Biosynthesis of biodegradable polyesters from renewable carbon sources by recombinant bacteria

Based on the metabolic pathways for polyhydroxyalkanoate (PHA) biosynthesis, we succeeded in establishing the recombinant Pseudomonas sp 61-3 strains that synthesize random copolyesters consisting of (R)-3-hydroxybutyrate (3HB) and (R)-medium-chain-length 3-hydroxyalkanoate (mcl-3HA) units, P(3HB-co-3HA), with very high 3HB compositions (up to 94 mol%) from glucose. The mechanical properties of P(94% 3HB-co-3HA) copolyester were very similar to those of low-density polyethylene. We carried out the molecular cloning and characterization of a PhaG(Ps) encoding (R)-3-hydroxyacyl-acyl carrier protein coenzyme A transferase of Pseudomonas sp 61-3. It was concluded that the PhaGPs gene product is involved in providing mcl-3HA-CoA from glucose in the original strain. Heterologous expression of the PhaGPs gene with the PhaC1(Ps) gene encoding PHA synthase from Pseudomonas sp 61-3 was performed in the PhbC(Re) negative mutant (PHB(-)4) of Ralstonia eutropha. The recombinant PHB-4 strain successfully produced PHA copolyesters consisting of 3HB and mcl-3HA units of 6-12 carbon atoms from sugars. The 3HB fraction in copolyesters was very high (95-97 mol%). The PHA content in the recombinant strain could further be increased by the additional introduction of the PhbAB(Re) genes from R eutropha encoding beta-ketothiolase and NADPH-dependent acetoacetyl-coenzyme A reductase. Moreover, we have established an in vivo assay system to analyze mutational effects of R eutropha synthase (PhbCRe) on the level of PHB accumulation in recombinant strains of Escherichia coli. The activity of the PhbCRe could be efficiently estimated using the in vivo system constructed here, and would be useful for in vitro evolution of PhbCRe. (C) 2002 Society of Chemical Industry.