基于“分子感应-CRISPRi”动态调控的酪醇类衍生物异源高效合成

酪醇类衍生物作为多种珍贵中草药植物的主要活性成分，在医疗、食品保健、化妆品等行业具有广泛应用。利用合成生物学手段构建细胞工厂为其工业化生产提供了可行策略，然而底盘微生物中产物合成与中心代谢流存在前体物竞争，致使产物合成与细胞生长相互制约，因此如何平衡两者之间的关系成为酪醇衍生物高效合成的关键难题。本项目拟建立基于分子感应-CRISPRi的动态调控策略，通过时间调控促进碳代谢流的高效转化。首先依据前期发现及文献报道的芳香醇细胞毒性及在细胞生理调控中的重要作用，采用转录组学方法挖掘酪醇感应元件；进而通过分子定向改造，优化感应元件及CRISPRi系统的感应阈值及调控幅度；最终将该策略应用于羟基酪醇路径工程实例中，通过对酪醇浓度变化的自动感应实现细胞生长与产物合成的平衡发展。该工作将有助于深入解析酿酒酵母中的酪醇调控机制，为其高效生物制造奠定基础，同时为更多复杂代谢网络的时空精细调控提供方法借鉴。

Tyrosol derivatives are the main bioactive ingredients in a number of highly valued herb plants, which are widely used in the food, pharmaceutical, and cosmetics industries. Construction of microbial cell factory via the employment of synthetic biology provides a feasible approach for their large-scale production. However, the biosynthetic efficiency is largely restricted by the precursor competition conflict between product accumulation and cell growth. Targeting at this bottleneck, we aim to develop a sensor-regulator and CRISPRi-based dynamic regulation strategy for the temporal control of gene expression. Firstly, based on the cytotoxicity of tyrosol and its important role in the physiological regulation of yeast in our preliminary work and previous reports, tyrosol-sensing elements will be mined by transcriptomics analysis. The threshold values and regulation ranges of targeted sensors as well as the CRISPRi system will be then optimized by molecular directed evolution. Finally, as a proof of concept, this dynamic regulation strategy will be applied in microbial production of hydroxytyrosol whereby a two-stage culture mode will be developed to balance cell growth and product accumulation. This work will help get insight into the regulation mechanism of tyrosol in Saccharomyces cerevisiae and provide methodological support for the regulation of other complex metabolic networks for the synthesis of high-value products.