产姜黄素大肠杆菌工程菌的构建

Production of curcumin by engineered Escherichia coli

摘要:

姜黄素是姜科植物的特征性成分，具有重要的药理活性。文中利用姜黄素生物合成关键酶β-酮酰辅酶A合酶 (Diketide-CoA synthase，DCS) 基因和姜黄素合酶 (Curcumin synthase，CURS) 基因构建非天然融合基因DCS::CURS，并将其与4-香豆酰辅酶A连接酶 (4-coumarate coenzyme A ligase，4CL) 和乙酰辅酶A羧化酶 (Acetyl coenzyme A carboxylase，ACC) 基因共同引入大肠杆菌Escherichia coli，构建由阿魏酸合成姜黄素的工程大肠杆菌，利用LB培养基和改良的M9培养基进行两步发酵。通过对生长培养阶段诱导蛋白表达方法和发酵培养阶段接种菌体量、培养基组成及发酵培养时间等条件的优化，并在发酵培养阶段加入大孔吸附树脂降低产物在培养液中的累积等方法，姜黄素的合成产率达到386.8 mg/L。本研究首次将非天然融合基因DCS::CURS用于合成姜黄素工程菌的构建，得到了姜黄素产率较高的大肠杆菌菌株，为后续通过进一步复杂的代谢网络优化、构建姜黄素合成能力更强的工程菌提供参考。

Abstract:

Curcumin is exclusively isolated from Zingiberaceae plants with a broad spectrum of bioactivities. In the present study, we used the diketide-CoA synthase (DCS) and curcumin synthase (CURS) genes to construct a non-natural fusion gene encoding diketide-CoA synthase::curcumin synthase (DCS::CURS). This fusion protein, together with the acetyl coenzyme A carboxylase (ACC) and the 4-coumarate coenzyme A ligase (4CL), were introduced into Escherichia coli for the production of curcumin from ferulic acid. The process is divided into two stages, the growth stage using LB medium and the fermentation stage using the modified M9 medium. The yield of curcumin reached 386.8 mg/L by optimizing the induction of protein expression in the growth stage, and optimizing the inoculum volume, medium composition and fermentation time in the fermentation stage, as well as the addition of macroporous resin AB-8 into the second medium to attenuate the toxicity of the end product. The exploitation of the non-natural fusion protein DCS::CURS for the production of curcumin provides a new alternative to further promoting the production of curcumin and the related analogues.