Metabolic engineering for the production of biodegradable polymer using 2-hydroxy acids as a monomer

Abstract

PHAs are polymerized by PHA synthase which uses various hydroxyacyl-CoAs as substrates. Among various hydroxyacyl-CoAs, natural PHA synthases generally accept 3-hydroxyacyl-CoAs (3HA-CoAs) as the most favorable substrates as well as 4-, 5- and 6-hydroxyacyl-CoAs. However, there has not been any report about 2-hydroxyacid containing PHA in vivo by natural PHA producing bacteria. Natural PHA synthases screened up to date have shown very low or negligible in vitro activities towards 2-hydroxyacyl-CoAs compared with other HA-CoAs. Here we report the biosynthesis of 2-hydroxyacid including glycolate, lactate, 2-hydroxybutyrate (2HB), 2-hydroxyisovalerate (2HIV), 2-hydroxyisocaprate (2HIC) by direct fermentation of metabolically engineered Escherichia coli. For the synthesis of PLGA, heterologous metabolic pathways for the generation of lactyl-CoA and glycolyl-CoA, the substrates of engineered PhaC1Ps6-19, were constructed by the expression of evolved propionyl-CoA transferase and Pasteurella multocida glycerate dehydrogenase. And then lactyl-CoA and glycolyl-CoA were polymerized into PLGA by engineered PhaC1Ps6-19. Furthermore, to enhance PLGA biosynthesis, E. coli metabolism was engineered by knocking out the pflB, frdABCD, adhE genes and by replacing the promoters of the ldhA and acs genes with the trc promoter to increase lactate precursors and was further engineered to increase glycolate pools by knocking out the iclR and aceB genes and by replacing the promoters of the aceA gene with the trc promoter based on in silico genome-scale metabolic flux analysis. Using this engineered strain, P(38.4mol%LA-co-61.6mol%GA) could be accumulated up to 32.6 wt% of polymer content from glucose. Next, biosynthesis of 2HB, 2HIV, or 2HIC containing polymer was examined in the recombinant E. coli ldhA mutant XLdh strain expressing PhaC1Ps6-19 and PctCp by varying the concentrations of 2HB, 2HIV, or 2HIC and 3HB added to the defined medium containing 20 g/L of glucose. PHAs cons...