长路径异源产物人工两菌分工研究

Long metabolic pathway is required to be built in artificial cells for producing the precursor of an important anticancer drug Taxol. The interaction among functional modules, metabolic burden and other issues exist, which will result in low yield of final products. Thus, the applicant envisages taking the biosynthesis of 5α, 13α- hydroxylation taxadiene, one of the key precursors of taxol, as an example to study the following issues. 1) First, we will design and construct a two-strain system of Saccharomyces cerevisiae - Yarrowia lipolytica. The construction bases on the characteristics of pathway intermediate (taxadiene) which can cross cell membranes and be secreted to the extracellular medium, and the advantages of Y. lipolytica which can use oil as carbon source; 2) We try to take the taxadiene“out”and“into”cells as a breakthrough to analyze key factors related to the metabolic balance and growth coordination. The effect of consortium structure and nutrition will be quantitatively analyzed by systems analysis. The relationship between the productivity and the key molecules will be explored to determine the impact of the key metabolites and proteins that affect the cell-cell collaboration. And the labor division and regulation models will be established; 3) Through single-target and multi-target regulation, the two strains are reconstructed, respectively, to strengthen their cooperation. And a better two-strain system will be obtained. This study will provide new ideas for building and optimizing the synthetic microbial consortia for heterologous product expression through a long pathway.

利用人工细胞生产抗癌药物紫杉醇等要构建长代谢路径，往往产生多个模块功能交叉、细胞代谢负荷重等问题，导致产量低。故申请者设想设计构建两菌体系，使菌株分工协作实现长路径异源产物表达。本研究以人工合成紫杉醇重要前体5α,13α-羟化紫杉二烯为例，主要研究：1）首先根据中间代谢物紫杉二烯在酿酒酵母中易分泌且易溶于油脂、解脂酵母能代谢油脂等特性，将路径设计构建于酿酒酵母-解脂酵母两菌体系; 2）尝试以紫杉二烯“出”“入”细胞作为切入点，分析影响两菌代谢平衡和生长协调的关键因素，结合菌群结构、营养因素等对两菌协作影响的系统定量解析，探索两菌中与合成路径直接相关的代谢物和蛋白与体系生产能力的关系，确定两菌协作的关键靶点，建立两菌分工协作和调控模型；3）再通过单靶点和多靶点调控手段对两菌协作进行精确调控，强化两菌关系，获得更优的两菌体系。本研究对通过长路径合成异源产物的人工多菌体系的构建和优化提供新思路。

利用人工细胞生产化学品、营养品等需要构建异源长代谢路径，往往产生多个模块功能交叉、细胞代谢负荷重等问题。通过设计构建混菌体系，可使菌株分工协作实现异源产物的合成。本研究为了构建高效、稳定的混菌体系，首先通过组合设计代谢途径，构建产香叶基香叶醇（Geranylgeraniol，GGOH）的酿酒酵母底盘细胞，进而获得高通量GGPP的酵母底盘，为构建高产紫杉二烯的混菌体系奠定基础。为了获得人工混菌体系的设计构建原则和调控机制，进行了混菌体系的设计重构及调控研究。首先，以VC生产混菌体系为例，通过构建高产VC前体的菌株，以促进其与伴生菌之间的相互作用，提高混菌体系发酵水平，为获得混菌互作关系的潜在调控机制奠定基础。第二，基于劳动分工原则，重构VC混菌发酵体系，实现VC两菌一步发酵，结合菌群结构、营养因素等对混菌协作影响的系统定量解析，对其进行优化调控，使原来两菌间的偏利共生和竞争关系转变为互利共生关系，实现了对两菌关系的人工调控。通过构建硫辛酸功能模块，强化了VC两菌一步发酵体系，减少了伴生菌的竞争效应，提高了两菌一步发酵体系的转化率。第三，设计构建了VC三菌一步发酵体系，通过分析影响混菌代谢平衡和生长协调的关键 因素，探索混菌中与合成路径直接相关的代谢物和蛋白与体系生产能力的关系，确定混菌协作的关键靶点；基于群体感应系统设计构建了混菌体系中群体感应程序性死亡模块，解除了微生物间的竞争关系，强化了群体的互利共生。本研究对通过长路径合成异源产物的人工混菌体系的构建和优化提供了新思路。