title: Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose.
abstract: Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(-)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L(-1) of optically pure D(-)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 μmoles min(-1) (mg protein)(-1). By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(-) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates.
authors: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA.
authors: Qingzhao
authors: Q
authors: Wang
authors: Lonnie O
authors: LO
authors: Ingram
authors: K T
authors: KT
authors: Shanmugam
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D019143:Evolution, Molecular; D005818:Genetic Engineering / Q000379:methods; D006863:Hydrogen-Ion Concentration; D045022:Lactate Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D019344:Lactic Acid / Q000096:biosynthesis*; D008031:Lignin / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013401:Sugar Alcohol Dehydrogenases / Q000096:biosynthesis / Q000235:genetics*; D013696:Temperature; D014169:Transformation, Bacterial / Q000235:genetics
chemical_list: C036909:lignocellulose; D019344:Lactic Acid; D008031:Lignin; D045022:Lactate Dehydrogenases; D013401:Sugar Alcohol Dehydrogenases; C071195:D-lactate dehydrogenase; C020672:glycerol dehydrogenase