| Summary: | 碩士 === 國立臺灣海洋大學 === 生命科學暨生物科技學系 === 104 === Secondary metabolites produced from bacteria, fungi, plants,mollusks, algae, insects etc. have been found. The metabolites show pharmacological activities and have potential in the new drug development. Polyketides, a type of secondary metabolites, produced by polyketide synthase (PKS) have medicine application including immune inhibitor, antibiotics, anticholesterol, antitumor, etc. PKSs contain multiple functional domain to synthesize polyketides. Organic extract from fungus NTOU 806 have been demonstrated a biological activity in the inhibiting the production of nitric oxide and lipopolysaccharide-induced inflammation, but not containing cytotoxicity, in RAW264.7 macrophage cells. Whole genome sequencing of NTOU 806ws performed and six PKS genes were chosen: Hyb-zero, PKS2271, PKS2675, PKS3010, PKS3034A, and PKS3034B.We used long range PCR to obtain target genes and these DNA were cloned into pENTR/D-TOPO cloning vector. The PKS genes were cloned into yeast expression vectors using LR reaction technologyin the Gateway cloning system. These vector vectors contain GPD and GAL promoter and consist four different selection makers, The expression vectors were transformed into Saccharomyces cerevisiae, and the induction protein were detected by western blot. HPLC was performed to analyze new compounds in induction medium by NTOU 806 PKSs, observing the new products in the PKS2271, 3034A, 3034B and Hybrid-zero. In protein expression analysis, we found PKS3010, PKS2271, and PKS2675 (MT-ER-KR-ACP) by western blot using anti-GFP antibody.
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