Biosynthesis of polylactic acid and its copolymers using evolved propionate CoA transferase and PHA synthase

For the synthesis of polylactic acid (PLA) and its copolymers by one-step fermentation process, heterologous pathways involving Clostridium propionicum propionate CoA transferase (Pct_Cp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1_Ps6-19) were introduced into Escherichia coli for the generation of lactyl-CoA endogenously and incorporation of lactyl-CoA into the polymer, respectively. Since the wild-type PhaC1P_s6-19 did not efficiently accept lactyl-CoA as a substrate, site directed mutagenesis as well as saturation mutagenesis were performed to improve the enzyme. The wild-type Pct_Cp was not able to efficiently convert lactate to lactyl-CoA and was found to exert inhibitory effect on cell growth, random mutagenesis by error-prone PCR was carried out. By employing engineered PhaC1P_s6-19 and Pct_Cp, poly(3- hydroxybutyrateco-lactate), P(3HB-co-LA), containing 20-49 mol% lactate could be produced up to 62wt% from glucose and 3HB. By controlling the 3HB concentration in the medium, PLA homopolymer and P(3HB-co-LA) containing lactate as a major monomer unit could be synthesized. Also, P(3HBco-LA) copolymers containing various lactate fractions could be produced from glucose alone by introducing the Cupria-vidus necator β-ketothiolase and acetoacetyl-CoA reductase genes. Fed-batch cultures were performed to produce P(3HBco-LA) copolymers having 9-64mol% of lactate, and their molecular weights, thermal properties, and melt flow properties were determined.