CLONING OF PHBC GENE, ENCODING PHB POLYMERASE ENZYME, IN ALCALIGENES EUTROPHUS PTCC 1615

Background and Aim:Alcaligenes eutrophus PTCC 1615 is well known for its polyhydroxybutyrate production capability. PHB polymerase which encoded by phbC gene is a key enzyme in PHB biosynthesis in A. eutrophus.Methods:For isolation of the target DNA including phbC gene, its promoter and terminator region, primers were designed with Oligo7 and Gene runner software and then evaluated by NCBI Primer Blast. pET28(a) vector which isolated from E. coli DH5α, cleaved by BamHI/HindIII and ligated with the double digested target DNA fragment. Ligation product was transferred in to A. eutrophus PTCC 1615 by CaCl2 and heat shock method. Transformants were selected on LB-agar plate containing kanamycin (200 μg/ml). Plasmid DNAs were isolated from some colonies and PCR was performed for identifying of recombinant plasmids.Results:The size and sequence of isolated DNA fragment were confirmed by agarose gel electrophoresis and the sequencing data, respectively. Also, according to the results of PCR and sequencing, the colonies harboring recombinant DNA molecule were identified.Conclusion:PHB polymerase which encoded by phbC gene, is the most critical enzyme in PHB biosynthesis pathway. Many studies have been indicated that overexpression of phbC gene in PHB producing bacteria, resulted in increased PHB accumulation. In this study, the isolated phbC gene was introduced in to the parent A. eutrophus PTCC 1615 by transformation process. The existence of promoter and terminator regions in isolated DNA fragment is appropriate for overexpression in A. eutrophus at the same time with its own phbC gene, without using an inducer.

Keywords:

cloning , PHB polymerase , PHB , Alcaligenes eutrophus