脂肪酸聚酮合酶的催化机制解析及二十碳五烯酸的定向合成

Directional synthesis of unsaturated fatty acids is one of the methods to solve the low productivity problem of some high nutritional scarce fatty acids. Polyunsaturated fatty acid/polyketide synthase (PUFA/PKS) can directly synthesize different kinds of polyunsaturated fatty acids. Compared with the elongase and desaturase pathway, PUFA/PKS pathway has higher energy utilization efficiency and modification space. In this proposal, taking the docosahexaenoic acid and eicosapentaenoic acid biosynthesis gene clusters as the research objects, we want to synthesize the natural low-efficiency EPA in the industrialized mature DHA producing strain Schizochytrium using the strategies of in vitro reaction and in vivo domain swap. The double bonds retention mechanism of the fatty acid chain by extension domain KS and modification domain ER and the substrate preference of the final release module AT will be investigated by in vitro reaction with acyl-ACP with different chain length and saturation. The specificity domain which can introduce the double bond at the ω-3 position and the domain which can release the EPA-ACP will be obtained through site-directed mutagenesis; the mutant strains of Schizochytrium will be constructed to synthesize EPA by constructing the hybrid PUFA/PKS synthase through the complement and domain swap of the specific domains, such as the single domain of KS, ER, AT or different combinations of these domains. In addition, the ability of the mutant strains to synthesize EPA will also be enhanced by optimizing the cultivation conditions. This project will develop a method for the synthesis of specific fatty acids by in vitro testing and in vivo modification, which will provide a new strategy for the biological production of natural low-efficiency fatty acid products and encourage the innovation in the field of fatty acids biosynthesis.

不饱和脂肪酸的定向合成是解决高营养稀缺脂肪酸低效率合成的关键，脂肪酸聚酮合酶可从头合成不饱和脂肪酸，较延长去饱和途径，具有更高的能量利用率和改造空间。本项目以产二十二碳六烯酸DHA和二十碳五烯酸EPA的脂肪酸聚酮合酶为研究对象，基于in vitro体外反应和in vivo体内功能域置换的研究思路，拟在产业化成熟的DHA生产菌裂殖壶菌体内合成EPA。通过研究脂肪酸链延伸模块KS和修饰模块ER对双键的保留机制以及潜在释放模块AT对终产物脂肪酸的底物偏好性，解析不饱和脂肪酸的从头合成途径对脂酰结构前体的合成和调控机制，获得在ω-3位特异性引入双键及专一性释放EPA的模块蛋白，在裂殖壶菌中重构EPA合成途径，获得特定链长和双键的脂肪酸产品。发展一种基于体外测试、体内改造定向控制脂肪酸双键位置和链长结构的方法，为天然低效率合成的脂肪酸产品制备提供新的技术手段，推动脂肪酸类化合物合成技术的创新。

本项目针对高营养稀缺脂肪酸低效率合成的问题，从代谢途径和酶催化层面来调控从乙酰 CoA到不饱和脂肪酸合成的代谢过程。从多不饱和脂肪酸合成酶的催化机制解析层面探究脂肪酸链长和双键位置控制的机制。（1）脂肪酸聚酮合酶功能模块的体外表达：发现来自裂殖壶菌HX-308的BER和CER主要是负责长链不饱和脂酰ACP双键的还原的。在大肠杆菌中表达了来源于裂殖壶菌和希瓦氏菌的四个脱水酶功能域，发现DH12功能域可以提高不饱和脂肪酸的比例，DH3可以促进饱和脂肪酸的积累。（2）裂殖壶菌的基因工程平台构建和脂肪酸聚酮合酶的体内表达、替换：在裂殖壶菌中成功建立了一套稳定的遗传转化体系；将体外构建的来自希瓦氏菌的 Shew-AT 表达框导入裂殖壶菌，EPA占总脂肪酸的含量由原来的 1.04%提高到 3.84%，比原始菌株提高了72.9%。（3）转录组学解析不饱和脂肪酸合成机制：完成了裂殖壶菌分别在高温胁迫下、强光胁迫下、高盐胁迫下的转录组学分析实验，整体分析裂殖壶菌的脂肪酸聚酮合酶基因在胁迫条件下的表达情况。（4）裂殖壶菌的定向驯化：对裂殖壶菌分别进行了耐高温驯化、强光驯化、过氧化氢驯化以及强光和过氧化氢交替驯化实验。结果显示，上述条件均一定程度上提高了DHA以及EPA的产量；添加过氧化氢后EPA的含量较对照组增加了7.35倍。本项目通过全方面的研究解析脂肪酸聚酮合酶合成机制，为促进长链多不饱和脂肪酸在微生物体内高效合成奠定理论基础。

内容获取失败，请点击重试