许多芳烃的细菌代谢途径已得到透彻解析,而古菌代谢芳烃研究屈指可数,且完整代谢途径无分子水平的研究。3-苯丙酸广泛存在于自然界,是木质素代谢中间产物和重要工业原料。目前细菌中只发现通过邻苯二酚代谢3-苯丙酸,而古菌Haloferax sp. strain D1227有证据表明通过龙胆酸代谢,但其完整代谢途径无遗传和生化水平报道。本研究拟以古菌D1227为研究对象,通过基因组测序、生物信息学分析或基因组文库预测通过龙胆酸代谢3-苯丙酸的基因簇；利用古菌遗传表达系统进行基因克隆表达并分析酶促反应产物鉴定代谢基因簇的功能；纯化顺序催化3-苯丙酸生成龙胆酸及龙胆酸代谢的关键酶或新型酶,并进行动力学研究。本研究将首次从分子、生化和遗传学水平阐明原核生物(包括古菌)通过龙胆酸代谢3-苯丙酸的完整代谢途径及龙胆酸途径的多样性,从而阐明原核生物代谢芳烃的多样性及进化策略,并揭示古菌和细菌代谢芳烃的可能差异。

The mechanisms of bacterial catabolism of aromatic compounds have been illustrated in many cases. In contrast, no single catabolic pathway for aromatics degradation in archaea has been identified at the molecular level. 3-Phenylpropionate, an important intermediate of lignin degradation and various industries, widely and abundantly exists in nature. It is known that bacteria degrade 3-phenylpropionate only via catechol as the ring-cleavage intermediate. Haloferax sp. strain D1227, a halophilic archaea, is able to catabolize 3-phenylpropionate via gentisate as the ring-cleavage intermediate. However, the complete catabolic pathway has not been reported at biochemical and genetic level. In this proposed study, the complete 3-phenylpropionate catabolic gene cluster involved in the degradation of 3-Phenylpropionate via gentisate in strain D1227 will be identified by genome sequencing together with bioinformatics analysis and/or genomic library screening. The physiological and biochemical functions of catabolic genes involved in this pathway will be characterized through gene clone, protein expression in vitro, product identification and so on, combined with archaea unique genetic manipulation systems. The functions and properties of the key enzymes in this pathway will be investigated in great details. In the case of archaea, the findings from this study will hopefully reveal, for the first time, the pathway for 3-phenylpropionate catabolism via gentisate at molecular, biochemical, and genetic levels in prokaryotes. In particular, the diversity of gentisate pathway in prokaryotes will also be elucidated. It is also of great help to explore the catabolic diversity for the aromatic degradation and illustrate the adaptive evolution strategies of prokaryote in their catabolism, as well as the possible difference in aromatic compounds degradation between archaea and bacteria.