phoRP双组份系统对生防枯草芽孢杆菌NCD-2中fengycin合成的调控机理

Lipopeptide fengycin is a major antifungal active compound produced by biocontrol agent Bacillus subtilis strain NCD-2, and the phoRP two component system indirectly regulated the production of fengycin in strain NCD-2. Recent studies have shown that the phoRP regulated the pentose phosphate pathway in strain NCD-2, that is the major carbohydrate metabolism pathway in the production of Acetyl-CoA under phosphate starvation. Acetyl-CoA is the precursor for the synthesis of fatty acyl and the fatty acyl is the backbone of lipopeptides, therefore, we speculate that the regulation of phoRP on the synthesis of fengycin is mediated by pentose phospate metabolism. To confirm this speculation, the transcriptome will be conducted to explore the genes up-regulated by PhoP under phosphate starvation. The metabolic intermediates regulated by PhoP in the carbohydrate metabolism will be identified by LC-MS/MS combined with the KEGG pathway analysis. The target genes that regulated by PhoP and involved in carbohydrate metabolism will be selected by transcriptome and LC-MS/MS analysis. The regulation of PhoP on the target genes will be confirmed with RT-qPCR and the DNA binding will be assayed using a gel retardation assay. Then, the function of target genes on the synthesis of fengycin will be clarified by gene in-frame deletion and complementation, and the key genes for the synthesis of fengycin will be identified. By overexpression of the key genes, the production of fengycin will be expected to be improved and then increase the antifungal acitivity and biocontrol efficiency of strain NCD-2. The results of this project will clarify the mechanism of phoRP on the regulation of fengycin synthesis, that will be important for the directional fermentation and the genetic modification of strain NCD-2.

Fengycin在枯草芽胞杆菌NCD-2菌株生防功能中发挥主要作用，phoRP双组份系统间接调控fengycin合成。近期研究发现phoRP调控NCD-2菌株磷酸戊糖途径，由于磷酸戊糖途径是合成脂肽类抗生素骨架物质—脂肪酸的主要途径，据此推测phoRP通过调控磷酸戊糖途径而影响fengycin合成。本项目拟通过转录组测序、LC-MS/MS结合KEGG通径分析确定NCD-2菌株中受调控因子PhoP调控并与糖代谢相关的目的基因，通过凝胶阻滞试验明确PhoP与目的基因的结合方式；对目的基因进行突变和互补分析，明确目的基因对fengycin合成的影响，确定影响fengycin合成的关键基因；对关键基因进行过量表达，以提高NCD-2菌株fengycin合成量和抑菌效果。预期结果将明确phoRP双组份系统对fengycin合成的调控机理，为更加科学合理的利用NCD-2菌株和菌株的遗传改良提供科学依据。

枯草芽胞杆菌NCD-2菌株能有效的防治多种植物病害，该菌株可以产生脂肽类抗生素fengycin和surfactin。前期研究发现，PhoRP双组份系统（PhoRP-TCS）正调控NCD-2菌株fengycin的合成。本项目围绕PhoRP -TCS调控fengycin合成的机理开展研究，获得以下研究成果：（1）明确了脂肽类抗生素fengycin和surfactin在NCD-2菌株防病功能中的作用。（2）PhoRP -TCS调控fengycin和surfactin的合成以及NCD-2菌株解磷能力、菌落形态以及芽胞的形成。（3）利用转录组学和差异蛋白质组学技术，获得了NCD-2菌株野生型与PhoRP双组份系统突变子之间差异表达的基因和蛋白，发现支链氨基酸的合成与脂肽类抗生素的合成有关。通过非靶向代谢组学，证明PhoRP-TCS主要影响了NCD-2菌株对氨基酸的合成和代谢。外源添加3种支链氨基酸混合液可以提高NCD-2菌株脂肽类抗生素的产量。突变支链氨基酸代谢关键基因lpdV显著降低了NCD-2菌株脂肽产生能力。（4）对支链氨基酸代谢途径中的关键基因citB、oxdC、abrB基因进行功能验证，突变citB降低了NCD-2菌株生fengycin的合成，但突变abrB提高了NCD-2菌株fengycin的产量。突变phoR基因和phoP基因后增强了NCD-2菌株对某些抗生素，尤其是靶标30S核糖体的抗生素的抗性。转录组学分析发现，突变phoRP双组份系统提高了kstSTU操纵子，ABC-transporters等基因的表达。在NCD-2菌株中过表达kstSTU可以增强NCD-2菌株对抗生素的抗性。通过本项目的实施，明确了枯草芽胞杆菌中脂肽类抗生素的合成途径，为通过遗传改良或定向发酵来提高脂肽类抗生素的产量提供依据。初步明确了枯草芽胞杆菌抗药性的分子机制，丰富了对细菌抗药性的了解。