野油菜黄单胞菌辅酶Q生物合成途径中3-八异戊烯基-4-羟基苯甲酸(HP8BA)脱羧酶基因鉴定和功能研究

Xanthomonas campestris pv. campestris (Xcc) causes black rot disease of crucifers that affects all cultivated brassicas throughout the world. Coenzyme Q (ubiquinone or CoQ) is a redox active lipid that plays a role in several essential cell processes including respiration, gene regulation and oxidative stress management. We have demonstrated that Xcc produces CoQ8. Xcc genome contains most of the homologs of the genes required for E. coli CoQ8 biosynthesis, ubiABEFGH, but not those of ubiC encoding chorismate-pyruvate lyase and ubiD/ubiX encoding the 3-polyprenyl-4-hydroxybenzoic acid (HP8BA) decarboxylase. We have identified a novel bifunctional chorismatase XanB2, which functions similarly as UbiC, converting chorismate to the direct precursor of CoQ8, 4-hydroxybenzoic acid, indicating that Xcc employs a unique biosynthetic pathway to produce CoQ8, which is independent on ubiC and UbiD/UbiX. However, the CoQ biosynthetic gene encoding the HP8BA decarboxylase remains unknown in Xcc. In this study, we use Xcc as the model organism to address the above-mentioned question. Three specific aims are planned to be achieved: (1) to identify the gene(s) encoding the HP8BA decarboxylase, through the Xcc genome-wide mutant library analysis, construction and screening of the Xcc genomic library and systematical deletion of the decarboxylase genes; (2) to characterize the biological functions, the enzymatic activity and catalytic mechanism of this novel decarboxylase by performing the in-vitro emzymatic reaction analysis, point mutation analysis and other biochemical methods; (3) to investigate the conservativeness of the HP8BA decarboxylase among all Xanthomonas bacteria, species belonging to other bacterial genus and the organisms belonging to higher kingdoms by multiple sequence alignment analysis and conserved domain/motif analysis. Unraveling these questions will provide a better understanding on the biosynthetic mechanism of CoQ8 in the phytopathogen Xanthomonas, which will facilitate the development of new antimicrobial agents to control Xanthomonas infections.

野油菜黄单胞菌(Xcc)能够侵染所有十字花科植物，引起黑腐病。辅酶Q是生物体内广泛存在的脂溶性醌类化合物，与黄单胞菌的生长发育和侵染致病密切相关。Xcc产生的辅酶Q是CoQ8。Xcc基因组包含大部分大肠杆菌CoQ8生物合成所需同源基因，但是缺少分支酸-丙酮酸裂解酶编码基因ubiC和HP8BA脱羧酶编码基因ubiD/ubiX。我们已经证明XanB2是一种新型分支酸-丙酮酸裂解酶；然而与UbiD/UbiX功能类似的HP8BA脱羧酶基因仍然未知。本项目以Xcc为模式菌株，(1)通过分析Xcc全基因组突变体库、构建Xcc基因组文库回补大肠杆菌ubiD突变体以及系统敲除Xcc基因组中的脱羧酶基因等方法，鉴定HP8BA脱羧酶基因；(2)研究新型HP8BA脱羧酶的生化功能和催化机理；(3)探讨这种新型HP8BA脱羧酶在细菌以及高等生物中的保守性。预期成果将为研发新型黄单胞菌防治措施奠定理论基础。

野油菜黄单胞菌(Xcc)能够侵染所有十字花科植物，引起黑腐病。辅酶Q(CoQ)是生物体内广泛存在的具有氧化还原活性的醌类化合物，在有氧呼吸电子传递链中处于中心位置，参与生物体的有氧呼吸生长和能量产生。因此，CoQ的生物合成与黄单胞菌的生长发育和侵染致病密切相关。Xcc产生CoQ8，其基因组包含大部分大肠杆菌CoQ8生物合成所需同源基因，但是缺少分支酸-丙酮酸裂解酶编码基因ubiC、HP8BA脱羧酶编码基因ubiD、C6-羟化酶编码基因xbiF、以及辅酶Q合成辅助基因ubiX和ubiJ。前期实验结果经证明XanB2是一种新型分支酸-丙酮酸裂解酶，然而具有ubiD、ubiX、ubiF及ubiJ相似功能的相关基因仍然未知。.本项目以Xcc为模式菌株，系统鉴定Xcc菌株中CoQ生物合成相关基因及其功能。通过突变体库筛选，鉴定了C6-羟化酶编码基因xbiF (Xc_0489)。我们发现xbiF敲除突变菌株的CoQ8含量显著下降，体内大量积累CoQ8合成中间产物DMQ8 (6-脱甲基辅酶Q8)。功能域分析结果揭示：XbiF并不属于原核生物辅酶Q生物合成途径中的“UbiI/UbiH/UbiF/COQ6羟化酶家族”，却与真核生物酿酒酵母 COQ7以及秀丽线虫CLK-1同属“双铁蛋白超家族”。点突变和蛋白模拟实验初步阐述了XbiF的三维结构和催化机理。此外，通过位置特异性查找，鉴定了辅酶Q生物合成相关基因xbiJ (Xc_0233)。xbiJ与大肠杆菌ubiJ基因虽不同源，与大肠杆菌ubiJ-ubiB共转录模式相似，xbiJ与ubiB在Xcc基因组上形成一个操纵子。敲除xbiJ或ubiB基因均导致Xcc的CoQ8产量下降，积累少量的CoQ合成中间产物3-八异戊烯基-4-羟基苯甲酸(HP8BA)。回补试验显示：由XbiJ碳端80个氨基酸构成的肽链即可回补xbiJ敲除突变体中的CoQ合成缺陷，但黄单胞菌XbiJ与大肠杆菌UbiJ之间不能相互替代回补。最后，生物信息学分析揭示，在不同属细菌中，CoQ生物合成相关基因具有遗传多样性。.本项目的研究结果填补黄单胞菌CoQ生物合成基因的空白，并为系统阐明细菌中CoQ合成机理奠定了基础。