Analysis of in vivo substrate specificity of the PHA synthase from Ralstonia eutropha: formation of novel copolyesters in recombinant Escherichia coli

In order to investigate the in vivo substrate specificity of the type I pol yhydroxyalkanoate (PHA) synthase from Ralstonia eutropha, we functionally e xpressed the PHA synthase gene in various Escherichia coli mutants affected in fatty acid beta-oxidation and the wild-type. The PHA synthase gene was expressed either solely (pBHR70) or in addition to the R. eutropha genes en coding beta-ketothiolase and acetoacetyl-coenzyme A (CoA) reductase compris ing the entire PHB operon (pBHR68) as well as in combination with the phaCl gene (pBHR77) from Pseudomonas aeruginosa encoding type II PHA synthase. T he fatty acid P-oxidation route was employed to provide various 3-hydroxyac yl-CoA thioesters, depending on the carbon source, as in vivo substrate for the PHA synthase. In vivo PHA synthase activity was indicated by PHA accum ulation and substrate specificity was revealed by analysis of the comonomer composition of the respective polyester. Only in recombinant E. coli fad m utants harboring plasmid pBHR68, the ii. eutropha PHA synthase led to accum ulation of poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) (poly(3HB-co-3HO)) and poly(3HB-co-3HO-co-3-hydroxydodecanoate (3HDD)), when octanoate and de canoate or dodecanoate were provided as carbon source, respectively. Coexpr ession of phaCl from P. aeruginosa indicated and confirmed the provision of PHA precursor via the P-oxidation pathway and led to the accumulation of a blend of two different PHAs in the respective E. coli strain. These data s trongly suggested that R. eutropha PHA synthase accepts, besides the main s ubstrate 3-hydroxybutyryl-CoA, also the CoA thioesters of 3HO and 3HDD. (C) 2000 Federation of European Microbiological Societies. Published by Elsevi er Science B.V. All rights reserved.