产酶溶杆菌中尿嘧啶核苷酸合成酶LePyrF调控抗菌物质HSAF合成的机制研究

产酶溶杆菌OH11合成并分泌的HSAF具有广谱高效的抗病原真菌活性，但原始产量低，是规模化生产的主要瓶颈。申请人前期通过突变体库筛选发现OH11中的LePyrF不仅具有ODCase酶活性参与UMP的合成，还可与调控HSAF合成的第二信使c-di-GMP的受体蛋白CdgL互作影响HSAF的生物合成，推测LePyrF可根据细胞内c-di-GMP浓度变化控制UMP和HSAF的合成。据此，本项目将通过遗传分析、ODCase酶活检测、DNA Affinity等技术解析LePyrF与CdgL互作的生物学意义，揭示LePyrF协同调控次级代谢产物HSAF和初级代谢产物UMP合成的分子机制，阐明c-di-GMP在该调控系统中的开关功能。从而为通过代谢流控制提高HSAF产量提供理论依据和技术支撑，并有望为ODCase的多功能性、初级与次级代谢产物合成和代谢流分配的调控机制等多个方面带来新认识。

Heat-Stable Antifungal Factor (HSAF) secreted by the biocontrol bacterium Lysobacter enzymogenes OH11 has broad-spectrum and highly efficient anti-fungal activity. However, low HSAF yield in the original OH11 strain considerably limits fermentation-based large-scale production. Studying the biosynthesis regulations of HSAF would lay an important foundation for strain engineering toward improved HSAF production. Via mutant library screening, we have found that LePyrF, an orotidine-5'-phosphate decarboxylase enzyme (ODCase) catalyzing a pivotal step of the UMP de novo biosynthesis pathway, significantly impacts HSAF yield, and more importantly, interacts with the c-di-GMP receptor CdgL, a secondary messenger which also regulates HSAF biosynthesis. We hypothesize that LePyrF is a dual-function protein which regulates HSAF biosynthesis via both its metabolic impact on UMP biosynthesis and its regulatory effect on c-di-GMP signaling. This research project aims to test this hypothesis via genetic modification, enzyme activity assay and DNA affinity. The study will unravel mechanisms underlying the cooperative regulatory functions of LePyrF and CdgL on HSAF biosynthesis, and the tertiary interaction effects of LePyrF, CdgL and c-di-GMP in signal transduction. The results will be critical to understand HSAF production regulation, which would lay an important foundation for improving HSAF production via metabolic flow design and will be important for understand the dual functions of bacterial ODCases.