ANALYSIS OF POLYHYDROXYALKANOIC ACID-BIOSYNTHESIS GENES OF ANOXYGENICPHOTOTROPHIC BACTERIA REVEALS SYNTHESIS OF A POLYESTER EXHIBITING AN UNUSUAL COMPOSITION

From genomic libraries of purple sulphur bacteria, fragments were clon ed that encoded for proteins involved in the synthesis of poly(3-hydro xyalkanoic acids), PHA. A 12.5- and a 15.0- plus a 15.6-kbp EcoRI-rest riction fragment of Ectothiorhodospira shaposhnikovii or Thiocapsa pfe nnigii, respectively, which hybridized with a fragment encoding the Al caligenes eutrophus PHA-biosynthesis operon, were identified in lambda L47 libraries, whereas an 18.0-kbp EcoRI fragment of Lamprocystis ros eopersicina, which phenotypically complemented a PHA-negative mutant o f A, eutrophus, was identified in a pVK100 cosmid library. Hybridizati on studies and enzymatic analysis of crude extracts derived from trans conjugants of Escherichia coli and A. eutrophus harbouring these fragm ents revealed the presence of the genes for NADH-dependent acetoacetyl -CoA reductase and/or PHA synthase. The PHA-biosynthesis genes of T. p fennigii and L. roseopersicina as well as of Chromatium vinosum, Thioc ystis violacea, Rhodospirillum rubrum and Rhodobacter sphaeroides were then analysed for their ability to confer synthesis of PHA other than poly(3-hydroxybutyric acid) to PHA-negative mutants of PHA-accumulati ng bacteria. The most striking result was that a fragment harbouring t he PHA-synthase gene of T. pfennigii conferred the ability to synthesi ze a polymer consisting of almost equimolar amounts of 3-hydroxybutyra te (48.5 mol%) and 3-hydroxyhexanoate (47.3 mol%) plus a small amount of 3-hydroxyoctanoate (4.2 mol%) to a PHA-negative mutant of Pseudomon as putida. A biosynthetic polyester with this composition has not been described before.