嗜热脱氮地芽孢杆菌NG80-2的厌氧解烃机制研究

The interaction mechanism between microorganisms and petroleum hydrocarbon in extreme environment of reservoir is an important part of studying the mechanism of hydrosphere microorganisms driving the transformation and mobilization of earth bioessential elements. Petroleum hydrocarbon can be anaerobically degraded in the reservoir extreme environment, which mechanisms include the alkane addition to fumarate, hydroxylation followed by carboxylation, and intra-hydroxylation and the related research is scarce. Our research group isolated a facultative anaerobic thermophilic Geobacillus thermodenitrificans NG80-2 from the reservoir and found that the long-chain n-alkanes hydroxylase LadA, which played a key role in the aerobic degradation process of alkanes, also participated in alkanes anaerobic degradation, and its anaerobic activity in vitro is related to the addition of nitrate, which may be a new mechanism of hydrocarbon anaerobic degradation. Therefore, combining the methods of genomics, transcriptoomics, proteomics, metabonomics, molecular biology and bioinformatics, this study will further elucidate the anaerobic hydrocarbon degradation pathway, identify the novel anaerobic oil-biodegradation/regulation genes, clarify the catalytic mechanism of long-chain n-alkanes hydroxylase LadA. In addition, the coupling mechanism of anaerobic oxidation of petroleum hydrocarbons with the reduction of nitrate to dinitrogen will be focused. This study may reveal a new physiological mechanism of hydrocarbon microbial degradation under extreme conditions. It is an important supplement to the microbial driving mechanism of carbon transformation and mobilization under extreme circumstance and of important theoretical significance and application value.

油藏极端环境中微生物与石油烃的互作机制是研究水圈微生物驱动地球元素循环机制的重要组成部分，石油烃可在油藏极端环境下厌氧降解，解烃机制包括延胡索酸加成反应、羧化反应，以及内部供氧机制，相关研究比较匮乏。课题组从油藏中分离得到一株兼性厌氧嗜热脱氮地芽孢杆菌NG80-2，研究发现在烷烃有氧降解过程中起关键作用的长链烷烃羟化酶（LadA）也同样参与烷烃厌氧降解，且其厌氧体外活性与硝酸盐的添加有关，这可能是一种未报道的厌氧解烃新机制。因此，本研究在此基础上将利用转录组、蛋白组学、代谢组学、分子生物学和生物信息学等研究手段，进一步识别菌株NG80-2厌氧解烃通路，揭示石油烃厌氧降解的功能基因和调控基因，阐明LadA的作用机制，重点解析脱氮途径与烷烃氧化的偶联机制。该研究可揭示极端条件下一种新的微生物解烃生理机制，是极端环境碳循环微生物驱动机制的重要补充，具有重要的理论意义和应用价值。

油藏极端环境中微生物与石油烃的互作机制是研究水圈微生物驱动地球元素循环的重要组成部分，石油烃可在油藏极端环境下厌氧降解，解烃机制包括延胡索酸加成反应、羧化反应、以及内部供氧机制，相关研究比较匮乏。课题组从油藏中分离得到一株兼性厌氧嗜热脱氮地芽孢杆菌NG80-2，本项目通过分子生物学及转录组学等手段识别了菌株NG80-2厌氧解烃通路，揭示了石油烃厌氧降解功能基因和调控基因，并进一步解析了脱氮途径与烷烃氧化途径的偶联机制。.嗜热脱氮地芽孢杆菌NG80-2能够以硝酸盐为末端电子受体对正构烷烃进行厌氧氧化。它采用了类似于正构烷烃好氧降解的途径，首先利用长链烷烃羟化酶（LadA）激活烷烃生成相应的脂肪醇，然后由醇脱氢酶进一步催化并逐步生成脂肪醛和脂肪酸，从而进入β-氧化途径。NG80-2在厌氧途径中使用了与有氧途径不同且具有更高厌氧催化活性的DhaT型III类含铁醇脱氢酶。通过响应氧气的有无，HU和MarR家族的调控因子可调节不同类型醇脱氢酶的转录来控制有氧和厌氧降解途径的转换，该策略提高了生活在氧气浓度不断变化环境中细胞的适应性。此外，NG80-2是通过基因水平转移获得了ladA这一起始其有氧及厌氧烃降解的关键酶，其通过GntR家族的两个调控蛋白协同激活来响应烷烃及中间代谢产物的诱导。类似地，本研究将ladA基因转移到与NG80-2同种属具有类似后续烷烃降解途径的兼性厌氧菌中，可有效地赋予了其好氧和厌氧降解烷烃的能力，这扩展了对缺氧条件下整个碳氢化合物降解途径的理解，并对厌氧解烃工程菌的构建提供了指导思路。同时，厌氧条件下烷烃活化的O原子来源于脱氮途径，且NO的产生对LadA催化烷烃到烷醇的过程至关重要，这表明LadA很可能是偶联脱氮途径中产生的NO在厌氧条件下激活烷烃的降解途径，从而起始一种新的极端条件下微生物解烃生理机制，是极端环境碳循环机制的重要补充，具有重要的理论意义和应用价值。.本项目发表SCI论文7篇，申请国家发明专利2项，获得科研奖励3项。培养研究生6人，其中博士3人，硕士3人。