萜类工程菌生物合成途径调控及其表达机制研究

Pathway optimization is very important for improving the production of plant derived terpenoids in enginnerd yeast. It has been found that coordinated gene expression within a synthetic pathway can significantly enhance the production of target compound by avoiding metabolic burden and accumulation of toxic intermediate metabolites.In our work, promoter libraries of Saccharomyces cerevisiae will be constructed and used for fine tuning of gene expression of the isopentenyl pyrophosphate (IPP) and geranylgeranyl diphosphate(GGPP) synthetic pathway through multiple gene homologous recombination. The strains harboring the optimized IPP and GGPP biosynthesis pathway will be tested with squalene and miltiradiene synthetic modules. Additionally, high-level production of squalene (triterpenoid ) will be further tested with 20(S)-protopanaxadiol synthetic module. Finally, the coordination mechanism will be analyzed, according to the gene expression profiling of pathway (by the methods of RT-PCR ) and its metabolites ( by GC-MS and LC-QTOF-MS/MS) . This work will provide a basis for constructing efficient biosynthetic pathway for production of plant derived compounds in yeast.

高效生物合成途径的建立，是提高酵母工程菌合成植物源萜类产量的重要基础。研究发现合成途径中各基因的协调性高表达能有效降低代谢中间体的积累所导致的代谢负荷和细胞毒性，显著提高目的产物的产量。本研究拟用酵母启动子文库，结合多基因体内同源重组方法，对酿酒酵母IPP 和GGPP生物合成途径的基因表达进行系统的精细调控。在此基础上用鲨烯（三萜）和次丹参酮二烯的生物合成模块分别对IPP和GGPP底盘工程菌进行测评；新获得的三萜底盘工程菌用20(S)-原人参二醇的生物合成模块进行进一步测评；最后结合RT-PCR分析代谢途径中各基因的表达谱，GC-MS和LC-QTOF-MS/MS分析其化学代谢物来探讨高效生物合成途径中基因表达的协调机制。本工作将为在酵母中建立高效生产植物源化合物的合成途径奠定基础。

该项目综合运用系统生物学与合成生物学技术，以“人参酵母”等系列细胞工厂创建为研究案例，在药用萜类成分生物合成关键机制解析，及高效人工细胞工厂的实现等方面主要完成如下工作：①在萜类生物途径关键步骤解析及应用方面，发现三萜合成的关键酶鲨烯环氧酶（SQE），二萜合成的关键酶3-羟基-3-甲基戊二酰辅酶A还原酶（HMGR）及牻牛儿基牻牛儿基焦磷酸合酶（GGPS）；②开发出“即插即用”生物合成途径解析平台，能成功用于二萜和三萜等物质的生物合成途径高通量解析；③国际首次发起并实施完成基于中药复杂性组分概念的第一代“人参酵母”细胞工厂创建工作。初步建立了以高效微生物细胞工厂为基础的中药有效成分生产新模式，奠定了中药有效成分与合成生物学领域的研究基础。