Cloning And Expression Of Poly-β-hydroxybutyrate Polymerase Gene From Koreensis Pseudomonas UVCN18

PHA is a polyester synthesized intracellularly imbalance in the ratio of carbon and nitrogen conditions, such as polyester carbon and energy accumulated in the cells. PHB is the most studied PHA family by far, the most thorough typical member has good biodegradability, biocompatibility and thermal processing characteristics. Its decomposition products can be used by all the microorganisms, the environment will not cause any pollution, environmental protection, medicine, agriculture, food industry and other fields has a very broad application prospects.However, due to low yield strain PHB production and substrate conversion rate, process complexity, high cost and subsequent application products less product development. It has seriously hampered the widespread use of PHB and industrial development. In order to reduce production costs, PHB production methods become a hot topic, including the use of microbial genetic engineering is the main direction of the current study, the use of a strain of E.coli as an expression construct engineered bacteria. It using not only the rapid growth of E.coli, medium wide source of raw materials etc., and the production of genetically engineered strains PHB significantly improves the yield, showing great potential for development.In this study, the strain Pseudomonas koreensis UVCN 18 genomic DNA as a template, successfully cloned strain Pseudomonas koreensis UVCN18 the PHB polymerase gene.Construction of the cloning vectors pMD18-T-phbC and expression vectors pET-28a(+)-phbC, and build engineering bacteria by IPTG induction of expression products by SDS-PAGE and Western blotting identified the strain Pseudomonas koreensis UVCN18 of PHB polymerase gene phbC achieve a heterologous protein expression.Specific contents of this study are as follows:(1) According to the published NCBI Pseudomonas phbC gene primers were designed. In strain Pseudomonas koreensis UVCN18 genomic DNA as a template, successfully cloned strain Pseudomonas koreensis UVCN18 the PHB polymerase gene. Bioinformatics analysis showed that the full-length gene is 1704 bp, The sequence comparison by BlastP, the amino acid that gene expression belongs to the family PHA polymerase, and bacteria Pseudomonas putida(AHG99486) homology of 96%. The gene sequences obtained submitted to the NCBI, accession number obtained KT716020.(2) Cloning vector: PHB polymerase gene target gene after the PCR amplification is connected with pMD18-T vector, recombinant plasmids pMD18T-phbC, and success will plasmid pMD18T-phbC transferred into E. coli JM109, obtaining cloning strains.(3) Expression Vector: PHB polymerase gene cloning vector is connected to the prokaryotic expression vector pET-28a(+) of prokaryotic recombinant plasmid pET-28a(+)-phbC, transforming competent BL21 strain by containing kanamycin medium screening recombinant strain E. coliBL21-pET-28a(+)- phbC.(4) Identification of Expression: Recombinant expression strain E. coliBL21-pET-28a(+)-phbC after induction by IPTG was extracted crude enzyme solution, by SDS-PAGE electrophoresis and Western blotting identified results PHB polymerase consistent theoretical molecular weight 63 KDa. It proved to achieve the expression of heterologous proteins.