Polylactic acid (PLA) is a promising biomass-derived polymer, but is currently synthesized by a two-step process: fermentative production of lactic acid followed by chemical polymerization. Here we report production of PLA homopolymer and its copolymer, poly(3-hydroxybutyrate-co-lactate), P(3HB-co-LA), by direct fermentation of metabolically engineered Escherichia coli. As shown in an accompanying paper, introduction of the heterologous metabolic pathways involving engineered propionate CoA-transferase and polyhydroxyalkanoate (PHA) synthase for the efficient generation of lactyl-CoA and incorporation of lactyl-CoA into the polymer, respectively, allowed synthesis of PLA and P(3HB-co-LA) in E. coli, but at relatively low efficiency. In this study, the metabolic pathways of E. coli were further engineered by knocking out the ackA, ppc, and adhE genes and by replacing the promoters of the ldhA and acs genes with the trc promoter based on in silico genome-scale metabolic flux analysis in addition to rational approach. Using this engineered strain, PLA homopolymer could be produced up to 11 wt% from glucose. Also, P(3HB-co-LA) copolymers containing 55–86 mol% lactate could be produced up to 56 wt% from glucose and 3HB. P(3HB-co-LA) copolymers containing up to 70 mol% lactate could be produced to 46 wt% from glucose alone by introducing the Cupriavidus necator β-ketothiolase and acetoacetyl-CoA reductase genes. Thus, the strategy of combined metabolic engineering and enzyme engineering allowed efficient bio-based one-step production of PLA and its copolymers. This strategy should be generally useful for developing other engineered organisms capable of producing various unnatural polymers by direct fermentation from renewable resources. Biotechnol. Bioeng. 2010; 105: 161–171. © 2009 Wiley Periodicals, Inc.