BIOCATALYSTS FOR PRODUCTION OF D-LACTIC ACID FROM GLYCEROL TECHNICAL FIELD

The present invention relates to a method for producing D-lactic acid from glycerol with high yield, productivity and purity by using a bacterium, and a bacterium capable of producing D- lactic acid by the fermentation of glycerol with high yield, productivity and purity.

BACKGROUND ART

[0001] Lactic acid, or 2-hydroxypropionic acid, is a versatile industrial chemical that is a common product of natural bioprocesses. The presence of both hydroxyl and carboxylic acid functional groups in lactic acid enable it to undergo a variety of industrially useful chemical reactions such as esterification with organic acids, dehydration to lactide and acrylic acid and hydrogenation to propylene glycol. Lactic acid occurs in three forms, namely, laevorotatory L (+) lactic acid, dextrorotatory D (-) lactic acid, and the optically inactive racemic mixture DL-lactic acid.

[0002] Lactic acid is produced commercially typically for its use in the polymer industry for making polylactic acid (PLA) polymers.

Lactic acid can be manufactured both by chemical synthesis and by microbial fermentation methods. Although many microorganisms are able to produce lactic acid by fermentation, the lactic acid bacteria belonging to the genus Lactobacillus are amongst the best studied for homofermentative lactic acid production. However, in spite of their high lactate production rates and acid tolerance, the industrial applicability of lactic acid bacteria is so far limited due to their high nutrient demand and low growth rates. Additionally, formation of by-products such as acetic acid, ethanol, acetoin etc. lowers the overall yield and purity of lactate produced. Genetic modification of lactic acid bacteria to reduce by-products formation has so far only led to marginal improvements in lactate yield and production rates.

[0003] Alternative promising hosts include strains of Rhizopus, Aspergillus, Saccharomyces, Kluyveromyces, Bacillus and Escherichia coli. Of these, E. coli, the workhorse of modern biotechnology, possesses many advantages such as simplicity of use, safety, elucidated genome information and having well-established genetic manipulation techniques to produce recombinant proteins and to knockout by-product pathways. However, during sugar fermentation, E. coli essentially carries out mixed-acid fermentation to produce formate, acetate, succinate and ethanol as major products in addition to D-lactate. These by-products with functional groups such as hydroxyl group and/or carboxyl group are known to hinder the polymerization of lactic acid. Disruption of pathways that lead to by-products formation along with amplification of the pathway leading to D-lactate formation in E. coli can enable it to carry out homolactate fermentations from glucose (Chang et al., 1999; Zhou et al., 2003; Wada et al., 2007; Zhu et al, 2007). Reduction of by-product formation is important from an industrial perspective. For example, acetic acid, carrying a carboxyl group, can be incorporated as a monomer during polymerization, and terminates the polymerization. Therefore, the presence of acetic acid affects the property of the resulting lactic acid polymer through the termination of the polymerization. The influence of acetic acid on thermal stability of the lactic acid polymer has been reported (Japanese Patent Application Laid-Open (JP-A) No. 2012-12322).

[0004] E. coli can readily utilize a variety of biomass-derived hexose and pentose sugars for growth and fermentation. Other than sugars such as glucose and xylose, E. coli can also metabolize glycerol as a primary carbon source for its growth and production of fermentation products. However, obtaining high productivity of D-lactate from glycerol is still a challenge.

[0005] A method for producing lactic acid from glycerol under microaerobic conditions is described in patent document WO 2010/051324 Al and in Mazumdar et al., 2010. For the case of lactic acid production, overexpression of the E. coli endogenous aerobic pathway including glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (glpD) and the anaerobic pathway including glycerol dehydrogenase (gldA) and phosphoenolpyruvate (PEP) dependent- dihydroxyacetone kinase (dhaKLM), both of which lead to the formation of dihydroxyacetone phosphate (DHAP) from glycerol, were compared. In addition, acetate, succinate and ethanol byproduct pathways were disrupted by deletions or mutations in pta gene involved in acetate formation, the frdABCD operon involved in succinate formation and the adhe gene involved in ethanol formation, respectively. Also, the use of microaerobic conditions was described to reduce co factor imbalance caused by glycerol metabolism. The highest titer of D-lactic acid reported by Mazumdar et al. using overexpressed glpK-glpD route was 32.3 g/L in 60 h with a product yield of 0.83 g of D-lactate/g of glycerol consumed.

Patent document US 2007/0065930 Al describes the engineering of E. coli to ferment glucose to D-lactic acid. A strain was constructed by disrupting the pyruvate formate lyase (pfl), malate dehydrogenase (mdh), aspartate ammonia lyase (aspA) and FAD-dependent D-lactate dehydrogenase (did) genes and enhancing the activity of ldhA by means of replacing the native promoter of ldhA on the genome of E. coli with a constitutively expressed glycolytic promoter. D-lactic acid was selectively produced to a concentration of 98 g/L with reduced by-product formation from glucose, but the fermentation from glycerol was not reported here.

[0006] For the anaerobic route, it has been reported that the simultaneous overexpression of both enzymes of the two-step anaerobic glycerol-metabolic pathway is necessary for any observable improvement of glycerol consumption in E. coli (Yazdani and Gonzalez, 2008). In this report, a 3.4-fold increase in amount of glycerol fermented by E. coli was observed by simultaneously overexpressing gldA and dhaKLM enzymes. However, individual overexpression of either gldA or dhaKLM did not have a significant effect on glycerol fermentation.

[0007] A method for producing succinic acid from glycerol by E. coli has been reported

(Gonzalez et ah, 2008). In the report, the method of introducing ATP-dependent

dihydroxyacetone kinase was employed so that PEP would not be used as the phosphate group donor for phosphorylation of DHA to DHAP with concomitant conversion of PEP to pyruvic acid. PEP is an intermediate in the succinic acid synthetic route and its conversion to pyruvic acid, which is not an intermediate in the succinic acid synthetic route, is not favorable for succinic acid production.

DISCLOSURE OF INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0008] As described above, conventional methods using the existing glycerol-fermenting and D- lactate-producing bacteria have low productivity of D-lactate. In order for the commercial production of D-lactate from glycerol to be feasible, higher titers and shorter production times are necessary. Hence, further engineering in terms of strain and/or process development is required to establish a method that is industrially feasible.

Another equally important aspect from an industrial point-of-view is to minimize the formation of by-products which would otherwise require additional purification processes for removal and the resulting increase in manufacturing costs would challenge the economic feasibility of the fermentation process.

[0009] The present invention was made under the above-described circumstances, and aims to provide a method for producing D-lactate from glycerol by which high titers of D-lactate can be produced from glycerol with low production of by-product organic acids acetate and succinate, and which uses a bacterium, and to provide a bacterium which can produce high titers of D- lactate from glycerol with low production of by-product organic acids acetate and succinate. MEANS FOR SOLVING THE PROBLEMS

[0010] More specifically, aspects of the present invention include the following.

<1> A method for producing D-lactic acid, comprising contacting a lactic acid-producing bacterium with a glycerol-containing carbon source,

wherein the lactic acid-producing bacterium comprises an imparted or overexpressed NADH-dependent D-lactate dehydrogenase gene and an imparted or overexpressed ATP- dependent dihydroxyacetone kinase gene, and, in the lactic acid-producing bacterium, expression of an endogenous malate dehydrogenase gene is reduced or inactivated, and a NAD ⁺ -dependent glycerol dehydrogenase gene is neither imparted nor overexpressed.

<2> The method for producing D-lactic acid as described in <1>, wherein the lactic acid- producing bacterium further comprises an imparted or overexpressed triosephosphate isomerase gene.

<3> The method for producing D-lactic acid as described in <1> or <2>, wherein, in the lactic acid-producing bacterium, expression of an endogenous gene selected from the group consisting of pyruvate formate lyase, FAD-dependent D-lactate dehydrogenase and aspartate ammonia lyase is reduced or inactivated.

<4> The method for producing D-lactic acid as described in any one of <1> to <3>, wherein the ATP-dependent dihydroxyacetone kinase gene is derived from a microorganism.

<5> The method for producing D-lactic acid as described in any one of <1> to <4>, wherein the ATP-dependent dihydroxyacetone kinase gene is derived from a bacterium or a eukaryote. <6> The method for producing D-lactic acid as described in any one of <1> to <5>, wherein the ATP-dependent dihydroxyacetone kinase gene is derived from a microorganism belonging to a genus selected from the group consisting of Klebsiella, Enterobacter, Citrobacter, Shigella, Rahnella, Pantoea, Rhizobium, Rhodobacter, Saccharomyces, Schizosaccharomyces and

Candida.

<7> The method for producing D-lactic acid as described in any one of <1> to <6>, wherein the ATP-dependent dihydroxyacetone kinase gene is derived from an organism selected from the group consisting of Citrobacter freundii, Klebsiella pneumoniae, Enterobacter aerogenes and Citrobacter youngae. <8> The method for producing D-lactic acid as described in any one of <1> to <7>, wherein the lactic acid-producing bacterium is a bacterium of the genus Escherichia.

<9> A lactic acid-producing bacterium comprising an imparted or overexpressed NADH- dependent D-lactate dehydrogenase gene and an imparted or overexpressed ATP-dependent dihydroxyacetone kinase gene, wherein expression of an endogenous malate dehydrogenase gene is reduced or inactivated, and a NAD ⁺ -dependent glycerol dehydrogenase gene is neither imparted nor overexpressed.

<10> The lactic acid-producing bacterium as described in <9>, further comprising an imparted or overexpressed triosephosphate isomerase gene.

<11> The lactic acid-producing bacterium as described in <9> or <10>, wherein expression of an endogenous gene selected from the group consisting of pyruvate formate lyase, FAD- dependent D-lactate dehydrogenase and aspartate ammonia lyase is reduced or inactivated.

<12> The lactic acid-producing bacterium as described in any one of <9> to <11>, wherein the ATP-dependent dihydroxyacetone kinase gene is derived from a microorganism.

<13> The lactic acid-producing bacterium as described in any one of <9> to <12>, wherein the ATP-dependent dihydroxyacetone kinase gene is derived from a bacterium or a eukaryote.

<14> The method for producing D-lactic acid as described in any one of <9> to <13>, wherein the ATP-dependent dihydroxyacetone kinase gene is derived from a microorganism belonging to a genus selected from the group consisting of Klebsiella, Enterobacter, Citrobacter, Shigella, Rahnella, Pantoea, Rhizobium, Rhodobacter, Saccharomyces, Schizosaccharomyces and Candida.

<15> The method for producing D-lactic acid as described in any one of <9> to <14>, wherein the ATP-dependent dihydroxyacetone kinase gene is derived from an organism selected from the group consisting of Citrobacter fre ndii, Klebsiella pneumoniae, Enterobacter aerogenes and Citrobacter youngae .

<16> The lactic acid-producing bacterium as described in any one of <9> to <15>, which is a bacterium of the genus Escherichia.

ADVANTAGEOUS EFFECTS OF INVENTION

[0011] According to the present invention, a method for producing D-lactate from glycerol by which high titers of D-lactate can be produced from glycerol with low production of by-product organic acids acetate and succinate, and which uses a bacterium, can be provided, and a bacterium which can produce high titers of D-lactate from glycerol with low production of byproduct organic acids acetate and succinate, can also be provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Fig. 1 is a graph which compares the time course for D-lactic acid production by the various recombinant E. coli strains prepared in Inventive Examples and Comparative Examples.

DESCRIPTION OF EMBODIMENTS

[0013] The method for producing D-lactic acid according to the present invention includes contacting a lactic acid-producing bacterium with a glycerol-containing carbon source. The lactic acid-producing bacterium to be used in this method has an imparted or overexpressed NADH- dependent D-lactate dehydrogenase gene and an imparted or overexpressed ATP-dependent dihydroxyacetone kinase gene, and, in the lactic acid-producing bacterium, expression of an endogenous malate dehydrogenase gene is reduced or inactivated, and a NAD ⁺ -dependent glycerol dehydrogenase gene is neither imparted nor overexpressed.

[0014] The lactic acid-producing bacterium according to the present invention, which can be used in the method for producing D-lactic acid according to the present invention, has an imparted or overexpressed NADH-dependent D-lactate dehydrogenase gene and an imparted or overexpressed ATP-dependent dihydroxyacetone kinase gene, wherein expression of an endogenous malate dehydrogenase gene is reduced or inactivated, and a NAD ⁺ -dependent glycerol dehydrogenase gene is not imparted or overexpressed, as described above.

[0015] In the lactic acid-producing bacterium according to the present invention, since a NADH- dependent D-lactate dehydrogenase is imparted or overexpressed, an ATP-dependent

dihydroxyacetone kinase gene is imparted or overexpressed, the expression of an endogenous malate dehydrogenase gene is reduced or inactivated, and a NAD ⁺ -dependent glycerol dehydrogenase gene is not imparted or overexpressed, generation of by-products can be suppressed, and lactic acid can be produced efficiently, as compared to a case in which, for example, the host bacterium is E. coli and has an enhanced endogenous PEP-dependent dihydroxyacetone kinase gene.

Hereinafter, the present invention will be explained in detail.

[0016] 'Imparting or overexpressing' a gene encoding an enzyme refers to at least one of (i) introducing an enzyme gene from the outside of a host bacterium that does not have a gene corresponding to the enzyme into the inside thereof, (ii) introducing an enzyme gene having a higher activity than that of a corresponding native enzyme gene of the host bacterium from the outside of the host bacterium into the inside thereof, (iii) enhancing the activity of a promoter of the enzyme gene retained in the genome of a host bacterium, and (iv) replacing the promoter of the enzyme gene with another promoter to cause overexpression of the enzyme gene.

[0017] 'Reduced gene expression' in the present invention refers to a state in which the activity of an endogenous or exogenous enzyme or protein in the host bacterium is decreased by mutation of a gene encoding the enzyme or protein or by genetic recombination methods to a level lower than the state before the modification (mutation or genetic recombination). Reduced gene expression may refer to a state in which the expressed activity of the endogenous or exogenous enzyme or protein is decreased by mutation or genetic recombination of a promoter that controls the expression of the gene encoding the enzyme or protein.

[0018] 'Inactivated gene expression' in the present invention refers to a state in which the activity of an endogenous or exogenous enzyme or protein in the host bacterium is decreased to below the detection limit by the conventional measurement technique by mutation of a gene encoding the enzyme or protein or by genetic recombination methods well known to those skilled in the art. Inactivated gene expression may refer to a state in which the expressed activity of the endogenous or exogenous enzyme or protein is decreased below the detection limit by the conventional measurement technique by mutation or genetic recombination of a promoter that controls the expression of the gene encoding the enzyme or protein.

[0019] 'Gene not imparted or overexpressed' refers to a state in which the activity of an endogenous enzyme or protein in the host bacterium is maintained at its naturally-occurring level without being altered by mutation of a gene encoding the enzyme or protein or by genetic recombination methods well known to those skilled in the art, and without introduction of an equivalent enzyme from outside the host bacterium.

[0020] "Genetic recombination" in the present invention encompasses any technique whereby a change in the nucleotide base sequence is introduced via insertion of another DNA into the base sequence of a native gene, substitution and/or deletion of a part of the gene, or a combination thereof. For example, the change in the base sequence may result from mutation.

[0021] The vector used in the present invention is not limited to a specific vector, and any expression vectors known in the art may be used. In the present invention, the term

'transformation' indicates the process of introducing an endogenous or exogenous DNA into a host bacterium, thereby allowing the introduced DNA to be expressed in the host bacterium. The transformed gene or DNA may be present either on a chromosome of the host bacterium or present as an autonomously replicating DNA as long as the transformed gene or DNA is capable of being expressed in the host bacterium.

[0022] 'Imparting' an activity encompasses introducing a gene encoding an enzyme from the outside of a host bacterium into the inside thereof, as well as enhancing the activity of a promoter of an enzyme gene retained in the genome of a host bacterium, and replacing the promoter with another promoter to cause overexpression of the enzyme gene.

[0023] 'Altered activity' in the present invention refers to a state in which the activity of an endogenous or exogenous enzyme or protein is either enhanced, reduced or completely inactivated in the host bacterium so as to achieve a desirable response in the host bacterium by methods well known to those skilled in the art, for instance, by genetic modification, mutagenesis or evolutionary engineering techniques.

[0024] 'Enhanced activity' in the present invention refers to a state in which the activity of an endogenous or exogenous enzyme or protein in the host bacterium is increased by mutation of a gene encoding the enzyme or protein or by genetic recombination methods to a level higher than the state before the modification (mutation or genetic recombination).

[0025] 'Reduced activity' in the present invention refers to a state in which the activity of an endogenous or exogenous enzyme or protein in the host bacterium is decreased by mutation of a gene encoding the enzyme or protein or by genetic recombination methods to a level lower than the state before the modification (mutation or genetic recombination).

[0026] 'Inactivated' in the present invention refers to a state in which the activity of an endogenous or exogenous enzyme or protein in the host bacterium is decreased to below the detection limit by the conventional measurement technique by mutation of a gene encoding the enzyme or protein or by genetic recombination methods well known to those skilled in the art.

[0027] 'Expressed from genome' in the present invention refers to a state in which the activity of an endogenous or exogenous enzyme or protein in the host bacterium is expressed by having a copy or multiple copies of a gene encoding the enzyme or protein in the chromosome of the bacterium, and the control of expression of which is driven by a suitably located endogenous or exogenous promoter element on the genome.

[0028] In the present invention, the "host" bacterium refers to a bacterium that has been made capable of exerting the effect of the invention as a result of introduction of one or more genes from the outside of the bacterium and/or modification of its endogenous genes by mutation or genetic recombination.

[0029] The lactic acid-producing bacterium has an imparted or overexpressed NADH-dependent fermentative D-lactate dehydrogenase gene.

[0030] Fermentative D-lactate dehydrogenase (ldhA) is classified into the enzyme number 1.1.1.28 and refers to a generic name of an enzyme that catalyzes the NADH-dependent conversion of pyruvate to D-lactate. Examples of such enzymes include those derived from bacteria of the genus Lactobacillus, the genus Staphylococcus, the genus Enterococcus, the genus Enterobacter, and the genus Escherichia. Among them, Lactobacillus delbruckii, Lactobacillus pentosus, Lactobacillus plantarum, Staphylococcus or E. coli etc, are preferable from the viewpoints of high production efficiency and ease of handling, and E. coli is further preferable.

[0031] The lactic acid-producing bacterium has an imparted or overexpressed ATP-dependent dihydroxyacetone kinase gene. ATP-dependent dihydroxyacetone kinase is classified into the enzyme number 2.7.1.29 and refers to a generic name of an enzyme that catalyzes the

phosphorylation of dihydroxyacetone to dihydroxyacetone phosphate using ATP as cofactor. The ATP-dependent dihydroxyacetone kinase may be derived from a microorganism, and may be derived from a bacterium or a eukaryote. Examples of the ATP-dependent dihydroxyacetone kinase include those derived from: bacteria of the genus Klebsiella such as Klebsiella

pneumoniae, Klebsiella variicola and Klebsiella oxytoca; bacteria of the genus Citrobacter such as Citrobacter freundii, Citrobacter youngae, Citrobacter koseri and Citrobacter werkmanii; bacteria of the genus Enterobacter such as Enterobacter aerogenes, Enterobacter radicincitans and Enterobacter cloacae; bacteria of the genus Clostridium such as Clostridium butyricum; bacteria of the genera Shigella, Rahnella, Pantoea, Rhizobium and Roseobacter; and eukaryotes such as Hansenula polymorpha, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida boidinii, Candida methylica and Pichia pastoris. The ATP-dependent

dihydroxyacetone kinase is distinct from PEP-dependent dihydroxyacetone kinase. Although PEP-dependent dihydroxyacetone kinase also catalyzes a reaction of the phosphorylation of dihydroxyacetone to dihydroxyacetone phosphate, PEP-dependent dihydroxyacetone kinase utilizes phosphoenolpyruvate (PEP) as the phosphate source instead of ATP.

[0032] PEP-dependent dihydroxyacetone kinase concomitantly produces pyruvic acid which is a precursor of lactic acid. Examples of PEP-dependent dihydroxyacetone kinase include those derived from bacteria of the genus Escherichia such as Escherichia coli, bacteria of the genus Sinorhizobium such as Sinorhizobium meliloti, and bacteria of the genus Lactococcus such as Lactococcus lactis.

[0033] Although ATP-dependent dihydroxyacetone kinase and PEP-dependent dihydroxyacetone kinase catalyze similar reactions, ATP-dependent dihydroxyacetone kinase provides significantly improved results in terms of lactic acid production especially with respect to lactic acid production efficiency as well as reduction of generation of by-products (particularly, acetic acid), as compared with PEP-dependent dihydroxyacetone kinase. The present invention, for the first time, clarified that ATP-dependent dihydroxyacetone kinase enables not only more efficient production of lactic acid but also reduction of generation of by-products (particularly, acetic acid), as compared with PEP-dependent dihydroxyacetone kinase.

[0034] In the lactic acid-producing bacterium, a glycerol dehydrogenase gene is neither imparted nor overexpressed. Enhancement of the expression of glycerol dehydrogenase gene results in a decrease in the overall lactic acid production efficiency of the lactic acid production method.

[0035] Glycerol dehydrogenase is classified into the enzyme number 1.1.1.6 and refers to a generic name of an enzyme that catalyzes the NAD ⁺ -dependent oxidation of glycerol to dihydroxyacetone. For example, Escherichia coli has gldA as a gene encoding glycerol dehydrogenase, and Bacillus megaterium has dhaD as a gene encoding glycerol dehydrogenase.

[0036] In the lactic acid-producing bacterium, the expression of the endogenous malate dehydrogenase activity is reduced or inactivated.

[0037] Malate dehydrogenase (mdh) is classified into the enzyme number 1.1.1.37 and refers to a generic name of an enzyme that catalyzes the conversion of oxaloacetate to malate in the

Tricarboxylic acid cycle.

[0038] Further, the lactic acid-producing bacterium may have an imparted or overexpressed triosephosphate isomerase gene, in view of the lactic acid production efficiency and suppression of the generation of by-products.

[0039] Triosephosphate isomerase (tpiA) is classified into the enzyme number 5.3.1.1 and refers to a generic name of an enzyme that catalyzes the isomerization of dihydroxyacetone phosphate to glyceraldehyde-3 -phosphate and is found in most microorganisms. Examples of

triosephosphate isomerase include those derived from bacteria of the genus Escherichia, Bacillus, Klebsiella, Lactococcus, Lactobacillus, or Staphylococcus. Triosephosphate isomerase derived from Escherichia coli is preferable from the viewpoint of compatibility with the bacterial host when the bacterial host is E. coli. [0040] Although depending on the type of the host bacterium, in the lactic acid-producing bacterium, it is preferable that the expression of at least one endogenous gene selected from the group consisting of pyruvate formate lyase, FAD-dependent D-lactate dehydrogenase and aspartate ammonia lyase is reduced or inactivated, from the viewpoint of increasing the titer and yield of D-lactic acid produced from glycerol. Reduction or inactivation of at least one of such endogenous genes tends to increase the efficiency of lactic acid production, and tends to suppress the generation of by-products. Although effects can be produced even when only one of pyruvate formate lyase activity, FAD-dependent D-lactate dehydrogenase activity and aspartate ammonia lyase activity is reduced or inactivated, reduction or inactivation of at least two of pyruvate formate lyase activity, FAD-dependent D-lactate dehydrogenase activity and aspartate ammonia lyase activity may be carried out, and reduction or inactivation of the activity of all of the three genes is most preferable.

[0041] Pyruvate formate lyase or formate acetyltransferase (pfl) is classified into the enzyme number 2.3.1.54 and refers to a generic name of an enzyme that catalyzes the conversion of pyruvate to acetyl-coA and formate.

[0042] D-lactate dehydrogenase (did) is classified into the enzyme number 1.1.1.28 and refers to a generic name of an enzyme that catalyzes the conversion of D-lactate to pyruvate.

[0043] Aspartate ammonia lyase (aspA) is classified into the enzyme number 4.3.1.1 and refers to a generic name of an enzyme that catalyzes the conversion of aspartate to fumarate.

[0044] The activity of each of these enzymes in the invention may be introduced from the outside of the host bacterium into the inside of the host bacterium, or, alternatively, the activity of each of these enzymes in the invention may be provided by overexpression of the enzyme genes by enhancement of activity of a promoter or promoters of the enzyme genes retained in the genome of the host bacterium or replacement of the promoter(s) with another promoter(s) to cause overexpression of the enzyme genes. Further, with respect to each of the enzymes employed in the present specification, it is possible to use only one enzymatic gene derived from an organism, but it is also possible to use two or more enzymatic genes derived from different organisms in combination.

[0045] Any promoter may be used as the promoter used for enhancement of the promoter activity or overexpression of the enzyme gene as long as the promoter is capable of working in the host bacterium. Examples of the promoter include constitutive promoters and inducible promoters, of which constitutive promoters are more preferable from the viewpoint of having continuous D- lactic acid production from glycerol during all growth phases resulting in higher D-lactate productivity and also lowering of production costs by virtue of having inducer-free processes. Examples of constitutive promoters and inducible promoters include promoters derived from E. coli or phages, such as the trp promoter, lac promoter, P _L promoter and P _R promoter; promoters artificially designed or modified, such as the tac promoter; the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter as employed in Inventive Examples; lipoprotein (Ipp) promoter; glutamate decarboxylase A (gadA) promoter; and serine hydroxymethyltransferase (glyA) promoter. These may be appropriately selected depending on the origins and types of the enzymes used.

[0046] These promoters may be introduced into the host cell according to a conventional method such that the target enzyme gene may be expressed by, for example, ligating the promoter(s) with a vector to which the target enzyme gene is ligated, followed by introduction of the vector carrying the promoter(s) and the enzyme gene into the host cell.

[0047] The enhancement of activity of a gene (e.g., IdhA) in the present invention may be achieved by means of replacing the promoter of the gene on the genome of the bacterium with an exogenous or endogenous promoter of another gene which controls the expression of an enzyme of the glycolytic pathway. However, the exogenous or endogenous promoter is not limited to a promoter of a glycolytic enzyme. Other examples may include promoters for enzymes of nucleic acid biosynthesis pathway or amino acid biosynthesis pathways that can function constantly in the bacterium and are less susceptible to suppression of expression even in the presence of glucose. The promoter used for driving the gene (e.g., IdhA) expression in the present invention may be the promoter of glyceraldehdyde-3-phosphate dehydrogenase gene. The enhancement of gene activity in this manner provides a more stable enhancement of the gene activity, compared to bacteria in which the gene is expressed from a plasmid.

[0048] In the invention, the host bacterium is prokaryotic. Examples of such bacteria include bacteria of the genus Escherichia, Lactobacillus, Lactococcus, Sporolactobacillus, Bacillus, Corynebacterium, Enterococcus; and Escherichia coli. Escherichia coli, which is especially convenient and has yielded plenty of results in industrial uses, is preferably used.

[0049] Escherichia coli that are provided with the activity of the respective enzyme genes described above can preferably be used in the present invention. Examples thereof include mutants aiming at increased expression or inactivation of activity of at least some of the above- described essential or optional enzymes. Examples of such mutants include E. coli MG1655ApflAdldAmdhAaspA/GAPldhA strain, E. coli

MG1655Apf dldAmdhAaspA/GAPldhA/pGAP-dhaK (C.freundii), E. coli

MG 1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (K. pneumoniae), E. coli

MG 1655ApflAdldAmdhAaspA/GAPldhA/pGAP-tpiA, E. coli

MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (K. pneumoniae)-tpi ^' A, E. coli

MG 1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (E. aerogenes) and E. coli

MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (C. youngae) strains. These mutant strains can be used suitably for producing D-lactate from glycerol or mixtures of carbon sources containing glycerol as a component.

[0050] In order to allow the lactic acid-producing bacterium of the present invention to grow, original growth conditions for the host bacterium can be employed as they are. The nutrition sources employed for the growth of the lactic acid-producing bacterium is not particularly limited as long as the nutrition sources provide nutrition required by the host bacterium, and may be a glycerol-containing nutrition source. The specifics thereof are described below.

[0051] The method of the invention for producing lactic acid includes contacting the lactic acid- producing bacterium with a glycerol-containing carbon source. Specifically, the method for producing lactic acid includes culturing the lactic acid-producing bacterium in the state of being contacted with a glycerol-containing carbon source (culturing process), and recovering lactic acid produced by the culturing (recovery process). By using the lactic acid-producing bacterium as described above, lactic acid can be produced from a glycerol-containing carbon source with high efficiency and suppressed generation of by-products.

Thereby, recovery of D-lactate can be simplified compared to use of conventional glycerol- fermenting and D-lactate-producing microorganisms.

[0052] The glycerol to be used as a carbon source may be crude glycerol obtained as a byproduct from biodiesel manufacturing, distilled glycerol, or pure glycerol, or glycerol obtained from any other naturally or commercially available source, provided that glycerol is contained as a component. The glycerol may also be present in the form of a mixture of glycerol with one or more other carbon sources that can be used by the bacterium for growth and fermentation.

[0053] The glycerol-containing carbon source may include a carbon source other than glycerol. Other carbon sources may include carbohydrate substrates such as glucose, sucrose, fructose, lactose, galactose, maltose, starch, cellulose and fats or fatty acids. However, carbon sources are not limited to these carbon sources. [0054] This production method includes a method including assimilating glycerol-containing material by culturing the lactic acid-producing bacterium of the invention in a mixture containing the lactic acid-producing bacterium and the glycerol-containing material, and, after a certain period of time, purifying D-lactate secreted in the culture medium using a known technique(s) such as distillation, solvent extraction, ion exchange, electrodialysis or the like or a combination thereof.

[0055] The culture in the present invention refers to microbial biomass, a culture solution and a treatment product thereof.

[0056] The culturing in the present invention refers to culturing the bacterium described in the present invention using a medium which is usually liquid. In this case, the medium comprises glycerol and is not particularly limited if it is a medium containing organic components, nucleic acids, vitamins or the like which are required for the growth of the bacterium and production of lactic acid. In the present invention, the medium may comprise natural products such as yeast extracts, casamino acids, peptone, tryptone, whey, blackstrap molasses, corn steep liquor and the like or hydrolysates of natural product extracts. To obtain more preferable results, the medium may comprise preferably from 0.5 % to 20 % and further preferably from 2 % to 15 % of at least one kind selected from yeast extracts, peptone, tryptone, whey, blackstrap molasses and corn steep liquor, or mixtures thereof. Especially, the addition of corn steep liquor is beneficial and better results may be obtained thereby. The glycerol concentration in the medium is preferably maintained such that the concentration does not exceed 8 % at any time and more preferably does not exceed 5 % at any time during the culture period. Depending on the glycerol consumption rate by the culture, this can be achieved by continuous, fed-batch and/or batch-wise addition of glycerol to the liquid medium at a rate such that the concentration does not exceed the maximum limit.

[0057] Corn steep liquor is obtained by concentrating an immersion fluid which contains soluble components eluted during soaking of maize and components produced by lactic acid fermentation. According to Microbiol. Mol. Biol. Rev., Dec 1948; Vol.12: pp.297-31 1, its components are, for example: 45% by mass to 55% by mass of water; 2.7% by mass to 4.5% by mass of the total nitrogen content; 1.0% by mass to 1.8% by mass of amino nitrogen; 0.15% by mass to 0.40% by mass of volatile nitrogen; 0.1% by mass to 11.0% by mass of reducing sugars; 5% by mass to 15% by mass of lactic acid; 9% by mass to 10% by mass of the ash content; 0.1 % by mass to 0.3% by mass of volatile acids; and 0.009%) by mass to 0.015% by mass of sulfur dioxide, based on the total mass of the corn steep liquor. Corn steep liquor is normally used at a concentration within the range of 0.1 to 20% (w/w), and preferably used at a concentration within the range of 0.5 to 7% (w/w).

[0058] In addition, the culture medium may contain other additive components which are usually added to media for bacteria, such as antibiotics, at concentrations at which they are usually used. An antifoaming agent is preferably added to the culture medium at an appropriate amount for suppressing foaming.

[0059] Examples of the culture medium used in the invention include: liquid media based on an aqueous medium; and solid media based on a solid phase such as agarose. The medium is preferably a liquid medium, from the viewpoint of suitability for industrial production. Examples of the aqueous medium which forms the liquid medium include those usually used, such as distilled water and buffers.

[0060] The method of the invention for production of D-lactate from glycerol may include pre- culturing to attain an appropriate bacterial cell number and/or an appropriate level of the active state of the lactic acid-producing bacterium to be used, before the culturing for producing D- lactate. The pre-culturing may employ a culture condition which is normally used and depends on the type of the lactic acid-producing bacterium.

[0061] In the culture, a fermentor is generally used for culturing, which can usually control temperature, pH, aeration and agitation. However, the culture of the present invention is not limited to the use of the fermentor. In the case of culture using the fermentor, if necessary, seed culture may be carried out in advance as pre-culture and this may be inoculated in a needed amount on a medium in a fermentor prepared in advance. The medium used for growth of seed culture may comprise organic components, nucleic acids, vitamins, buffering agents or the like which are required for the growth of the bacterium. In the present invention, the pre-culture medium may comprise natural products such as yeast extracts, casamino acids, peptone, tryptone, whey, blackstrap molasses, corn steep liquor and the like or hydrolysates of natural product extracts. To obtain more preferable results, the pre-culture medium used can be Luria Bertani medium or Terrific Broth medium. Especially, the use of Terrific Broth medium is beneficial and better results may be obtained thereby.

[0062] In the present invention, the culture for fermentation of glycerol or a glycerol-containing mixture may be conducted with a culture apparatus (fermentor) of any type and any size, under any culture conditions. The conditions, including the composition of the culture medium, the agitation speed, the aeration rate, the culture temperature, the pH, etc. can be appropriately changed within the above-described ranges, in consideration of the size of the fermentor. For example, the culture for fermentation of glycerol or a glycerol-containing mixture may be conducted using 3 L capacity fermentor manufactured by ABLE Corporation but is not limited to the use of the fermentor or the conditions described herein. In an exemplary embodiment, preferable results can be obtained by culturing in 900 g culture medium comprising 5 % corn steep liquor, at an agitation speed of 350 rpm, a temperature of 35 °C, an aeration rate of 0.5 wm and pH of 7.5 maintained with ammonia, sodium hydroxide, calcium hydroxide, calcium carbonate or the like. Use of calcium hydroxide as alkali can provide better results, and thus is preferable.

[0063] In the culture process, glycerol as a carbon source may be supplementarily added to the reaction mixture in order to compensate for the glycerol consumed during lactic acid production. The supplemental addition of glycerol enables continuous production of lactic acid, and further improves the production efficiency. Regarding the amount of glycerol to be supplementarily added, from the viewpoint of productivity, the glycerol may be added to the fermentation medium at periodic intervals to maintain sufficient glycerol in the medium and at the same time not exceeding a glycerol concentration of over 8 %. Preferable results may be obtained by continuous feeding of glycerol to maintain glycerol concentration below 5 % at any time point during the fermentation.

[0064] The culture time is not particularly limited. The culture time is set to be a time necessary to grow the microbial biomass sufficiently, and further produce lactic acid. The present invention is, however, not limited to the use of these conditions, and includes similar conditions employed within an acceptable range for the parameters that is evident to those skilled in the art.

[0065] The culture conditions may be varied depending on the bacterium prepared and the culture apparatus. In general, the culture temperature during culture is preferably from 20°C to 40°C, more preferably from 25°C to 35°C, and still more preferably from 30°C to 37°C. The pH during culture is preferably adjusted to be from 4 to 9, more preferably from 6.0 to 8.0, and still more preferably from 7.0 to 8.0, and further more preferably from 7.3 to 7.7. The pH adjuster employed for adjusting the pH to be within the above range is not particularly limited, and pH may adjusted by using Ca(OH) ₂ , CaC0 ₃ , NaOH, ammonia, or the like. The culture time is not particularly limited, and is a period of time necessary for the bacterium to grow sufficiently and produce lactic acid. Further, glycerol may be introduced to the culture medium by batch, fed- batch and/or continuous modes of addition.

[0066] The culture is generally carried out using a culture vessel capable of controlling the temperature, pH, aerobic conditions, and agitation speed. However, the use of a culture vessel is not essential in the culture according to the invention. In a case in which culture is conducted using a culture vessel, if necessary, seed culture may be carried out in advance as a pre-culture, and a required amount of the resultant culture may be inoculated into a medium in a culture vessel that has been prepared in advance.

[0067] In the present invention, the agitation speed is preferably from 200 rpm to 800 rpm and more preferably from 300 rpm to 500 rpm. Further, the air supplied may be in the range of 0.1 vvm to 1.0 wm, while more preferable results may be obtained using aeration rate in the range of 0.1 yvm to 0.5 wm. By doing these, there can be obtained an effect of increasing consumption of glycerol or mixtures of carbon sources comprising glycerol and production of lactic acid. The condition as described above does not need to be carried out throughout from the start to the end of the culture, and carrying out in part of the culture process can also give preferable results.

[0068] The culturing may be continued from the beginning of the culture until the glycerol is consumed, or until the activity of the lactic acid-producing bacterium is lost. The time period of the culturing varies depending on the number and activity of the lactic acid-producing bacterium as well as the amount of glycerol, and may be generally 1 hour or more, and be preferably 4 hours or more. On the other hand, although the culturing period may be unlimitedly extended by further providing glycerol and/or the lactic acid-producing bacterium, it may be generally 5 days or less, preferably 48 hours or less, in view of the processing efficiency.

[0069] A process for recovering lactic acid accumulated in the obtained culture as described above is not particularly limited. However, there can be adopted conventionally known methods, for example, direct distillation after acidification, a method of lactide formation and distillation, a method of distillation after adding alcohol and catalyst for esterification, a method of extraction in an organic solvent, a method of isolation with an ion exchange column, a method of concentration and isolation by electrodialysis or the like or a combination thereof. In addition, since the microbial mass produced by the method of the present invention produces an enzyme group which is suitable for lactic acid production from glycerol, producing lactic acid from glycerol further by using the enzyme group and recovering the produced lactic acid is also regarded as a part of the method of recovering lactic acid from the culture. EXAMPLES

[0070] Examples of the invention will now be described below, although the invention is not limited thereby.

[0071] PREPARATION EXAMPLE 1

Construction of E. coli MG1655Adld strain

The sequence provided for the FAD-dependent D-lactate dehydrogenase gene (did) from E. coli strain MG1655 was obtained using NCBI-Gene ID 946653 and used to design primers with SEQ ID Nos. 1, 2, 3 and 4 for PCR amplification of the regions adjacent to did gene on the genome of E. coli MG1655.

[0072] The genomic DNA of E. coli strain MG1655 was prepared by the method described in Current Protocols in Molecular Biology (John Wiley & Sons). The extracted genomic DNA was used as template for PCR amplification of two 1.1 kb fragments flanking the did gene using primers with SEQ ID Nos. 1 and 2 and SEQ ID Nos. 3 and 4 under standard thermal cycling conditions. These fragments were gel electrophoresed, purified and subsequently digested with HmdIII and Pstl, and Pstl and Xbal, respectively. The temperature-sensitive plasmid pTH18csl (Hashimoto-Gotoh, T., et al., 2000, Gene, Vol. 241(1), pp 185-191) was digested with HmdIII and Xbal and then ligated with the digested fragments. The ligated mixture was transformed to E. coli DH5a competent cells and allowed to grow at 30 °C on LB-agar plates containing 10 μg/mL of chloramphenicol to maintain the plasmid. The constructed plasmid was extracted from an overnight culture of a positive transformant in LB chloramphenicol (10 μg/mL) medium. The extracted plasmid was transformed into E. coli strain MG1655 and positive transformants were selected on LB-agar plate containing 10 μg/mL of chloramphenicol at 30 °C. The obtained transformant was plated on to an agar plate and cultured overnight at 30 °C. Next, in order to obtain cultured microbial mass, the cultured transformant was plated on to an LB-agar plate containing 10 μg/mL chloramphenicol and incubated at 42 °C. The grown colonies were re- plated on to a fresh LB-agar plate containing 10 μg mL chloramphenicol and incubated at 42 °C for homologous recombination of the temperature-sensitive plasmid. The integration of the plasmid was confirmed by PCR using genomic DNA extracted from the obtained strain. The confirmed strain was cultured overnight on an LB-agar plate at 30 °C, and then inoculated into 3 ml LB liquid medium and allowed to grow at 42 °C for 3 to 4 hours with shaking. This culture was suitably diluted and plated on to a LB-agar plate and allowed to grow overnight at 42 °C. One hundred of the grown colonies were replica-plated onto a fresh LB-agar plate and a LB-agar plate containing 10 μg/mL of chloramphenicol. Chloramphenicol-sensitive colonies which could grow only on the LB-agar plate were selected and screened by PCR for deletion of the did gene. The positive did deleted strain was designated as E. coli MG1655Adld.

[0073] PREPARATION EXAMPLE 2

Construction of E. coli MG1655ApflAdld strain

The sequence provided for the pyruvate formate lyase gene (pfl) from E. coli strain MG1655 was obtained using GenBank accession number AE000192 and used to design primers with SEQ ID Nos. 5, 6, 7 and 8 for PCR amplification of the regions adjacent to pfl gene on the genome of E. coli MG1655.

The genomic DNA of E. coli strain MG1655 was used as template for PCR amplification of 1.8 kb and 1.3 kb fragments flanking the pfl gene using primers with SEQ ID Nos. 5 and 6, and SEQ ID Nos. 7 and 8 under standard thermal cycling conditions. These fragments were gel electrophoresed, purified and subsequently digested with Hindlll and Sphl, and Sphl and Pstl, respectively. The temperature-sensitive plasmid pTH18csl (Hashimoto-Gotoh, T., et al., 2000, Gene, Vol. 241(1), pp 185-191) was digested with Hwdlll and Pstl and then ligated with the digested fragments. The ligated mixture was transformed to E. coli DH5a competent cells and allowed to grow at 30 °C on solid LB medium plates containing 10 μg/mL of chloramphenicol in order to maintain the plasmid.

[0074] The constructed plasmid was extracted from an overnight culture of the positive transformant in LB chloramphenicol (10 μξ/ηύ,) medium. The plasmid was transformed into E. coli strain MG1655Adld described in Preparation Example 1 and positive transformants were selected on LB-agar plate containing 10 μg/mL of chloramphenicol at 30 °C. The obtained transformant was plated on to an agar plate and cultured overnight at 30 °C. Next, in order to obtain cultured microbial mass, the cultured transformant was plated on to an LB-agar plate containing 10 μg/mL chloramphenicol and incubated at 42 °C. Subsequently, the procedure described in Preparation Example 1 for homologous recombination was followed in the same manner to disrupt the did gene, thereby obtaining E. coli MG1655ApflAdld strain.

[0075] PREPARATION EXAMPLE 3

Construction of E. coli MG1655ApflAdldAmdh strain

The sequence provided for the malate dehydrogenase gene (mdh) from E. coli strain MG 1655 was obtained using NCBI-Gene ID 947854 and used to design primers with SEQ ID Nos. 9, 10, 1 1 and 12 for PCR amplification of the regions adjacent to mdh gene on the genome of E. coli MG1655.

[0076] The genomic DNA of E. coli strain MG1655 was used as template for PCR amplification of 0.8 kb and 1 kb fragments flanking the mdh gene using primers with SEQ ID Nos. 9 and 10, and SEQ ID Nos. 11 and 12 under standard thermal cycling conditions. These fragments were gel electrophoresed, purified and subsequently digested with Kpnl and BamHl, and BamHl and Xbal, respectively. The temperature-sensitive plasmid pTH18csl (Hashimoto-Gotoh, T., et al., 2000, Gene, Vol. 241(1), pp 185-191) was digested with Kpnl and Xbal and then ligated with the digested fragments. The ligated mixture was transformed to E. coli DH5a competent cells and allowed to grow at 30 °C on solid LB medium plates containing 10 μg/mL of chloramphenicol in order to maintain the plasmid. The constructed plasmid was extracted from an overnight culture of the positive transformant in LB chloramphenicol (10 μg/mL) medium. The plasmid was transformed into E. coli strain MG1655ApflAdld described in Preparation Example 2 and positive transformants were selected on LB-agar plate containing 10 μg/mL of chloramphenicol at 30 °C. Subsequently, the procedure described in Preparation Example 1 for homologous recombination was followed in the same manner to disrupt the mdh gene, thereby obtaining E. coli

MG1655ApflAdldAmdh strain.

[0077] PREPARATION EXAMPLE 4

Construction of E. coli MG1655ApflAdldAmdhAaspA strain

The sequence provided for the aspartate ammonia lyase gene (aspA) from E. coli strain MG1655 was obtained using NCBI-Gene ID 948658 and used to design primers with SEQ ID Nos. 13, 14, 15 and 16 for PCR amplification of the regions adjacent to asp A gene on the genome of E. coli MG1655.

[0078] The genomic DNA of E. coli strain MG1655 was used as template for PCR amplification of 0.91 kb and 1.1 kb fragments flanking the aspA gene using primers with SEQ ID Nos. 13 and 14, and SEQ ID Nos. 15 and 16 under standard thermal cycling conditions. These fragments were gel electrophoresed, purified and subsequently blunt-ended using DNA Blunting Kit (Takara Bio Inc., Japan). The 5' termini of the blunt-ended DNA fragments were phosphorylated using T4 polynucleotide kinase. The temperature-sensitive plasmid pTH18csl (Hashimoto-Gotoh, T., et al., 2000, Gene, Vol. 241(1), pp 185-191) was digested with Smal and then dephosphorylated using alkaline phosphatase treatment. The two phosphorylated fragments and the dephosphorylated plasmid were ligated and transformed to E. coli DH5a competent cells and allowed to grow at 30 °C on solid LB medium plates containing 10 μg/mL of chloramphenicol in order to maintain the plasmid. The constructed plasmid was extracted from an overnight culture of the positive transformant in LB chloramphenicol (10 μg/mL) medium. The plasmid was transformed into E. coli strain MG1655ApflAdldAmdh described in Preparation Example 3 and positive

transformants were selected on LB-agar plate containing 10 μg/mL of chloramphenicol at 30 °C. Subsequently, the procedure described in Preparation Example 1 for homologous recombination was followed in the same manner to disrupt the aspA gene, thereby obtaining E. coli

MG1655ApflAdldAmdhAaspA strain.

[0079] PREPARATION EXAMPLE 5

Construction of E. coli MG1655ApflAdldAmdhAaspA/GAPldhA strain

The upstream promoter sequence for the glyceraldehyde-3-phosphate dehydrogenase gene (gapA) from E. coli strain MG1655 was obtained using NCBI-Gene ID 947679 and used to design primers with SEQ ID Nos. 17 and 18 for PCR amplification of the GAPDH promoter from the genome of E. coli MG1655. Additionally, the sequence provided for the fermentative D-lactate dehydrogenase gene {IdhA) from E. coli strain MG1655 was obtained using NCBI-Gene ID 946315 and used to design primers with SEQ ID Nos. 19 and 20 for PCR amplification of the IdhA gene from the genome of E. coli MG1655.

[0080] The genomic DNA of E. coli strain MG1655 was used as template for PCR amplification of 0.1 kb GAPDH promoter and 1 kb IdhA gene using primers with SEQ ID Nos. 17 and 18, and SEQ ID Nos. 19 and 20, respectively, under standard thermal cycling conditions. These fragments were gel electrophoresed, purified and subsequently digested with EcoKl, and EcoRl and Hindlll, respectively. The plasmid pUC18 was digested with EcoRl and Hwdlll and then ligated with the digested fragments. The ligated mixture was transformed to E. coli DH5a competent cells and positive clones were screened on solid LB medium plates containing 50 μg/mL of ampicillin at 30 °C. The constructed plasmid was extracted from an overnight culture of the positive transformant in LB ampicillin (50 g/mL) medium and designated as pGAPldhA.

[0081] For replacement of the native IdhA promoter on the genome of the constructed E. coli MG1655ApflAdldAmdhAaspA strain from Preparation Example 4 with the GAPDH promoter, the sequence provided for the fermentative D-lactate dehydrogenase gene {IdhA) from E. coli strain MG1655 was obtained using NCBI-Gene ID 946315 and used to design primers with SEQ ID Nos: 21 and 22 for PCR amplification of a 1 kb 5'-adjacent region of the IdhA gene from the genome of E. coli MG1655. Additionally, primers with SEQ ID Nos. 23 and 24 were designed complementary to the GAPDH promoter and the IdhA gene to PCR amplify a 0.85 kb fragment comprising the GAPDH promoter and initiation codon-adjacent region of the IdhA gene from plasmid pGAPldhA described above. These fragments were gel electrophoresed, purified and subsequently digested with Kpnl and Xbal, and Xbal and Pstl, respectively. The temperature- sensitive plasmid pTH18csl (Hashimoto-Gotoh, T., et al., 2000, Gene, Vol. 241(1), pp 185-191) was digested with Kpnl and Pstl and then ligated with the digested fragments. The ligated mixture was transformed to E. coli DH5ot competent cells and allowed to grow at 30 °C on solid LB medium plates containing 10 μg/mL of chloramphenicol in order to maintain the plasmid. The constructed plasmid was extracted from an overnight culture of the positive transformant in LB chloramphenicol (10 μg/mL) medium. The plasmid was transformed into E. coli

MG1655ApflAdldAmdhAaspA described in Preparation Example 4 and positive transformants were selected on LB-agar plate containing 10 μg/mL of chloramphenicol at 30 °C. Subsequently, the procedure described in Preparation Example 1 for homologous recombination was followed in the same manner, thereby obtaining E. coli MG1655ApflAdldAmdhAaspA/GAPldhA strain, the genome of which contains the IdhA promoter replaced with the GAPDH promoter, .

[0082] COMPARATIVE EXAMPLE 1

Production of D-lactic acid from glycerol by E. coli MG1655ApflAdldAmdhAaspA/GAPldhA strain

E. coli MG1655Apf dldAmdhAaspA/GAPldhA strain described in Preparation Example 5 was first inoculated into 100 mL Terrific Broth (TB) culture medium taken in a 500 mL capacity conical flask equipped with baffles. The pre-culture was grown at 35 °C in a rotary incubator with shaking at the rate of 200 rpm for 20.5 h. Forty five milliliters of the pre-culture was used to inoculate 900 g of the fermentation medium with the starting composition of 50 g/kg medium Corn Steep Liquor (CSL) and 30 g/kg medium glycerol contained in 3 L volume fermentor (BMS-03NP3, culture apparatus manufactured by ABLE Corporation, Japan). Fermentation of glycerol was conducted for 46.5 h at a temperature of 35 °C, agitation rate of 350 rpm, aeration rate of 0.5 wm and pH of 7.5 maintained using 25 % (w/v) slurry of calcium hydroxide. A continuous supply of 50 % (w/w) glycerol solution was fed to the fermentation vessel starting from 6 h at the rate of 0.08 g/min. The consumption of glycerol and production of lactic acid and by-products such as acetic acid, succinic acid and ethanol were monitored at different time intervals by High Performance Liquid Chromatography (HPLC) technique using Ultron PS-80H (300 x 8.0 mm) column (Shinwa Chemical Industries Ltd., Japan) operated under standard conditions. The optical purity of D-lactic acid was also estimated by HPLC using the Sumichiral OA-5000 column (Sumika Chemical Analysis Service, Japan) operated under standard conditions.

[0083] E. coli MG1655ApflAdldAmdhAaspA/GAPldhA strain consumed 66.5 g/kg medium of glycerol in 46.5 h to produce 52.6 g/kg medium of lactic acid with a product yield of 0.79 g lactic acid/g glycerol consumed and productivity of 1.131 g lactic acid/kg medium/h. The major byproducts produced were acetate - 6.84 g/kg medium, succinate - 2.85 g/kg medium and ethanol - 0.30 g/kg medium. The optical purity of D-lactic acid was 100 %. Fig. 1 and Table 1 give a comparison of the fermentation performance of this control strain with respect to the strains constructed in the present invention for the fermentation of glycerol to D-lactic acid and formation of by-products acetate and succinate.

[0084] COMPARATIVE EXAMPLE 2

Construction of E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaKLM strain

The sequence provided for the PEP-dependent dihydroxyacetone kinase genes operon (dhaKLM) from E. coli strain MG1655 was obtained using NCBI-Gene ID's 945747, 945748 and 945749 which were used to design primers with SEQ ID nos. 25 and 26 for PCR amplification of

dhaKLM operon. The primer with SEQ ID No. 25 has a Xbal restriction site at the 5' terminus while, the primer with SEQ ID No. 26 has a HmdIII restriction enzyme site at the 5' terminus for cloning the dhaKLM operon in to an expression vector. The 5' region of primer SEQ ID No. 25 additionally contains a suitable Shine-Dalgarno sequence downstream of the restriction enzyme site for efficient translation of dhaK, dhaL and dhaM proteins in the host strain.

[0085] The genomic DNA of E. coli strain MG1655 was extracted using the DNeasy Blood and Tissue Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). The extracted genomic DNA was used as template for PCR amplification of dhaKLM operon using primers SEQ ID Nos. 25 and 26 under standard thermal cycling conditions for KOD Plus DNA polymerase (Toyobo, Japan). The around 3.1 kb dhaKLM operon was gel electrophoresed and purified using the QIAquick Gel extraction Kit supplied by Qiagen (Hilden, Germany) and subsequently digested with Xbal and HmdIII. Additionally, for gene expression, the upstream promoter sequence for the glyceraldehyde-3-phosphate dehydrogenase gene (gapA) from E. coli strain MG1655 was obtained using NCBI-Gene ID 947679 and used to design primers with SEQ ID Nos. 27 and 28 for PCR amplification of the GAPDH promoter from the genome of E. coli MG1655. The genomic DNA of E. coli strain MG 1655 was used as template for PCR

amplification of 0.1 kb GAPDH promoter using primers with SEQ ID Nos. 27 and 28 under standard thermal cycling conditions. The amplified GAPDH promoter was gel electrophoresed, purified and subsequently digested with Ndel. The plasmid vector pBR322 (GenBank accession number JO 1749) was digested with Ndel and Pvull and then ligated with the digested GAPDH promoter fragment. The ligated mixture was transformed to E. coli DH5a competent cells and positive clones were screened on solid LB medium plates containing 50 μg/mL of ampicillin at 37 °C. The constructed plasmid was extracted from an overnight culture of the positive transformant in LB ampicillin (50 μg mL) medium and designated as pBRgapP. Plasmid pBRgapP was digested with Xbal- and HmdIII and then ligated with the digested dhaKLM fragment. The ligated plasmid mixture was then transformed to E. coli NEB5a competent cells supplied by New England Biolabs (MA, USA) and allowed to grow at 37 °C on solid LB medium plates containing 100 μg/mL of ampicillin. Positive colonies harboring the plasmid with dhaKLM genes inserted were screened for by PCR. The constructed plasmid was extracted from E. coli NEB 5a and designated as pGAP-dhaKLM. The presence of the complete ORF's of the dhaKLM genes was confirmed by DNA sequencing analysis using the Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems, USA).

[0086] Competent cells of E. coli MG1655ApflAdldAmdhAaspA/GAPldhA strain described in

Preparation Example 5, prepared by the standard calcium chloride method provided in Sambrook and Russell (Cold Spring Harbor Laboratory Press, 2001), were transformed with pGAP- dhaKLM and positive transformants were selected by screening for growth on solid LB medium plates containing 100 μg/mL ampicillin at 35 °C. The present strain was designated as E. coli

MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaKLM.

[0087] COMPARATIVE EXAMPLE 3

Production of D-lactic acid from glycerol by E. coli

MG 1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaKLM strain

The strain E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaKLM constructed as described in Comparative Example 2 was tested for the production of D-lactic acid from glycerol. E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaKLM strain was first inoculated into 100 mL TB culture medium containing 100 μg mL ampicillin taken in a 500 mL capacity conical flask equipped with baffles. The pre-culture was grown at 35 °C in a rotary incubator with shaking at the rate of 200 rpm for 20.5 h. Forty five milliliters of the pre-culture was used to inoculate 900 g of the fermentation medium with the starting composition of 50 g/kg medium Corn Steep Liquor (CSL), 30 g/kg medium glycerol and 100 μg/ml ampicillin contained in 3 L volume fermentor (BMS-03NP3, culture apparatus manufactured by ABLE Corporation, Japan). Fermentation of glycerol was conducted for 46:5 h at a temperature of 35 °C, agitation rate of 350 rpm, aeration rate of 0.5 vvm and pH of 7.5 maintained using 25 % (w/v) slurry of calcium hydroxide. A continuous supply of 50 % (w/w) glycerol solution was fed to the fermentation vessel starting from 6 h at the rate of 0.08 g/min to maintain glycerol in the medium in the range of 30 to 40 g/kg medium. The optical purity, consumption of glycerol and production of lactic acid and by-products such as acetic acid, succinic acid and ethanol were monitored as described in Comparative Example 1.

[0088] E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaKLM strain consumed 74.0 g/kg medium of glycerol in 46.5 h to produce 62.3 g/kg medium of lactic acid with a product yield of 0.84 g lactic acid/g glycerol consumed and productivity of 1.339 g lactic acid/kg medium/h. The major by-products produced were acetate - 8.26 g/kg medium, succinate - 1.63 g/kg medium and ethanol - 0.44 g/kg medium. The optical purity of D-lactic acid was 97.32 %. Fig. 1 and Table 1 give a comparison of the fermentation performance of this strain with respect to the strains constructed in the present invention for the fermentation of glycerol to D-lactic acid and formation of by-products acetate and succinate.

[0089] INVENTIVE EXAMPLE 1

Construction of E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK {C.freundii) strain The sequence provided for the ATP-dependent dihydroxyacetone kinase gene (dhaK) from Citrobacter freundii was obtained using GenBank accession number U09771 and used to design primers with SEQ ID nos. 29 and 30 for PCR amplification of dhaK (C. freundii). The primer with SEQ ID No. 29 has a Sail restriction site at the 5' terminus while, the primer with SEQ ID No. 30 has a Hwdlll restriction enzyme site at the 5' terminus for cloning the dhaK (C.freundii) gene in to an expression vector. The 5' region of primer SEQ ID No. 29 additionally contains a suitable Shine-Dalgarno sequence downstream of the restriction enzyme site for efficient translation of dhaK (C. freundii) protein in the host strain.

[0090] The genomic DNA of C.freundii strain NBRC 12681 was extracted using the DNeasy Blood and Tissue Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). The extracted genomic DNA was used as template for PCR amplification of dhaK (C. freundii) gene using primers SEQ ID Nos. 29 and 30 under standard thermal cycling conditions for KOD Plus DNA polymerase (Toyobo, Japan). The around 1.6 kb dhaK (C.freundii) gene was gel electrophoresed and purified using the QIAquick Gel extraction Kit supplied by Qiagen (Hilden, Germany) and subsequently digested with Sail and HmdIII. The plasmid vector pBRgapP constructed in Comparative Example 2 was also digested with the same two restriction enzymes and then ligated with the Sail- and HmdIII-digested dhaK {C.freundii) gene. The ligated plasmid mixture was transformed to E. coli NEB5a competent cells supplied by New England Biolabs (MA, USA) and allowed to grow at 37 °C on solid LB medium plates containing 100 μg/mL of ampicillin. Positive colonies harboring the plasmid with dhaK (C. freundii) inserted were screened for by PCR. The constructed plasmid was extracted from E. coli NEB5a and designated as pGAP-dhaK {C.freundii). The presence of the complete ORF of the dhaK (C. freundii) gene was confirmed by DNA sequencing analysis using the Applied Biosystems 3130 Genetic

Analyzer (Applied Biosystems, USA). The nucleotide sequence of the cloned dhaK {C. freundii) gene was 99 % identical to the dhaK gene from C.freundii strain CECT 4626 and the translated protein sequence was 99 % identical to the C.freundii dhaK sequence reported in GenBank accession number ABF06666.1.

[0091] Competent cells of E. coli MG1655ApflAdldAmdhAaspA/GAPldhA strain described in Preparation Example 5, prepared by the standard calcium chloride method provided in Sambrook and Russell (Cold Spring Harbor Laboratory Press, 2001), were transformed with pGAP-dhaK (C. freundii) and positive transformants were selected by screening for growth on solid LB medium plates containing 100 g/mL ampicillin at 35 °C. The present strain was designated as E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK {C. freundii).

[0092] INVENTIVE EXAMPLE 2

Production of D-lactic acid from glycerol by E. coli

MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK {C.freundii) strain

The strain E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK {C.freundii) constructed as described in Inventive Example 1 was tested for the production of D-lactic acid from glycerol. E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK {C.freundii) strain was first inoculated into 100 mL TB culture medium containing 100 μg/mL ampicillin taken in a 500 mL capacity conical flask equipped with baffles. The pre-culture was grown at 35 °C in a rotary incubator with shaking at the rate of 200 rpm for 20.5 h. Forty five milliliters of the pre-culture was used to inoculate 900 g of the fermentation medium with the starting composition of 50 g/kg medium Corn Steep Liquor (CSL), 30 g/kg medium glycerol and 100 μg/ml ampicillin contained in 3 L volume fermentor (BMS-03NP3, culture apparatus manufactured by ABLE Corporation, Japan). Fermentation of glycerol was conducted for 46.5 h at a temperature of 35 °C, agitation rate of 350 rpm, aeration rate of 0.5 wm and pH of 7.5 maintained using 25 % (w/v) slurry of calcium hydroxide. A continuous supply of 50 % (w/w) glycerol solution was fed to the fermentation vessel starting from 6 h at the rate of 0.08 g/min to maintain glycerol in the medium in the range of 30 to 40 g/kg medium. The optical purity, consumption of glycerol and production of lactic acid and by-products such as acetic acid, succinic acid and ethanol were monitored as described in Comparative Example 1.

[0093] E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (C.freundii) strain consumed 79.9 g/kg medium of glycerol in 46.5 h to produce 76.5 g/kg medium of lactic acid with a product yield of 0.96 g lactic acid/g glycerol consumed and productivity of 1.645 g lactic acid/kg medium/h. The major by-products produced were acetate - 3.04 g/kg medium, succinate - 0.85 g/kg medium and ethanol - 0.52 g/kg medium. The optical purity of D-lactic acid was 99.47 %. Fig. 1 and Table 1 give a comparison of the fermentation performance of this strain with respect to the control and other strains constructed in the present invention for the fermentation of glycerol to D-lactic acid and formation of by-products acetate and succinate.

[0094] INVENTIVE EXAMPLE 3

Construction of E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (K. pneumoniae) strain

The sequence provided for the glycerol dehydrogenase gene (dhaD) from Bacillus megaterium QM B1551 strain was obtained using NCBI-Gene ID 8986243 and used to design primers with SEQ ID Nos. 31 and 32 for PCR amplification oidhaD. The primer with SEQ ID No. 31 has a EcoRl restriction site at the 5' terminus while, the primer with SEQ ID No. 32 has a Spel restriction enzyme site at the 5' terminus for cloning the dhaD gene in to an expression vector. The 5' region of primer SEQ ID No. 31 additionally contains a suitable Shine-Dalgarno sequence downstream of the restriction enzyme site for efficient translation of dhaD protein in the host strain.

[0095] The genomic DNA of B. megaterium PV361 strain was extracted using the DNeasy Blood and Tissue Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). The extracted genomic DNA was used as template for PCR amplification of dhaD gene using primers SEQ ID Nos. 31 and 32 under standard thermal cycling conditions for KOD Plus DNA polymerase (Toyobo, Japan). The around 1.13 kb dhaD gene was gel electrophoresed and purified using the QIAquick Gel extraction Kit supplied by Qiagen (Hilden, Germany) and subsequently digested with EcoRl and Spel. The plasmid vector pBRgapP constructed in Comparative Example 2 was digested with EcoRl and Xbal and then ligated with the EcoRl - and S el-digested dhaD gene. The ligated plasmid mixture was transformed to E. coli NEB5a competent cells supplied by New England Biolabs (MA, USA) and allowed to grow at 37 °C on solid LB medium plates containing 100 μg/mL of ampicillin. Positive colonies harboring the plasmid with dhaD inserted were screened for by PCR. The constructed plasmid was extracted from E. coli NEB5a and designated as pGAP-dhaD. The presence of the complete ORF of the dhaD gene was confirmed by DNA sequencing analysis using the Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems, USA).

[0096] To construct the vector overexpressing the gene for ATP -dependent dihydroxyacetone kinase from Klebsiella pneumoniae, the sequence provided for the dihydroxyacetone kinase gene (dhaK) from Klebsiella pneumoniae 342 strain was obtained using NCBI-Gene ID 6936606 and used to design primers with SEQ ID Nos. 33 and 34 for PCR amplification of dhaK (K.

pneumoniae). The primer with SEQ ID No. 34 has a Hindlll restriction enzyme site and an added linker with incorporating restriction sites for Sad and Xmal at the 5' terminus. The 5' region of primer SEQ ID No. 33 additionally contains a suitable Shine-Dalgarno sequence for efficient translation of dhaK (K: pneumoniae) protein in the host strain.

[0097] The genomic DNA of K. pneumoniae 342 strain was extracted using the DNeasy Blood and Tissue Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). The extracted genomic DNA was used as template for PCR amplification of dhaK (K. pneumoniae) gene using primers SEQ ID Nos. 33 and 34 under standard thermal cycling conditions for KOD Plus DNA polymerase (Toyobo, Japan). The around 1.6 kb dhaK (K. pneumoniae) gene was gel electrophoresed and purified using the QIAquick Gel extraction Kit supplied by Qiagen (Hilden, Germany) and subsequently phosphorylated at the 5' end using T4 Polynucleotide kinase and then digested with Hindlll. The plasmid vector pGAP-dhaD described above was digested with Sail, blunt ended with DNA Blunting Kit, and then digested with H «dIII to inactivate the dhaD gene. The digested vector was ligated with the phosphorylated and Hwdlll-digested dhaK (K. pneumoniae) gene. The ligated plasmid mixture was transformed to E. coli NEB5a competent cells supplied by New England Biolabs (MA, USA) and allowed to grow at 37 °C on solid LB medium plates containing 100 μg/mL of ampicillin. Positive colonies harboring the plasmid with dhaK (K. pneumoniae) inserted were screened for by PCR. The constructed plasmid was extracted from E. coli NEB5a and designated as pGAP-dhaK (K. pneumoniae). The presence of the complete ORF of the dhaK (K. pneumoniae) gene and inactivation of the dhaD gene was confirmed by DNA sequencing analysis using the Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems, USA). The nucleotide sequence of the cloned dhaK (K. pneumoniae) gene was 99 % identical to the dhaK gene from K. pneumoniae strain KCTC 2242 and the translated protein sequence was 100 % identical to the K. pneumoniae strain MGH 78578 dhaK sequence.

[0098] Competent cells of E. coli MG1655ApflAdldAmdhAaspA/GAPldhA strain described in Preparation Example 5, prepared by the standard calcium chloride method provided in Sambrook and Russell (Cold Spring Harbor Laboratory Press, 2001), were transformed with pGAP-dhaK (K. pneumoniae) and positive transformants were selected by screening for growth on solid LB medium plates containing 100 μg/mL ampicillin at 35 °C. The present strain was designated as E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (K. pneumoniae).

[0099] INVENTIVE EXAMPLE 4

Production of D-lactic acid from glycerol by E. coli

MG 1655ApflAdldAmdhAaspA/ GAPldhA/pGAP-dhaK (K. pneumoniae) strain

The strain E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (K. pneumoniae) constructed as described in Inventive Example 3 was tested for the production of D-lactic acid from glycerol. E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (K. pneumoniae) strain was first inoculated into 100 mL TB culture medium containing 100 μg/mL ampicillin taken in a 500 mL capacity conical flask equipped with baffles. The pre-culture was grown at 35 °C in a rotary incubator with shaking at the rate of 200 rpm for 20.5 h. Forty five milliliters of the pre-culture was used to inoculate 900 g of the fermentation medium with the starting composition of 50 g/kg medium Corn Steep Liquor (CSL), 30 g/kg medium glycerol and 100 μg/ml ampicillin contained in 3 L volume fermentor (BMS-03NP3, culture apparatus manufactured by ABLE Corporation, Japan). Fermentation of glycerol was conducted for 46.5 h at a temperature of 35 °C, agitation rate of 350 rpm, aeration rate of 0.5 vvm and pH of 7.5 maintained using 25 % (w/v) slurry of calcium hydroxide. A continuous supply of 50 % (w/w) glycerol solution was fed to the fermentation vessel starting from 6 h at the rate of 0.08 g/min to maintain glycerol in the medium in the range of 30 to 40 g/kg medium. The optical purity, consumption of glycerol and production of lactic acid and by-products such as acetic acid, succinic acid and ethanol were monitored as described in Comparative Example 1.

[0100] E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (K. pneumoniae) strain consumed 85.6 g/kg medium of glycerol in 46.5 h to produce 86.0 g/kg medium of lactic acid with a product yield of 1.00 g lactic acid/g glycerol consumed and productivity of 1.848 g lactic acid/kg medium/h. The major by-products produced were acetate - 2.75 g/kg medium, succinate - 0.76 g/kg medium and ethanol - 0.44 g/kg medium. The optical purity of D-lactic acid was 99.77 %. Fig. 1 and Table 1 give a comparison of the fermentation performance of this strain with respect to the control and other strains constructed in the present invention for the fermentation of glycerol to D-lactic acid and formation of by-products acetate and succinate.

[0101] COMPARATIVE EXAMPLE 4

Construction of E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-tpiA strain

The sequence provided for the triosephosphate isomerase gene {tpiA) from E. coli strain MG1655 was obtained using NCBI-Gene ID 948409 and used to design primers with SEQ ID Nos. 35 and 36 for PCR amplification of tpiA. The primer with SEQ ID No. 35 has an Xmal restriction site at the 5' terminus while, the primer with SEQ ID No. 36 has a Hiwdlll restriction enzyme site at the 5' terminus for cloning the tpiA gene. The 5' region of primer SEQ ID No. 35 additionally contains a suitable Shine-Dalgarno sequence downstream of the restriction enzyme site for efficient translation of tpiA protein in the host strain.

[0102] The genomic DNA of E. coli MG1655 strain was extracted using the DNeasy Blood and Tissue Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). The extracted genomic DNA was used as template for PCR amplification of tpiA gene using primers SEQ ID Nos. 35 and 36 under standard thermal cycling conditions for KOD Plus DNA polymerase (Toyobo, Japan). The around 0.77 kb tpiA gene was gel electrophoresed and purified using the QIAquick Gel extraction Kit supplied by Qiagen (Hilden, Germany) and subsequently digested with Xmal and Hz ^' wdlll. The plasmid vector pGAP-dhaK (K. pneumoniae) constructed in Inventive Example 3 was digested with Hwdlll and Xmal to inactivate dhaK (K. pneumoniae). The digested vector was ligated with the digested tpiA gene. The ligated plasmid mixture was transformed to E. coli NEB5a competent cells supplied by New England Biolabs (MA, USA) and allowed to grow at 37 °C on solid LB medium plates containing 100 μg/mL of ampicillin.

Positive colonies harboring the plasmid with tpiA inserted and dhaK (K. pneumoniae) inactivated were screened for by PCR. The constructed plasmid was extracted from E. coli NEB5a and designated as pGAP-tpiA. The presence of the complete ORF of the tpiA gene was confirmed by DNA sequencing analysis using the Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems, USA).

[0103] Competent cells of E. coli MG 1655ApflAdldAmdhAaspA/GAPldhA strain described in Preparation Example 5, prepared by the standard calcium chloride method provided in Sambrook and Russell (Cold Spring Harbor Laboratory Press, 2001), were transformed with pGAP-tpiA plasmid and positive transformants were selected by screening for growth on solid LB medium plates containing 100 μg/mL ampicillin at 35 °C. The present strain was designated as E. coli

MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-tpiA.

[0104] COMPARATIVE EXAMPLE 5

Production of D-lactic acid from glycerol by E. coli

MG 1655ApflAdldAmdhAaspA/GAPldhA/pGAP-tpi A strain

The strain E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-tpiA constructed as described in Comparative Example 4 was tested for the production of D-lactic acid from glycerol. E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-tpiA strain was first inoculated into 100 mL TB culture medium containing 100 μg/mL ampicillin taken in a 500 mL capacity conical flask equipped with baffles. The pre-culture was grown at 35 °C in a rotary incubator with shaking at the rate of 200 rpm for 20.5 h. Forty five milliliters of the pre-culture was used to inoculate 900 g of the fermentation medium with the starting composition of 50 g/kg medium Corn Steep Liquor (CSL), 30 g/kg medium glycerol and 100 μg/ml ampicillin contained in 3 L volume fermentor (BMS-03NP3, culture apparatus manufactured by ABLE Corporation, Japan). Fermentation of glycerol was conducted for 46 h at a temperature of 35 °C, agitation rate of 350 rpm, aeration rate of 0.5 vvm and pH of 7.5 maintained using 25 % (w/v) slurry of calcium hydroxide. A continuous supply of 50 % (w/w) glycerol solution was fed to the fermentation vessel starting from 6 h at the rate of 0.08 g/min to maintain glycerol in the medium in the range of 40 to 50 g/kg medium. The optical purity, consumption of glycerol and production of lactic acid and byproducts such as acetic acid, succinic acid and ethanol were monitored as described in

Comparative Example 1.

[0105] E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-tpiA strain consumed 75.0 g/kg medium of glycerol in 46.0 h to produce 67.2 g/kg medium of lactic acid with a product yield of 0.90 g lactic acid/g glycerol consumed and productivity of 1.462 g lactic acid/kg medium/h. The major by-products produced were acetate - 7.84 g/kg medium, succinate - 1.39 g/kg medium and ethanol - 0.67 g/kg medium. The optical purity of D-lactic acid was 100 %. Fig. 1 and Table 1 give a comparison of the fermentation performance of this strain with respect to the strains constructed in the present invention for the fermentation of glycerol to D-lactic acid and formation of by-products acetate and succinate.

[0106] COMPARATIVE EXAMPLE 6 Construction οϊΕ. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaD-dhaK (K.

pneumoniae)-tpiA strain

The tpiA gene from E. coli MG1655 was amplified by PCR under standard thermal cycling conditions using primers with SEQ ID Nos. 37 and 38 from plasmid pGAP-tpiA constructed in Comparative Example 4. The primer with SEQ ID No. 37 has a Sail restriction site at the 5' terminus while, the primer with SEQ ID No. 38 has a Sphl restriction enzyme site at the 5' terminus for cloning the tpiA gene. The 5' region of primer SEQ ID No. 37 additionally contains a suitable Shine-Dalgarno sequence and additional BamHl and Sad restriction enzyme sites downstream of the Sail site. The around 0.77 kb tpiA gene was gel electrophoresed and purified using the QIAquick Gel extraction Kit supplied by Qiagen (Hilden, Germany) and subsequently digested with Sail and Sphl. The plasmid vector pGAP-dhaD constructed in Inventive Example 3 was digested with Sail- and Sphl and then ligated with the double-digested tpiA gene. The ligated plasmid mixture was transformed to E. coli NEB5a competent cells supplied by New England Biolabs (MA, USA) and allowed to grow at 37 °C on solid LB medium plates containing 100 μg/mL of ampicillin. Positive colonies harboring the plasmid with tpiA inserted were screened for by PCR. The constructed plasmid was extracted from E. coli NEB5a and designated as pGAP- dhaD-tpiA. The presence of the complete ORF of the tpiA gene was confirmed by DNA sequencing analysis using the Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems, USA).

[0107] Next, the dhaK (K. pneumoniae) gene was amplified by PCR under standard thermal cycling conditions using primers with SEQ ID Nos. 39 and 40 from plasmid pGAP-dhaK (K. pneumoniae) constructed in Inventive Example 3. The primer with SEQ ID No. 39 has a BamHl restriction site at the 5' terminus while, the primer with SEQ ID No. 40 has a Sacl restriction enzyme site at the 5' terminus for cloning the dhaK (K. pneumoniae) gene. The 5' region of primer SEQ ID No. 39 additionally contains a suitable Shine-Dalgarno sequence downstream of the BamHl site. The around 1.6 kb dhaK (K. pneumoniae) gene was gel electrophoresed and purified using the QIAquick Gel extraction Kit supplied by Qiagen (Hilden, Germany) and subsequently digested with BamHl and Sacl. The plasmid vector pGAP-dhaD-tpiA described above was digested with BamHl- and Sacl and then ligated with the double-digested dhaK (K. pneumoniae) gene. The ligated plasmid mixture was transformed to E. coli NEB5a competent cells supplied by New England Biolabs (MA, USA) and allowed to grow at 37 °C on solid LB medium plates containing 100 μg/mL of ampicillin. Positive colonies harboring the plasmid with dhaK (K. pneumoniae) inserted were screened for by PCR. The constructed plasmid was extracted from E. coli NEB5a and designated as pGAP-dhaD-dhaK (K. pneumoniae)-Xp K. The presence of the complete ORF of the dhaK (K pneumoniae) gene was confirmed by DNA sequencing analysis using the Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems, USA).

[0108] Competent cells of E. coli MG 1655ApflAdldAmdhAaspA/GAPldhA strain described in Preparation Example 5, prepared by the standard calcium chloride method provided in Sambrook and Russell (Cold Spring Harbor Laboratory Press, 2001), were transformed with pGAP-dhaD- dhaK {K. and positive transformants were selected by screening for growth on solid LB medium plates containing 100 μg/mL ampicillin at 35 °C. The present strain was designated as E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaD-dhaK (K.

pneumoniae)-tp\A.

[0109] COMPARATIVE EXAMPLE 7

Production of D-lactic acid from glycerol by E. coli

MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaD-dhaK (A:.^«eMwo« ae)-tpiA strain The strain E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaD-dha (K. pneumoniae)- tpiA constructed as described in Comparative Example 6 was tested for the production of D- lactic acid from glycerol. E. coli MG1655ApflAd!dAmdhAaspA/GAPldhA/pGAP-dhaD-dhaK (K. pneumoniae)-tpiA strain was first inoculated into 100 mL TB culture medium containing 100 μg/mL ampicillin taken in a 500 mL capacity conical flask equipped with baffles. The pre-culture was grown at 35 °C in a rotary incubator with shaking at the rate of 200 rpm for 20.5 h. Forty five milliliters of the pre-culture was used to inoculate 900 g of the fermentation medium with the starting composition of 50 g/kg medium Corn Steep Liquor (CSL), 30 g/kg medium glycerol and 100 μg/ml ampicillin contained in 3 L volume fermentor (BMS-03NP3, culture apparatus manufactured by ABLE Corporation, Japan). Fermentation of glycerol was conducted for 22.5 hours at a temperature of 35 °C, agitation rate of 350 rpm, aeration rate of 0.5 vvm and pH of 7.5 maintained using 25 % (w/v) slurry of calcium hydroxide. A continuous supply of 50 % (w/w) glycerol solution was fed to the fermentation vessel starting from 6 h at the rate of 0.02 g/min. The consumption of glycerol and production of lactic acid and by-products such as acetic acid, succinic acid and ethanol were monitored as described in Comparative Example 1.

[01 10] E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaD-dhaK (K. pneumoniae)- tpiA strain consumed only 9.0 g/kg medium of glycerol during 22.5 h of fermentation. The strain showed only minimal growth and products formation during this time. Therefore, overexpression of dhaD in addition to dhaK (K. pneumoniae) and tpiA genes was observed to have a toxic effect on the cell metabolism in the presence of glycerol. Fig. 1 and Table 1 give a comparison of the fermentation performance of this strain with respect to the strains constructed in the present invention for the fermentation of glycerol to D-lactic acid and formation of by-products acetate and succinate.

[011 1] INVENTIVE EXAMPLE 5

Construction of E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (K. pneumoniae)- tpiA strain

The plasmid pGAP-dhaD-dhaK (K. constructed as described in Comparative Example 6 was digested with the restriction enzymes EcoRl and Bam HI to excise out the B. megaterium dhaD gene. The around 5 kb double-digested vector backbone was gel

electrophoresed and purified using the QIAquick Gel extraction Kit supplied by Qiagen (Hilden, Germany) and subsequently blunt ends were created using the Blunting High kit (Toyobo, Japan). The blunt-ended vector was self-ligated and the ligation mixture was transformed to E. coli NEB5a competent cells supplied by New England Biolabs (MA, USA) and allowed to grow at 37 °C on solid LB medium plates containing 100 μg/mL of ampicillin. Positive colonies harboring the plasmid with dhaD gene removed were screened for by PCR. The constructed plasmid was extracted from E. coli NEB5a and designated as pGAP-dhaK (K. pneumoniae)-Xp\ ^' A. The presence of the complete ORF's of the dhaK (K. pneumoniae) and tpiA genes was re-confirmed by DNA sequencing analysis using the Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems, USA).

[0112] Competent cells of E. coli MG 1655ApflAdldAmdhAaspA/ GAPldhA strain described in Preparation Example 5, prepared by the standard calcium chloride method provided in Sambrook and Russell (Cold Spring Harbor Laboratory Press, 2001), were transformed with pGAP-dhaK (K. pneumoniae)-tp\A and positive transformants were selected by screening for growth on solid LB medium plates containing 100 μg/mL ampicillin at 35 °C. The present strain was designated as E. coli MG 1655ApflAdldAmdhAaspA/GAPIdhA/pGAP-dhaK (K. pneumoniae)-tpiA.

[0113] INVENTIVE EXAMPLE 6

Production of D-lactic acid from glycerol by E. coli

MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (K. strain The strain E. coli MG 1655 Δρίΐ AdldAmdhAasp A/ G AP ldh A/pGAP-dhaK (K. pnettmoniae)-tpiA constructed as described in Inventive Example 5 was tested for the production of D-lactic acid from glycerol. E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (K. pneumoniae)- tpiA strain was first inoculated into 100 mL TB culture medium containing 100 μg/mL ampicillin taken in a 500 mL capacity conical flask equipped with baffles. The pre-culture was grown at 35 °C in a rotary incubator with shaking at the rate of 200 rpm for 20.5 h. Forty five milliliters of the pre-culture was used to inoculate 900 g of the fermentation medium with the starting composition of 50 g/kg medium Corn Steep Liquor (CSL), 30 g/kg medium glycerol and 100 μg/ml ampicillin contained in 3 L volume fermentor (BMS-03NP3, culture apparatus manufactured by ABLE Corporation, Japan). Fermentation of glycerol was conducted for 46.5 h at a temperature of 35 °C, agitation rate of 350 rpm, aeration rate of 0.5 wm and pH of 7.5 maintained using 25 % (w/v) slurry of calcium hydroxide. A continuous supply of 50 % (w/w) glycerol solution was fed to the fermentation vessel starting from 6 h at the rate of 0.08 g/min to maintain glycerol in the medium in the range of 30 to 40 g/kg medium. The optical purity, consumption of glycerol and production of lactic acid and by-products such as acetic acid, succinic acid and ethanol were monitored as described in Comparative Example 1.

[0114] E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (K. pneumoniae)-tpiA strain consumed 80.8 g/kg medium of glycerol in 46.5 h to produce 79.7 g/kg medium of lactic acid with a product yield of 0.99 g lactic acid/g glycerol consumed and productivity of 1.714 g lactic acid/kg medium/h. The major by-products produced were acetate - 3.18 g/kg medium, succinate - 0.68 g/kg medium and ethanol - 0.54 g/kg medium. The optical purity of D-lactic acid was 99.02 %. Fig. 1 and Table 1 give a comparison of the fermentation performance of this strain with respect to the control and other strains constructed in the present invention for the fermentation of glycerol to D-lactic acid and formation of by-products acetate and succinate.

[0115] INVENTIVE EXAMPLE 7

Construction of E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (E. aerogenes) strain

The sequence provided for the dihydroxyacetone kinase gene (dhaK) from Enterobacter aerogenes was obtained using NCBI-Gene ID 10790941 and used to design primers with SEQ ID Nos. 41 and 42 for PCR amplification of dhaK (E. aerogenes). The primer with SEQ ID No. 41 has a Sail restriction site at the 5' terminus while, the primer with SEQ ID No. 42 has a HmdIII restriction enzyme site at the 5' terminus for cloning the dhaK {E. aerogenes) gene in to an expression vector. The 5' region of primer SEQ ID No. 41 additionally contains a suitable Shine- Dalgarno sequence downstream of the restriction enzyme site for efficient translation of dhaK (E. aerogenes) protein in the host strain.

[01 16] The genomic DNA of E. aerogenes strain ATCC 13048 was extracted using the DNeasy Blood and Tissue Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). The extracted genomic DNA was used as template for PCR amplification of dhaK (E. aerogenes) gene using primers SEQ ID Nos. 41 and 42 under standard thermal cycling conditions for KOD Plus DNA polymerase (Toyobo, Japan). The around 1.6 kb dhaK (E. aerogenes) gene was gel electrophoresed and purified using the QIAquick Gel extraction Kit supplied by Qiagen (Hilden, Germany) and subsequently digested with Sail and Hindlll. The plasmid vector pBRgapP constructed in Comparative Example 2 was also digested with the same two restriction enzymes and then ligated with the Sail- and HmdIII-digested dhaK (E. aerogenes) gene. The ligated plasmid mixture was transformed to E. colt NEB5a competent cells supplied by New England Biolabs (MA, USA) and allowed to grow at 37 °C on solid LB medium plates containing 100 μg/mL of ampicillin. Positive colonies harboring the plasmid with dhaK (E. aerogenes) inserted were screened for by PCR. The constructed plasmid was extracted from E. coli NEB 5a and designated as pGAP-dhaK (E. aerogenes). The presence of the complete ORF of the dhaK (E. aerogenes) gene was confirmed by DNA sequencing analysis using the Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems, USA).

[0117] Competent cells of E. coli MG1655ApflAdldAmdhAaspA/GAPldhA strain described in Preparation Example 5, prepared by the standard calcium chloride method provided in Sambrook and Russell (Cold Spring Harbor Laboratory Press, 2001), were transformed with pGAP-dhaK (E. aerogenes) and positive transformants were selected by screening for growth on solid LB medium plates containing 100 μg/mL ampicillin at 35 °C. The present strain was designated as E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (E. aerogenes).

[0118] INVENTIVE EXAMPLE 8

Production of D-lactic acid from glycerol by E. coli

MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (E. aerogenes) strain

The strain E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (E. aerogenes)

constructed as described in Inventive Example 7 was tested for the production of D-lactic acid from glycerol. E. coli MG 1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (E. aerogenes) strain was first inoculated into 100 mL TB culture medium containing 100 μg/mL ampicillin taken in a 500 mL capacity conical flask equipped with baffles. The pre-culture was grown at 35 °C in a rotary incubator with shaking at the rate of 200 rpm for 20.5 h. Forty five milliliters of the pre-culture was used to inocu late 900 g of the fermentation medium with the starting composition of 50 g/kg medium Corn Steep Liquor (CSL), 30 g/kg medium glycerol and 100 μg/ml ampicillin contained in 3 L volume fermentor (BMS-03NP3, culture apparatus manufactured by ABLE Corporation, Japan). Fermentation of glycerol was conducted for 46.5 h at a temperature of 35 °C, agitation rate of 350 rpm, aeration rate of 0.5 wm and pH of 7.5 maintained using 25 % (w/v) slurry of calcium hydroxide. A continuous supply of 50 % (w/w) glycerol solution was fed to the fermentation vessel starting from 6 h at the rate of 0.08 g/min to maintain glycerol in the medium in the range of 30 to 40 g/kg medium. The optical purity, consumption of glycerol and production of lactic acid and by-products such as acetic acid, succinic acid and ethanol were monitored as described in Comparative Example 1.

[01 19] E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (E. aerogenes) strain consumed 80.9 g/kg medium of glycerol in 46.5 h to produce 79.4 g/kg medium of lactic acid with a product yield of 0.98 g lactic acid/g glycerol consumed and productivity of 1.708 g lactic acid/kg medium/h. The major by-products produced were acetate - 3.43 g/kg medium, succinate - 0.83 g/kg medium and ethanol - 0.54 g/kg medium. The optical purity of D-lactic acid was 99.63 %. Fig. 1 and Table 1 give a comparison of the fermentation performance of this strain with respect to the control and other strains constructed in the present invention for the

fermentation of glycerol to D-lactic acid and formation of by-products acetate and succinate.

[0120] INVENTIVE EXAMPLE 9

Construction of E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK {C. youngae) strain The sequence provided for the dihydroxyacetone kinase gene (dhaK) from Citrobacter youngae was obtained using NCBI-GI 283836372 and used to design primers with SEQ ID Nos. 43 and 44 for PCR amplification of dhaK (C. youngae). The primer with SEQ ID No. 43 has a Sail restriction site at the 5' terminus while, the primer with SEQ ID No. 44 has a H/wdlll restriction enzyme site at the 5' terminus for cloning the dhaK (C. youngae) gene in to an expression vector. The 5' region of primer SEQ ID No. 43 additionally contains a suitable Shine-Dalgarno sequence downstream of the restriction enzyme site for efficient translation of dhaK (C. youngae) protein in the host strain.

[0121] The genomic DNA of C. youngae strain ATCC 29220 was extracted using the DNeasy Blood and Tissue Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). The extracted genomic DNA was used as template for PCR amplification of dhaK (C. youngae) gene using primers SEQ ID Nos. 43 and 44 under standard thermal cycling conditions for KOD Plus DNA polymerase (Toyobo, Japan). The around 1.6 kb dhaK (C. youngae) gene was gel electrophoresed and purified using the QIAquick Gel extraction Kit supplied by Qiagen (Hilden, Germany) and subsequently digested with Sail and H «dIII. The plasmid vector pBRgapP constructed in Comparative Example 2 was also digested with the same two restriction enzymes and then ligated with the Sail- and Hz ^' wdlll-digested dhaK (C. youngae) gene. The ligated plasmid mixture was transformed to E. coli NEB5a competent cells supplied by New England Biolabs (MA, USA) and allowed to grow at 37 °C on solid LB medium plates containing 100 μg/mL of ampicillin. Positive colonies harboring the plasmid with dhaK (C. youngae) inserted were screened for by PCR. The constructed plasmid was extracted from E. coli NEB5a and designated as pGAP-dhaK (C. youngae). The presence of the complete ORF of the dhaK (C. youngae) gene was confirmed by DNA sequencing analysis using the Applied Biosystems 3130 Genetic

Analyzer (Applied Biosystems, USA).

[0122] Competent cells of E. coli MG 1655 ApflAdldAmdhAaspA/ G APldhA strain described in Preparation Example 5, prepared by the standard calcium chloride method provided in Sambrook and Russell (Cold Spring Harbor Laboratory Press, 2001), were transformed with pGAP-dhaK (C. youngae) and positive transformants were selected by screening for growth on solid LB medium plates containing 100 μg/mL ampicillin at 35 °C. The present strain was designated as E. coli MG 1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (C. youngae).

[0123] INVENTIVE EXAMPLE 10

Production of D-lactic acid from glycerol by E. coli

MG 1655ApflAdldAmdhAaspA/GAPldh A/pGAP-dhaK (C. youngae) strain

The strain E. coli MG 1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (C. youngae)

constructed as described in Inventive Example 9 was tested for the production of D-lactic acid from glycerol. E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (C. youngae) strain was first inoculated into 100 mL TB culture medium containing 100 μg/mL ampicillin taken in a 500 mL capacity conical flask equipped with baffles. The pre-culture was grown at 35 °C in a rotary incubator with shaking at the rate of 200 rpm for 20.5 h. Forty five milliliters of the pre- culture was used to inoculate 900 g of the fermentation medium with the starting composition of 50 g/kg medium Corn Steep Liquor (CSL), 30 g/kg medium glycerol and 100 μg/ml ampicillin contained in 3 L volume fermentor (BMS-03NP3, culture apparatus manufactured by ABLE Corporation, Japan). Fermentation of glycerol was conducted for 46.5 h at a temperature of 35 °C, agitation rate of 350 rpm, aeration rate of 0.5 vvm and pH of 7.5 maintained using 25 % (w/v) slurry of calcium hydroxide. A continuous supply of 50 % (w/w) glycerol solution was fed to the fermentation vessel starting from 6 h at the rate of 0.08 g/min to maintain glycerol in the medium in the range of 30 to 40 g/kg medium. The optical purity, consumption of glycerol and production of lactic acid and by-products such as acetic acid, succinic acid and ethanol were monitored as described in Comparative Example 1.

[0124] E. coli MG 1655 ApflAdldAmdhAaspA/ GAPldhA/pGAP-dhaK (C. youngae) strain consumed 80.5 g/kg medium of glycerol in 46.5 h to produce 77.9 g/kg medium of lactic acid with a product yield of 0.97 g lactic acid/g glycerol consumed and productivity of 1.675 g lactic acid/kg medium/h. The major by-products produced were acetate - 2.91 g/kg medium, succinate - 0.79 g/kg medium and ethanol - 0.54 g/kg medium. The optical purity of D-lactic acid was 99.51 %. Fig. 1 and Table 1 give a comparison of the fermentation performance of this strain with respect to the control and other strains constructed in the present invention for the

fermentation of glycerol to D-lactic acid and formation of by-products acetate and succinate.

[0125] The results of the lactic acid production by the respective strains are summarized in the following Table 1 and Fig. 1.

[0126] In Fig. 1, the black square symbol represents the data for strain E. coli

MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (K. pneumoniae) strain in Inventive Example 4; black triangle symbol represents the data for strain E. coli

MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (K. pneumoniae)-tpiA strain in Inventive Example 6; black circle symbol represents the data for strain E. coli

MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (C.freundii) strain in Inventive Example 2; black diamond symbol represents the data for strain E. coli

MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (E. aerogenes) strain in Inventive

Example 8; white diamond symbol represents the data for strain E. coli

MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (C. youngae) strain in Inventive Example 10; white square symbol represents the data for strain E. coli

MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-tpiA strain in Comparative Example 5; white triangle symbol represents the data for strain E. coli

MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaKLM strain in Comparative Example 3; white circle symbol represents the data for strain E. coli MG1655ApflAdldAmdhAaspA/GAPldhA strain in Comparative Example 1 and cross symbol represents the data for strain E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaD-dhaK (K. pneumoniae)-tpi ^' A strain in Comparative Example 7.

[0127]

TABLE 1

Fermentation time was 46.5 h unless specified

Strain background: E. coli MG1655ApflAdIdAmdhAaspA/GAPldhA

Comparative Example 1 : Control (no overexpression)

E. coli MG 1655 ApflAdldAmdhAaspA/ G APldh A strain

Comparative Example 3: "E. coli dhaKLM"

E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaKLM strain

Comparative Example 5: "E. coli tpiA" (Fermentation time of 46.0 h)

E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-tpiA strain

Comparative Example 7: "K. pneumoniae dhaK + B. megaterium dhaD + E. coli tpiA" (Fermentation time of 22.5 h)

E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaD-dhaK (K. pneumoniae)-tpiA strain

Inventive Example 2: "C.freundii dhaK"

E. coli MG1655ApflAdldAmdhAaspA/G APldh A/pGAP-dhaK {C.freundii) strain

Inventive Example 4: " K. pneumoniae dhaK"

E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (K. pneumoniae) strain Inventive Example 6: "K. pneumoniae dhaK + E. coli tpiA " E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (K. pneumoniae)-Vp\K strain Inventive Example 8: "E. aerogenes dhaK"

E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dha (E. aerogenes) strain

Inventive Example 10: "C. yoimgae dhaK"

E. coli MG1655ApflAdldAmdhAaspA/GAPldhA/pGAP-dhaK (C. youngae) strain

[0128] As shown in Table 1 and Fig. 1 , it is demonstrated that the method for producing D-lactic acid according to the invention and the lactic acid-producing bacterium according to the invention enables production of D-lactic acid with higher efficiency and lower production of byproducts.

[0129] The disclosure of Japanese Patent Application No. 2012-078184, filed March 29, 2012, is incorporated herein by reference.

All publications, patent applications, and technical standards mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or technical standard was specifically and individually indicated to be incorporated by reference.