中链烷烃生物合成新途径的构建与调控研究

Producing bioenergy from renewable resource is an important approach to solve the energy problem. Compare to ethanol and biodiesel, alkanes show high energy density and low freezing point, are the best liquid fuels. The productivity of the reported alkane biosynthesis pathway was low, and the products could not be controlled. The utilization of this pathway is very limited. In this proposal, we will design a novel metabolic pathway, to overcome above-mentioned drawbacks. The current alkane biosynthesis pathway is initiated by acyl-ACP reductase, forming main product palmitic aldehyde. After decarbonylation, the final product pentadecane is produced. Acyl-ACP reductases distribute mainly in bacteria with low diversity. And the selectivity is major towards long-chain fatty acyl-ACP. In contrary, fatty acyl-ACP thioesterases from plants have large diversity and are able to distinguish different chain-length of fatty acyl-ACP from short chain to long chain. For each thioesterase the specificity can be very high. For example, the thioesterase from Umbellularia californica produces lauric acid and myristic acid by expression in E. coli. The fatty acids are than ligated to CoA by acyl-CoA ligase. After reduction by Fatty acyl-CoA reductase catalyzes the fatty acyl-CoA to the fatty aldehyde. Final, undecane and tridecane are obtained by decarbonylation of fatty aldehyde, which is catalyzed by decarbonylase. After construction of this novel alkane biosynthesis pathway, we will regulate energy balance in order to enhance cofactor supplement. This project will provide fundament for accomplishment of novel cell factor by global optimization on genomic level, to produce different kind of alkanes for various utilization.

利用可再生资源生产生物能源是目前解决能源危机的重要办法。相比乙醇等生物燃料，烷烃有能量密度等优点，是最好的液体燃料。但已报道的烷烃生物合成途径产量低，产物不可设计，使用范围局限。本项目提出一种全新的代谢途径，以解决上述两个问题。已知的烷烃合成途径由酯酰ACP还原酶起始，主产物为十六烷醛，脱去羰基合成十五烷。酯酰ACP还原酶主要存在于细菌中，缺乏底物选择性。很多植物会积累特定链长的脂肪酸，例如月桂树积累月桂酸等中链脂肪酸，机理为特异性酯酰ACP硫酯酶造成的特定链长酯酰ACP的水解。以此为基础，大肠杆菌表达的月桂树硫酯酶会积累月桂酸及肉豆蔻酸。通过酯酰CoA连接酶，脂肪酸转化为酰基CoA，在酰基CoA还原酶的还原下，形成相应脂肪醛，被脱羰基酶脱去羰基为终产物十一、十三烷。构建该途径后，通过调控能量平衡，为实现产各种烷烃燃料的细胞工厂提供初步工作基础和理论依据

本课题利用基因工程的手段，调控烷烃合成过程中相关酶的表达，在微生物细胞内构建了全新的代谢途径以合成烷烃，是生产可再生能源的前沿技术。初步构建代谢途径后，气相质谱检测表明，烷烃产量约在5mg/g，经过代谢途径优化后，产量达到6mg/g。该方法以可再生生物质为原料，为传统石化燃料供给不足、新型生物燃料不能完全替代石化燃料等问题提供了一个有潜力的解决方案。与其他菌株相比，大肠杆菌具有遗传背景清楚、易于改造、生长速度快、适合高密度发酵等优点，是微生物法合成化学品和燃料的理想菌株。鉴于烷烃合成代谢需要消耗大量ATP，ATP/ADP比例的检测有助于了解细胞代谢过程，为进一步代谢调控提供实验基础，本课题采用表面增强拉曼散射技术开发了ATP/ADP的快速检测方法。