酿酒酵母丙二酰辅酶A动态感应元件的构建及其调控的基因组体内连续进化系统的设计

Malonyl-CoA is a key precursor for a variety of pharmaceuticals, chemicals, biofuels and other products. It is involved in the biosynthesis of many important compounds such as fatty acid derived products, flavonoids, polyketides, 3-hydroxypropionic acid, etc. However, the concentration of malonyl-CoA is very low in cytosol due to its tight regulation, thus it largely limited the synthesis of its derived products. Although many rational metabolic engineering strategies have been performed, the low malonyl-CoA pool is still a bottleneck to produce its derived chemicals. In this project, we aim to obtain platform strains for the high level accumulation of malonyl-CoA by irrational in vivo continuous mutagenesis and growth-based screening. Basis on this, we will identify the key mechanism affecting malonyl-CoA flux. Specifically, we express the proofreading-deficient DNA polymerase to develop an in vivo continuous evolution system, then construct the malonyl-CoA biosensor, and use the biosensor to couple the concentration of malonyl-CoA to the cell growth rate in order to establish the growth based selection system. The efficient malonyl-CoA synthesis strains are then enriched and selected using the in vivo continuous evolution and growth based high-throughput screening. The key mutations that affect the malonyl-CoA accumulation are then identified through inverse metabolic engineering. The project has great significance for the breakthrough a common metabolic engineering problem---low malonyl-CoA synthesis flux and improve the synthesis of a variety of malonyl-CoA derived products.

丙二酰辅酶A作为多种医药、化工、生物能源等产品的关键前体，参与重要化合物如脂肪酸衍生物、黄酮类、聚酮类、3-羟基丙酸等的合成，但其需求量高与代谢受到严格调控、合成通量低矛盾突出，而现有的理性代谢工程改造对该矛盾的解决有限，迫切需要非理性的策略提高其合成通量。本项目拟利用丙二酰辅酶A动态感应元件建立非理性筛选平台，通过体内连续突变进化和基于生长的筛选，获得高丙二酰辅酶Ａ积累菌株，并在此基础上挖掘影响其通量的关键机理。具体来讲，通过表达校正活性缺陷的DNA聚合酶，进行体内连续突变进化；并构建丙二酰辅酶Ａ动态感应元件，利用该元件偶联丙二酰辅酶A浓度与生长速率，建立基于生长的筛选系统，进行丙二酰辅酶A高积累菌株的连续进化和富集筛选；高产菌株再通过反向代谢工程揭示影响其积累的关键机理。该项目的完成对于突破代谢工程改造中丙二酰辅酶A合成通量低这一共性难题，提高多种衍生产物的合成都具有重要意义。

丙二酰辅酶A作为多种医药、化工、生物能源等产品的关键前体，参与重要化合物如脂肪酸衍生物、黄酮类、聚酮类、3-羟基丙酸等的合成，但其需求量高与代谢受到严格调控、合成通量低矛盾突出，而现有的理性代谢工程改造对该矛盾的解决依然有限。本项目通过建立体内连续突变进化和基于生长的筛选的方法，获得高丙二酰辅酶Ａ积累的平台菌株，并在此基础上挖掘了影响其通量的关键机理。具体来讲，通过表达校正活性缺陷的DNA聚合酶，并改造错配修复系统，增加DNA复制过程中的突变率，进行体内连续突变进化；与此同时，构建丙二酰辅酶Ａ动态感应元件，利用感应元件将丙二酰辅酶A浓度与生长速率偶联，建立基于生长的筛选系统；结合体内突变系统和生长筛选系统进行丙二酰辅酶A高合成菌株的连续进化和富集筛选；再通过基因组测序、转录组分析，以及反向代谢工程验证，揭示影响丙二酰辅酶A合成通量的关键突变基因，发现了储藏性碳源合成和赖氨酸、精氨酸代谢的调控可以显著提高丙二酰辅酶A的合成通量。这些发现的新机理、新靶点可用于丙二酰辅酶A下游产物的代谢工程改造，因此该项目的完成对于突破代谢工程改造中丙二酰辅酶A合成通量低这一共性难题，提高多种衍生产物的合成都具有重要意义。