THE FUNCTION OF ACKA AND PTA GENES IS NECESSARY FOR POLY(3-HYDROXYBUTYRATE-CO-3-HYDROXYVALERATE) SYNTHESIS IN RECOMBINANT PHA(+) ESCHERICHIA-COLI

In Escherichia coli carrying the poly(3-hydroxyalkanoate) (PHA) biosynthesis pathway on a plasmid (pha(+)), the function of the ackA (acetate kinase) and pra (phosphotransacetylase) genes is necessary for efficient incorporation of 3-hydroxyvalerate (3-HV) into the copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)). Recombinant pha(+) E, coli fadR atoC(Con) strains possessing mutations in ackA, pta, or both ackA and pla exhibited substantially reduced levels of 3-HV formation. Conversely, the same strains carrying the ackA gene on a multicopy plasmid exhibited an increase in 3-HV formation concomitant with a large increase in acetate kinase activity. However, if the strain possessing the multicopy ackA(+) plasmid was mutant at the pra locus, it lost the ability to incorporate significant amounts of 3-HV into P(3HB-co-3HV). In addition to the ackA pta pathway, there is an inducible activity that can also mediate the incorporation of 3-HV into P(3HB-co-3HV). This pathway is repressed by glucose and is not normally operative in P(3HB-co-3HV) production in recombinant pha(+) E. coli strains that are grown using glucose as the major carbon source. It appears likely that this activity is due to an inducible acetyl-CoA synthetase that converts propionate to propionyl-CoA.