Studies on polyhydroxyalkanoate (PHA) accumulation in a PHA synthase I-negative mutant of Burkholderia cepacia generated by homogenotization

In the genome of Burkholderia cepacia strain IPT64, which accumulates a ble nd of the two homopolyesters poly(3-hydroxybutyrate), poly(3HB), and poly(3 -hydroxy-4-pentenoic acid), poly(3H4PE), from sucrose or gluconate as singl e carbon source, the polyhydroxyalkanoate (PHA) synthase structural gene wa s disrupted by the insertion of a chloramphenicol-resistant gene cassette ( phaCl::Cm). The suicide vector pSUP202 harboring phaCl::Cm was transferred to B. cepacia by conjugation. The inactivated gene was integrated into the chromosome of B. cepacia by homologous recombination. This mutant and also 15 N-methyl-N'-nitrosoguanidine (NMG)-induced mutants still accumulated low amounts of PHAs and expressed low PHA synthase activity. The analysis of t he mutant phaCl::Cm showed that it accumulated about 1% of PHA consisting o f 68.2 mol% 3HB and 31.8 mol% 3H4PE from gluconate. The wild-type, in contr ast, accumulated 49.3% of PHA consisting of 96.5 mol% 3HB and 3.5 mol% 3H4P E. Our results indicated that the genome of B. cepacia possesses at least t wo PHA synthase genes, which probably have different substrate specificitie s. (C) 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.