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

Poly(3-hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli harbouring Streptomyces aureofaciens PHB biosynthesis genes: Effect of various carbon and nitrogen sources

Recombinant Escherichia coli (ATCC:PTA–1579) harbouring poly(3-hydroxybutyrate) (PHB) synthesising genes from Streptomyces aureofaciens NRRL 2209 accumulates PHB. Effects of different carbon and nitrogen sources on PHB accumulation by recombinant E. coli were studied. Among the carbon sources used glycerol, glucose, palm oil and ethanol supported PHB accumulation. No PHB accumulated in recombinant cells when sucrose or molasses were used as carbon source. Yeast extract, peptone, a combination of yeast extract and peptone, and corn steep liquor were used as nitrogen sources. The maximum PHB accumulation (60% of cell dry weight) was measured after 48 h of cell growth at 37°C in a medium with glycerol as the sole carbon source, and yeast extract and peptone as nitrogen sources. Scanning electron microscopy of the PHB granules isolated from recombinant E. coli revealed these to be spherical in shape with a diameter ranging from 0.11 to 0.35 μm with the mean value of 0.23 ± 0.06 μm