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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摘要： 4-香豆酰辅酶A连接酶（4-coumarate-CoA ligase, 4CL ）和芪合酶（stilbene synthase, STS）是白藜芦醇苯 丙氨酸代谢合成的最后两个关键酶。运用悬挂PCR （overlap PCR）的方法将烟草4CL基因（Nt4CL）和虎杖 STS基因（PcPKS5）用3个中性氨基酸链连接，得到融合 基因Nt4CL-PcPKS5，将其插入原核表达载体中，构建 pET30a-Nt4CL-PcPKS5重组质粒，表达Nt4CL-PcPKS5融合 蛋白。经Ni2+纯化和PD-10柱脱盐后，得到可溶性纯化蛋 白。体外酶促反应结果表明该融合酶具有4CL和STS的双 重活性，其催化产物为白藜芦醇。酶促反应最适条件为 ：pH 6.5，反应温度为45℃。研究结果获得了有效催化 白藜芦醇生物合成的双功能融合酶，为进一步利用融合 酶基因转化工程菌株实现白藜芦醇工业化生产奠定了基 础。

关键词: 白藜芦醇, 融合基因, 原核表达, 酶促反应