# Patent

## Title
Variants of glycerol dehydrogenase having D-lactate dehydrogenase activity and uses thereof

## Abstract

The present invention provides methods of designing and generating glycerol dehydrogenase (GlyDH) variants that have altered function as compared to a parent polypeptide. The present invention further provides nucleic acids encoding GlyDH polypeptide variants having altered function as compared to the parent polypeptide. Host cells comprising polynucleotides encoding GlyDH variants and methods of producing lactic acids are also provided in various aspects of the invention.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application Ser. No. 61/543,003, filed Oct. 4, 2011, the disclosure of which is hereby incorporated by reference in its entirety, including all figures, tables and amino acid or nucleic acid sequences.


This invention was made with government support awarded by Department of Energy (DE-FG36-04G014019 and DE-FG36-08G088142) and U.S. Department of Agriculture, National Institute of Food and Agriculture (2011-10006-30358). The government has certain rights in the invention.


BACKGROUND OF THE INVENTION
Petroleum serves as the primary source of automotive fuel and as the dominating feedstock for organic chemicals and plastics. The finite nature of the petroleum reserves and the negative environmental impact of petroleum use have shifted attention towards alternatives from renewable feedstocks (1-4). Fermentation of carbohydrates has been shown to produce short chain hydroxy acids as well as other chemicals that can be polymerized into plastics and replace petroleum (5). Commercial production of lactic acid using pure bacterial cultures started as early as 1895 (6) and current annual production is >300,000 metric tons. Although lactic acid is primarily used by food and pharmaceutical industries, its application in production of polylactic acid biopolymers (PLA) is expected to exceed other uses, provided the cost of PLA production can be lowered (7, 8).
Lactic acid is condensed into lactide dimers, purified and polymerized into a thermoplastic (7, 9). Blending D(−)- and L(+)-lactic acid polymers provides substantial control of the physical and thermochemical properties and the rate of biodegradation (9). Although lactic acid can be synthesized from petroleum, chemical synthesis produces a mixture of isomers that are not suitable for PLA. Optically pure lactic acid required for PLA synthesis can be readily produced by microbial fermentation (8). L(+)-lactic acid is produced commercially by lactic acid bacteria such as Lactobacillus, Lactococcus, etc. at high yield and titers from glucose and sucrose at temperatures between 30° C. and 40° C. (8, 10). Derivatives of Escherichia coli are the only known commercial D(−)-lactic acid producers (11, 12) and these also operate optimally at 40° C. or lower.
Alternative sources of fermentable sugars such as lignocellulosic biomass and improved microbial biocatalysts are needed to eliminate the use of food carbohydrates (glucose, sucrose) for lactic acid production (13). With cellulose as a feedstock, however, commercial fungal cellulases represent a significant process cost. This cost could be reduced by the development of thermotolerant biocatalysts that effectively ferment under conditions that are optimal (pH 5.0, 50° C.) for fungal cellulases (14, 15).
Lactic acid biocatalysts used by industry metabolize pentose sugars (from hemicellulose) by the phosphoketolase pathway, preventing efficient conversion of these sugars to lactic acid. Lactic acid produced from pentoses using this pathway is contaminated with an equimolar amount of acetic acid (FIG. 1) (8, 16-18). Attempts to improve the xylose fermentation properties of these lactic acid bacteria have met with limited success (18, 19). Although all the pentoses in hemicellulose are efficiently fermented by the E. coli derivatives to D(−)- or L(+)-lactic acid through the pentose-phosphate pathway, the temperature or pH tolerance of this biocatalyst is insufficient to permit cellulosic fermentations at the optimal temperature (50° C.) or pH (5.0) for commercial cellulases (20).
Bacillus coagulans has many desirable properties for the fermentation of lignocellulosic sugars into lactic acid. This bacterium ferments both hexoses and pentoses to L(+)-lactic acid using the highly efficient pentose phosphate pathway (FIG. 1) at 50-55° C. and pH 5.0, conditions that are optimal for commercial fungal cellulases (15, 17, 21). Native strains produce optically pure L(+)-lactic acid at concentrations as high as 180 g L−1 in fed-batch fermentations from either glucose or xylose, and perform well during simultaneous saccharification and fermentation (SSF) of cellulose using fungal cellulases (21, 22). This match in optima both for the fermentation of B. coagulans and for fungal cellulase activity allowed a 4-fold reduction in cellulase usage in comparison to SSF with mesophilic bacterial biocatalysts (21). B. coagulans also grows and ferments sugars in mineral salt medium with inexpensive corn steep liquor (0.25%, w/v) supplementation in contrast to lactic acid bacteria that require complex nutrients (8, 15, 18). Although B. coagulans has excellent potential as a biocatalyst for the conversion of cellulose to optically pure L(+)-lactic acid, an equivalent microbe for production of D(−)-isomer of lactic acid has not been previously described.
BRIEF SUMMARY OF THE INVENTION
The present invention provides methods of designing and generating glycerol dehydrogenase (GlyDH) variants that have altered function as compared to a parent polypeptide. The present invention further provides nucleic acids encoding GlyDH polypeptide variants having altered function as compared to the parent polypeptide. Host cells comprising polynucleotides encoding GlyDH variants and methods of producing lactic acids are also provided in various aspects of the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication, with color drawing(s), will be provided by the Office upon request and payment of the necessary fee.
FIG. 1. Anaerobic metabolic pathways of glucose and xylose in B. coagulans strain P4-102B. Thickness of the arrow indicates glucose or xylose flux to L-lactate (broken line) as the preferred route during anaerobic growth of wild type and to D-lactate in strain QZ19 constructed in this study. Broken arrows (L-LDH and ALS) represent mutations in the two pathways introduced in this study. Xylose fermentation pathway in lactic acid bacteria, such as L. lactis utilizing phosphoketolase pathway resulting in an equimolar lactate and acetate, is also presented for comparison. See Patel et al. for details on pentose fermentation by B. coagulans and L. lactis (17).
FIG. 2. Fermentation profile of D-lactate producing B. coagulans strain QZ19. Glucose concentration for the fermentation at 50° C. was 110 g L−1 (0.6M) and the medium pH at 5.0 was maintained by addition of Ca(OH)2.
FIG. 3. Simultaneous saccharification and fermentation of crystalline cellulose to D-lactic acid by B. coagulans strain QZ19 at 50° C., pH 5.0. Lactococcus lactis data was from Ou et al. (22) and included for comparison. Initial cellulose (Solka Floc) concentration was 40 g L−1.
FIG. 4. SDS-PAGE of proteins from the crude extracts of various B. coagulans strains on the path to D-lactic acid producing strain QZ19. Cultures grown in LB+glucose (30 g L−1) in pH-controlled (5.0) fermentations were harvested during mid- to late-exponential phase of growth and cell extracts were prepared and analyzed. Left lane, molecular weight standards. Numbers on the left represent the corresponding molecular mass of the proteins in kilodaltons. Right lane, pure GlyDH* from strain QZ19.
FIG. 5. Insertion of DNA in the upstream region of gldA gene of B. coagulans strain QZ13 that enhanced gldA expression. An insert DNA (1,821 bp) indicated in italics starts after “C” at −62 of the gldA gene (“A” in ATG as +1) and continues upstream. An eleven base direct repeat (enclosed within the oval) is present at the ends of the insertion, one at the 5-end of the insert and the second at the 5-end of the gldA gene immediately after the 3-end of the insert. ORF1, a putative DeoR family transcriptional regulator; ORF2 and ORF3 in the insert DNA, putative transposase; gldA, glycerol dehydrogenase. Putative −10, −35 and Shine-Dalgarno sequences (SD) are indicated.
FIGS. 6A, 6B, 6C. Fermentation profile of B. coagulans wild type P4-102B (FIG. 6A) and its ldh (strain QZ4, FIG. 6B), and ldh, als (strain QZ5, FIG. 6C) mutants. Fermentations were at 50° C. in small fermenters with pH control at 5.0 by automatic addition of KOH in LB+glucose (0.16M).
FIG. 7. Metabolic evolution of B. coagulans strain QZ5 to D-lactate producing strain QZ19 in small fermenters. Lactic acid titer during metabolic evolution was determined every three days. See text for other details.
FIG. 8. Metabolic evolution of B. coagulans strain QZ14 in small fermenters in LB+glucose at pH 7.0 with increasing glucose concentration. Medium also contained 0.2M CaCO3. Starting glucose concentration was 50 g L−1 (0.28M). After the third transfer, glucose concentration was increased to 60 g L−1 (0.33M). Glucose concentration of the medium was 100 g (0.56M) after the fifth transfer. Lactic acid titer of the culture was determined after 3 days of each transfer. After 60 days of incubation, strain QZ15 that produced about 0.8 M lactic acid was isolated. Strain QZ19 was isolated after an additional 63 days of metabolic evolution.
FIG. 9. Fed-batch fermentation of glucose to D-lactic acid by B. coagulans strain QZ19, at pH 5.0 and 50° C. Fermentations with pH control in LB+glucose were started with 100 g (0.56M) of glucose and at 72 hours, another 100 g L−1 (0.56M) of glucose was added. Culture pH was maintained at 5.0 by Ca(OH)2 addition.
FIG. 10. Fermentation of xylose to D-lactic acid by B. coagulans strain QZ19 at 50° C. and pH 5.0. LB medium contained 80 g (0.53M) of xylose and 0.2 M CaCO3.
FIG. 11. Amino acid sequence of glycerol dehydrogenase with D-lactate dehydrogenase activity from B. coagulans strain QZ19. The amino acids in uppercase were identified after trypsin digestion and LC-MS/MS of fragments.
FIG. 12. Amino acid sequence of glycerol dehydrogenase (GlyDH) (SEQ ID NO: 3) from B. coagulans wild type strain P4-102B and its D-lactate producing derivative, strain QZ19 (SEQ ID NO:1), which contains two mutational changes in the GlyDH (GlyDH*).
FIG. 13. Model of B. coagulans GlyDH based on B. stearothermophilus GlyDH (PDB 1JPU) constructed by Swiss-Model. Native enzyme is colored green and the enzyme from QZ19 (GlyDH*) is in cyan and superimposed. Amino acids 121 and 245 are highlighted, native amino acid in yellow and the altered amino acid (strain QZ19) in magenta. Bottom: Only the amino acids at the active site, based on B. stearthermophilus enzyme structure, are listed. Left, native GlyDH with glycerol at the active site; right, GlyDH* (D121N, F245S) with pyruvate at the active site.
FIG. 14 provides an alignment of various GlyDH polypeptides (SEQ ID NOs: 5-32). As indicated in the alignment, the aspartic acid and phenylalanine found at the positions corresponding to amino acids 121 and 245 of SEQ ID NO: 3 are conserved among all the polypeptides.
FIG. 15. Glycerol dehydrogenase activity of native protein and altered forms of the enzyme obtained during growth-based selection of B. coagulans for D-lactic acid production.
FIG. 16. Lactate dehydrogenase activity of native protein and altered forms of the enzyme obtained during growth-based selection of B. coagulans for D-lactic acid production.
FIG. 17. Insertion sequence upstream of the gldA gene in Bacillus coagulans strain QZ19 (SEQ ID NO: 53).
FIG. 18. Insertion sequence upstream of the gldA gene of Bacillus coagulans strain QZ19 with the gldA gene included. Lowercase letters represent the insertion sequence and uppercase letters represent the gldA sequence and its immediate upstream sequence up to the location of the insertion (after −62 with “A” in “ATG” as +1). The sequence “ttgaca” in lower case, bold, italicized and underlined —————— (positions 1822-1827 in SEQ ID NO: 54) is the putative −35 sequence that was introduced into the gldA upstream sequence by the insertion sequence. The native upstream sequence of gldA did not have the putative −35 sequence. Introduction of this new −35 sequence through the insertion enabled the gldA gene to be expressed and the protein produced at a high level in strain QZ13 and its descendents. The putative −10 sequence from the native gldA gene is also highlighted (bold, italics and double-underlined).
FIG. 19. Native gldA sequence without the insertion sequence. Position of insertion is marked by an arrow (after base 219 of SEQ ID NO: 55).



DETAILED DISCLOSURE OF THE INVENTION
“Nucleotide sequence”, “polynucleotide” or “nucleic acid” can be used interchangeably and are understood to mean, according to the present invention, either a double-stranded DNA, a single-stranded DNA or products of transcription of the said DNAs (e.g., RNA molecules). It should also be understood that the present invention does not relate to genomic polynucleotide sequences in their natural environment or natural state. The nucleic acid, polynucleotide, or nucleotide sequences of the invention can be isolated, purified (or partially purified), by separation methods including, but not limited to, ion-exchange chromatography or molecular size exclusion chromatography, or by genetic engineering methods such as amplification, subtractive hybridization, cloning, subcloning or chemical synthesis, or combinations of these genetic engineering methods.
Both protein and nucleic acid sequence homologies may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW. Sequence comparisons are typically conducted using default parameters provided by the vendor or using those parameters set forth in the above-identified references, which are hereby incorporated by reference in their entireties. As discussed below, GlyDH sequences can be aligned to identify phenylalanine and aspartic acid amino acid residues that correspond to those found at positions 245 and 121, respectively, of SEQ ID NO: 3, and those amino acids can be subsequently substituted by another amino acid, such as serine or asparagine.
The subject invention also provides genetic constructs comprising a polynucleotide sequence encoding a variant GlyDH polypeptide. Genetic constructs as disclosed herein can contain additional regulatory elements such as promoters and enhancers and, optionally, selectable markers. Also within the scope of the subject invention are vectors or expression cassettes containing genetic constructs as set forth herein or polynucleotides encoding the variant GlyDH polypeptides disclosed herein operably linked to regulatory elements. The vectors and expression cassettes may contain additional transcriptional control sequences as well. The vectors and expression cassettes may further comprise selectable markers. The expression cassettes may contain at least one additional gene, operably linked to control elements, to be co-transformed into the organism. Alternatively, the additional gene(s) and control element(s) can be provided on multiple expression cassettes. Such expression cassettes are provided with a plurality of restriction sites for insertion of the sequences of the invention to be under the transcriptional regulation of the regulatory regions. The expression cassette(s) may additionally contain selectable marker genes operably linked to control elements.
The expression cassette will include, in the 5′-3′ direction of transcription, a transcriptional and translational initiation region, a DNA sequence of the invention, and a transcriptional and translational termination region. The transcriptional initiation region, the promoter, may be native or analogous, or foreign or heterologous, to the host cell. By “foreign” is intended that the transcriptional initiation region is not found in the organism into which the transcriptional initiation region is introduced.
The subject invention also provides for the expression of a variant GlyDH polypeptide, encoded by a polynucleotide sequence disclosed herein comprising the culture of a host cell transformed with a polynucleotide encoding said variant GlyDH polypeptide under conditions that allow for the expression of the polypeptide and, optionally, recovering the expressed polypeptide.
Yet another aspect of the invention provides polynucleotides encoding a variant GlyDH (e.g., SEQ ID NO: 1), as well as recombinant vectors and recombinant host cells comprising the nucleic acids or recombinant vectors encoding a variant GlyDH. In one embodiment, the polynucleotide encoding the variant GlyDH of SEQ ID NO: 1 comprises SEQ ID NO: 2.
Yet another aspect of the invention provides variant GlyDH polypeptides having D-lactate dehydrogenase activity. In various embodiments of this aspect of the invention, a GlyDH polypeptide is mutated (altered) such that a phenylalanine corresponding to position 245 of SEQ ID NO: 3 is substituted with a serine (F245S) and/or an aspartic acid corresponding to position 121 of SEQ ID NO: 3 is substituted with an asparagine (D121N). In one embodiment, the variant GlyDH has a single amino acid substitution (F245S or D121N). Other embodiments provide a variant GlyDH that has two amino acid substitutions (F245S or D121N). Yet another embodiment provides a variant GlyDH that comprises SEQ ID NO: 1, or a fragment thereof that has D-lactate dehydrogenase activity. In certain preferred embodiments, fragments of the variant GlyDH polypeptides disclosed herein retain at least one property or activity of the full-length variant GlyDH polypeptide. Such a property or activity is D-lactate dehydrogenase activity. Another embodiment of this aspect of the invention provides for the substitution of the amino acid corresponding to the phenylalanine at position 245 with an amino acid that is synonymous to serine (e.g., Thr, Gly or Asn) and the aspartic acid at corresponding to position 121 of SEQ ID NO: 3 is substituted with an amino acid that is synonymous to asparagine (e.g., Gln).
Other aspects of the invention provide compositions comprising the GlyDH variants disclosed herein. In various embodiments, such compositions typically comprise a carrier, such as a buffer or other liquid medium, in combination with a variant GlyDH as disclosed herein.
Yet another aspect of the invention provides a promoter that causes increased constitutive expression of nucleic acid sequences that are operably linked to the promoter. The new reconstructed promoter (FIG. 5) appears to function constitutively and the level of transcription from this promoter is high (Table 2; gldA mRNA level; Protein level in the SDS-PAGE gel—FIG. 4). This promoter functions in Bacillus coagulans (Gram-positive) and E. coli (Gram-negative). The “−10 and -35” sequences are essentially E. coli consensus and derived by insertion of the transposon. In some embodiments, the nucleotides cga aaattacacc gagcaagtaa agcacggtag ccgaaagtgt acaaaaagag tgtcttggaa tacccgatat ttaaaccttg tcgagaaaaa cttgacacat acCAAAAATA AGTTATGATG GAATTGTGCT TGTTATATTT TTCAC (SEQ ID NO: 56) or tcgagaaaaa cttgacacat acCAAAAATA AGTTATGATG GAATTGTGCT TGTTATATTT TTCAC (nucleotides 84-148 of SEQ ID NO: 56) can be operably linked to a heterologous sequence in order to drive expression of a heterologous gene sequence.
Yet other aspects of the invention provide bacterial cells, fungal cells and yeast cells that demonstrate increased production of D-lactic acid (have increased D-lactate dehydrogenase activity), as compared to reference bacterial, fungal or yeast cells (cells not producing a variant GlyDH polypeptide as disclosed herein). Bacterial cells can be either Gram-negative bacteria or Gram-positive bacteria. In this aspect of the invention, the Gram-negative bacterial cell can be selected from a genus selected from the group consisting of Escherichia, Zymomonas, Acinetobacter, Gluconobacter, Geobacter, Shewanella, Salmonella, Enterobacter and Klebsiella. Gram-positive bacteria can be selected from the group consisting of Bacillus, Clostridium, Corynebacterium, Lactobacillus, Lactococcus, Oenococcus, Streptococcus and Eubacterial cells. Various thermophilic bacterial cells, such as Thermoanaerobes (e.g., Thermoanaerobacterium saccharolyticum), can also be manipulated to increase lactic acid production by way of expression of a variant GlyDH polypeptide as disclosed herein. Other thermophilic microorganisms include, but are not limited to, Bacillus spp., e.g., Bacillus coagulans strains, Bacillus licheniformis strains, Bacillus subtilis strains, Bacillus amyloliquifaciens strains, Bacillus megaterium strains, Bacillus macerans strains, Paenibacillus spp. strains or Geobacillus spp. such as Geobacillus stearothermophilus strains which can be genetically modified. Other Bacillus strains can be obtained from culture collections such as ATCC (American Type Culture Collection) and modified (transformed with a polynucleotide encoding one or more variant GlyDH polypeptides) to have increased lactate dehydrogenase activity by expression of one or more of the variant GlyDH polypeptides disclosed herein. Certain embodiments specifically exclude B. coagulans strain QZ19 (deposited Nov. 4, 2010 with the Agricultural Research Service Culture Collection, 1815 N. University Street, Peoria, Ill., 61604 U.S.A as accession number NRRL B-50443) in its native (untransformed) state.
Other embodiments provide for a yeast cell or fungal cell having increased D-lactate dehydrogenase activity. The yeast cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.
In other embodiments, the cell having increased D-lactate dehydrogenase activity may be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, Oomycota and all mitosporic fungi. A fungal cell may be a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
The fungal host cell may be a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell. For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.
Various other aspects of the invention provide methods of producing lactic acid. In these aspects of the invention, known bacterial, fungal or yeast cells are transformed with a polynucleotide that encodes a variant GlyDH as disclosed herein and then used to produce D-lactic acid. In various embodiments, the methods comprise culturing a bacterial, fungal or yeast cell comprising a polynucleotide encoding a variant GlyDH as disclosed herein under conditions that allow for the production of D-lactic acid.
As used herein, “isolated” refers to bacterial, fungal or yeast cells partially or completely free from contamination by other bacteria. An isolated bacterial, fungal or yeast cell can exist in the presence of a small fraction of other bacteria which do not interfere with the properties and function of the isolated bacterial, fungal or yeast cell (e.g., a bacterial, fungal or yeast cell having increased D-lactate dehydrogenase activity). An isolated bacterial, fungal or yeast cell will generally be at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% pure. Preferably, an isolated bacterial, fungal or yeast cell according to the invention will be at least 98% or at least 99% pure. A “recombinant cell” is a bacterial, fungal or yeast cell that contains a heterologous polynucleotide sequence (e.g., a polynucleotide encoding a variant GlyDH as disclosed herein).
A wild-type bacterial, fungal or yeast cell is the typical form of an organism or strain, for example a bacterial cell, as it occurs in nature, in the absence of mutations. Wild-type refers to the most common phenotype in the natural population. “Parental bacterial, fungal or yeast strain”, “parental bacterial strain”, “parental fungal strain” or “parental yeast strain” is the standard of reference for the genotype and phenotype of a given bacterial, fungal or yeast cell and may be referred to as a “reference strain” or “reference bacterial, fungal or yeast cell”. A “parental bacterial, fungal or yeast strain” may have been genetically manipulated or be a “wild-type” bacterial cell depending on the context in which the term is used. Where D-lactate dehydrogenase expression is increased in genetically modified bacterial, fungal or yeast cells, the reference strain or reference bacterial, fungal or yeast cell will be a wild-type bacterial, fungal or yeast cell.
In various embodiments, compositions comprising microbial cells, such as a bacterial, fungal or yeast cells, transformed with a polynucleotide encoding a variant GlyDH polypeptide as disclosed herein are provided. These compositions can comprise a culture medium appropriate for a particular microbial cell which has been transformed with a polynucleotide encoding a variant GlyDH polypeptide as disclosed herein.
The terms “increasing”, “increase”, “increased” or “increases” refer to increasing by at least 5%, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 100% or more, a particular activity (e.g., increased D-lactate dehydrogenase activity or the increased production of D-lactic acid).
Various aspects of the invention provide for the use of a variety of hydrolysates for the production of D-lactic acid, including, but not limited to, hydrolysate derived from a biomass, a hemicellulosic biomass, a lignocellulosic biomass or a cellulosic biomass. Yet other aspects of the invention provide a bacterial, fungal or yeast cell with increased D-lactate dehydrogenase activity that produces D-lactic acid.
Another aspect of the present invention provides methods of designing and generating glycerol dehydrogenase (GlyDH) variants that have altered function as compared to a parent polypeptide. The present invention further provides nucleic acids encoding GlyDH polypeptide variants having altered function as compared to the parent polypeptide (e.g., alteration of a native GlyDH polypeptide within a host cell or another GlyDH polypeptide such that phenylalanine corresponding to the phenylalanine at position 245 of SEQ ID NO: 3 is changed to serine, glycine, threonine or asparagine and/or the aspartic acid corresponding to the aspartic acid at position 121 of SEQ ID NO: 3 is changed to an asparagine or glutamine). Host cells comprising polynucleotides encoding GlyDH variants and methods of producing lactic acids are also provided in various aspects of the invention.
One aspect of the present invention provides methods of designing and generating GlyDH variants that have altered function as compared to a parent polypeptide. The methods generally involve: a) providing a library of GlyDH polypeptides and aligning the amino acid sequences; b) selecting one or more of the GlyDH polypeptides; c) altering an amino acid corresponding to phenylalanine at position 245 of SEQ ID NO: 3 to serine, glycine, threonine or asparagine and/or altering an amino acid corresponding to aspartic acid at position 121 of SEQ ID NO: 3 to an asparagine or glutamine; and d) optionally testing the altered GlyDH polypeptide for the ability to produce lactic acid. In some embodiments, the individual effect of a single amino acid substitution (at either a phenylalanine corresponding to position 245 or an aspartic acid corresponding to position 121 of SEQ ID NO: 3) on the function of a GlyDH polypeptide is determined. In other embodiments, the effect of two amino acid substitutions (substitutions at a phenylalanine corresponding to position 245 and an aspartic acid corresponding to position 121 of SEQ ID NO: 3) on the function of a GlyDH polypeptide is determined.
In another aspect of the invention, the variant GlyDH polypeptides have altered substrate specificity and/or altered activity as compared to the parent GlyDH polypeptide. Particularly, the variant GlyDH polypeptides can be used to produce D-lactate (D-lactic acid). One embodiment of this aspect of the invention provides a polypeptide comprising SEQ ID NO: 1, or fragments thereof that catalyze the formation of D-lactic acid or have D-lactate dehydrogenase activity.
Finally, the terms “comprising”, “consisting of” and “consisting essentially of” are defined according to their standard meaning. The terms may be substituted for one another throughout the instant application in order to attach the specific meaning associated with each term. The phrases “isolated” or “biologically pure” refer to material that is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.
All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
Example 1
Materials and Methods
Bacterial Strains and Plasmids
Bacterial strains, plasmids and primers used in this study are listed in Tables 3 and 4. B. coagulans strain P4-102B used as the wild-type strain was described previously (17). Plasmid pGK12 replicates in several Gram-positive bacteria and E. coli (30, 31) and its replication is naturally restricted to temperatures ≦42° C. This temperature-sensitive nature of plasmid pGK12 replication at 50° C. provides an opportunity to select for chromosomal DNA integrants of B. coagulans that can grow at 50-55° C.
Medium and Growth Condition
Growth and fermentation conditions have been described previously (15, 21-23). Cultures were grown in L-broth (LB) at pH 5.0 or 7.0, as needed. Sterile glucose was added before inoculation.
Gene Deletions in B. coagulans 
Construction of deletion mutants of B. coagulans was based on previously described methods (32). Plasmid pGK12 was used as the primary vehicle for transfer of DNA to B. coagulans for deletion construction. Presence of plasmid in each cell in a population (109 CFU ml−1) at 37° C. helps to overcome the low transformation efficiency of plasmid DNA into this bacterium and chromosomal integrants in the population were readily identified when the plasmid was eliminated by growth at 50-55° C.
Metabolic Evolution
Metabolic evolution was carried out by sequentially subculturing in small pH-controlled fermenters under indicated conditions. Sequential transfers (2% inoculum; v/v) were made every 1-3 days, as growth permitted.
Enzyme Assays
Cultures were grown (LB-glucose medium in pH-controlled fermenters) to mid-exponential phase, harvested, and used for enzyme assays as previously described (23). Glycerol dehydrogenase from strain QZ19 was purified using differential ammonium sulfate precipitation followed by anion-exchange and hydrophobic interaction chromatography. GlyDH and GlyDH* were also purified as N-terminal His-tagged enzymes (33). GlyDH and LDH were assayed using standard methods (34, 35).
Analytical Methods
Glucose and fermentation products were determined by HPLC as previously described (36). Optical isomers of D-(−)- and L-(+)-lactic acids were determined by HPLC (Chirex 31266(D)-penicillamine column; 150×4.6 mm, 5 micron; Phenomenex) using 2 mM CuSO4 as mobile phase, and enzymatically using D-lactate dehydrogenase (Sigma Chemical Co., St. Louis, Mo.).
Lactate Dehydrogenase Genes in B. coagulans 
B. coagulans strain P4-102B typically produces L-lactate with low (15, 17) or undetectable level of the D-isomer (Table 1). Using the annotated genome sequence of a related B. coagulans strain 36D1 (GenBank Accession number CP003056) as a guide, homologous genes encoding both L-lactate dehydrogenase (ldh) and D-lactate dehydrogenase (ldhA) activities in strain P4-102B were amplified by PCR and confirmed by sequencing. The gene encoding D-lactate dehydrogenase in strain P4-102B was also identified by its ability to suppress anaerobic growth defect of an E. coli mutant (ldhA, pflB) followed by sequencing the corresponding cloned ldhA gene. With appropriate deletions to block competing pathways (FIG. 1), this organism has the potential to produce either D(−)-lactate or L(+)-lactate using only native genes.
Deletion of Native L-(+)-Lactate Dehydrogenase (ldh) and Acetolactate Synthase (alsS) Genes
B. coagulans strain P4-102B produced L-lactic acid as the primary fermentation product at pH 5.0 (Table 1; FIG. 6) together with small amounts of acetate and ethanol. Deletion of the ldh gene encoding L(+)-lactate dehydrogenase (L-LDH) (strain QZ4) using methods developed for B. coagulans eliminated the primary route for NADH oxidation, slowing growth and sugar metabolism. However, production of 2,3-butanediol was increased dramatically with only a small increase in D(−)-lactate (Table 1; FIG. 6). At pH 7.0, the primary product of fermentation was ethanol as seen previously with an ldh mutant of a related strain 36D1 (23).
Strain QZ4 was further modified by deleting acetolactate synthase (alsS), essential for 2,3-butanediol production (FIG. 1). The resulting strain (QZ5) metabolized glucose rapidly at pH 7.0 with ethanol, acetate and formate as primary products (Table 1). In this strain and at this pH, pyruvate flux to D-lactate was only about 5% of the total. However, strain QZ5 did not grow anaerobically when cultured at pH 5.0 in the same medium. Fermentation profiles of strain QZ5 at pH 5.0 were determined by starting cultures with air in the gas phase to support growth. Depletion of O2 in the medium, due to increasing cell density, initiated and maintained anaerobic metabolism of the bacterium. The specific rate of glucose consumption by strain QZ5 at pH 5.0 during the O2-limited phase (0.9 mmoles h−1 g cells−1) was about 15% of the pH 7.0 culture (5.9 mmoles h−1 g cells−1) under similar conditions. The 6-fold lower specific glucose consumption rate and formate titer (Table 1) suggest that pyruvate formate-lyase (PFL) activity limits anaerobic growth of strain QZ5 at pH 5.0. Strain QZ5 also had higher D-LDH activity and a corresponding increase in ldhA mRNA as compared to strain QZ4 (Table 2). However, the D(−)-lactate titer was only about 10 mM in the QZ5 broth irrespective of the culture pH with yields of <0.15 lactate per glucose metabolized (theoretical yield of 2 per glucose).
Since the double mutant produced significant amounts of formate and other products of PFL activity, a triple mutant lacking PFL was constructed. However, this triple mutant lacking L-LDH, ALS and PFL activities failed to grow anaerobically under all conditions tested and was not investigated further.
Growth-Based Selection Increased D(−)-Lactate Production
Sugar metabolism, ATP production and anaerobic growth are apparently constrained in strain QZ5, especially at pH 5.0, by inadequate routes for NADH oxidation (Table 1; FIG. 1). Increasing the level of D(−)-lactate produced by strain QZ5 is expected to restore anaerobic growth of the double mutant to a level similar to that of the parent during L(+)-lactate production. Growth-based metabolic evolution is a powerful tool for selection of advantageous mutations (24) but this requires design of appropriate selection. Since strain QZ5 produced low levels of PFL activity at pH 5.0, as determined by the relative concentration of formate in the broth (Table 1), metabolic evolution of this strain for anaerobic growth at this pH offers a route to co-select increased production of D-lactate.
Metabolic evolution of strain QZ5 at pH 5.0 for higher cell yield in LB+glucose fermentation yielded a derivative with improved growth after 113 days of selection. Cell yield of this strain (QZ13) was higher than the wild type, with a 20-fold increase in D-(−)-lactate production (Table 1; FIG. 7). To further increase the D-lactate yield and titer, strain QZ13 was further evolved at pH 7.0 in LB+glucose fermentations with CaCO3. Addition of CaCO3 to the medium was previously shown to overcome lactate inhibition of B. coagulans fermentation and permit titers approaching 2M in fed-batch fermentations (22). Since solid CaCO3 buffered the medium better at pH 7.0 than at pH 5.0, further metabolic evolutions were at this higher pH. After 42 days of selection, strain QZ14 was isolated and tested. With strain QZ14, D(−)-lactate represented over 90% of the total fermentation products with a yield of 88% of the fermented glucose (Table 1).
Strain QZ15 was isolated after an additional 60 days of serial transfers at pH 7.0 with increasing glucose concentrations (FIGS. 7 and 8). The D(−)-lactic acid titer of strain QZ15 at pH 7.0 was close to 1 M in 72 hours (Table 1). Continued metabolic evolution of strain QZ15 using 100 g L−1 (0.56 M) glucose led to further increase in glucose flux to lactic acid. Strain QZ19 was isolated after 63 days of transfer (FIG. 8) and this strain produced about 1.0 M D(−)-lactate in less than 48 hours at pH 7.0 (Table 1). Fermentation of glucose by strain QZ19 at pH 5.0 started after a short lag with a titer of 1.1 M lactate in 72 hours (FIG. 2). Formate was not detected in the fermentation broth of strains QZ15 and QZ19 producing high levels of lactate. Formate is an indicator of PFL activity since these strains lack formate hydrogen-lyase activity. The small amount of ethanol and acetate (<5% of glucose carbon) produced by strain QZ19 is probably derived from acetyl-CoA produced by the pyruvate dehydrogenase complex (25).
These results show that deletion of the ldh and alsS genes combined with metabolic evolution led to alteration of the primary fermentation product of B. coagulans from L(+)-lactate to D(−)-lactate. Although the pfl genes are still present and transcribed in strain QZ19 at levels that are comparable to that of strain QZ4 (about 1.2 ng ml−1 of total RNA), glucose flux through PFL was minimal as seen by the absence of formate in the fermentation broth. This is apparently a consequence of increasing metabolic flux to D-lactate that supplanted any pyruvate flux through PFL to acetyl-CoA during growth-based selection (Table 1).
Fermentation Characteristics of D-Lactate Producing Strain QZ19
Strain QZ19 produced close to 2M D-lactate in a fed-batch fermentation of glucose at pH 5.0 (FIG. 9). In such a fed-batch fermentation, the first 100 g L−1 (0.56 M) of glucose was fermented in about 50 hours and fermentation of the second 100 g L−1 (0.56 M) of glucose required an additional 72 hours, although the rate of lactate production was linear during this phase. Strain QZ19 also fermented xylose to D-lactic acid after a short lag. Approximately 80 g L−1 (0.53 M) of xylose was converted to D(−)-lactate in 72 hours at 90% yield by weight (FIG. 10).
Simultaneous Saccharification and Fermentation (SSF) of Cellulose to D-Lactic Acid by Strain QZ19
One of the advantages of using B. coagulans as a microbial biocatalyst for lactic acid production is to lower the cellulase enzyme loading in SSF of cellulose to lactic acid due to its higher operating temperature and lower pH that match the optimum for commercial cellulase activity (50° C. and pH 5.0) (14, 15). Strain QZ19 reached the highest volumetric productivity for D-lactate with crystalline cellulose as feedstock at about 7.5 FPU (g cellulose)−1 (FIG. 3) With this enzyme loading, the productivity of a typical lactic acid bacterium Lactococcus lactis at 40° C. was only 1/3 that of strain QZ19. These results are in agreement with previous studies on lower cellulase requirements for optimal L-lactate production by unmodified B. coagulans compared to other lactic acid bacteria (21). Strain QZ19 is better suited for SSF of cellulose to D-lactate and requires significantly lower levels of cellulase for cost-effective metabolism of this non-food carbohydrate.
Identification of Glycerol Dehydrogenase as the Source of D(−)-LDH Activity in Strain QZ19
Cell-free extracts of strain QZ19 had D(−)-lactate dehydrogenase activity (0.2 unit per mg protein) while the wild-type B. coagulans had no detectable D-LDH activity (Table 2). An observed 30-fold increase in D-LDH activity in strain QZ19 compared to strain QZ5 did not correlate with the 1.3-fold increase in the mRNA of the ldhA gene identified as encoding D-LDH. To identify potential mutation(s) in the D-LDH protein that could be responsible for the unusually high D-lactate dehydrogenase activity, the ldhA and flanking DNA from strains P4-102B and QZ19 were sequenced. However, both sequences were identical, indicating that ldhA-encoded D-LDH is not responsible for the large increase in D(−)-lactate production in strain QZ19. Only a single copy of ldhA was also found in the chromosome of strains QZ19 and P4-102B, eliminating gene duplication as a potential basis for D(−)-lactate production. This conclusion was further supported by comparable ldhA mRNA levels of strain QZ19 and its pre-evolution parent, strain QZ5 (Table 2). Higher levels of ldhA mRNA in these strains compared to the wild type could be responsible for the small amount of D(−)-lactate produced by strain QZ5 and other evolved strains, but do not explain the high D-lactate titer and yield observed with strain QZ19. The highest level of ldhA mRNA (D-LDH) in strain QZ19 was only about 10% of the level of ldh mRNA encoding L(+)-LDH, the primary fermentation route in the parent (FIG. 1; Table 2). These results suggest that another enzyme with D-LDH activity is present in strain QZ19, contributing to high D-lactate titer.
Cell-free extracts from strain QZ13 through QZ19 contained an abundant protein (apparent molecular mass of 37 KDa) that was absent in the wild-type strain P4-102B and in the pre-evolution strain QZ5 (FIG. 4). This protein was calculated to represent 11% of the total proteins in the extract of these strains. A protein with D-LDH activity was purified from strain QZ19. The size of this purified protein, 37 KDa, was comparable to the new abundant protein found in the evolved strains. After trypsin digestion of the protein with D-LDH activity, the fragments were separated by LC-MS/MS and identified. Of the 22 peptides generated, 9 were identical to predicted tryptic peptides from a protein in the B. coagulans strain 36D1 genome (Bcoa_1919; Accession number AEP01106.1) that was identified as glycerol dehydrogenase (gldA) by comparative sequence analysis (26) (FIG. 11). Based on the gldA gene sequence of B. coagulans strain 36D1, the gldA genes of strains P4-102B and QZ19 were amplified, cloned and sequenced. The gldA gene from strain QZ19 had two mutations compared to the native gene: G361A and T734C. These two mutations led to amino acid changes, D121N and F245S; the allele is designated as gldA101 and the protein as GlyDH* (FIG. 12). In addition to these two mutations in the coding region, a 1,821 bp DNA fragment was found to be inserted upstream of −62 (“A” in ATG as +1) of the gldA gene in QZ19 (FIG. 5). Two ORFs were identified within this insert (103 and 232 amino acids in length), both with similarity to a putative transposase (ISLhe15) from Lactobacillus helveticus strain H10 (25% identity and 48% similarity) (27).
The purified protein from strain QZ19 corresponding to the 37 KDa protein in the SDS-PAGE gel had both glycerol oxidation and pyruvate reduction activities. The GDH activity (glycerol oxidation to dihydroxyacetone) was 1.2 units (μmole min−1) per mg protein and the pyruvate reduction to D-lactate activity was 0.8 unit per mg protein. The native enzyme purified from recombinant E. coli had a GlyDH activity of 7.1 units and an LDH activity of 0.01 unit per mg protein. These results suggest that a GlyDH in the B. coagulans genome acquired D-LDH activity through two mutations during the metabolic evolution of strain QZ19. The high level of expression of this protein apparently resulted from the upstream transposon insertion.
GlyDH* from Strain QZ19 Produced D-LDH Activity in E. coli 
To confirm that the gldA101 allele in strain QZ19 encodes D-LDH activity, the gene was cloned from strain QZ19 (plasmid pQZ115) and introduced into E. coli strain AH242. E. coli strain AH242 is anaerobic minus due to mutations in ldhA and pflB that abolished the ability to oxidize NADH produced during glycolysis (25). Anaerobic growth of strain AH242 was restored by plasmid pQZ115 with D(−)-lactate as the fermentation product. Strain AH242 carrying plasmid pQZ109 that lacks the C-terminal 10 amino acids of the GlyDH* did not grow anaerobically, indicating that the truncated form of this protein lacks D-LDH activity. These results establish that GlyDH* from B. coagulans strain QZ19 has D(−)-LDH activity.
To further confirm that the gldA101 from strain QZ19 encodes a protein with D-LDH activity, the gene was cloned from strain QZ19 (plasmid pQZ113) and expressed in recombinant E. coli with N-terminal His-tag for protein purification. The recombinant GlyDH* also had both GlyDH and D-LDH activities. The specific activity of the enzyme with glycerol and NAD+ was 0.68 unit per mg protein. With pyruvate and NADH as substrates (LDH reaction), the specific activity of the protein was an unexpectedly high 6.9 units per mg protein and the reaction product was identified as D-lactic acid by HPLC. In the reverse reaction, the enzyme was active only with D(−)-lactate and NAD+ as substrates (specific activity of 0.17 unit per mg protein). Activity with L(+)-lactate as substrate was undetectable.
These results show that during metabolic evolution of strain QZ5, the native GlyDH acquired D-LDH activity. Native GlyDH from B. stearothermophilus has been reported to lack LDH activity (28) and in agreement with this, the LDH activity of B. coagulans native GlyDH purified from recombinant E. coli had less than 0.15% of the GlyDH activity.
Boosting gldA Transcription by Insertion of DNA in the D-Lactate Producing Strains
In addition to evolution of the enzyme as D-LDH, the level of expression of gldA101 in strain QZ19 was the highest of the genes analyzed in this study (Table 2). This increase in transcription is apparently due to the 1,821 bp DNA insertion upstream of the gldA101 gene contributing a strong promoter for gldA101 expression in the evolved strain (FIG. 5).
Although consensus sequences corresponding to Shine-Dalgarno (GGAG) and −10 region (TATGAT) can be identified in the upstream DNA of gldA gene of the wild type, a corresponding −35 region could not be discerned. An inverted repeat with a 6 base pair stem and 11 base pair loop was found at the projected −35 region, suggesting that expression of this gene is only moderate, at best, in the native bacterium. Insertion of the 1,821 bp sequence in the upstream region of the gldA gene in strain QZ19 introduced a consensus −35 sequence to the gldA gene (FIG. 5). The high level of expression of the gldA gene and GDH* protein in strain QZ19 (Table 2) is apparently due to this reconstruction of the gldA upstream region in strain QZ13 and its derivatives yielding a strong promoter.
Evolutionary Path to D-Lactate Production in Strain QZ19
There are apparently three seminal events that occurred in the evolutionary path of B. coagulans strain QZ5 to strain QZ19 that produced over 90 g of D-lactate in about 48 hours: two mutations in the GlyDH (D121N and F245S) and the change in the promoter structure of gldA101 by insertion. DNA sequence analysis of the various intermediate strains in the evolutionary path revealed that the transposon DNA insertion occurred first in strain QZ13. This is in agreement with the elevated levels of gldA mRNA and GlyDH activity in strain QZ13 (about 100-fold and 50-fold, respectively, over the levels of strain QZ5) and other derivatives of this strain (Table 2). The higher GlyDH activity in strain QZ13 also led to a slight increase in D-LDH activity. However, the ratio of D-LDH to GlyDH activity in this strain was only 0.003, suggesting that the GlyDH still lacks significant D-LDH activity. The first GlyDH mutation (F245 S) was detected in strain QZ15 after the increase in transcription of gldA and this mutation apparently increased D-LDH/GlyDH ratio by about 7-fold to 0.02 (Table 2). The second mutation (D121N) occurred during the evolution of strain QZ19 from strain QZ15 and this second mutation increased the D-LDH/GlyDH ratio by about 10-fold to 0.22. It is interesting to note that with the increase in D-LDH activity, the enzyme lost part of its GlyDH activity. A 10-fold increase in D-LDH activity of the enzyme due to the two mutations (QZ19 vs QZ14) in strain QZ19 lowered the GlyDH activity by about 7-fold from that of strain QZ14 (Table 2). This loss in GlyDH activity in the extracts of strain QZ19 is apparently not due to lower protein levels in the cell since this protein band still accounted for about 11% of the total proteins (FIG. 4).
The mutations in the GlyDH of strain QZ19 occurred in two of nine critical amino acids forming a deep pocket where the nicotinamide ring of NAD+ binds in the enzyme from B. stearothermophilus (29). A model of the GlyDH of B. coagulans also shows the deep cleft between the two domains of the enzyme with the mutated amino acids in the edge (FIG. 13). Glycerol C1 and C3 are reported to be stabilized in the B. stearothermophilus GlyDH by van der Walls interactions with the benzyl ring of Phe247. Changing the corresponding Phe245 of B. coagulans GlyDH to serine is expected to abolish this interaction, resulting in the observed reduction in glycerol oxidation activity of the enzyme from strain QZ19. The Asn121 and Ser245 probably stabilize pyruvate at the active site through their interactions with C1 and C2 of pyruvate (FIG. 13), resulting in the observed LDH activity.
These results show that an increase in the expression level of gldA and acquisition of D-LDH activity by the GlyDH through two mutations are responsible for the increase in D-lactate titer of B. coagulans strain QZ19 fermentations (Table 1).
Biochemical Properties of the Native and Various Mutated Forms of Glycerol Dehydrogenase from B. coagulans 
During the development of a D-lactate producing B. coagulans derivative (strain QZ19), a glycerol dehydrogenase (GDH) with two mutations (D121N and F245S) evolved to catalyze the reduction of pyruvate to D-lactate (Table 1 and FIG. 7). The first derivative that produced a significant D-lactate titer, strain QZ15, carried a single mutation that changed the amino acid phenylalanine at position 245 to serine. As strain QZ15 was further selected for higher lactate productivity, the GDH protein acquired a second mutation in amino acid position 121 (D121N) (FIG. 15). Combination of these two mutations led strain QZ19 to produce D-lactic acid at a titer close to 100 g/L in 48 hours at pH 5.0 and 50° C. in pH-controlled fermentations. In order to evaluate the role of each of these two mutations in the catalysis of pyruvate reduction to D-lactate, GDH/LDH enzyme with either of the two substitutions (D121N or F245S) and the protein with both changes were expressed in E. coli, purified, and their biochemical properties were determined.
The native GDH oxidized glycerol at a specific activity of about 22.5 units (μmole min−1 mg protein−1) at 55° C. (Table 5). The specific activity of this enzyme increased from about 8.6 units at room temperature to the 22.5 units at the optimum temperature for activity of 55° C., which is also the optimum growth temperature for B. coagulans. The GDH activity of the native enzyme saturated at about 20 mM glycerol which is in agreement with an apparent Km for this enzyme of 11.7 mM glycerol (FIG. 16; Table 6).
The native enzyme also oxidized several other substrates (Table 5). Although the levels of activity with ethane-1,2-diol and propane-1,2-diol are significantly lower than the activity with glycerol, specific activity with 2,3-butanediol was almost 2 times higher than the rate of glycerol oxidation. 1,2-butanediol and 1,3-butanediol also served as substrates for the native enzyme. These results suggest that the GDH enzyme from B. coagulans has broader substrate specificity. Although this enzyme exhibited pyruvate reduction activity (D-LDH), this activity was less than 1.5% of the glycerol oxidation activity (LDH to GDH activity ratio of 0.013). In addition to reducing pyruvate, this enzyme also had a very low but detectable level of 2-ketobutyric acid reduction (to 2-hydroxybutyric acid) activity. The low pyruvate reduction activity of this enzyme could be attributed to the very high apparent Km for pyruvate (1M; about 100-fold higher than that for glycerol) (Table 6). The native enzyme is about 1,000 times more efficient at oxidizing glycerol with NAD+ as an electron acceptor than reducing pyruvate using NADH.
B. coagulans strain QZ15, a derivative of strain QZ5, fermented glucose at 55° C. and produced about 80 g/L D-lactic acid in about 72 hours. The GDH in this strain carries an amino acid change, phenylalanine to serine, at position 245 (FIG. 7). This change significantly altered the ratio of LDH to GDH to 0.16 from that of 0.01 for the native enzyme with minimal reduction in GDH activity (Table 5; FIG. 15). In addition to gaining significant pyruvate reduction activity (LDH) (FIG. 16), this F245S form of the enzyme also had higher activity with 1,2-propanediol and 1,2-butanediol as substrates compared to the native enzyme (Table 5). These differences in the GDH and LDH activities of the F245S enzyme could be attributed to an increase in apparent Km for glycerol (78.6 mM compared to the value of 11.7 for the native enzyme) and a reduction in apparent Km for pyruvate (0.23 M vs 1.0 M for the native enzyme) (Table 6).
Further growth- and fermentation-based selection of B. coagulans strain QZ15 led to strain QZ19, which produced close to 100 g/L D-lactic acid in about 48 hours at pH 5.0 and 55° C. The GDH enzyme from this strain carries a second alteration, D121N, in addition to the F245S. GDH with this change alone had significantly lower activity with glycerol and other diols tested (Table 5). A slight increase in apparent Km for glycerol could not explain this almost 7-fold reduction in specific activity (Table 6). Although the D121N mutation decreased the glycerol oxidation activity, this change had no detectable effect on pyruvate reduction activity (D-LDH) of the enzyme that was comparable to that of the native enzyme.
The negative effect of the D121N mutation on the oxidation of glycerol and other diols is retained even when this mutation was introduced into the F245S form of the enzyme. The enzyme with both alterations (F245S and D121N) had an LDH/GDH activity ratio of about 1.8 compared to the value of 0.01 for the native enzyme (Table 5). The D121N mutation also lowered the apparent Km for pyruvate from 231 mM for the enzyme with the F245S mutation alone to 51 mM for the enzyme with both changes (Table 6), probably the primary cause of the higher catalytic efficiency with pyruvate as substrate. These results show that both these changes are essential for the enzyme to function as a D-LDH in strain QZ19 developed for D-LDH production. The F245S mutation increased the D-LDH activity of the enzyme while the D121N mutation greatly lowered the GDH activity of the protein that further enhanced the D-LDH activity.
Based on the crystal structure of a GDH from Geobacillus stearothermophilus (29) that is 47% identical to the B. coagulans GDH amino acid sequence, the aspartate at position 121 forms a hydrogen bond with the oxygen in the C2 position of glycerol. This secondary alcohol (H—C—OH) at the C2 position of glycerol is the one that is oxidized to a C═O in the oxidation of glycerol to dihydroxyacetone. Changing this aspartate to asparagine apparently minimized this interaction with the substrate glycerol. In addition, the phenylalanine at 245 is expected to interact with the C1 and C3 of glycerol while the C2-O is interacting with D121. Replacing the phenylalanine with serine could minimize this interaction of the enzyme with the substrate glycerol and this could account for the higher observed apparent Km in these altered forms of the enzymes. However, these observations do not predict that the F245S alone or with D121N would support interaction of the GDH enzyme with pyruvate as a substrate and the altered enzyme would function as a D-LDH.
These results suggest that the native enzyme is indeed a polyol dehydrogenase with broad substrate specificity. The higher activity of the F245S enzyme with various short-chain diols coupled with their thermophilic characteristics (optimum of 55° C.) makes these enzymes useful in bio-conversion of various diols and polyols to corresponding ketones with stereoselectivity. For example, 1,3-butanediol can be oxidized to 4-hydroxy-2-butanone, a potential pharmaceutical intermediate of commercial interest, which is currently produced by the chemical industry.







TABLE 1







Fermentation profiles of B. coagulans derivatives on the path to D(−)-lactic acid production at 50° C.












Glucose

Product (mM)
















Culture
Cell
Consumed

Lactate

Yield†





















Strain
Genotype
pH
Yield
(mM)
Qs*
L(+)-
D(−)
Pyruvate
Acetate
Succ
Formate
Ethanol
Lactate
Total
























P4-102B
wild type
5.0
3.0
144.3
5.7
255.6
UD
UD
5.7
0.6
UD
10.5
0.89
1.00




7.0
7.1
188.6
18.4
336.4
UD
UD
15.9
0.4
UD
4.6
0.89
0.96


QZ4‡
Δldh
5.0
1.8
53.8
0.8
UD
1.0
UD
UD
1.0
6.5
20.3
0.01
1.03




7.0
8.1
226.0
4.3
UD
9.7
UD
42.3
6.7
90.6
224.5
0.02
1.17


QZ5
Δldh Δ alsD
5.0
2.5
32.8
0.5
UD
9.8
4.5
4.6
0.1
25.3
25.6
0.15
0.72




7.0
5.8
152.5
6.8
UD
12.8
10.1
95.4
2.1
154.6
164.5
0.04
0.94


QZ13
Evolved§
5.0
4.3
128.8
2.4
UD
212.4
6.1
2.7
0.6
24.3
23.4
0.82
0.95




7.0
6.0
162.6
4.0
UD
219.8
9.3
14.8
0.4
66.9
58.1
0.68
0.93


QZ14
Evolved§
7.0
ND¶
265.7
5.4
UD
468.4
1.6
UD
1.3
21.5
28.4
0.88
0.93


QZ15
Evolved§
7.0
ND¶
562.6
17.0
UD
928.2
UD
22.6
8.1
UD
97.5
0.83
0.94


QZ19
Evolved§
5.0∥
ND 
580.0
12.5
UD
1,108.1
UD
10.2
UD
UD
40.9
0.96
1.00




7.0
ND¶
590.0
19.4
UD
993.0
UD
45.3
5.7
UD
84.2
0.84
0.96





All fermentations were in LB medium with glucose and the reported values were after 72 h, unless indicated otherwise. Cell yield is expressed in OD420 nm.


*Qs, rate of glucose consumption - mmoles glucose consumed L−1 h−1.


†Product yield is presented as a fraction of theoretical yield from glucose (for lactate, the theoretical yield is 2 per glucose).


‡Strain QZ4 also produced acetoin and 2,3-butanediol; pH 5.0 culture, 44.5 mM 2,3-butanediol; pH 7.0 culture, 31.6 mM acetoin and 93.1 mM 2,3-butanediol. These two products were not detected in the broths from other cultures.


§Evolved, various stages in evolution (FIG. 7).


¶Due to the presence of CaCO3 in the medium, the cell density of these cultures was not determined.


∥Fermentation pH was maintained by addition of Ca(OH)2 and cell density was not determined due to the presence of Ca-salts.


UD—undetectable, less than 0.5 mM; Succ, succinate.













TABLE 2







Enzyme and mRNA levels of B. coagulans strains during


evolution for high level of D-lactate production











mRNA level
Enzyme Activity
Ratio














Strain
Genotype
ldh
gldA
ldhA
GlyDH
D-LDH
D-LDH/GlyDH

















P4-102B
Wild type
61.90
1.13
0.12
0.19
<0.001
<0.005


QZ4
Δldh
0.02
1.29
1.08
0.13
0.002
0.015


QZ5
Δldh, ΔalsS
UD
1.51
4.87
0.13
0.007
0.050


QZ13
Evolution
UD
148.50
3.62
7.06
0.028
0.003


QZ14
Evolution
UD
175.20
3.77
7.12
0.021
0.003


QZ15
Evolution
UD
176.70
5.12
5.60
0.112
0.020


QZ19
Evolution
UD
243.50
6.12
1.01
0.221
0.219





All cultures were grown in pH-controlled fermentations in LB + glucose (30 g/L) at pH 5.0. mRNA levels are ng ml−1 of total RNA. ldh and ldhA represent the mRNA encoding L-LDH and D-LDH, respectively. gldA, glycerol dehydrogenase mRNA.


Enzyme activities were determined in cell extracts and expressed as μmoles min−1 mg protein−1.


UD—Undetectable, <0.01 ng ml−1 of total RNA.













TABLE 3







Bacterial strains and plasmids used in this study











Source or



Relevant genotype
Reference















Strain





B. coagulans
Wild type
(17)



P4-102B



E. coli
mcrA Δ(mrr-hsdRMS-mcrBC)
Invitrogen



Top 10
φ80lacZΔM15 ΔlacX74 nupG




recA1 araD139 Δ(ara-leu)7697




galE15 galK16 rpsL(StrR) endA1



E. coli
ΔldhA, Δ(focA-pflB)
(25)



AH242



B. subtilis
SPβ2Δ 2::Tn917::pSK10Δ6
(37)



HB1000
attSPβ



QZ3
P4-102B ldh::pQZA4, EmR
This work



QZ4
QZ3 Δldh
This work



QZ5
QZ4 ΔalsS
This work



QZ13
QZ5 evolved at pH 5.0 for higher
This work




cell yield



QZ14
QZ13 evolved at pH 7.0 for higher
This work




lactic acid titer



QZ15
QZ14 evolved for higher sugar use
This work



QZ19
QZ15 further evolved for higher
This work




rate of lactate production.



Plasmid



pGK12
Broad host-range, CmR, EmR
(38)



pUC19
Plasmid vector ApR
Lab stock



pQZ44
pGK12 with promoterless ldh (P4-
This work




102B) with 100 bp deletion



pQZ45
pUC19 with P4-102B alsSD
This work



PQZ45-1
pUC19 with 2,380 bp promoterless
This work




P4-102B alsSD



pQZ54
pQZ45-1 with 596 bp alsS deletion
This work




with EmR gene insertion



pQZ64
pGK12 with 506 bp alsS deletion
This work




with EmR gene insertion



PQZ109
pUC19 with 3,185 bp fragment
This work




from QZ19 with truncated gldA



PQZ113
pET15b with gldA from QZ19
This work



PQZ115
pUC19 with 3,422 bp fragment
This work




from QZ19 with gldA

















TABLE 4







Primers used in this study








Primer name
Sequence(5′-3′)





Primer9
ccctacgtaTTGGAACGGGTGCAGTTGGT (SEQ ID NO: 33)


 


Primer10
cccgaattcCCGGGTTGCTGGCAACAAGA (SEQ ID NO: 34)


 


Primer11
cccgaattcTTTGAGCGCCCAATTTGGAA (SEQ ID NO: 35)


 


Primer12
cccaggcctCCGGAACGCCAACGTACACA (SEQ ID NO: 36)


 


Primer17
ACGAGCCGCTGACACTGGAT (SEQ ID NO: 37)


 


Primer18
GCCGTCTTCGCCTTCGTTCA (SEQ ID NO: 38)


 


Primer21
TGTCATAAGTCGCCGAACCG (SEQ ID NO: 39)


 


Primer22
TGATTGTATGCCGCCACGAA (SEQ ID NO: 40)


 


Primer23
GGTGTTGCAGAAGAGCTTGT (SEQ ID NO: 41)


 


Primer24
GTGCCGCAATCGGAATAATC (SEQ ID NO: 42)


 


Primer29
AGATCTTAAGCCGTGTGGAG (SEQ ID NO: 43)


 


Primer30
CGCAACAATACTGCCGATTC (SEQ ID NO: 44)


 


Primer33
TTGGAGGCGAACAAAGAACA (SEQ ID NO: 45)


 


Primer34
CGGCAATGGAAAAAGAAATG (SEQ ID NO: 46)


 


Primer GDH10-R
AGTCCGACACTCAGGCAGAA (SEQ ID NO: 47)


 


Primer GDH11-F
GGCTTACCGTGCTCGAAGAA (SEQ ID NO: 48)


 


D-LDH-3
GCGTTCTTCTGCTAACATCC (SEQ ID NO: 49)


 


GDH-6
GTGCTCGCTTCCTATATCGT (SEQ ID NO: 50)


 


GDH-7
gggctcgagATGACGAAAATCATTACCTC (SEQ ID NO: 51)


 


GDH-12
gggggatccGTGCTCGCTTCCTATATCGT (SEQ ID NO: 52)





Capital letters represent B. coagulans sequence. Lower case letters indicate the restriction enzyme recognition sequence and 5′extensions for optimum cleavage of the PCR-amplified product by the respective enzyme.













TABLE 5







Specific activity of B. coagulans GDH


and its derivatives on various substrates









Specific Activity (μmole · min−1 · mg protein−1)















F245S


Substrate
GDH
F245S
D121N
D121N














Glycerol
22.5
16.8
3.5
2.6


Ethane-1,2-diol
4.5
4.3
0.6
1.8


Propane-1,2-diol
4.3
45.2
2.9
8.4


2,3-butanediol
46.1
68.0
1.1
9.0


1,2-butanediol
4.7
42.9
6.6
10.5


1,3-butanediol
10.8
4.0
3.0
0.6


1-butanol
UD
UD
UD
UD


2-butanol
UD
UD
UD
UD


1,4-butanediol
0.2
0.2
0.01
0.03


Pyruvate*
0.3
2.70
0.2
4.6


2-ketobutyric acid*
0.1
2.70
0.2
3.7


Lactate*
UD
UD
UD
1.8


Malate*
0.02
UD
0.1
UD


LDH/GDH
0.01
0.16
0.06
1.77





[Substrate], 100 mM; Temperature, 55° C.


*With pyruvate and 2-ketobutyric acid the forward reaction was monitored and with lactate and malate, the reverse reaction was followed.


UD, undetectable, less than 0.01 unit of activity.













TABLE 6







Kinetic properties of native and mutated forms


of Glycerol dehydrogenase of B. coagulans











Substrate
Native
F245S
D121N
S245S, D121N










Km(nM)











Glycerol
11.7
78.6
132.5
141.4


Pyruvate
1,030.0
231.0
683.0
51.4


NAD+
0.6
0.6
0.2
0.4


NADH
0.4
0.4
0.4
0.5


D-lactate
ND
ND
243.0
72.9







Kcat


(s−1)











Glycerol
28.0
62.5
13.2
16.7


Pyruvate
3.3
19.5
2.0
35.5


D-lactate
ND
ND
1.9
14.5







Kcat/Km


(s−1 · mM−1)











Glycerol
2.4
0.8
0.1
0.1


Pyruvate
0.003
0.08
0.003
0.7


D-lactate
ND
ND
0.008
0.2





Kinetic properties were determined using glycerol oxidation, pyruvate reduction or lactate oxidation reactions at 55° C.


ND, not determined due to low activity.






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## Claims

1. An isolated polypeptide comprising the sequence of a glycerol dehydrogenase selected from SEQ ID NOs: 5-32 in which a phenylalanine has been substituted with an amino acid selected from serine, threonine, glycine or asparagine and, optionally, an aspartic acid has been substituted with an amino acid selected from glutamine or asparagine, wherein said phenylalanine corresponds to the phenylalanine at position 245 of SEQ ID NO: 5 and said aspartic acid corresponds to the aspartic acid at position 121 of SEQ ID NO: 5, and wherein the isolated polypeptide has D-lactate dehydrogenase activity.

2. The isolated polypeptide according to claim 1, wherein said polypeptide comprises SEQ ID NO: 1.

3. The isolated polypeptide according to claim 1, wherein said polypeptide comprises the sequence of a glycerol dehydrogenase selected from SEQ ID NOs: 5-32 in which a phenylalanine has been substituted with an amino acid selected from serine, threonine, glycine or asparagine and an aspartic acid has been substituted with an amino acid selected from glutamine or asparagine, wherein said phenylalanine corresponds to the phenylalanine at position 245 of SEQ ID NO: 5 and said aspartic acid corresponds to the aspartic acid at position 121 of SEQ ID NO: 5, and wherein the isolated polypeptide has D-lactate dehydrogenase activity.

4. The isolated polypeptide according to claim 1, wherein only the phenylalanine that corresponds to the phenylalanine at position 245 of SEQ ID NO: 5 has been substituted with an amino acid selected from serine, threonine, glycine or asparagine.

5. The isolated polypeptide according to claim 1, wherein said polypeptide comprises amino acid substitutions selected from:
a) a phenylalanine corresponding to the phenylalanine at position 245 of SEQ ID NO: 5 has been substituted with serine and an aspartic acid corresponding to the aspartic acid at position 121 of SEQ ID NO: 5 has been substituted with glutamine;
b) a phenylalanine corresponding to the phenylalanine at position 245 of SEQ ID NO: 5 has been substituted with threonine and an aspartic acid corresponding to the aspartic acid at position 121 of SEQ ID NO: 5 has been substituted with glutamine;
c) a phenylalanine corresponding to the phenylalanine at position 245 of SEQ ID NO: 5 has been substituted with threonine and an aspartic acid corresponding to the aspartic acid at position 121 of SEQ ID NO: 5 has been substituted with asparagine;
d) a phenylalanine corresponding to the phenylalanine at position 245 of SEQ ID NO: 5 has been substituted with glycine and an aspartic acid corresponding to the aspartic acid at position 121 of SEQ ID NO: 5 has been substituted with glutamine;
e) a phenylalanine corresponding to the phenylalanine at position 245 of SEQ ID NO: 5 has been substituted with glycine and an aspartic acid corresponding to the aspartic acid at position 121 of SEQ ID NO: 5 has been substituted with asparagine;
f) a phenylalanine corresponding to the phenylalanine at position 245 of SEQ ID NO: 5 has been substituted with asparagine and an aspartic acid corresponding to the aspartic acid at position 121 of SEQ ID NO: 5 has been substituted with glutamine;
g) a phenylalanine corresponding to the phenylalanine at position 245 of SEQ ID NO: 5 has been substituted with asparagine and an aspartic acid corresponding to the aspartic acid at position 121 of SEQ ID NO: 5 has been substituted with asparagine; and
h) only a phenylalanine corresponding to the phenylalanine at position 245 of SEQ ID NO: 5 has been substituted with an amino acid selected from serine, threonine, glycine or asparagine in said polypeptide.

6. The isolated polypeptide according to claim 1, wherein said polypeptide consists of the sequence of the glycerol dehydrogenase with amino acid substitutions selected from:
a) a phenylalanine corresponding to the phenylalanine at position 245 of SEQ ID NO: 5 has been substituted with serine and an aspartic acid corresponding to the aspartic acid at position 121 of SEQ ID NO: 5 has been substituted with glutamine;
b) a phenylalanine corresponding to the phenylalanine at position 245 of SEQ ID NO: 5 has been substituted with threonine and an aspartic acid corresponding to the aspartic acid at position 121 of SEQ ID NO: 5 has been substituted with glutamine;
c) a phenylalanine corresponding to the phenylalanine at position 245 of SEQ ID NO: 5 has been substituted with threonine and an aspartic acid corresponding to the aspartic acid at position 121 of SEQ ID NO: 5 has been substituted with asparagine;
d) a phenylalanine corresponding to the phenylalanine at position 245 of SEQ ID NO: 5 has been substituted with glycine and an aspartic acid corresponding to the aspartic acid at position 121 of SEQ ID NO: 5 has been substituted with glutamine;
e) a phenylalanine corresponding to the phenylalanine at position 245 of SEQ ID NO: 5 has been substituted with glycine and an aspartic acid corresponding to the aspartic acid at position 121 of SEQ ID NO: 5 has been substituted with asparagine;
f) a phenylalanine corresponding to the phenylalanine at position 245 of SEQ ID NO: 5 has been substituted with asparagine and an aspartic acid corresponding to the aspartic acid at position 121 of SEQ ID NO: 5 has been substituted with glutamine;
g) a phenylalanine corresponding to the phenylalanine at position 245 of SEQ ID NO: 5 has been substituted with asparagine and an aspartic acid corresponding to the aspartic acid at position 121 of SEQ ID NO: 5 has been substituted with asparagine; and
h) a phenylalanine corresponding to the phenylalanine at position 245 of SEQ ID NO: 5 has been substituted with an amino acid selected from serine, threonine, glycine or asparagine in said polypeptide.

7. A composition comprising a carrier and a polypeptide according to claim 1.

8. The composition according to claim 7, wherein said carrier comprises a hydrolysate derived from a biomass, a hemicellulosic biomass, a lignocellulosic biomass or a cellulosic biomass.

9. A composition comprising a diol and/or polyol and a polypeptide according to claim 1.

10. The composition according to claim 9, wherein the diol is 1,3-butanediol.

11. A method of producing 4-hydroxy-2-butanone comprising contacting 1,3-butanediol with a polypeptide according to claim 1 under conditions that result in the oxidation of 1,3-butanediol to 4-hydroxy-2-butanone.

12. An isolated nucleic acid comprising a polynucleotide encoding a polypeptide according to claim 1.

13. A vector or genetic construct comprising a polynucleotide according to claim 12.

14. An isolated microbial cell transformed with a nucleic acid according to claim 12.

15. The isolated microbial cell according to claim 14, wherein said microbial cell comprises a vector or genetic construct comprising said nucleic acid.

16. The isolated microbial cell according to claim 14, wherein said microbial cell is a Gram-negative or a Gram-positive bacterial cell.

17. The isolated microbial cell according to claim 16, wherein the Gram-negative bacterial cell is a bacterial cell selected from the genera of Escherichia, Zymomonas, Acinetobacter, Gluconobacter, Geobacter, Shewanella, Salmonella, Enterobacter or Klebsiella and the Gram-positive bacterial cell is a bacterial cell selected from the genera of Bacillus, Clostridium, Corynebacteria, Lactobacillus, Lactococcus, Oenococcus, Streptococcus and Eubacteria.

18. The isolated microbial cell according to claim 14, wherein the microbial cell is Escherichia coli or Klebsiella oxytoca.

19. The isolated microbial cell according to claim 14, wherein the microbial cell is selected from Thermoanaerobes, Bacillus spp., Paenibacillus spp. or Geobacillus spp.

20. The isolated microbial cell according to claim 14, wherein the microbial cell is a yeast cell selected from a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

21. The isolated microbial cell according to claim 20, wherein the yeast cell is Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica.

22. The isolated microbial cell according to claim 14, wherein said microbial cell is a fungal cell selected from an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

23. The isolated microbial cell according to claim 22, wherein said fungal cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

24. A composition comprising culture medium and a transformed microbial cell according to claim 14.

25. The composition according to claim 24, wherein said culture medium comprises a hydrolysate derived from a biomass, a hemicellulosic biomass, a lignocellulosic biomass or a cellulosic biomass.

26. A method of making D-lactic acid comprising culturing a transformed microbial cell according to claim 14 in a culture medium under conditions that allow for the production of D-lactic acid.

27. The method according to claim 26, wherein said culture medium comprises a hydrolysate derived from a biomass, a hemicellulosic biomass, a lignocellulosic biomass or a cellulosic biomass.