Engineering of stable recombinant bacteria for production of chiral medium-chain-length poly-3-hydroxyalkanoates

In order to scale up medium-chain-length polyhydroxyalkanoate (mcl-PHA) pro duction in recombinant microorganisms, we generated and investigated differ ent recombinant bacteria containing a stable regulated expression system fo r phaC1, which encodes one of the mcl-PHA polymerases of Pseudomonas oleovo rans. We used the mini-Tn5 system as a tool to construct Escherichia coli 1 93MC1 and P. oleovorans POMC1, which had stable antibiotic resistance and P HA production phenotypes when they were cultured in a bioreactor in the abs ence of antibiotic selection. The molecular weight and the polydispersity i ndex of the polymer varied, depending on the inducer level. E. coli 193MC1 produced considerably shorter polyesters than P. oleovorans produced; the w eight average molecular weight ranged from 67,000 to 70,000, and the polydi spersity index was 2,7. Lower amounts of inducer added to the media shifted the molecular weight to a higher value and resulted in a broader molecular mass distribution. In addition, we found that E. coli 193MC1 incorporated exclusively the R configuration of the 3-hydroxyoctanoate monomer into the polymer, which corroborated the enantioselectivity of the PhaC1 polymerase enzyme.