假单胞菌PCA01经原儿茶酸途径和丛毛单胞菌GEN05经龙胆酸途径代谢3-甲基苯酚的分子机理研究

Compound 3-cresol is one of highly toxic and carcinogenic alkylphenols. It has been reported to be metabolized by bacteria via three pathways, respectively forming protocatechuate, gentisate and 3-methyl-catechol. The genes and enzymes involved in the 3-methyl-catechol pathway has been identified, but the studies at molecular level on the other two pathways (protocatechuate and gentisate) are still lacking. In this study, The degradation studies at molecular level on newly isolated 3-methyl-catechol utilizers Pseudomonas sp. PCA01 (via protocatechuate) and Comamonas sp. GEN05 (via gentisate) will be carried out. Through genomic sequencing and library construction, the entire 3-cresol catabolic gene cluster of protocatechuate and gentisate pathways in these strains will be obtained. The function of each catabolic gene will be identified through gene knockout and complementation, expression in vitro, product identification and enzyme assay. The function and character of the key enzymes involved in the catabolism will be investigated after purification. The differences and relationships among the different cresol catabolic pathways will be illustrated. We hope that this study will elucidate, at molecular, biochemical, and genetic levels, the mechanism for the degradation of 3-cresol via protocatechuate and gentisate pathways. In addition, the different functions and evolutionary relationships will be further resolved by the three metabolic pathways in different environments. And the diversities of cresol catabolism will also be explored. The data present here will be greatly helpful to explore the diverse capability for the microbial aromatic degradation and illustrate the adaptive evolution strategies of microbes using cresol as a model.

3-甲基苯酚是高毒性和潜在致癌性的烷基酚污染物的重要代表。3-甲基苯酚的微生物代谢途径有三种，即分别形成原儿茶酸、龙胆酸或3-甲基邻苯二酚进行代谢，其中，3-甲基邻苯二酚途径的相关基因和酶学已被鉴定，而另外的两条途径仍缺乏相关分子生物学研究。本项目以工业废水中筛选到经原儿茶酸途径代谢3-甲基苯酚的Pseudomonas sp. PCA01和经龙胆酸途径代谢的Comamonas sp. GEN05为研究对象，通过基因组分析、文库构建等方法获得完整的经由原儿茶酸和龙胆酸代谢3-甲基苯酚的基因簇，结合基因敲除、体外表达、酶活测定和产物鉴定等方法鉴定其生理功能，并对关键酶进行纯化和酶学研究。从分子、生化和遗传学水平上阐明3-甲基苯酚完整的代谢途径，并进一步解析不同环境中这三条代谢途径的功能差异和进化关系，确定它们的生态学意义。探索微生物代谢芳烃污染物的代谢机理的多样性及其适应性进化策略。

3-甲基苯酚是高毒性和潜在致癌性的烷基酚污染物的重要代表。3-甲基苯酚的微生物代谢途径有三种，即分别形成原儿茶酸、龙胆酸或3-甲基邻苯二酚进行开环代谢，其中，3-甲基邻苯二酚途径的相关基因和酶学已被鉴定，而另外的两条途径仍缺乏相关分子生物学研究。本项目以工业废水中筛选到经原儿茶酸途径代谢3-甲基苯酚的Pseudomonas sp. PCA01和经龙胆酸途径代谢的Comamonas sp. GEN05为研究对象，考虑到这两株菌代谢途径的重叠，我们优先进行GEN05菌株中代谢途径的研究。通过基因组分析和转录分析，我们找到了可能参与3-甲基苯酚代谢mchAB基因和mchC基因，进一步通过体外表达、酶活测定和产物鉴定等方法鉴定了它们的生理功能，结果表明，mchA和mchB基因编码一个双组份的3-甲基苯酚甲基羟化酶，该酶可以催化2-甲基苯酚、3-甲基苯酚和4-甲基苯酚，最适底物为3-甲基苯酚，将其催化生成3-羟基苯甲醇，再氧化成3-羟基苯甲醛；mchC基因编码3-羟基苯甲醛脱氢酶，催化3-羟基苯甲醛生成3-羟基苯甲酸，它可以进一步通过龙胆酸或原儿茶酸进行开环代谢。本研究从分子生物学、生物化学和遗传学水平上阐明一条新的3-甲基苯酚代谢途径，表明了3-甲基苯酚代谢的多样性，为3-甲基苯酚以及三种单甲基苯酚的生物修复奠定了基础。