一条全新的细菌生物素前体的生物合成途径研究

As a fatty acid derivative sulfur-containing vitamin, biotin is an essential enzyme cofactor in bacteria because it plays critical roles in central metabolic processes. The biosynthesis of the precursor molecule (pimelic acid carbon skeleton) is a key step in bacterial biotin synthesis, which is diverse and provides new targets to develop antibacterial drugs targeting different bacteria. However, only the E. coli BioC-BioH pathway is thoroughly understood so far. Our preliminary results showed that the bioZ gene from two different bacteria in Rhizobiales can genetically complement E. coli bioC and bioH mutants, respectively, showing that the bioZ gene is involved in a novel biosynthetic pathway for biotin precursor in bacteria. In this project, A. tumefaciens is used as the research object, and the following three aspects will be studied via in vitro functional analysis and in vivo gene knockout: (1) BioZ function and its role in biotin biosynthesis; 2) molecular basis for BioZ substrate recognition; and (3) identification of other synthetase in the BioZ pathway. The aim of this project is to elucidate a new biosynthesis mechanism of bacterial biotin precursor, to enrich and improve the physiological and metabolic mechanisms of bacteria, and to provide a theoretical basis for the development of new antibacterial agents targeting biotin biosynthesis.

生物素作为一种脂肪酸衍生物类含硫维生素，是细菌生长代谢所必需的辅酶因子。生物素前体（庚二酸碳骨架）的生物合成是细菌生物素合成的关键步骤，且在细菌中存在广泛的多样性，这为研发针对不同细菌的抗菌药物提供特异性靶点。但迄今为止研究透彻的生物素前体合成只有大肠杆菌的BioC-BioH途径。本项目前期研究发现根瘤菌目中2种不同细菌的bioZ基因可分别异体遗传互补大肠杆菌bioC和bioH突变株，初步表明存在一条bioZ参与的细菌生物素前体的合成新途径。本项目以根癌农杆菌为研究对象，采用体外功能分析和体内基因敲除两种策略，开展以下三方面的研究：（1）BioZ的生化功能及其在生物素合成中的作用；（2）BioZ底物识别的分子基础；（3）BioZ途径中其他聚合酶的鉴定。该项目旨在阐明细菌生物素前体生物合成的一种新机制，丰富和完善细菌生理代谢机理，为研发针对生物素合成途径的新型抗菌药物提供理论基础。

细菌生物素的生物合成途径是极具潜力的研发新型抗菌药物的靶标。生物素合成的关键是其前体七碳单位庚二酸碳骨架的来源，不同细菌其生物素前体的合成代谢途径具有多样性。到目前为止，大肠杆菌BioC-BioH途径和芽孢杆菌BioI-BioW途径研究得较为清楚，但植物致病菌根癌农杆菌(Agrobacterium tumefaciens)中BioZ途径如何合成庚二酸单酰ACP仍不清楚。本项目以根癌农杆菌为研究对象，采用体外功能分析和体内基因敲除两种策略，研究BioZ的生化功能以及BioZ底物识别的分子基础。取得的主要成果有：（1）鉴定了影响BioZ酶活的6个关键氨基酸残基，包括W47, L90, V265, S286, T290 和 A315F，并验证了关键氨基酸差异并不能解释底物识别特异性。（2）解析了BioZ 和FabH底物识别差异性的分子机制，将α9-β10 loop环和残基Ala317从AtBioZ移植到EcFabH，导致FabH的底物偏好从acetyl-CoA转变到galutaryl-CoA，反之亦然，证实了它们是底物特异性和结合的结构决定因素。（3）鉴定了存在于立克次体和卡他莫拉菌生物素前体合成途径中关键酶BioT、BioU以及BtsA，并验证BtsA参与卡他莫拉菌的毒力。该项目共发表SCI论文4篇，培养硕博士研究生3名。总之，该项目阐明细菌生物素前体生物合成中BioZ的酶学催化新机制，丰富和完善了细菌生理代谢机理，为研发针对生物素合成途径的新型抗菌药物提供理论基础。