迪茨氏属菌降解烷烃过程中底物响应的分子机制研究

Bacteria belonging to the genus Dietzia are typical alkane degraders that can degrade a broad range of alkanes. Our present research revealed that upon growth on n-hexadecane, n-tetracosane, and n-hexatriacontane, Dietzia sp. DQ12-45-1b could produce different glycolipids, phospholipids, and lipopeptides.The cell surface hydrophobic activities and cell behaviors were also different. Further resutls showed that alkane hydroxylase AlkW1 showed high transcriptional level during growth on alkanes but it could not be induced by alkanes directly. The results suggested different mechanisms of the responses to alkanes with different carbon chain length in Dietzia. Alkanes might regulate the expresion of downstream genes via novel signal tranduction pathways. We sequenced and compared 25 genomes of Dietzia strains. The core gene Dz_1341 that was encoded for the histidine kinase of two-component system in Dietzia sp. DQ12-45-1b was significantly induced during alkane metabolism. So we hypothesized that the two-component system Dz_1341/Dz_1342 might participate in the responses to alkane substrates in Dietzia sp. DQ12-45-1b and regulate the expression of downstream genes during alkane metabolism. In this study, we will investigate the relationship between Dz_1341/Dz_1342 two-component system and the responses to different alkanes with gene knockout, transriptional analysis and measurement of kinase activities, and further determine the target promoter and binding site of the two-component reponse regulator Dz_1342 with EMSA and ChIP-seq methods. This study aims to reveal the molecular mechanism for the reponses to alkane substrates of Dietzia sp. DQ12-45-1b during alkane metabolism. It will also provide us new insight into the mechanisms of global metabolic regulation of microbial degradation of alkanes.

迪茨氏属菌是典型的广谱烷烃降解菌，在降解不同烷烃时表现出不同的烷烃接触行为和代谢特征。进一步研究发现烷烃并不能直接诱导烷烃羟化酶AlkW1的转录。以上结果表明迪茨氏属菌对不同烷烃具有不同的响应机制，细胞感应烷烃后可能通过一系列的信号传导途径调节下游基因的表达。通过对迪茨氏属菌25个不同菌株的核心基因组分析和基因转录分析，我们发现核心基因Dz_1341在烷烃代谢过程中被显著诱导，它负责编码双组分系统(TCS)中的组氨酸激酶。因此我们推测双组分系统Dz_1341/Dz_1342可能与底物响应有关，迪茨氏菌通过其感应不同烷烃调控下游基因的表达。本项目将通过基因敲除，基因转录分析和激酶活性研究与不同烷烃底物的对应关系；通过EMSA，ChIP-seq研究TCS调控蛋白的调控序列位点。本研究将初步揭示迪茨氏属菌烷烃降解过程中底物响应的分子机制，为全面了解烷烃降解的分子机制和全局代谢调控提供了新的思路。

烷烃的微生物降解是自然界普遍存在的一种现象，对于石油污染修复和微生物提高石油采收率技术十分重要。迪茨氏属菌DQ12-45-1b是一株从石油采出液中分离获得的烷烃降解菌，在前期研究中发现该菌能够利用C8-C40正构烷烃、支链烷烃及部分芳香烃作为唯一碳源生长，具有很高的应用前景。迪茨氏属菌是典型的广谱烷烃降解防线菌，在降解不同烷烃时表现出不同的烷烃接触行为和代谢特征。进一步研究发现烷烃并不能直接诱导烷烃羟化酶AlkW1的转录。以上结果表明迪茨氏属菌对不同烷烃具有不同的响应机制，细胞感应烷烃后可能通过一系列的信号传导途径调节下游基因的表达。本项目在前期工作基础上，通过基因敲除、EMSA等方法对迪茨氏属烷烃降解过程中与不同底物响应的分子机制展开了研究。通过研究我们建立了迪茨氏属菌通用遗传操作体系，菌株的重组效率能够达到8×10-5，阳性率大于30%。在此基础上，阐明了Dietzia sp. DQ12-45-1b菌株中烷烃羟化酶基因alkW1和CYP153受转录调控蛋白AlkX和CypR的诱导调控机制，该调控机制在放线菌中具有普遍性。建立了菌株Dietzia sp. DQ12-45-1b中TCS系统的基因敲除菌株，并鉴定了TCS与烷烃代谢、细胞表面性质和溶剂耐受的关系，为进一步研究TCS在菌株石油环境适应性中的作用提供了依据。