Construction of a Streptomyces sp.–Escherichia coli conjugative shuttle vector and its application for recombinant biosynthesis of poly(3-hydroxyalkanoic acid)

pGTR760 and pGTR761, two new shuttle vectors, withmultiple cloning sites and capable of conjugal transfer from E. coli to Streptomyces sp. were constructed. The poly-3-hydroxybutyrate (PHB) biosynthetic polycistron from Ralstonia eutropha was cloned into the pGTR760 vector to derive the pCABRe plasmid. The pCABRe plasmid was conjugally transferred from E. coli S17-1 to Streptomyces lividans TK64. Fluorescence microscopy of the recombinant and the untransformed S. lividans TK64 revealed presence of polyhydroxyalkanoates (PHAs) in both cell types. GC/GC-MS analysis revealed the accumulated polymer to be polyhydroxyoctanoate (PHO). While the untransformed S. lividans cells accumulate 3.5% PHO of cell dry wt, the recombinant cells accumulate 8% PHO of the cell dry wt. The transformation of S. lividans, however, resulted in slower growth rate, delayed sporulation and impaired pigment formation. Scanning electron microscope analysis revealed broken mycelia probably due to release of accumulated PHO granules from the cells