红平红球菌KB1低温降解烷烃关键基因的确定及调控机制研究

Microbial bioremediation is the main pathway to degrade petroleum hydrocarbons in the environment, and has been the focus of environmental science research. In the study, a strain of Rhodococcus erythropolis KB1, which was isolated from Yumen Oil Field, Gansu, and could degrade petroleum hydrocarbons efficiently at low temperature, will be selected as the study object. The complete genome information will be obtained using whole genome sequencing technology. The potential alkanes oxidase genes, gene cluster structure and transcriptional regulation factors will be analysed by bioinformatics. The function and metabolism mechanisms of alkanes oxidase genes will be illustrated by gene mutation technology combined with metabonomics technology. Furthermore, the potential novel transcriptional regulation factors will be screened using DNA affinity purification technology. The interaction between promoter sequences of alkanes oxidase genes and transcriptional regulation factors will be verified by gel retardation assay. Based on these results, the transcriptional regulation factors genes deletion mutation strains will be constructed according to the intention to explain detailedly the regulatory mechanisms of the petroleum hydrocarbons degradation. Therefore, new research thoughts focused on full developments of microbial functional genes, explanation of microbial degradation mechanisms of petroleum hydrocarbons, and design of genetic engineering strains with high metabolic activities will be likely come true according to the abovementioned results. As a consequence, Rhodococcus erythropolis KB1 has very important theoretical research and practical application values.

利用微生物为主体的生物修复技术是降解环境中石油烃的主要途径，已成为当今环境科学研究的热点。本项目以从甘肃玉门油田分离的一株在低温下具有高效石油烃降解能力的红平红球菌KB1为研究对象，拟采用全基因组测序技术获取完整基因组信息，借助生物信息学方法分析其潜在的烷烃氧化酶基因、基因簇结构和转录调控因子，结合基因突变技术和代谢组学技术研究烷烃氧化酶基因的功能和代谢机制；同时利用DNA亲和纯化技术进一步筛选新的转录调控因子，凝胶阻滞实验验证烷烃氧化酶基因启动子序列与转录调控因子间的相互作用，进一步构建转录调控因子基因的缺失菌株，研究其低温下的石油烃降解调控机制。该研究结果可为充分挖掘微生物功能基因资源，诠释微生物降解石油烃的分子机制和设计高代谢活性的基因工程菌株等提供新的研究思路，具有重要的理论研究和实际应用价值。

基于多相分类学法对KB1进行了鉴定，结果表明，KB1菌株为革兰氏阳性好氧微生物，呈短杆状或杆状。特征性氨基酸组分为meso-DAP（二氨基庚二酸）、Ala（丙氨酸）、Glu（谷氨酸）、Gly（甘氨酸）和Asp（天冬氨酸）；全细胞水解物特征性糖组分为阿拉伯糖、半乳糖、甘露糖和核糖；主要极性脂为双磷脂酰甘油、磷脂酰乙醇胺、磷脂酰肌醇、磷脂酰肌醇甘露糖苷和一种未知磷脂酰极性脂；非极性类脂主要由甲基萘醌组成。进一步采用16S rDNA序列和gyrB序列构建系统发育树，从进化分支上显示该菌株属于红平红球菌，命名为Rhodococcus erythropolis KB1。联用基因组、转录组和代谢组技术，从多维度揭示了KB1菌株高效石油烃降解能力、良好的低温耐受性以及植物促生能力等主要生物学特性的遗传学基础。PacBio单分子长测序结果表明，KB1菌株的遗传物质主要包含1条染色体和3个质粒，共计6837个基因；编码单加氧酶、双加氧酶、芳基乙醇脱氢酶等石油烃降解相关的基因约731个。其中，82个基因在二甲苯、氯环己烷、氯苯、萘等确定的石油烃组分降解通路中发挥着关键的作用。基于antiSMASH预测出KB1菌株中含有NPRS、Ectoine、RiPP-like等18个次级代谢产物生物合成基因簇，在遗传水平上提示KB1菌株能够产生铁载体，具有良好的盐胁迫、干燥、高温和低温等逆境适应性。不同温度条件下石油烃降解的比较转录组分析结果提示，低温下KB1菌株可通过上调ATP合成、氧化磷酸化和脂肪酸代谢等基因（FC>8）表达量，代偿性地产生能量或改变膜脂组分比例来“御寒”，且温度差异并没有引起石油烃各组分降解相关基因表达强度的显著逆转。不同温度条件下石油烃降解的菌体代谢组学结果则显示，相比10 ℃，在16 ℃下处理，能确保KB1菌株的低温应激系统更好地充分发挥作用，但随着温差的加大，磷脂酰乙醇胺的表达上调在数量和强度上都会增加，且磷脂酰乙醇胺的表达上调是响应低温的主要代谢模式，这与广泛报道的磷脂酰乙醇胺发挥重要的膜脂成分功能相符。.