构建人工菌群系统合成植物黄酮糖苷

Constructing artificial biosystem for efficient production of complex natural products is the frontier of synthetic microbiology. Here, taking puerarin as an example of plant flavonoid glycosides, we distribute the total metabolic pathway to two Escherichia coli strains BMC and BMP, which produces precursor 4-coumaric acid and puerarin, respectively. The combination of strains BMC and BMP forms the artificial bacterial consortium. By screening and designer modification of genes and expression patterns for puerarin biosynthesis, we would optimize the heterologous modules compatible with the chassis, and develop the artificial metabolic pathway for puerarin production. After engineering different utilization of xylose and glucose to reduce competition for carbon source and modifying amino acid metabolism, two strains would form a syntrophic consortium system. The interactive relationship of the consortium and the productivity of puerarin would be regulated via the inoculation ratio and the ratio of glucose to xylose, in addition to the supplementation of malonate. To probe the interaction between strains BMC and BMP, we would systematically analyze the dynamic changes of the populations, intermediates and puerarin in fermentation process, and finally, establish the stable and controllable consortium for efficient biosynthesis of puerarin. The project would not only bring forth new theoretical knowledge for synthetic microbiology, but also create novel biotechnology for production of puerarin, which shows scientific significance and valuable application.

构建人工生物系统，高效合成结构复杂的天然产物是合成微生物学研究前沿。本项目以葛根素为植物黄酮糖苷的模式，将整个代谢途径分配到两个大肠杆菌中，分别合成前体对香豆酸(BMC菌株)和葛根素(BMP菌株)，构成人工菌群系统。通过表达筛选和设计改造，优化葛根素生物合成基因元件及表达模式和模块适配，创建从糖到葛根素的人工代谢途径。通过改造木糖和葡萄糖的利用能力，减少竞争。通过改造氨基酸代谢，使两株菌形成互养共生模式。通过葡萄糖和木糖比例、接菌比例、添加丙二酸，调控人工菌群系统的双菌相互作用和葛根素生物合成。综合分析菌群、中间代谢物、终产物的动态变化，研究两个菌株之间的相互作用关系，建立稳定可控的人工菌群系统并高效合成葛根素。本项目既为合成微生物学积累新原理，也为葛根素制造创新技术，具有重大科学意义和重要应用价值。

植物黄酮及黄酮糖苷是一类非常重要的天然产物，具有抗病毒、抗氧化损伤抗菌等生物活性，广泛应用于医药、保健品、化妆品及食品工业中。目前，黄酮及衍生化合物黄酮糖苷在微生物合成的代谢途径冗长、酶催化效率低、前体供应不足、细胞生长和生产的冲突是功能实现的关键科学问题。本项目探究了黄酮和黄酮糖苷的代谢途径重构、模块与底盘细胞的适配性及菌群的稳定性和可控性问题。通过对关键酶的表达筛选、不同启动子、编码序列的设计和组合，建立根皮酸、根皮素、柚皮素、甘草素、染料木素、大豆苷元、山奈素、白藜芦醇、淫羊藿次苷D2和甜菜黄素等植物黄酮及黄酮糖苷的人工代谢途径。针对不同代谢途径特点，将生物合成途径模块化并分布到不同菌中，建立了中立、互利共生和偏利共生的双菌及三菌大肠杆菌共培养系统，实现微生物共培养合成植物黄酮及黄酮糖苷。通过对细胞生长、中间代谢物的积累、发酵工艺调控等研究，探索了不同黄酮途径与共培养系统的相互作用关系，阐明了适配性调控机制。本项目的研究结果为构建人工菌群系统合成植物天然产物提供理论与技术参考。