the strain  E. coli  Δedd-eda ΔgloA, ΔaldA, ΔgldA (pME101VB01-yqhD*(G149E), was performed by the technique of transduction with phage P1. IPTG was added to the culture to promote the expression of the gene borne on the plasmid. 
     The modifications mgsA*(H21Q)::Km and the others deletion were checked using the oligonucleotides described in Table 5. 
     The strain obtained was named  E. coli  MG1655 mgsA*(H21Q)::Km Ptrc01-gapA::cm Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA pME101-VB01-yqhD*(G149E). 
     3. Assessment of Acetol Production in Two  E. coli  Isogenic Strains Differing Only in the yqhD Alleles 
     The two strains described above were cultivated in an Erlenmeyer flask assay (500 ml flasks with 50 ml of medium) under aerobic conditions in minimal medium MML11PG1 — 100 (see composition in Table 9) with 20 g/l glucose as sole carbon source. Spectinomycin was added at a concentration of 50 mg/l. 
     The culture was carried out at 37° C. and the pH was maintained by buffering the culture medium with MOPS. 
     At the end of the culture, acetol and residual glucose in the fermentation broth were analysed by HPLC using a Biorad HPX 97H column for the separation and a refractometer for the detection. The biomass concentration was recorded as dry cell weight. The 1,2-propanediol specific productions were then calculated 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 production of acetol in minimal medium 
               
               
                 with glucose as carbon source. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 acetol specific 
               
               
                   
                 Carbon 
                 acetol titer 
                 production 
               
               
                 Strain 
                 source 
                 (g/l) 
                 (g/g biomass) 
               
               
                   
               
               
                   E. coli  MG1655 
                 glucose 
                 1.90 +/− 0.18 
                 0.933 +/− 0.043 
               
               
                 mgsA*(H21Q)::Km 
                   
                 (n = 2) 
                 (n = 2) 
               
               
                 ΔgloA Δedd-eda ΔaldA 
               
               
                 ΔaldB ΔgldA 
               
               
                 (pME101VB01-yqhD) 
               
               
                   E. coli  MG1655 
                 glucose 
                 2.23 +/− 0.23 
                 0.968 +/− 0.056 
               
               
                 mgsA*(H21Q)::Km 
                   
                 (n = 2) 
                 (n = 2) 
               
               
                 ΔgloA Δedd-eda ΔaldA 
               
               
                 ΔaldB ΔgldA 
               
               
                 (pME101VB01-yqhD* 
               
               
                 (G149E) 
               
               
                   
               
               
                 n is the number of cultures of different clones of the same strain - The figures given are the mean and standard deviation of the figures obtained for n cultures. 
               
            
           
         
       
     
     The titer and the specific production of acetol in the  E. coli  strain with a mutant YqhD were improved as compared with the isogenic strain with a native YqhD. 
     The improved properties of the mutant YqhD enzyme shown in Example 3 will lead to a more significant improvement of the performances of the strain with a mutant YqhD over the isogenic strain when compared in fed-batch culture. 
     Example 7 
     Construction of a  E. coli  Strain with a Mutant MGS and a Mutant YqhD 
     1. Construction of the Modified Strain  E. coli  MG1655 Ptrc01-gapA::cm, mgsA*(H21Q), Δedd-eda, ΔgloA, ΔaldA, ΔaldB, ΔgldA, pME101-VB01-yqhD*(G149E), (pJB137-PgapA-ppsA) 
     The construction of this strain was described in Example 6 
     2. Construction of the Modified Strain  E. coli  MG1655 Ptrc01-gapA::cm, mgsA*(H21Q), Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA pME101-VB01-yqhD*(G149E) pBBR1MCS5-cscBKAR 
     The plasmid pBBR1MCS5-cscBKAR (see Example 5) was introduced by electroporation in the strain  E. coli  MG1655 Ptrc01-gapA::cm, mgsA*(H21Q), Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA pME101-VB01-yqhD*(G149E). 
     The resulting strain was named  E. coli  MG1655 Ptrc01-gapA::cm, mgsA*(H21Q), Δedd-eda ΔgloA, ΔaldA, ΔaldB, ΔgldA pME101-VB01-yqhD*(G149E) pBBR1MCS5-cscBKAR. 
     Example 8 
     Construction of Two  E. coli  Ethylene Glycol Producing Strains Differing Only in the Overexpression of the Gene Coding for yqhD 
     1. Construction of the Modified Strain  E. coli  MG1655 ΔsdaA ΔsdaB ΔpykF Ptrcl8-gpmA Ptrcl8-gpmB (pME101-kivDll-yqhD-TT07) 
     The construction of the strain  E. coli  MG1655 ΔsdaA ΔsdaB ΔpykF Ptrcl8-gpmA Ptrcl8-gpmB pME101-kivDll-yqhD*(G149E)-TT07 is already described in patent application WO2010076324. 
     2. Construction of the Modified Strain  E. coli  MG1655 ΔsdaA ΔsdaB ΔpykF Ptrcl8-gpmA Ptrcl8-gpmB (pME101-kivDll-yqhD*(G149E)-TT07) 
     2.1. Construction of the Plasmid pME101-kivDll-yqhD*(G149E)-TT07 
     The PCR amplified fragment on genomic DNA  E. coli  MG1655 using the oligonucleotides yqhD Rbis and yqhD Fbis is cloned in pSCB(Strataclone®). 
     
       
         
           
               
               
            
               
                   
                 yqhD F bis (SEQ ID NO 27) 
               
               
                   
                 T  cccagcaaagggagcaagtaatgaacaac 
               
            
           
         
       
         
         
           
             a region for addition of a SnaBI restriction site (italic bold upper case) 
             a region (lower case) homologous to the  E. coli  MG1655 yqhD region from 3153357 to 3153385 
           
         
       
    
     
       
         
           
               
               
            
               
                   
                 yqhD R bis (SEQ ID NO 28) 
               
               
                   
                 a  TTAGCGGGCGGCTTCGTATATAC 
               
            
           
         
       
         
         
           
             a region (upper case) homologous to the  E. coli  MG1655 yqhD region from 3154540 to 3154518 
             a region for addition of a BglII restriction site (italic bold lower case). 
           
         
       
    
     The resulted plasmid is named pSCB-rbsyqhD. A directed mutagenesis is performed on this plasmid with the following oligonucleotides: yqhD*G149EmutDirF (SEQ ID NO 24) and yqhD*G149EmutDirR (SEQ ID NO 25). The two oligonucleotides are homologous to the region 3153803 to 3153850. In bold face letter, bases which are changed to create the mutation G149E and capital letter, the base which are change to create EcoRV restriction site. The resulting plasmid is named pSCB-rbsyqhD*(G149E). 
     The plasmid pSCB-rbsyqhD*(G149E) is cut with the restriction enzymes SnaBI and BglII. the fragment containing yqhD*(G149E) is cloned into the SnaBI-BgkII sites of the vector pME101-kivDll-TT07 (see WO2010076324) giving vector pME101-kivDll-yqhD*(G149E)-TT07. 
     2.2. Construction of the Modified Strain MG1655 ΔsdaA ΔsdaB ΔpykF Ptrcl8-gpmA Ptrcl8-gpmB (pME101-kivDll-yqhD*(G149E)-TT07) 
     The pME101-kivDll-yqhD*(G149E)-TT07 plasmid is then introduced into the strain MG1655 ΔsdaA ΔsdaB ΔpykF Ptrcl8-gpmA Ptrcl8-gpmB. 
     The strain obtained is named  E. coli  MG1655 ΔsdaA ΔsdaB ΔpykF Ptrcl8-gpmA Ptrcl8-gpmB pME101-kivDll-yqhD*(G149E)-TT07. 
     Example 9 
     Construction of Two  E. coli  1,4-Butanediol Producing Strains Differing Only in the Overexpression of the Gene Coding for YqhD 
     1. Construction of the Modified Strain  E. coli  MG1655 ΔsucCD ΔaceBAK ΔarcA ΔgdhA (pUC19-Ptrc01/OP01/RBS01-adhE2ca-prpE-TT02) (pME101-kivDll-yqhD-TT07) 
     The construction of the strain  E. coli  MG1655 ΔsucCD ΔaceBAK ΔarcA ΔgdhA (pUC19-Ptrc01/OP01/RBS01-adhE2ca-prpE-TT02) (pME101-kivDll-yqhD-TT07) is already described in patent application WO2010076324. 
     2. Construction of the Modified Strain  E. coli  MG1655 ΔsucCD ΔaceBAK ΔarcA ΔgdhA (pUC19-Ptrc01/OP01/RBS01-adhE2ca-prpE-TT02) (pME101-kivDll-yqhD*(G149E)-TT07) 
     The pUC19-Ptrc01/OP01/RBS01-adhE2ca-prpE-TTO2 (see WO2010076324) and the pME101-kivDll-*(G149E)-TT07 (see Example 8) plasmids are then introduced into the strain MG1655 ΔsucCD ΔaceBAK ΔarcA ΔgdhA. 
     The strain obtained is named  E. coli  MG1655 ΔsucCD ΔaceBAK ΔarcA ΔgdhA (pUC19-Ptrc01/OP01/RBS01-adhE2ca-prpE-TT02)(pME101-kivDll-yqhD*(G149E)-TT07) 
     Example 10 
     Construction of Two  Saccharomyces cerevisiae  1,2-Propanediol Producer Strains 
     1—Construction of Two  S. cerevisiae  Strains CENPK Δgpd2, Δtpi1, gldA*(A160T), yqhD, mgsA*(H21Q) and CENPK Δgpd2, Δtpi1, gldA*(A160T), yqhD*(G149E), mgsA*(H21Q) 
     1-1. Construction of  S. cerevisiae  Strain CENPK Δgpd2, gldA*(A160T). 
     The  S. cerevisiae  strain used was CEN.PK2-1C (MATa; ura3-52; trp1-289; leu2-3,112; his3 Δ1; MAL2-8C; SUC2) from Euroscarf. 
     The gene GPD2 was inactivated by transforming the strain CEN.PK2-1C with a PCR fragment corresponding to pTDH3-gldA*(A160T)-CYCt-pTEF1-ble-TEF1t cassette, built using the “short flanking homology” (SFH) method described by Guldener et al. (1996). 
     The pTDH3-gldA*(A160T)-CYCt-pTEF1-ble-TEF1t cassette was constructed using long PCR-based fusion of several fragments as described by Shevchuk et al. (2004). 
     pTDH3 and CYCt were amplified from the plasmid p406TDH3 (Addgene) using pTDH3/GPD2 F and pTDH3 R primers and CYCt/gldA F and CYCt/Zeo R primers respectively. gldA*(A160T)—was amplified from pSCB gldA*(A160T)—using primers gldA/TDH3F and gldA/CYCtR. 
     pTEF1-ble-TEF1t was amplified from the plasmid pUG66 from Euroscarf using Zeo/CYCt F and ZEO/GPD2 R as primers. 
     All fragments were amplified using primers having overlapping ends as described in Table 13. Each fragment was then purified. 
     100 ng of each fragment was used in a PCR experiment without primers, using low annealing conditions allowing their simultaneous fusion. 
     The unpurified product obtained in this step was used as a matrix a in a PCR experiment at high Tm, using pTDH3/GPD2 F and ZEO/GPD2 R primers having an extension of 40 bp homologous to the 40 first and 40 last by of the GPD2 locus (Table 13). 
     This fragments was integrated in the GPD2 locus, replacing the GPD2 open reading frame. The transformation method used was the lithium acetate method described by Schiestl and Gietz (1989). The strain CENPK, Δgpd2, gldA*(A160T) was selected on YEPD rich medium (1% bacto yeast extract, 2% bactopeptone, 2% glucose) supplemented with 75 μg/ml of phleomycin (Cayla, France). The integration of gldA*(A160T) and the deletion of GPD2 gene were confirmed by PCR on genomic DNA extracted, using GPD2 ver F and GPD2 ver R primers (Table 13). 
     This resulted in the heterologous expression of gldA*(A160T) and deletion of GPD2. The resulting strain was named CENPK Δgpd2, gldA*(A160T). 
     
       
         
           
               
               
               
             
               
                 TABLE 13 
               
               
                   
               
               
                 Primer Name 
                 Sequence 
                 Description 
               
               
                   
               
             
            
               
                 pTDH3/GPD2 F 
                 
                   ATG CTT GCT GTC AGA AGA TTA ACA 
                 
                 The underlined 40 
               
               
                   
                 
                   AGA TAC ACA TTC CTT A 
                   GT TTA TCA 
                 
                 nucleotides correspond to 
               
               
                   
                 
                   TTA TCA ATA CTC G 
                 
                 the 40 first bp of GPD2 
               
               
                   
                 (SEQ ID NO 29) 
                 gene 
               
               
                   
                   
                 21 nucleotides in bold are 
               
               
                   
                   
                 homologous to the 21 first 
               
               
                   
                   
                 bp of pTDH3 
               
               
                   
               
               
                 pTDH3 R 
                 
                   ATCCTCGAAACTAAGTTCTTGGT 
                 
                 23 nucleotides homologous 
               
               
                   
                 (SEQ ID NO 30) 
                 to the 23 last bp of pTDH3 
               
               
                   
               
               
                 gldA/TDH3F 
                 
                   AAA CAC CAG AAC TTA GTT TCG A 
                   AC 
                 
                 The underlined 22 
               
               
                   
                 
                   TAG TTT ATT CCC ACT CTT 
                 
                 nucleotides underlined 
               
               
                   
                 (SEQ ID NO 31) 
                 correspond to the last bp 
               
               
                   
                   
                 of pTDH3 
               
               
                   
                   
                 20 nucleotides in bold are 
               
               
                   
                   
                 homologous to the 20 first 
               
               
                   
                   
                 bp of gldA* (A160T) 
               
               
                   
               
               
                 gldA/CYCtR 
                 
                   TGA AAT ATA AAT AAC GTT CTT AAT 
                 
                 The underlined 42 
               
               
                   
                 
                   ACT AAC ATA ACT ATA AAC  
                   TAG TAT 
                 
                 nucleotides correspond to 
               
               
                   
                 
                   GGA CCG CAT TAT TC 
                 
                 the 42 first bp of CYCt 
               
               
                   
                 (SEQ ID NO 32) 
                 20 nucleotides in bold are 
               
               
                   
                   
                 homologous to the 20 last 
               
               
                   
                   
                 bp of gldA*(A160T) 
               
               
                   
               
               
                 CYCt/gldA F 
                 
                   CCT GGA TGT ATT TAC CCG GTG ATT 
                 
                 The underlined 46 
               
               
                   
                 
                   GAA TAA TGC GGT CCA TAC TAG T 
                   TT 
                 
                 nucleotides correspond to 
               
               
                   
                 
                   ATA GTT ATG TTA GTA TTA 
                 
                 the 46 last bp of gldA or 
               
               
                   
                 (SEQ ID NO 33) 
                 gldA 
               
               
                   
                   
                 20 nucleotides in bold are 
               
               
                   
                   
                 homologous to the 20 first 
               
               
                   
                   
                 bp of CYCt 
               
               
                   
               
               
                 CYCt/Zeo R 
                 
                   GAG GCA AGC TAA ACA GAT CTC TAG 
                 
                 The underlined 40 
               
               
                   
                 
                   ACC TAG GTA CCC GCC G 
                   GC AAA TTA 
                 
                 nucleotides correspondto 
               
               
                   
                 
                   AAG CCT TCG AGC 
                 
                 the 40 first bp of ble gene 
               
               
                   
                 (SEQ ID NO 34) 
                 20 nucleotides in bold are 
               
               
                   
                   
                 homologous to the 20 last 
               
               
                   
                   
                 bp of CYCt 
               
               
                   
               
               
                 Zeo/CYCt F 
                 
                   GCT TGA GAA GGT TTT GGG ACG CTC 
                 
                 The underlined 40 
               
               
                   
                 
                   GAA GGC TTT AAT TTG C 
                   TA GGT CTA 
                 
                 nucleotides correspond to 
               
               
                   
                 
                   GAG ATC TGT TTA GC 
                 
                 the 40 last bp of CYCt 
               
               
                   
                 (SEQ ID NO 35) 
                 22 nucleotides in bold are 
               
               
                   
                   
                 homologous to the 22 first 
               
               
                   
                   
                 bp of TEF1p 
               
               
                   
               
               
                 ZEO/GPD2 R 
                 
                   CTA TTC GTC ATC GAT GTC TAG CTC 
                 
                 The underlined 40 
               
               
                   
                 
                   TTC AAT CAT CTC CGG T 
                   CC ACT AGT 
                 
                 nucleotides correspond to 
               
               
                   
                 
                   GGA TCT GAT ATC ACC T 
                 
                 the 40 last bp of GPD2 gene 
               
               
                   
                 (SEQ ID NO 36) 
                 24 nucleotides in bold are 
               
               
                   
                   
                 homologous to the 24 last 
               
               
                   
                   
                 bp of TEF1t 
               
               
                   
               
               
                 GPD2 ver F 
                 ATG CTT GCT GTC AGA AGA TT 
                 20 nucleotides homologous 
               
               
                   
                 (SEQ ID NO 37) 
                 to the 20 first bp of GPD2 
               
               
                   
                   
                 gene 
               
               
                   
               
               
                 GPD2 ver R 
                 TAG TAT GGA CCG CAT TAT TC 
                 20 nucleotides homologous 
               
               
                   
                 (SEQ ID NO 38) 
                 to the 20 last nucleotides 
               
               
                   
                   
                 of gldA*(A160T) 
               
               
                   
               
            
           
         
       
     
     1-2. Construction of Two  S. cerevisiae  Strains CENPK Δgpd2, gldA*(A160T), yqhD and CENPK Δgpd2, gldA*(A160T), yqhD*(G149E) 
     The strain used was CENPK, Δgpd2, gldA*(A160T), previously built. The expression of yqhD or yqhD* (G149E) was realised by transforming the strains with a PCR fragment corresponding to a pTEF1-yqhD-CYCt-pTEF1-nat1-TEF1t cassette or pTEF1-yqhD* (G149E)-CYCt-pTEF1-nat1-TEF1t cassette using the “short flanking homology” (SFH) method. 
     The pTEF1-yqhD-CYCt-pTEF1-nat1-TEF1t cassette or pTEF1-yqhD* (G149E)-CYCt-pTEF1-nat1-TEF1t cassette were constructed using long PCR-based fusion of several fragments. 
     pTEF1 and CYCt were amplified from the plasmid p405TEF1 (Addgene) using pTEF1/URA3 F and pTEF R primers and CYCt/yqhD F and CYCt/Nat1R primers respectively. yqhD and yqhD* were amplified respectively from pSCB-yqhD and pSCB yqhD* (G149E) using primers yqhD/TEF-F and yqhD/CYCtR. 
     pTEF1-nat1-TEF1t was amplified from the plasmid pAG35 from Euroscarf using Nat1/CYCt F and Nat1/Leu2 as primers. 
     All fragments were amplified using primers having overlapping ends as described in Table 14. Each fragment was then purified. 
     100 ng of each fragment was used in a PCR experiment without primers using low annealing conditions allowing their simultaneous fusion. 
     The unpurified product obtained in this step was used as a matrix in a PCR experiment with at high Tm, using pTEF1/LEU2 F and Nat1/Leu2 primers having an extension of 40 bp homologous to the 40 first and 40 last bp of the LEU2 locus (Table 14). 
     These fragments were integrated in the LEU2 locus, replacing the LEU2 open reading frame. 
     The transformation method used was the lithium acetate method. The strain CENPK, Δgpd2, gldA*(A160T) was transformed either by pTEF1-yqhD-CYCt-pTEF1-nat1-TEF1t cassette or by pTEF1-yqhD* (G149E)-CYCt-pTEF1-nat1-TEF1t to obtained CENPK, Δgpd2, gldA* (A160T), yqhD and CENPK, Δgpd2, gldA* (A160T), yqhD* (G149E). Transformants were selected on YEPD rich medium (1% bacto yeast extract, 2% bactopeptone, 2% glucose) supplemented with 50 μg/ml of nourseothricine (Weber bioagents, Germany). The integration of yqhD or yqhD* (G149E) were confirmed by PCR on genomic DNA extracted, using YQHD ver F and YQHD ver R primers (Table 14). This resulted in the heterologous expression of yqhD and yqhD* (G149E). The resulting strains were named CENPK Δgpd2, gldA*(A160T), yqhD and CENPK Δgpd2, gldA*(A160T), yqhD*(G149E). 
     
       
         
           
               
               
               
             
               
                 TABLE 14 
               
               
                   
               
               
                 Primer Name 
                 Sequence 
                 Description 
               
               
                   
               
             
            
               
                 pTEF1/URA3 F 
                 
                   ATG TCT GCC CCT AAG AAG ATC 
                 
                 The underlined 40 nucleotides 
               
               
                   
                 
                   GTC GTT TTG CCA GGT GAC C 
                   AG 
                 
                 correspond to the 40 first bp of 
               
               
                   
                 
                   CTG GAG CTC ATA GCT TCA 
                 
                 LEU2 gene 
               
               
                   
                 (SEQ ID NO 39) 
                 20 nucleotides in bold are 
               
               
                   
                   
                 homologous to the 20 first bp pTEF1 
               
               
                   
               
               
                 pTEF R 
                 
                   TCC GGG TTG GGG TGT GCA GAT 
                 
                 The underlined 40 nucleotides 
               
               
                   
                 
                   TAA AGT TGT TCA TAC TAG T 
                   GG 
                 
                 correspond to the 40 first bp of 
               
               
                   
                 
                   ATC CAC TAG TTC TAG AAA 
                 
                 yqhD or yqhD*(G419E) 
               
               
                   
                 (SEQ ID NO 40) 
                 20 nucleotides bold are homologous 
               
               
                   
                   
                 to the20 last bp of pTEF1 
               
               
                   
               
               
                 yqhD/TEF-F 
                 
                   CAT AGC AAT CTA ATC TAA GTT 
                 
                 The underlined 40 nucleotides 
               
               
                   
                 
                   TTC TAG AAC TAG T 
                   GG ATC CAC 
                 
                 correspond to the 40 last bp of 
               
               
                   
                 
                   TAG TAT GAA CAA CTT TAA 
                 
                 pTEF1 
               
               
                   
                 (SEQ ID NO 41) 
                 20 nucleotides in bold are 
               
               
                   
                   
                 homologous to the 20 first bp of 
               
               
                   
                   
                 yqhD or yqhD*(G419E) 
               
               
                   
               
               
                 yqhD/CYCtR 
                 
                   TGA AAT ATA AAT AAC GTT CTT 
                 
                 The underlined 40 nucleotides 
               
               
                   
                 
                   AAT ACT AAC ATA ACT ATA A 
                   AC 
                 
                 correspond to the 40 first bp of 
               
               
                   
                 
                   TAG TTT AGC GGG CGG CTT 
                 
                 CYCt 
               
               
                   
                 (SEQ ID NO 42) 
                 20 nucleotides in bold are 
               
               
                   
                   
                 homologous to the 20 last bp of yqhD 
               
               
                   
                   
                 or yqhD*(G419E) 
               
               
                   
               
               
                 CYCt/yqhD F 
                 
                   TGT CAG CCG CCG TAT ATA CGA 
                 
                 The underlined 40 nucleotides 
               
               
                   
                 
                   AGC CGC CCG CTA AAC TAG T 
                   TT 
                 
                 correspond to the 40 last bp of yqhD 
               
               
                   
                 
                   ATA GTT ATG TTA GTA TTA 
                 
                 or yqhD*(G419E) 
               
               
                   
                 (SEQ ID NO 43) 
                 20 nucleotides in bold are 
               
               
                   
                   
                 homologous to the 20 first bp of 
               
               
                   
                   
                 CYCt 
               
               
                   
               
               
                 CYCt/Nat1R 
                 
                   CTCCATGTCGCTGGCCGGGTGACCCGG 
                 
                 Theunderlined 40 nucleotides 
               
               
                   
                 
                   CGGGGACGAGGCA 
                   GCAAATTAAA 
                 
                 correspondto the 40 first bp of 
               
               
                   
                 
                   GCCTTCGAGC 
                 
                 pTEF1 
               
               
                   
                 (SEQ ID NO 44) 
                 20 nucleotides in bold are 
               
               
                   
                   
                 homologous to the 20 last bp of CYCt 
               
               
                   
               
               
                 Nat1/CYCt F 
                 
                   GCT TGA GAA GGT TTT GGG ACG 
                 
                 The underlined 40 nucleotides 
               
               
                   
                 
                   CTC GAA GGC TTT AAT TTG C 
                   TG 
                 
                 correspond to the 40 last bp of CYCt 
               
               
                   
                 
                   CCT CGT CCC CGC CGG GTC 
                 
                 20 nucleotides in bold are 
               
               
                   
                 (SEQ ID NO 45) 
                 homologous to the 20 first bp of 
               
               
                   
                   
                 pTEF1 
               
               
                   
               
               
                 Nat1/Leu2 
                 
                   TTA AGC AAG GAT TTT CTT AAC 
                 
                 The underlined 40 nucleotides 
               
               
                   
                 
                   TTC TTC GGC GAC AGC ATC A 
                   CA 
                 
                 correspond to the 40 last bp of LEU2 
               
               
                   
                 
                   GTA TAG CGA CCA GCA TTC 
                 
                 gene 
               
               
                   
                 (SEQ ID NO 46) 
                 20 nucleotides in bold are 
               
               
                   
                   
                 homologous to the 20 last bp of 
               
               
                   
                   
                 TEF1t 
               
               
                   
               
               
                 YQHD ver F 
                 ATG TCT GCC CCT AAG AAG ATC 
                 20 nucleotides homologous to the 20 
               
               
                   
                 (SEQ ID NO 47) 
                 first bp of LEU2 gene 
               
               
                   
               
               
                 YQHD ver R 
                 AC TAG TTT AGC GGG CGG CTT 
                 20 nucleotides homologous to the 20 
               
               
                   
                 (SEQ ID NO 48) 
                 last bp of yqhD or yqhD* (G149E) 
               
               
                   
               
            
           
         
       
     
     1-3. Construction of Two  S. cerevisiae  Strain CENPK Δgpd2, Δtpi1, gldA*(A160T), yqhD, mgsA*(H21Q) and CENPK Δgpd2, Δtpi1, gldA*(A160T), yqhD*(G149E), mgsA*(H21Q) 
     The two strains used was CENPK, Δgpd2, gldA*(A160T), yqhD or CENPK, Δgpd2, gldA*(A160T), yqhD*, previously built. 
     The gene TPI1 was inactivated by transforming the strains with a PCR fragment corresponding to a pTEF1-hph-TEF1t-pPGK1-msgA*(H21Q) cassette using the “short flanking homology” (SFH) method. 
     The pTEF1-hph-TEF1t-pPGK1-msgA*(H21Q) cassette was constructed using long PCR-based fusion of several fragments. 
     The pTEF1-hph-TEF1t-pPGK1 were amplified from the plasmid pAG35pPGK1 constructed from pAG35 (Euroscarf) using PGK1/TPI1F and PGK1/mgsAR mgsA*(H21Q) was amplified from pETTOPO mgsA*(H21Q) using the primers mgsA/PGK1F and mgsA/TPI R as primers. 
     All fragments were amplified using primers having overlapping ends as described in Table 15. Each fragment was then purified. 
     100 ng of each fragment was used in a PCR experiment without primers and using low annealing conditions, allowing their simultaneous fusion. 
     The unpurified product obtained in this step was used as a matrix in a PCR experiment at high Tm, using PGK1/TPI1F and mgsA/TPI R primers having an extension of 40 bp homologous to the 40 first and 40 last by of the TPI1 locus (Table 15). 
     This fragment was integrated in the TPI1 locus, replacing TPI1 open reading frame. 
     The transformation method used is the lithium acetate method. The strain CENPK, Δgpd2, gldA*(A160T), yqhD and the strain CENPK, Δgpd2, gldA*(A160T), yqhD* (G149E) was transformed by pTEF1-hph-TEF1t-pPGK1-msgA*(H21Q) cassette to obtained CENPK, Δgpd2, Δtpi1, gldA*(A160T), yqhD* (G419E), msgA*(H21Q), CENPK, Δgpd2, Δtpi1, gldA*(A16 Transformants were selected on YEPD rich medium (1% bacto yeast extract, 2% bactopeptone, 2% glucose) supplemented with 250 μg/ml of hygromycin (Sigma-Aldrich). 
     The integration of msgA*(H21Q) was confirmed by PCR on genomic DNA extracted, using mgsA ver F and mgsA ver R primers (Table 15). 
     This resulted in the heterologous expression of mgsA*(H21Q) and deletion of TPI1. The resulting strains were named CENPK Δgpd2, Δtpi1, gldA*(A160T), yqhD, mgsA*(H21Q) and CENPK Δgpd2, Δtpi1, gldA*(A160T), yqhD*(G149E), mgsA*(H21Q). 
     
       
         
           
               
               
               
             
               
                 TABLE 15 
               
               
                   
               
               
                 Primer Name 
                 Sequence 
                 Description 
               
               
                   
               
             
            
               
                 PG1/TPI1F 
                 
                   ATG TCG AAA GCT ACA TAT AAG 
                 
                 The underlined 40 nucleotides 
               
               
                   
                 
                   GAA CGT GCT GCT ACT CAT C 
                   GC 
                 
                 correspond to the 40 first bp of 
               
               
                   
                 
                   CAG ATC TGT TTA GCT TGC 
                 
                 TPI1 gene 
               
               
                   
                 (SEQ ID NO 49) 
                 20 nucleotides in bold are 
               
               
                   
                   
                 homologous to 20 the first bp pTEF1 
               
               
                   
               
               
                 PGK1/mgsAR 
                 
                   GTG CGA GTC GTC AGT TCC ATA 
                 
                 The underlined 21 nucleotides 
               
               
                   
                 
                   ATA CGC AAA CCG CCT CTC C 
                 
                 correspond to the 40 first bp of 
               
               
                   
                 (SEQ ID NO 50) 
                 mgsA or msgA*(H21Q) 
               
               
                   
                   
                 19 nucleotides in bold are 
               
               
                   
                   
                 homologous to 20 the last bp of 
               
               
                   
                   
                 pPGK1 
               
               
                   
               
               
                 mgsA/PGK1F 
                 
                   GGA GAG GCG GTT TGC GTA TTA 
                 
                 The underlined 22 nucleotides 
               
               
                   
                 
                   T 
                   GG AAC TGA CGA CTC GCA C 
                 
                 correspond to the 40 last bp of 
               
               
                   
                 (SEQ ID NO 51) 
                 pPGK1 
               
               
                   
                   
                 18 nucleotides in bold are 
               
               
                   
                   
                 homologous to the 20 first bp of 
               
               
                   
                   
                 mgsA or msgA*(H21Q) 
               
               
                   
               
               
                 mgsA/TPI R 
                 
                   TTA GTT TTG CTG GCC GCA TCT 
                 
                 The underlined 40 nucleotides 
               
               
                   
                 
                   TCT CAA ATA TGC TTC CCT T 
                   TA 
                 
                 correspondto the 40 last bp of TPI1 
               
               
                   
                 
                   CTT CAG ACG GTC CGC GAG 
                 
                 20 nucleotides in bold are 
               
               
                   
                 (SEQ ID NO 52) 
                 homologous to the 20 last bp of mgsA 
               
               
                   
                   
                 or msgA*(H21Q) 
               
               
                   
               
               
                 mgsA ver F 
                 GG AAC TGA CGA CTC GCA C 
                 20 nucleotides homologous to the 20 
               
               
                   
                 (SEQ ID NO 53) 
                 first bp of TPI1 gene 
               
               
                   
               
               
                 mgsA ver R 
                 TTAGTTTCTAGAGTTGATGA 
                 20 nucleotides homologous to the 20 
               
               
                   
                 (SEQ ID NO 54) 
                 last bp of msgA*(H21Q) 
               
               
                   
               
            
           
         
       
     
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