脂肪酶—P450脂肪酸脱羧酶人工代谢区室多酶复合体高效转化甘油酯生成脂肪烯烃新途径研究

The advanced biofuels fatty alkane and alkene are recognized as the best alternative fuels due to their similar properties to petroleum, such as high energy density, low water solubility. Thus, their biosynthetic pathways have received ever-increasing attention from both academies and industries. So far the reported pathways for fatty alkane and alkene biosynthesis have almost always involved multiple enzymatic steps based on fatty acid biosynthetic pathway, and used fatty acids or their activated forms (acyl-CoAs or acyl-ACPs) as starting materials. The high cost of fatty acids and their inefficient de novo biosynthesis have limited application of these biosynthetic pathways into industry scale production. To overcome this problem, in this project we propose to develop a novel and economic pathway for fatty alkene bioproduction from renewable and cost-effective triacylglycerols based on the lipase hydrolysis coupled to the P450 decarboxylation in the form of multiple enzyme complex (in vitro metabolon). One step purification and co-immobilization of a multi-enzyme complex was developed based on a mixture of cell extracts containing dockerin-containing enzymes and one cellulose-binding module (CBM) or anchor protein ice nuclear protein (INP)-containing scaffoldin through specific high-affinity interaction of dockerin and cohesion on low cost amorphous cellulose or cell surface. The key problems including substrate channeling of hydrolysis and decarboxylation, and the overall efficiency of fatty alkene bioproduction will be solved. The assembled synthetic metabolon offers advantages such as substrate channeling, controlled enzyme ratio and position, and good reusability. This proposed synthetic metabolon multiple enzyme complex would construct a novel, economic and effective pathway and catalytic system for alkene bioproduction. Since the novel pathway composed of lipase hydrolysis coupled to P450 decarboxylation in the form of synthetic metabolon only requires two enzymatic steps, it could fulfill the cost-effective conversion of renewable triglycerides to advanced biofuels. This has not only academic significance in the field of in vitro synthetic biology, but also great potential for industrial application.

脂肪烃作为优越先进生物燃料，目前其生物合成主要是围绕脂肪酸代谢途径进行遗传改造。而涉及多步酶促反应的脂肪酸从头合成效率低、成本高，制约了其进一步应用。本项目拟用廉价甘油酯为原料，通过模拟代谢区室多酶复合体的底物通道，构建共固定化多酶复合体，解决脂肪酶水解和P450脱羧偶联反应的底物对接、酶比例及位置可控、催化剂重复利用等脂肪烯烃生成效率中的关键问题。即利用纤维小体组件Dockerin、Cohesin、纤维素结合域（CBM）及锚定蛋白INP，以脂肪酶与P450脱羧酶为酶组分，借助Dockerin与Cohesin的特异性相互作用，CBM与纤维素基质的结合，以及基于INP的表面展示，通过体外一步纯化、共固定化自组装成具有底物通道效应、酶比例及位置易控、可重复利用的人工代谢区室多酶复合体纤维素基质（或细胞）—脂肪酶—P450脱羧酶，拟构建一条新的、低成本、高效的脂肪烯烃生物催化途径与系统。

脂肪酸族衍生物（脂肪酸酯、脂肪烃、脂肪醇）作为优越的生物燃料与化工原料，受到学术界与工业界的广泛关注。目前其生物合成主要是围绕脂肪酸代谢途径进行遗传改造。而涉及多步酶促反应的脂肪酸从头合成效率低，制约了其进一步应用。本项目以廉价甘油酯为原料，模拟代谢区室多酶底物通道，构建了多酶复合体，解决了脂肪酶水解和P450脱羧偶联反应的底物对接、酶比例及位置可控等脂肪烯烃生成效率中的关键问题。即利用纤维小体组件锚定域（Dockerin）、粘合域（Cohesin）、纤维素结合域（CBM），以脂肪酶与P450脱羧酶为酶组分，借助Dockerin与Cohesin的特异性相互作用，CBM与纤维素基质的结合，通过体外一步纯化、自组装成具有底物通道效应、酶比例及位置易控的纤维素基质—脂肪酶—P450脱羧酶多酶复合体。优化的多酶复合体具有明显的底物通道效应，催化油脂底物生成脂肪烯烃的初速度（提高9.2倍）与转化率（69%-72%）均显著高于当量的游离酶。在此基础上，以解脂耶氏酵母、大肠杆菌为底盘细胞，以单酶（脂肪酶），双酶（脂肪酶与P450脱羧酶），三酶（脂肪酶、脂肪酸还原酶、脂肪醛脱羧酶或脂肪醛还原酶）为酶组分，基于表面展示与Dockerin-Cohesin自组装， 构建了一系列单酶、双酶、三酶复合体。构建的解脂耶氏酵母—脂肪酶全细胞可转化91%的油脂生成脂肪酸酯，构建的解脂耶氏酵母—脂肪酶—P450脱羧酶双酶复合体、解脂耶氏酵母—脂肪酶—脂肪酸还原酶—脂肪醛脱羧酶三酶复合体可转化71-84％的油脂生成脂肪烃，构建的大肠杆菌—脂肪酶—脂肪酸还原酶—脂肪醛还原酶三酶复合体可转化73％的油脂生成脂肪醇，均显著高于当量的游离型单酶、双酶、三酶转化率。构建了基于解脂耶氏酵母脂质相关细胞器区室化的亚细胞生物合成脂肪酸酯、脂肪烃途径，其合成效率比传统的胞质合成效率提高了10-14倍。基于基因编程、个体化定制、自组装构建的人工代谢区室多酶复合体与区室化亚细胞工厂，不仅建立了新的、低成本、高效的脂脂肪酸族衍生物催化与合成体系，也为其他化学品的多酶级联先进生物催化与合成提供了平台。