4-香豆酰-CoA连接酶 (4CL) 和聚酮合酶 (PKS1) 的融合蛋白在莱茵衣藻中表达促进树莓酮积累

Overexpression of a fusion protein of 4-coumaroyl-CoA ligase and polyketide synthase for raspberry ketone production in Chlamydomonas reinhardtii

摘要:

树莓酮具有重要的医药价值，如抗流感、预防糖尿病等。为了在莱茵衣藻Chlamydomonas reinhardtii中获得树莓酮，本研究将树莓酮合成的最后两个步骤中的酶，即4-香豆酰-CoA连接酶 (4-coumaryl-CoA ligase，4CL) 和聚酮合酶 (Polyketide synthase，PKS1) 通过甘氨酸-丝氨酸-甘氨酸 (Gly-Ser-Gly，GSG) 三肽接头融合在一起，构建莱茵衣藻表达载体pChla-4CL-PKS1。通过电转化的方法将由PSAD启动子驱动的融合基因4CL-PKS1插入野生型莱茵衣藻 (CC125) 和细胞壁缺失型莱茵衣藻 (CC425) 中。结果在表达4CL-PKS1融合蛋白的野生型莱茵衣藻和细胞壁缺失型莱茵衣藻中，树莓酮含量分别为6.7 μg/g (鲜重) 和5.9 μg/g (鲜重)，高于天然植物中树莓酮的含量（2–4 μg/g）。

Abstract:

Raspberry ketones have important therapeutic properties such as anti-influenza and prevention of diabetes. In order to obtain raspberry ketone from Chlamydomonas reinhardtii, two enzymes catalyzing the last two steps of raspberry ketone synthesis, i.e. 4-coumaryl-CoA ligase (4CL) and polyketide synthase (PKS1), were fused using a glycine-serine-glycine (GSG) tripeptide linker to construct an expression vector pChla-4CL-PKS1. The fusion gene 4CL-PKS1 driven by a PSAD promoter was transformed into a wild-type (CC125) and a cell wall-deficient C. reinhardtii (CC425) by electroporation. The results showed the recombinant C. reinhardtii strain CC125 and CC425 with 4CL-PKS1 produced raspberry ketone at a level of 6.7 μg/g (fresh weight) and 5.9 μg/g (fresh weight), respectively, both were higher than that of the native raspberry ketone producing plants (2–4 μg/g).