产酶溶杆菌中小分子化学信号4-HBA调控抗菌物质HSAF合成的机制研究

HSAF (Heat-stable antifungal antibiotic) is a secondary metabolite with a broad-spectrum antifungal activity produced by Lysobacter enzymogenes, a biological control agent. HSAF has a chemical structure different from any existing antifungal drugs and shows a novel mode of action and a unique biosynthetic mechanism. HSAF is also safe to plants, animals and environment. These unique features undoubtedly confer HSAF to be an ideal candidate for developing bio-pesticide in agriculture. In our previous works, we identified 4-HBA (4-hydroxybenzoic acid) as a new diffusible molecule produced by strain OH11 to control HSAF production. In strain OH11, LenB2 encodes an oxygenase and converts chorismate, the end-product shikimate pathway, to 4-HBA. For functional performance, 4-HBA directly interacts with its receptor protein, LysR (a transcription regulator with DNA binding domain) to help the binding of LysR to pHSAF (promoter region of the HSAF biosynthetic gene operon), leading to a precise regulation of HSAF biosynthesis in L. enzymogenes OH11. These findings reveal that 4-HBA is a diffusible molecule connecting metabolic shikimate pathway to a unique antifungal metabolite HSAF biosynthesis in bacteria. In this project, we found that LysR could specifically interact with L. enzymogenes YajQ, whose homolog proteins are known to mediate virulence of bacterial pathogens. The role and working mechanism of YajQ family proteins have never been documented in Lysobacter and other bacterial biological control agents. Notably, we further found that the YajQ-LysR interaction enhances the binding of LysR to pHSAF in vitro and facilities HSAF production in vivo, but the underlying mechanism remains poorly understood. Here, we aimed to understand: (i) how LysR and YajQLe interact; (ii) what is the biochemical basis and genetic significance for YajQ to enhance the binding of LysR to pHSAF; and (iii) if 4-HBA has an important role in YajQ-LysR-pHSAF interaction or triplet complex formation. If yes, what is the underlying mechanism and whether this molecular effect contributes to the production of antimicrobial factors, i.e. HSAF expressed by L. enzymogenes OH11. Our results are expected to reveal a new 4-HBA-mediated regulatory mechanism in Lysoabcter, and are also greatly beneficial for improvement HSAF production by engineering the 4-HBA regulatory network in L. enzymogenes.

HSAF是生防产酶溶杆菌中新发现的一种结构新颖、生物合成和作用方式独特、对环境安全、广谱抗真菌和卵菌的次生代谢产物。4-HBA（4-羟基苯甲酸）是菌株OH11中发现的一种新型小分子化学信号。该信号与下游受体转录因子LysR互作，有利于LysR结合在HSAF合成基因簇的启动子区（pHSAF），指导该基因簇的表达。本项目新发现菌株OH11体内的YajQ蛋白能与LysR互作。通过这种互作，YajQ能显著增强LysR结合pHSAF的能力，促进HSAF的合成。本项目旨在深入解析YajQ与LysR互作分子机制；阐明YajQ增强LysR-pHSAF复合物形成的生化基础及遗传学意义；探明4-HBA对YajQ-LysR-pHSAF互作的影响、作用机制及其对HSAF等生防因子形成的贡献。研究结果将以溶杆菌为材料揭示4-HBA发挥调控功能的新机制，也有助于通过分子改良4-HBA信号网络来提升HSAF的产量。

4-HBA（4-羟基苯甲酸）是产酶溶杆菌OH11中发现的一种新型小分子化学信号。该信号与下游受体转录因子LysR互作，有利于LysR结合在HSAF合成基因簇的启动子区（pHSAF），指导该基因簇的表达。本项目前期发现菌株OH11体内的YajQ蛋白能与LysR互作。通过这种互作，YajQ能显著增强LysR结合pHSAF的能力，促进HSAF的合成。本项目旨在深入解析YajQ与LysR互作的分子机制；阐明YajQ增强LysR-pHSAF复合物形成的生化基础及遗传学意义；探明4-HBA对YajQ-LysR-pHSAF互作的影响、作用机制及其对HSAF等生防因子形成的贡献。. 项目利用蛋白质亲和层析在菌株OH11鉴定了一个c-di-GMP的新受体蛋白YajQ（后命名为CdgL）。研究发现CdgL能与4-HBA的受体蛋白LysR特异性结合形成CdgL-LysR的蛋白复合物。该蛋白复合物能显著增强LysR直接结合在HSAF合成基因簇启动子区的能力，促进HSAF合成基因表达并提升HSAF产量。C-di-GMP结合CdgL或4-HBA结合LysR均能解离CdgL-LysR-DNA这个三者复合体，通过释放CdgL或LysR来解除其对HSAF合成的促进作用，实现c-di-GMP和4-HBA对HSAF合成协同调控，由此揭示了是生防细菌中两种截然不同的小分子化学信号交叉对话的一种新途径。. 上述基础研究新发现在PLoS Pathogens, Environmental Microbiology等领域内主流刊物上共发表项目标注的SCI论文12篇。在此基础上，本项目通过敲除菌株OH11体内的c-di-GMP合成酶来解除c-di-GMP对HSAF合成的抑制作用，结合发酵优化，提升HSAF产量150倍，据此创制了HSAF的凝胶剂和种衣剂，授权国家发明专利2项，加速了HSAF作为绿色生物农药创制的进程。