海洋石油细菌T7-7降解烷烃的分子机制的研究和功能优化

Oil pollution in the ocean has attracted increasing attention for its threat to the environment and the economical development. The bioremediation using oil microorganisms has been considered as the effective solution for the treatment of oil pollution in the ocean. Pusillimonas sp. T7-7 was isolated from the benthal mud of a petroleum-contaminated site in Bohai Sea, China, and has the capability to degrade diesel oil. The ability to tolerate cold and salty conditions makes this strain a good candidate to be used for the bioremediation of oil spill in the ocean, In our previous research, the complete genome of T7-7 was sequenced, and the putative genes involved in the alkane degradation were predicted using bioinformatical analysis. However, no genes sharing homology with the characterized genes encoding alkane monooxygenase, the key enzyme of alkane degradation,,were found, suggestingn the presence of novel alkane monooxygenase gene in T7-7. In this study, putative genes involved in alkane degradation in T7-7 will be analyzed using proteomics, molecular biological and biochemical analysis,and the respective degradation pathway will be elucidated. Vectors suitable for gene knock-out and gene expression in T7-7 will be constructed. The gene for the key enzyme, alkane monooxygenase, will be subject to site-specific and/or random mutagenesis and the mutant enzymes with improved alkane degraqdation ability will be selected. Plasmids with mutant genes or/and other alkane-degrading genes will be transferred into T7-7 to improve the ability of the strain to degrade oils. Finally, molecular mechanisms for theenhanced oil degradation caused by the interaction between T7-7 and a Rhodococcus strain PR5 will be investigated by transcriptomic analysis, This study will help us to understand the physiological function of the ocean microorganisms and their adaptive mechanism against the oil pollutant, and provide the basis for the construction of bacterial strains with better oil degradation ability for the treatment of oil pollutions in ocean environment.

海洋石油污染严重威胁生态环境和经济发展。石油微生物修复技术是解决海洋石油污染的有效手段。本课题组从渤海污染海域的海底泥样中分离得到一株具有高效降解石油能力的耐冷、耐盐菌株T7-7。前期研究中，申请人完成了该菌全基因测序，并预测了烷烃降解相关基因，但未发现与已知烷烃降解关键酶-羟化酶基因相似的基因，推测该菌中可能存在新烷烃羟化酶基因。本项目将利用蛋白组学、分子生物学和生物化学方法对T7-7中潜在的烷烃降解相关基因进行鉴定和功能分析，确定烷烃降解途径；构建适用于T7-7菌株的遗传操作系统；通过随机突变和定向进化策略对羟化酶基因进行改造，优化其蛋白功能；通过引入并高效表达相关基因，提高T7-7的降油能力和效率。此外，还将研究红球菌与T7-7在降解烷烃过程中的协同作用机制。本项目对于认识海洋石油微生物的重要生理功能和环境适应机制具有重要意义，同时对于构建用于治理海洋石油污染的工程菌株具有重要价值。

海洋石油污染严重威胁生态环境和经济发展。石油微生物修复技术是解决海洋石油污染的有效手段。极小单孢菌T7-7（Pusillimonas sp. T7-7）是一株革兰氏阴性的，耐冷的，柴油降解菌株，该菌分离自中国渤海原油污染区域的海底淤泥中。T7-7 可以利用柴油（C5 到 C30 链长的烷烃）为唯一碳源和能源进行生长。.利用生物信息学方法、蛋白质组学和RT-PCR方法，对T7-7的烷烃降解途径进行鉴定，并对部分重要功能基因进行了克隆表达和酶学活性分析。构建了适用于T7-7的基因缺失系统和基因表达系统。对红平红球菌PR4和T7-7协同作用降解石油过程中的关键时间点进行转录组测序，并通过转录组测序结果分析，确定红平红球菌PR4和T7-7协同作用促进生长和提高石油降解率的机制，全面了解两种菌相互作用时基因转录本的变化，解析了协同作用的关键基因及协同机理，构建了两菌的烷烃代谢调控网络。本项目对于认识海洋石油微生物的重要生理功能和环境适应机制具有重要意义，同时利用菌株的协同作用治理海洋石油污染具有重要价值。