菌株Pigmentiphaga sp.H8对3,5-二溴-4-羟基苯甲酸的降解及脱溴机制

Brominated compounds are important environmental pollutants. In addition, brominated compounds are reported to be naturally produced in marine environment. In recently years, microbial debromination of brominated compounds becomes one of hot research topics among scientific research community. In our previous study, a bacterial strain named Pigmentiphaga sp. H8, which was capable of debrominating 3,5-dibromo-4-hydroxybenzoate (DBHB), was isolated. It was found that the debrominated metabolites were totally different from previously reported ones, indicating that novel debromination pathway and debromination mechanism exist. In this proposed project, we will emphasize on the study of the complete metabolic pathway of DBHB in strain H8, clone of the complete degrading gene (cluster), especially the debromination gene, involved in the metabolism of DBHB; and the illumination of the catalytic mechanism of debromination enzyme. The outcomes of this project will broaden the microbial debromination mechanism of brominated aromatics, provide technical support for biodegradation and bioremediation of brominated aromatic contaminated sites, and in some extent, provide theoretical foundation of the halogen cycle in marine ecosystem.

溴代化合物不仅是一类重要的环境污染物，而且是海洋环境中自发产生的一类物质，溴代化合物的微生物脱溴研究是国内外关注的热点。本研究小组前期筛选到一株3,5-二溴-4-羟基苯甲酸（DBHB）的脱溴菌株Pigmentiphaga sp. H8，研究发现其脱溴产物不同于以往报道的产物，暗示存在新的脱溴途径和脱溴机制。本项目以好氧菌株H8为研究对象，揭示其降解DBHB的完整代谢途径；克隆DBHB的降解基因（簇），尤其是脱溴酶基因；并阐明脱溴酶的催化机制。本项目的研究不仅能丰富溴代芳烃类化合物的微生物脱溴机制，为此类化合物的生物降解和污染修复提供技术支持，而且为研究海洋生态系统的卤素循环提供一定的理论基础。

3,5-二溴-4-羟基苯甲酸（3,5-dibromo-4-hydroxybenzoate, DBHB）是除草剂溴苯腈代谢的主要中间产物，同时许多海洋生物也会自发产生，在环境中被广泛检出。卤素原子的强电子吸附效应赋予卤代芳烃高毒性、持久性和生物富集性等特点。因此，DBHB对人类健康和生态安全具有严重的威胁。微生物降解是环境中DBHB消除的主要因素。然而，关于DBHB的微生物降解机制研究不够深入，已报道的降解途径只有厌氧还原脱卤和好氧还原脱卤两种。本项目中菌株Pigmentiphaga sp. H8可以高效降解DBHB，并能以DBHB为唯一碳源生长。通过比较转录组学和比较蛋白质组学技术，结合分子生物学实验，揭示了菌株H8中NADPH-依赖型的黄素单加氧酶OdcA、双加氧酶OdcB和马来酰乙酸还原酶OdcC参与DBHB的降解。通过液相色谱、质谱等技术，鉴定了DBHB降解途径中的各种中间产物。通过体外酶学实验研究了降解关键酶OdcA的酶学特性。最终，从生化和分子生物学角度阐明了菌株H8降解DBHB的分子机制：首先，NADPH-依赖型的黄素单加氧酶OdcA利用NAD(P)H作为电子供体将DBHB氧化脱羧为2,6-二溴对苯二酚（2,6-DBHQ）；随后，2,6-DBHQ在双加氧酶OdcB的作用下被转化为2-溴马来酰乙酸；2-溴马来酰乙酸被还原酶OdcC还原脱溴生成马来酰乙酸（maleylacetate），并进一步被还原为β-酮己二酸（β-ketoadipate）；最后，β-ketoadipate进入TCA循环。本项目揭示了DBHB微生物降解新的氧化脱羧途径，同时也丰富了卤代羟基苯甲酸的微生物降解研究，为此类化合物的生物降解和污染修复提供理论基础和技术依据。