ACCUMULATION OF POLY(3-HYDROXYBUTYRIC ACID CO-3-HYDROXYVALERIC ACID CO-4-HYDROXYVALERIC ACID) BY MUTANTS AND RECOMBINANT STRAINS OF ALCALIGENES-EUTROPHUS

Alcaligenes eutrophus accumulated a terpolyester of 3-hydroxybutyric a cid (3HB), 3-hydroxyvaleric acid (3HV), and 4-hydroxyvaleric acid (4HV ) during cultivation with 4HV as carbon and energy source under nitrog en starvation. The polyester accumulated by wild-type strains under th ese conditions contained 4HV at a molar fraction of approximately 5 mo l% only. A catabolic pathway of 4HV was postulated, which included the activation of 4HV to 4HV-CoA and a conversion of 4HV-CoA to 3HV-CoA. Tn5::mob-induced mutants were isolated from A. eutrophus HF39, which w ere affected in 4HV and/or valeric acid catabolism. Among 83 mutants w ere 27 4HV-negative or 4HV-leaky mutants; two mutants were identified which accumulated a terpolyester with a molar fraction of 10.1 to 22.7 mol% 4HV. In addition, a further increase in the molar fraction of 4H V in poly(3HB-co-3HV-co-4HV) and a two- to fourfold increase in the PH A synthase activity were monitored in these mutants or others and also in HF39, if the cells were complemented with the hybrid plasmid pHP10 14::PP1, which contained the PHA biosynthesis genes of A. eutrophus H1 6. Application of mutagenesis plus recombinant DNA techniques resulted in the accumulation of a terpolyester with up to 30 mol% 4HV and with approximately equimolar fractions of 3HB, 3HV, and 4HV.