设计构建新型生物调控元件及其在云杉新甙微生物合成中的应用

可响应微生物细胞内小分子化合物的生物调控元件是优化天然产物合成的重要工具。云杉新甙是白藜芦醇的糖基化产物，其更好的水溶性和稳定性提升了生物可利用性。本项目拟通过改造鼠伤寒沙门氏菌的转录调控蛋白RamR的诱导特异性定制云杉新甙的新型调控元件，用于其生物合成途径的优化研究。该元件能特异性识别云杉新甙并调控报告基因的表达，因此可用于构建其生物传感器，实时报告胞内云杉新甙浓度。用其作为云杉新甙的高通量筛选工具，可对合成途径中的限速酶糖基转移酶进行定向进化改造提升其活性，亦可对宿主菌基因组进行定向进化改造筛选影响合成效率的关键基因组靶点。此外，该生物调控元件还可用于动态调控产物外排系统的表达，根据云杉新甙胞内累积适时适量地调节外排，增强菌株耐受性。本项目研制的新型感应/调控元件，具备生物传感和动态调控双重功能，可拓展该策略在其它小分子化合物生物合成中的应用，对优化天然产物的生物合成有重要意义。

Small-molecular targeted biocircuit is of great importance in real-time monitoring intracellular concentrations of small molecular, thus, plays an important role in biosynthesis of natural products. Herein, a novel biocircuit is designed by modifying the specificity of a transcriptional protein, RamR, and applied in biosynthesis of piceid. Piceid is a resveratrol glucoside, possessing improved water-solubility and bioavailability compared with aglycone, but its microbial biosynthesis is low-efficient. The specificity of RamR, a transcriptional protein in Salmonella enterica serovar Typhimurium (S. Typhimurium), is modified to recognize piceid, via rational design and directed evolution combined approaches. As an indicator of the concentrations of intracellular piceid, the novel biosensor could be applied in engineering metabolic flux in biosynthesis of piceid, by improving the specific activity of pathway key enzyme, and through genome-wide screening of gene targets critical for the pathway flux. Furthermore, the efflux pump proteins of the piceid could be dynamically controlled by this biocircuit, which is beneficial to release the toxicity of accumulated products. This work demonstrates the feasibility of monitoring metabolic flux with the aid of biocircuit designed for key metabolites.