酿酒酵母关键前体乙酰辅酶A及其衍生产物合成和辅因子扰动的模块代谢工程研究

As a potential cell factory, Saccharomyces cerevisiae is used to produce various biotechnology products. However, the shortage of the precursors and redox cofactor imbalance significantly limit their productivity. Acetyl-CoA is the key precursor of many pharmaceutical drugs and chemicals. However, the large need of acetyl-CoA contradicts with its low flux in the cytosol. The cofactor perturbation is an effective strategy to overcome the cofactor imbalance, but the precise control of perturbation level is a challenge of metabolic engineering. To solve these two problems, this project plan to use module metabolic engineering to construct the acetyl-CoA synthesis module library and cofactor perturbation module library, and study the adaptive mechanism between these libraries and product modules. Specifically, acetyl-CoA synthesis module library is obtained through the introduction of various acetyl-CoA synthesis pathways and the disruption of the bypass. In the meanwhile, the redox perturbation module library is constructed by expressing the cofactor dependent enzymes to change the redox level or coulpe different cofactors. These two module libraries are then combined with acetyl-CoA derived product modules, and the productivity is analyzed to select the best acety-CoA synthesis and cofactor perturbation strategies. This project not only benefits to overcome the two general challenges of metabolic engineering: the shortage of the precursor supply and the redox cofactor imbalance, the results can also be used to optimize the yeast strains to produce biotechnology products.

关键前体供给不足和氧化还原辅因子不平衡是制约酿酒酵母高效生产生物基产品的瓶颈。乙酰辅酶A为多种医药及化工产品的关键前体，其需求量高与在细胞质中代谢通量低的矛盾突出；辅因子扰动是解决其失衡的有效策略，但扰动水平的精确调控为代谢工程的难点。针对上述问题，本项目拟通过模块代谢工程，构建乙酰辅酶A合成和辅因子扰动模块库，研究它们与乙酰辅酶A衍生产物合成的适配机制。具体来讲，通过代谢截流和不同乙酰辅酶A合成途径的构建，获得乙酰辅酶A合成模块库；同时，通过表达多种辅因子调控酶，构建辅因子扰动模块库，实现辅因子氧化还原态的转化或不同辅因子的偶联。进而对两种模块库与乙酰辅酶A衍生产物的合成模块进行组装，并分析不同组合形式下的产物得率，筛选最佳乙酰辅酶A合成以及辅因子扰动策略。本项目的完成，不仅有利于突破代谢工程中关键前体不足和辅因子不平衡这两个共性难题，且相关结论可以用于酿酒酵母生物基产品生产菌株的优化。

关键前体供给匮乏和氧化还原辅因子不平衡是制约酿酒酵母高效生产生物基产品的瓶颈。乙酰辅酶 A 为多种医药、化工产品的关键前体，其需求量高与在细胞质中代谢通量低的矛盾突出；辅因子扰动是解决其失衡的有效策略，但扰动水平的精确控制一直是代谢工程的难点。针对上述问题，本项目通过模块代谢工程进行途径的优化。首先在酿酒酵母中引入了异源的乙酰辅酶 A 合成途径，包括柠檬酸裂解酶途径和乙酰磷酸途径，并进一步改造内源途径以加强这些异源途径的通量。此外，还通过磷酸戊糖途径的强化等策略，进行辅因子的精细调控。在此基础上，研究了上述途径的改造对两种乙酰辅酶A 衍生产物--单萜香叶醇以及平台化合物3-羟基丙酸的合成的影响。通过上述策略，结合代谢的动态调控、转录调控、发酵优化等策略，实现了单萜香叶醇的高效合成。首次在酵母中合成单萜达到了克级。高效乙酰辅酶A合成菌株的构建，也可为其他乙酰辅酶A衍生产品的合成提供良好的平台。