酰基-ACP硫酯酶的底物链长选择机制及中链底物特异性改造策略研究

Acyl-ACP thioesterase catalyzes the termination of acyl-ACP elongation during the fatty acid synthesis process, which directly determines the chain length of the products. Medium chain (C8-C12) fatty acids and their derivatives have significant demand in chemical products and biofuels; however, the fatty acids produced by TE are usually C16 or longer in length. Tuning of the substrate carbon chain length selectivity is expected to raise a new strategy for medium chain fatty acids synthesis. Based on the previous studies on the TE structure and catalytic center, this proposal intends to reveal the substrate selectivity mechanism of TE and further reconstitute its specificity. Acyl-ACP thioesterase from the eukaryotic unicellular organism Chlamydomonas reinhaidtii (CrTE) is chosen as the research subject and the three-dimensional structure of the “frozen” intermediate complex of CrTE with its substrate will be resolved. Combining with site directed mutagenesis and enzymatic kinetics analysis, the mechanism of CrTE chain length selectivity will be revealed. Further analysis of the enzyme-substrate structure and function relationship is conducted to rationally guide the design of mutated CrTE with medium chain specificity. Finally, the strategy of thioesterase redesign with C8-C12 specificity by (C8-C12)-ACP pool selectivity will be validated by screening in vitro and by chloroplast transformation of mutated CrTE in vivo. This project will provide important theoretical foundation for realization of tailored medium chain fatty acid synthesis and development of low-carbon biofuels.

酰基-ACP硫酯酶(TE)催化酰基-ACP水解，直接决定脂肪酸合成产物的链长。中碳链(C8-C12)脂肪酸及其衍生物在化工及能源领域具有重要的应用需求，而TE通常仅识别C16及C18底物，改造TE底物链长选择性有望为中链脂肪酸合成提供新策略。本项目以解析的TE三维结构及催化活性中心工作为基础，拟深入揭示TE底物链长选择机制并进行中链底物特异性改造。选取真核单细胞生物莱茵衣藻酰基-ACP硫酯酶(CrTE)为研究对象，设计催化位点失活的CrTE与其底物结合复合物，捕获结合中间态并通过X射线衍射解析其晶体结构及底物结合口袋，阐释CrTE的底物链长选择机制。进一步以酶-底物构效关系为指导，理性设计中链底物特异性CrTE改造方案并在体外及体内系统性分析验证。最终获得C8、C10及C12底物特异性TE的定向改造策略。为实现中链脂肪酸定长合成及开发低碳生物燃料提供重要科学基础。

中碳链脂肪酸及其衍生物在能源化工领域具有重要的应用需求，酰基-ACP硫酯酶在脂肪酸合成过程识别底物酰基-ACP，直接决定产物分子链长。近年硫酯酶的链长特异性改造为脂肪酸定长合成领域的热点方向，然而硫酯酶对底物链长的选择性机制缺乏限制了其理性设计。本研究工作首次解析了高等植物来源酰基-ACP硫酯酶的晶体结构，明确了硫酯酶包含两个串联的hot-dog结构域。其中C端的hot-dog结构域包含DXNXH和XXEC两个motif构成酶的催化活性中心，而N端的hot-dog结构域包含了底物链长识别关键氨基酸如T137, M197及F201等。基于硫酯酶的结构分析、突变体设计及其对底物的构效关系，研究工作提出系列理性设计硫酯酶底物链长选择性的策略，并在体内和体外共同证实。（1）基于底物活性口袋末端空位位阻改造硫酯酶底物链长选择性的策略，通过硫酯酶UcFatB酰基结合口袋T137G突变体理性设计，实现C14选择性二倍以上提高，将T137G突变的UcFatB转入大肠杆菌K27菌株，获得的C14脂肪酸相比于野生型占比由19.5%提升至36.6%；（2）基于硫酯酶同ACP的相互作用调控脂肪酸碳流分配的策略，通过硫酯酶ChFatB2同ACP的计算模拟相互作用分析，设计并实验筛选ChFatB2表面多个潜在的同ACP相互作用的关键位点，最终获得硫酯酶表面I198位点，通过I198定点突变为I198E后，实现了大肠杆菌中主要合成脂肪酸从C8（85%）到C10（56.1%）的工程化改造；（3）基于硫酯酶底物口袋及帽子区域共同调控硫酯酶选择性的策略，设计硫酯酶CpTE的三位点突变体V162L/V166L/I177T，体外实现硫酯酶选择性从C8到C12的工程化改造，同时上述突变体转化微拟球藻，实现微拟球藻中C8-C12链长脂肪酸的定制。本研究工作解析的酰基-ACP硫酯酶的底物链长选择机制及提出的相关中链底物特异性改造策略将为中链长脂肪酸的定长合成奠定科学基础。