解脂耶氏酵母胞内外联动积累共轭亚油酸的脂肪酸氧化调控机制

Chemically-synthesized conjugated linoleic acid (CLA) is a mixture of isomers containing CLA monomers of uncertain function and safety. In recent years, in order to meet the requirements of the food industry, biological transformation has been widely investigated to selectively produce safe, green and highly specific CLA isomers. In our previous research, trans-10，cis-12-CLA was successfully created and secreted from exogenous plant oil by recombinant Yarrowia lipolytica strain. However, a large part of substrate and product were consumed by β-oxidation in Y. lipolytica. The objective of this research is to improve the synthesis of CLA through weakening β-oxidation of linoleic acid (LA) and CLA by comprehensive regulation of recombinant Y. lipolytica. Firstly, six POX genes regulating β-oxidation of fatty acids will be studied on substrate preference with different carbon lengths and unsaturated degrees. The key POX genes for catalyzing β-oxidation of LA and CLA will be determined according to their substrate preference. Secondly, the mechanisms by which deletion of different POX genes influences accumulation of CLA will be studied by comparing modified strains with the parental strain on cellular and molecular levels. The comprehensive metabolic regulation strategy will be determined to improve the intra-extracellular co-accumulation of CLA. The results of this research will lay a foundation for CLA industrial production by bio-transformation. Meanwhile, different from traditional methods for intracellular accumulation of functional fatty acids by microorganisms, we provide a new approach using intra-extracellular co-accumulation which has many advantages, such as easy separation of fatty acids and reusability of cells.

化学法合成共轭亚油酸（CLA）是多种异构体的混合物，其中包含生理功能及安全性未知的单体，而生物转化法可以选择性合成安全、环保的CLA活性单体，以满足食品领域的要求。在前期研究中，申请人利用重组解脂耶氏酵母转化外源植物油成功合成共轭亚油酸单体。但大量脂肪酸进入β氧化途径，造成底物及产物损失。本项目基于对脂肪酸β氧化途径的研究，通过对出发菌株的全面调控，削弱或阻断底物（LA）及产物（CLA）的β氧化，实现CLA高效合成。首先，通过分析脂肪酸β氧化途径上的六个POX基因对不同链长和不饱和度底物的偏好性，确定调控LA和CLAβ氧化途径的关键POX基因；通过分析POX敲除菌株从细胞水平到分子水平的变化，阐明其对CLA胞内外联动积累过程的影响机制，确定全面调控策略。本项目的完成为实现生物法替代化学法合成CLA提供了理论与实验基础，同时，本研究的胞内外联动积累策略为微生物法合成功能性脂肪酸提供了新思路。

本研究选择解脂耶氏酵母脂肪酸氧化代谢途径中POX2、POX3、PEX10作为调控位点，采用Cre-lox系统构建四种基因敲除菌：Δpox2、Δpox3、Δpox2& pox3、Δpex10，以油酸、椰子渣油、挥发性脂肪酸为唯一碳源进行培养，确定了三个基因对于脂质底物的偏好性：POX2编码的酰基CoA氧化酶底物范围覆盖中长链脂肪酸；POX3编码蛋白对短链脂肪酸有强活性；同时敲除POX2和POX3，菌株对于短、中、长链脂肪酸的氧化能力极大削弱；PEX10敲除菌株只保留了部分利用短链脂肪酸的能力。在四株敲除菌株的基础上，构建了带有PAI单拷贝的重组菌，western蛋白水平显示四株敲除菌株种PAI成功表达且表达量相当；与未敲除菌株相比，四株敲除菌株的生长出现了延迟，在外源添加底物LA的培养条件下，无脂质生物量二次增加量仅为2 g/L，而未敲除菌株为8 g/L，说明氧化分解利用LA能力受到限制；脂质分析表明Δpox2菌株和Δpox3菌株总脂、t10,c12-CLA产量及降解率均与未敲除对照菌株相似，证明单敲除POX2和POX3基因未减缓产物CLA的β氧化，而POX2&POX3双敲除菌和PEX10敲除菌对应产物降解速率为27.3 mg/L/h和20 mg/L/h，显著抑制底物消耗，提高t10,c12-CLA积累的稳定性。以Polf-Δpex10为出发菌株，构建了PAI多拷贝整合菌株进一步PAI表达量，通过PAI拷贝数、western以及GC分析筛选出一株高产t10,c12-CLA菌株Polf-Δpex10/1292 opai-6。外源添加底物红花籽油的培养条件下，t10,c12-CLA总产量提高到7.4 g/L，是单拷贝菌株的2倍。重组菌株CLA降解率为23.0 mg/L/h，是未敲除菌株六分之一。在5 L发酵罐模式下，采用两阶段分批补料策略，利用补加廉价碳源VFAs及外源添加底物红花籽油，t10,c12-CLA产量进一步提高至9.8 g/L，降解率为35.7 mg/L/h，实现了t10,c12-CLA的稳定高效积累，同时廉价碳源的使用节约了生产成本。