Construction of recombinant Bacillus subtilis strains for polyhydroxyalkanoates synthesis

Plastic wastes are considered to be a worldwide environmental problem and demand for biodegradable plastics has become a highly visible issue. One of the most important characteristics of microbial polyesters is that they are thermoplastic with environmentally degradable properties. In this experiment, pSG703/pha RBC and pSG703/pha PQRBC were cloned and transformed into φ105 prophage-based Bacillus subtilis. Construction of recombinant Bacillus was only partially successful. The pha PQRBC genes from B. megaterium were cloned into B. subtilis 1A304 (φ105 MU331). The pha genes is stable in the absence of selective pressure because the prophage is covalently inserted, in a single copy, into the host chromosome. The B. subtills 1A304 (φ105 MU331) carrying the pha genes (pha PQRBC) was subjected to fermentation and showed PHA accumulation, which was the first report of the expression of the pha genes of B. megaterium in B. subtilis. The cells was subjected for FT-IR and GC analysis and the product was identified to be a PHB homopolymer. The recombinant B. subtilis was expressed with heat shock process also had the PHA accumulation but with the yield lower than the same strain without heat shock that might be due to the RNA polymerase bound to phage promoter and native promoters of pha PQ gene, respectively, and then was run along in the opposite direction during the transcription. The results also showed that the recombinant B. subtilis could utilize the malt waste in the medium as a carbon source better than that of glucose and thus could substantially lowered the cost of production of PHA.