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 (PhaC1Ps6-19) 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 PhaC1Ps6-19 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 PhaC1Ps6-19 and PctCp, 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.
|Number of pages||11|
|Journal||Biotechnology and Bioengineering|
|State||Published - 1 Jan 2010|
- Enzyme evolution
- PHA synthase
- Polylactic acid
- Propionate CoA transferase