关键词: 白藜芦醇, 融合基因, 原核表达, 酶促反应

Abstract: 4-coumarate coenzyme A ligase (4CL) and stilbene synthase (STS) are two key final rate-limiting enzymes in the resveratrol phenylalanine metabolic synthesis. In this paper, Nicotiana tabacum 4CL and Polygonum cuspidatum PKS5 were fused together by overlap PCR with three amino acids to construct fusion gene Nt4CL-PcPKS5. The fusion gene was subcloned into pET30a, the recombinant plasmid pET30a-Nt4CL-PcPKS5 was analyzed through heterologous expression in Escherichia coli. The expression product was purified with Ni2+ affinity column and desalted through PD-10 column. The collected target fusion protein was used for enzymatic reaction in vitro. The enzymatic product analysis showed that the fusion protein showed the activities of both 4CL and STS. The final product was resveratrol. The optimum pH and reaction temperature were 6.5 and 45℃. In this study, a bifunctional fusion enzyme catalyzing resveratrol biosynthesis was obtained, which proved a basis for realizing industrial production of resveratrol.

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