SYNTHESIS OF POLY(3-HYDROXYALKANOATES) IN ESCHERICHIA-COLI EXPRESSINGTHE PHA SYNTHASE GENE PHAC2 FROM PSEUDOMONAS-AERUGINOSA - COMPARISON OF PHAC1 AND PHAC2

In order to obtain functional expression of PHA synthase gene phaC2 fr om Pseudomonas aeruginosa in Escherichia coli, the coding region of ph aC2 was subcloned, including the ribosomal binding site, into pBluescr ipt SK- collinear to the lac promoter. This plasmid pBHR71-C2 enabled functional expression of phaC2 in E. coli LS1298 (fadB) under Inc prom oter control, leading to PHA accumulation, when grown in LB medium con taining 0.5% (w/v) of various fatty acids (C-8-C-14) The strongest acc umulation of PHA was observed, when dodecanoate was provided as carbon source, and PHA contributed to 15% of cell dry weight, which was comp osed of 35 mol% 3-hydroxydodecanoate, 60 mol% 3-hydroxydecanoate and 5 mol% 3-hydroxyoctanoate. Plasmid pBHR78, which contained both genes p haC1 and phaC2 from P. aeruginosa under Inc promoter control in pBlues cript SK- led in E. coli LS1298 to PHA accumulation, which contributed to 13% of cell dry weight, when cells were grown on decanoate. Only s light differences in PHA composition compared with either PhaC1 or Pha C2 were obtained. The weight average molecular masses of PHA purified from decanoate-grown cells of E. coli LS1298 expressing PhaC1 or PhaC2 alone or both PHA synthases, were 106 x 10(3), 70 x 10(3) or 67 x 10( 3), respectively. This study clearly demonstrated that both PHA syntha ses from P. aeruginosa exhibit very similar properties resulting in si milar extent of PHA accumulation, similar composition and molecular ma ss, when expressed in E. coli and that fatty acid beta-oxidation provi des substrates for both PHA synthases.