红平红球菌中“硫饥饿”诱导蛋白和含硫氨基酸合成途径的调控机制研究

For most bacteria, the preferred sulfur source is inorganic sulfate. When the supply of their preferred sulfur sources is limited, a set of proteins known as sulfate starvation-induced (SSI) proteins are synthesized by several species of bacteria for scavenging sulfur from organic compounds. Rhodococcus erythropolis could efficiently desulfurize dibenzothiophene (DBT), the main sulfur compound that exists in fossil fuels, through the sulfur-specific pathway. The removed sulfur can be assimilated and further incorporated into cysteine. The unclear regulatory mechanisms of SSI proteins and biosynthesis of sulfur-contained amino acids greatly restricted the metabolic engineering strategies applied to engineer R. erythropolis and potential industrial application of desulfurization strains. Therefore, in this project, we plan to identify SSI proteins in R. erythropolis XP, our research object, using quantitative proteomics. Based on small regulatory RNA and CRISPR-AID, we will develop novel tools for genetic and metabolic engineering in Rhodococcus strains and confirm the biosynthesis pathway of sulfur-contained amino acids by constructing gene knockdown mutants. With the combination of DNA affinity pull-down and genome analysis of R. erythropolis XP, we screen transcriptional control systems in RNA-level and identify some regulatory factors in DNA-level to fully reveal the regulatory mechanisms of SSI proteins and biosynthesis of sulfur-contained amino acids. This study will provide more directional guidance for metabolic engineering of high-efficiency desulfurization strains and lay a foundation for further industrial application.

大多数微生物生长优先选择无机硫源，优先利用的硫源不充足时，一些细菌能合成一系列“硫饥饿”诱导蛋白，用于替代硫源的代谢。优先利用硫源不充足时，高效脱硫菌——红平红球菌能专一性脱下化石燃料中主要有机硫（二苯并噻吩）中的硫，进入硫同化和含硫氨基酸合成途径。红球菌中“硫饥饿”诱导和含硫氨基酸合成调控机制不清晰严重制约着代谢工程策略选择和脱硫菌株工业化应用。因此，我们以红平红球菌XP为研究对象，通过定量蛋白质组鉴定菌株中“硫饥饿”诱导蛋白，基于small regulatory RNA和CRISPR-AID建立红球菌属有效的基因失活方法并验证含硫氨基酸合成途径。在此基础上将“硫饥饿”诱导和含硫氨基酸合成结合起来，筛选RNA水平的转录调控系统和DNA水平的转录调控因子，充分阐明“硫饥饿”诱导和含硫氨基酸合成的调控机制。本研究将为高效脱硫菌的代谢工程改造提供更有方向性的指导，为进一步工业化应用打下基础。

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