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

Abstract

For the synthesis of polylactic acid (PLA) and its copolymers by one‐step fermentation process, heterologous pathways involving Clostridium propionicum propionate CoA transferase (PctCp) and Pseudomonas sp. MBEL 6‐19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps 6‐19) 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 PhaC1Ps 6‐19 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 PctCp 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 PhaC1Ps 6‐19 and PctCp, poly(3‐hydroxybutyrate‐co‐lactate), P(3HB‐co‐LA), containing 20–49 mol% lactate could be produced up to 62 wt% 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(3HB‐co‐LA) copolymers containing various lactate fractions could be produced from glucose alone by introducing the Cupriavidus necator β‐ketothiolase and acetoacetyl‐CoA reductase genes. Fed‐batch cultures were performed to produce P(3HB‐co‐LA) copolymers having 9–64 mol% of lactate, and their molecular weights, thermal properties, and melt flow properties were determined. Biotechnol. Bioeng. 2010; 105: 150–160. © 2009 Wiley Periodicals, Inc.