Hydrophila And Aeromonas Polyhydroxyalkanoate (pha) Biosynthesis, A Preliminary Study

To develop a system for gene replacement in Aeromonas hydrophila, conditions were varied systemically to develop an electroporation protocol. The optimal yield of transformant was approximately 4×10~2 CFU/μg DNA at 12.5 kV/ Cm and 1000 O, resulting in a time constant of approximately 5 ms. The A hydrophila transformants expressed plasmid-encoded resistance to chlormaphenicol. Plasmid DNA in the A hydrophila transformant was stably maintained. To our knowledge, this is the first report of genetic manipulation of A. hydrophila and certainly the first report of transformation in this species. The results of this study provide basic genetic tools with which to begin development of more advanced recombinant DNA methodologies and also facilitate the application of recombinant-DNA technology to A. hydrophila.Using the optimized condition, electroporation of suicide plasmid pFH5 (6.5 kb) harboring the disrupted type I polyhydroxyalkanoate (PHA) synthase gene (phaC::Cm) transformed A. hydrophila WQ through an in vivo homologous recombination process, genomic phaC of A. hydrophila was replaced by the disrupted gene and gene Cm was integrated into the genome of A. hydrophila, resulting in type I PHA synthase negative mutant harboring chloramphenicol resistance gene. This result strongly proved that A. hydrophila WQ, via a electroporation procedure, is amenable to genetic manipulation in a way similar to E. coli.Synthesis of medium-chain-length polyhydroxyalkanoates, abbreviated as mcl PHA, was observed in type I PHA synthase negative mutant of A. hydrophila WQ grown on lauric acid or glucose. As wild type A hydrophila WQ normally produced only copolyesters consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx), abbreviated as PHBHHx, the synthesis of mcl PHA in its mutant showed that there was another PHA synthase gene in the genome of wild type A hydrophila WQ.To clone this proposed mcl PHA synthase gene, genomic DNA of wild type A. hydrophila WQ was used as PCR template and a 2.9 kb fragment was obtained. Nucleotide sequences of this region showed a 1680 bp type II PHA synthase gene, which exhibited strong homology to known type II PHA synthase genes of Pseudomonas putida. Based on the DNA sequence information, the complete type II PHA synthase gene was PCR subcloned into plasmid pBBRlMCS2 and expressed in PHA-negative mutant of P. putida. The expression of the putative type II PHA synthase gene fragment allowed PHA-negative mutant of P. putida to synthesize mcl PHA consisting of 24 wt% 3-hydroxydecanoate (3HD) and 76 wt% 3-hydroxydodecanoate (3HDD) from gluconate.