古菌Haloferax sp. strain D1227 通过龙胆酸分解代谢3-苯丙酸的分子机理研究

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.

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。

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。本研究证明嗜盐古菌D1227利用苯甲酸(BA)、3-羟基苯甲酸(3HBA)、苯丙酸(3PP)和苯乙酸(PAA)为唯一碳源和氮源生长时，其中龙胆酸1,2-双加氧酶（GDO）活力不受3HBA诱导，而龙胆酸(GA)途径中的顺丁烯二酸单酰丙酮酸(MP)水解酶（Maleylpyruvate hydrolase, MPH）活力受其诱导。通过基因组测序和序列比对，在D1227基因组序列中发现了两个编码GDO的基因（hagA1和hagA2）和可能编码MPH的基因（hagF1和hagF2）。将目的基因在古菌宿主H1424中表达，纯化的HagA1和HagA2具有GDO活力，其中HagA1活性依赖于Fe2+而HagA2不依赖；HagF2具有MPH活力，而HagF1没有MP水解酶或异构酶活力。3个潜在的顺序催化3HBA生成龙胆酸的关键酶3-羟基苯甲酰辅酶A (3HBA-CoA)连接酶HagL，3HBA-CoA羟化酶HagH和龙胆酰辅酶A (GA-CoA)硫酯酶HagT的编码基因，共同转入大肠杆菌中做生物转化实验，9h后检测到3HBA浓度降低了85%。将HagL单独克隆到古菌宿主H1424中表达，其细胞抽提液对六种芳香酸具有活力，其为底物范围较广的芳香酸辅酶A连接酶。从而证明古菌D1227通过龙胆酸直接水解途径降解多种芳香酸。基因sodA和sodB在E.coli BL21(DE3)和Burkholderia sp. SJ98中分别异源表达均具有超氧化物歧化酶活力。在添加500mM NaCl的M9培养基，以葡萄糖或4-硝基苯酚为碳源时，菌株SJ98[pBBR-sodA]仍可正常生长和降解底物，而对照菌株的生长和降解能力几乎丧失。SodAD1227和SodBD1227均具有Fe/Mn-SOD家族的经典结构特征。另一株嗜盐古菌WFD11能以3HBA和3PP为唯一碳源和能源生长，具有GDO基因hagA，其粗酶液和纯化蛋白HagA均具GDO的活性，催化GA开环生成MP；HagA的GDO比活力为0.0248U/mg，且其活性不依赖于Fe2+，且其是组成型表达。本研究为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奠定了基础。