合成生物技术改造解脂耶氏酵母合成前列腺素F2α的代谢途径构建与调控研究

Prostaglandin F2α (PGF2α), which is a kind of endogenous natural products with special function, has got increasing attention due to its important application in the field of medicine health. Mammals and some organisms can synthesize PGF2α, however, these organisms produce a very low yield, which cannot meet the needs of people. At present, the chemical synthesis method is mainly used to synthesize PGF2α, however, some hindering obstacles, such as complex reaction routes, environmentally unfriendly and high cost, are still existed in this method. In order to solve the problem, in this project, based on the principle of synthetic biology, we are going to construct and optimize the PGF2α biosynthetic pathway in Yarrowia lipolytica, which is incapable of producing PGF2α. Here, based on the modular pathway engineering strategy, the PGF2α biosynthetic pathway is partitioned into three modules: module1 is composed of △8-elongation and desaturation pathway for arachidonic acid production; module2 is formed by AcoT7 for free arachidonic acid accumulation; module3 consisted of prostaglandin H synthase and prostaglandin F synthase for PGF2α biosynthesis. Moreover, the combined enzyme engineering and compartmentalization engineering is used to improve the heterologous PGF2α production through regulating the three metabolic modules, and the adaption mechanism of the synthetic metabolic pathway and chassis cell will be analyzed. The method of regulating the heterologous metabolic pathway through modular construction and spatial optimization strategy, developed in the present research, aiming at maximizing the metabolic flux to the target products, will provide theoretical guideline and technical support for bio-manufacturing endogenous natural products.

前列腺素F2α（PGF2α）是一种具有多种生理活性的内源性天然产物，因在医药健康领域有着重要应用而得到广泛关注。哺乳动物与一些生物体能够合成PGF2α，然而，产量极低，不能满足人们的需求。目前，主要靠化学法合成PGF2α，化学合成法却存在反应路线复杂、环境不友好、成本高等问题。本课题针对上述问题，基于合成生物学原理，拟在不能合成PGF2α的解脂耶氏酵母体内，构建并优化PGF2α合成途径。通过采用模块化途径工程思路将PGF2α合成途径分为花生四烯酸合成模块、游离花生四烯酸积累模块与PGF2α合成模块；在此基础上，采用酶工程技术手段与区域化工程技术手段组合调控三个代谢模块，实现PGF2α高效异源合成的同时，剖析人工构建的代谢途径与底盘细胞的适配机制。本研究发展一种通过模块化构建、区域化调控异源代谢途径，实现代谢流最大地导向目标产物的方法，将为内源性天然产物的生物制造提供理论指导与技术支撑。

前列腺素F2α（PGF2α）是一种具有多种生理活性的内源性天然产物，因在医药健康领域有着重要应用而得到广泛关注。哺乳动物与一些生物体能够合成PGF2α，然而，产量极低，不能满足人们的需求。目前，主要靠化学法合成PGF2α，化学合成法却存在反应路线复杂、环境不友好、成本高等问题。本课题针对上述问题，基于合成生物学原理，采用无细胞代谢工程策略探究了基于不同生物反应体系生成PGF2α情况，结果发现基于大肠杆菌体系转化花生四烯酸生成PGF2α含量为101 μg/L；相比之下，基于解脂耶氏酵母体系转化花生四烯酸生成PGF2α含量达3.828 mg/L。同时，本研究采用模块化思路设计PGF2α生物合成路径，将其分为花生四烯酸合成模块、花生四烯酸积累模块与PGF2α合成模块。首先，针对花生四烯酸合成模块，通过采用途径工程与酶融合策略优化ARA合成模块，使得解脂耶氏酵母工程菌RH-4从头合成花生四烯酸含量高达118.1 mg/L；其次，考虑到底盘细胞自身具有合成游离花生四烯酸能力，采用途径工程思路在解脂耶氏酵母体内组装花生四烯酸合成模块与PGF2α合成模块。为了优化PGF2α在底盘细胞中生产强度，采用区域化工程策略将PGF2α合成模块分别定位于底盘细胞自身内质网和脂滴，获得工程酵母菌YL-ER1与YL-OLE1，结果发现相比于YL-ER1，工程菌YL-OLE1生产PGF2α能力达125.1 pg/mL；在此基础之上，采用途径工程策略在底盘细胞中从头构建PGF2α生物合成途径，通过组装三个功能模块成功获得四株工程酵母菌株。工程菌发酵72 h后，发现工程菌YL-P1生产PGF2α能力达436.96 pg/mL，实现了PGF2α在解脂耶氏酵母体内的异源从头合成。本研究将为内源性天然产物的生物制造提供理论指导与技术支撑。