转录调控因子LmbU调控林可霉素生物合成的分子机制

Lincomycin has high medicinal value and widely used in clinic treatment and animal husbandry, although the fermentation level of lincomycin has been significantly increased since industrial production, it is still far lower than that of some antibiotics. Research on the biosynthesis pathway and regulatory mechanism of lincomycin are the key to the oriented transformation of high-yield strains. Our previous study firstly demonstrated that LmbU, a novel cluster-situated regulator (CSR) with no similarities to various typical CSRs reported on the sequences and structures, positively regulates the biosynthesis of lincomycin in Streptomyces lincolnensis NRRL 2936, and confirmed that LmbU is involved in the regulation of multiple biosynthetic genes in different ways. However, the molecular mechanism of LmbU regulation was not clarified. This project intends to focus on the regulatory mechanism of LmbU, exploring the sequence characteristics of its targeted DNA and its regulatory mode, screening the ligands which cooperate with LmbU and analyzing the synergy regulation mechanism of LmbU and its ligands, determining the crystal structure of the ligand-LmbU-target DNA complex, thereby elucidating the molecular regulation of LmbU on the biosynthesis of lincomycin. Then, the network diagram of biosynthetic regulation of lincomycin will be drawn, which lays a solid foundation for the realization of high-yield of lincomycin in industry in the future, and provides the theoretical basis for the functional research of LmbU family proteins.

林可霉素应用广泛且药用价值高，但发酵水平却远低于国内大宗抗生素，解析林可霉素生物合成途径和调控机制成为高产株定向改造的关键基础。申请者博士期间首次证实了LmbU为一种新型的簇内调控因子（cluster-situated regulator，CSR），与已报道的各类典型CSRs无序列和结构的相似性，能正调控林可霉素生物合成，且调控模式呈现复杂性与多样性，但其确切的分子调控机制还有待进一步研究。故本项目拟聚焦LmbU，采用凝胶迁移实验、qRT-PCR、HPLC-MS、Pull down和晶体结构解析等方法，筛查其调控作用的配体并解析其协同调控作用的分子机制，确定配体-LmbU-靶DNA复合物的晶体结构，探索LmbU靶向调控DNA的序列特征与调控模式，从而在分子水平阐明LmbU对林可霉素生物合成的调控机制，为未来高产林可霉素生产菌的理性设计奠定基础，也为LmbU家族蛋白的功能研究提供理论依据。

前期研究表明林可霉素簇内调控因子LmbU为新型的调控蛋白，不仅在序列和结构上并不类似于其他已鉴定的调控蛋白家族，且广泛分布于数百种放线菌中。本项目围绕LmbU调控功能的差异性分析、小分子配体的鉴定、关键功能域的鉴定与晶体结构的解析等开展研究，预期全面阐明LmbU调控林可霉素生物合成的分子机制。. 项目实施过程中，初步获得了制备LmbU-DNA复合物晶体的条件；证实林肯链霉菌发酵培养液中存在影响LmbU发挥调控功能的配体；DBD区整体作为LmbU转录调控作用的关键区域，且不依赖于特定氨基酸；RHH结构独立于DBD区，也对LmbU的调控起到重要作用。LmbU不仅调控lmb簇内的靶基因，还对至少8个簇外靶基因发挥调控功能，其中3个编码调控因子的靶基因（SLINC_RS02575、SLINC_RS05540和SLINC_RS42780）进一步证实参与林可霉素生物合成调控。此外，还拓展了林可霉素生物合成的调控网络：证实了Rex通过间接促进lmbU等lmb基因的表达，促进林可霉素生物合成；RamR调控菌丝发育的同时，间接负调控林可霉素生物合成，直接正调控初级代谢关键调控因子GlnR；σL直接激活lmbU等簇内基因和前体分支硫醇合成基因mshC的表达，从而促进林可霉素生物合成；DeoR通过识别保守基序CGATCR，直接抑制部分林可霉素合成与调控基因的表达，进而抑制林可霉素生物合成。. 本项目受疫情影响，LmbU结构解析方面未达成预期目标，其他研究内容基本完成任务，较全面的阐释了LmbU的调控功能，进一步拓展了林可霉素生物合成的调控网络。