细胞合成中链烷烃过程中酰基载体蛋白-硫酯酶适配性强化脂肪酸合成途径的研究

The development of synthetic biology gives us chance to transform genes into network, build targets synthesis module and enhancing target synthesis pathway by improving the adaptability between synthesis module and host cells. As fatty acid is an ideal precursor for bio-materials and bio-fuels, this research is focused on the key assistant protein - acyl carrier protein (ACP) in fatty acid synthesis pathway to deal with the bottleneck problem of low fatty acid chain length selectivity and yield. In this reseach, the adaptability among acyl carrier protein, FAS and thioesterase will be improved to enhance the fatty acid chain length selectivity and yield in biosynthesis of medium chain length alkanes process. Firstly, the adaptability among exogenetic acyl carrier protein, FAS and thioesterase was investigated by introducing different kinds of exogenetic acyl carrier protein, FAS and thioesterase genes into E. coli to find the essential matching regularity. Sencondly, the matching regularity was used for selecting the most suitable exogenetic acyl carrier protein, FAS according to the medium chain length fatty acid selective thioesterase to improve the fatty acid chain length’s (10-14 carbons) selectivity and yield in recombinant E. coli. Finally, the alkanes biosynthetic pathway was introduced into the recombinant E. coli with to get medium chain length alkanes with high middle chain length selectivity and yield.

合成生物学将基因连成网络，构建目标产物合成模块，通过分析及调试目标产物合成模块与宿主的适配性，强化目标产物合成途径。脂肪酸是生物材料、生物能源的优秀前体。本项目针对脂肪酸合成过程中所面临的选择性差的瓶颈问题，通过研究脂肪酸合成过程中关键辅助蛋白—酰基载体蛋白（Acyl Carrier Protein, ACP）、脂肪酸合成酶系及特异性硫酯酶间的匹配性，探讨提高重组细胞合成中链脂肪酸的链长选择性的机理。鉴于酰基载体蛋白在脂肪酸合成中的重要作用，首先探究外源ACP的介入对脂肪酸合成途径的物质流通路和碳链组成影响的基本规律；然后结合外源ACP对脂肪酸合成途径影响规律和ACP动力学匹配结果，对大肠杆菌的脂肪酸合成代谢途径网络进行改造，并组合中链脂肪酸合成途径与烷烃合成途径，最终在E.coli中实现高产量的中链烷烃的生产，验证适配性机制。将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。

脂肪酸是生物材料、生物能源的优秀前体，本研究探索了E.coli中外源脂肪酸合成辅助蛋白—酰基载体蛋白（ACP）的表达对底盘细胞和特定中链脂肪酸选择性硫酯酶表达条件下脂肪酸产量和碳链组成的影响，通过引入来自Umbellularia californica的链长特异性硫酯酶、麻疯树来源的酰基载体蛋白（ACP）基因和脂肪酸合成系统（FAS），对大肠杆菌的脂肪酸合成代谢途径网络进行了改造，最终在E.coli中实现了中链烷烃的较高选择性生产，改造获得的工程菌株中癸酸，月桂酸和肉豆蔻酸的浓度分别为0. 011mg/g，0.093mg/g和1.657mg/g；最终中链烷烃产物，壬烷，十一烷和十三烷的浓度分别为0.00062mg/g，0.0052mg/g和0.249mg/g。来自Umbellularia californica的硫酯酶将脂肪酸链长度控制在10-14个碳原子范围内，来自JC的带有完整FAS的ACP基因显著提高了中链脂肪酸的产量。