基于系统代谢工程的逆向脂肪酸β-氧化途径合成中链脂肪酸的调控机制研究

Compared with the long chain fatty acids, medium chain fatty acids have many important application values due to its characteristics such as fast absorption, reducing fat deposition, regulating energy metabolism, so the development of high efficient and low cost microbial production of medium chain fatty acid has attracting more and more attention. Reverse of beta oxidation pathway has become the main method of microbial production of medium chain fatty acid. However, current researches always focus on identifying pathway gene function, lacking research on identifying bottleneck steps, which leads to the lack of effective regulation mechanism of carbon flow and cofactor. In this project, based on previous systems metabolic engineering platform, the pathway bottleneck is identified by the quantitative strategy and multi-modular engineering strategy is employed to alleviate the bottleneck step; at the same time, the CRISPR interference system is employed to analyze the limiting factors affecting cofactor supply and further achieve the balance between the cell growth and pathway synthesis. The research results are helpful to understand the synthesizing mechanism for specific length fatty acid, providing important theoretical references for metabolic engineering of microorganisms for synthesizing various types of fatty acids and its derivatives.

与长链脂肪酸相比，中链脂肪酸具有消化吸收快、减少脂肪沉积、调节能量代谢等特性，有着重要的应用价值，因而发展绿色、高效、低成本的微生物法生产中链脂肪酸就受到越来越多的关注。逆向脂肪酸β-氧化途径因其合成中链脂肪酸的高效性而成为微生物法生产的主要方式，然而目前针对该途径的研究多集中在途径基因功能的鉴定上，对于合成途径存在的瓶颈步骤缺乏研究，并缺少有效的碳流和辅因子供给的调控机制。本课题利用申请人前期构建的系统代谢工程平台，通过靶向定量策略分析该合成途径中存在的瓶颈步骤，并运用多模块化改造策略缓解瓶颈步骤；同时运用CRISPR干扰技术分析代谢网络中影响合成途径所需辅因子供给的限制因子，实现细胞生长与途径合成所需辅因子的供需平衡，最终建立有效的碳流和辅因子供给的调控机制。研究结果有助于深入了解大肠杆菌的特异链长脂肪酸调控机制，为代谢工程改造微生物高效合成各类链长脂肪酸及其衍生物提供重要的理论参考。

中链脂肪酸在食品、医药及日用化工领域有着重要的应用价值，当前其主要来源是椰子油和棕榈仁油，无法在我国广泛栽培，目前我国所需要的中链脂肪酸主要依赖于进口，国家每年都花费大量外汇(7.5-10万元/吨)从国外进口，每年仅食品香料和医药行业就需要从国外进口约10000吨的中链油脂，随着社会大众对中链脂肪酸性质及特性的越来越了解，中链脂肪酸的需求量将越来越大。因此本项目研发的绿色、高效、可持续发展模式的微生物法合成中链脂肪酸机制将极大提高国内经济效益，摆脱对国外进口的依赖。. 申请人微生物体内重构中链脂肪酸合成途径，发现胞内较低的乙酰辅酶A浓度是途径的限制性因子。同年，发现NADH与乙酰辅酶A偶联供给能最大程度提高途径效率。近期，申请人研发出一种新的代谢模式-人工微氧代谢体系，极大降低了菌体内代谢负担及生产成本，由此中链脂肪酸的微生物发酵也首次在3-L发酵罐中进行了生产小试，产量进一步提高到了15.6 g/L，为目前国内外中链脂肪酸的最高产量。申请人于2017年发表的中链脂肪酸生物合成的前期研究成果，同年即被Nature重要子刊Nature Biotechnology(2017, 35(12):1158-1166)(IF=41.667)所引用，引起国内外学者的关注。