Cloning and heterologous expression of the polyketide synthase genes from Leptosphaeria sp. NTOU 806

• Main Authors: Yu, Kai-Chieh, 游凱傑
• Other Authors: Tang, Shye-Jye
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/shkuvu

碩士 === 國立臺灣海洋大學 === 生命科學暨生物科技學系 === 103 === Polyketides are belong to secondary metabolites obtained from bacteria, fungi, plants, mollusks, algae, insects, sponges etc. and contain diverse chemical structure and extensive bioactivity. They have been applied in the field of agriculture, industry and pharmaceuticals such as antibiotics erythromycin and tetracycline used in the human, veterinary antibiotic tylosine, anticancer drug doxorubicin, immunosuppressant rapamycin, or cholesterol-lowering drug lovastatin. Therefore, the discovery and producing of novel polyketides may have a high contribution in the development of new drugs. In order to establish a strategy for heterologous expression of polyketide synthases (PKS) that underline the biosynthesis of polyketide, we choice the PKS genes, pfaA, pfaB, pfaC, pfaD and pfaE, involving EPA biosynthesis in Shewanella oneidensis MR-1. These genes was analyzed by bioinformation to design cloning primers. For cloning EPA PKSs, we extracted genomic DNA and performed long-range PCR to obtain full-length EPA PKS genes. These genes were cloned into pENTER/D-TOPO cloning vector and then used gateway cloning system to construct yeast expression vectors in which harbor GAL1 promoter in their 5’ end and in-frame GFP in their 3’-end and contain ura3, leu, his or trp auxotrophic selection marker, respectively. These EPA PKS expression vectors were transformed into INVSc1 and BJ2168 Saccharomyces cerevisiae by lithium chloride method. After induction, the protein expression of EPA PKSs were analyzed by western blot using GFP antibody, and EPA production was detected by gas chromatography. A Leptosphaeria sp fungus, NTOU806, contains bioactive compounds that inhibited the production of nitric oxide and lipopolysaccharide-induced inflammation and showed low cytotoxicity in RAW264.7 cells. For identification of PKS genes and the biosynthesis of polyketides, we performed whole genome sequencing of NTOU806, finding that NTOU806 contains 10 genes of PKS and 3 genes of nonribosomal peptide synthetase. The PKS genes, not containing intron, of PKS2271, PKS2675, PKS3034A and PKS3034B were performed cloning from genomic DNA of NTOU806 and transformed into S. cerevisiae for expression. Moreover, PKS64 obtained from NTOU2362 were performed large scale culture to purify its polyketide. These yeast transformants were collected culture medium, extracted by ethyl acetate, evaporated to concentrate their products and dissolved by methanol. These collected material were analyzed and purified by high performance liquid chromatography, estimated molecular weight by mass spectrometry, and determined chemical structure by nuclear magnetic resonance. Accroding to analysis of HPLC which shows that gene PKS2271 and PKS2675 can produce new chemical compound. NTOU 2362(PKS 64 gene) can express the new protein by induction of S. cerevisiae BJ2168. We use HPLC analysis to ensure the production of the new compund. after the HPLC analysis,we use MS analysis to analyze the new compound. we found out that the new chemical compoud has the molecular weight 144 now we use the final analysis,NMR,and we found that the formula of the chemical compound is tryptophol.Our results show that PKS genes are successfully expressed in S. cerevisiae, and we establish a reliable and feasible strategy to purify and identify polyketides from heterologous expression.