Anaylsis of polyhydroxyalkanoic acid-biosynthesis genes of anoxygenic phototrophic bacteria reveals synthesis of a polyester exhibiting an unusal composition
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From genomic libraries of purple sulphur bacteria, fragments were cloned that encoded for proteins involved in the synthesis of poly(3-hydroxyalkanoic acids), PHA. A 12.5- and a 15.0- plus a 15.6-kbp EcoRI-restriction fragment of Ectothiordospira shaposhnikovii of Thiocapsa pfennigii, respectively, which hybridized with a fragment encoding the Alcaligenenes eutrophus PHA-biosynthesis operon, were identified in δL47 libraries, whereas an 18.0-kbp EcoRI fragment of Lamprocytis roseopersicina, which phenotypically complemented a PHA-neagative mutant of A. eutrophus, was identified in a pVK100 cosmid library. Hybridization studies and enzymatic analysis of crued extracts derived from transconjugants of Escherichia coli and A. eutrophus harbouring these fragments revealed the presence of the genes for NADH-dependent acetoacetyl-CoA reductase and/or PHA synthase. The PHA-biosynthesis genes of T. pfennigii and L. roseopersicina as wells as of Chromatium vinosum, Thiocystis violacea, Rhodospirillum rubrum and Rodobacter sphaeroides were then analysed for thire ability to confer synthesis of PHA other poly(3-hydroxybutric acid) to PHA-negative mutants of PHA-accumulating bacteria. The most striking result was that a fragment harbouring the PHA-synthase gene of T. pfennigii conferred the ability to synthesize a polymer consisting of almost equimolar amounts of 3-hydroxybutyrate (48.5 mol%) and 3-hydroxyhexanote (47.3%) plus a small amount of 3-hydroxyoctanoate (4.2 mol %) to a PHA-negative mutant of Pseudomonas putida. A niosynthetic polyester with this composition has not been described before.
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