新鞘氨醇属海洋细菌降解高分子量PAHs的分子转运与代谢调控机制研究

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in the environment generated by natural combustion processes and human activities. High-molecular-weight (HMW) PAHs with four or more fused benzene rings are of environmental concern due to the most carcinogenic and bioaccumulation potential. Novosphingobium pentaromativorans US6-1, originally isolated from oil-contaminated estuarine sediment could utilize a serial of HMW PAHs as sole carbon and energy source. In this study, with the first bacterial genome which can degrade benzo(a)pyrene, N. pentaromativorans US6-1 will be used as mode strain to elucidate the molecular background for the metabolism of HMW PAHs. Base on the genome sequence and proteome data we will utilize a high-throughput comparative transcirptome analysis to assess gene differential expression, intergenic region, co-expression network and metabolic pathway in N. pentaromativorans US6-1 of response to three different aromatic compounds, phenanthrene, pyrene, and benzo[a]pyrene. Together with biological verification tests using qRT-PCR, gene knockout and gene covering techniques, efforts to elucidate the gene structure, function and expression regulation mode on the metabolic pathway involved in HMW PAHs transportation and catabolism will be initiated. Our study from the standpoints of PAHs transport mechanism and gene regulation network for the metabolism of HMW PAHs, which could provide a system-wide perspective on the biodegradation of PAHs and insights on how N. pentaromativorans US6-1 satisfies the demands for unusual metabolic capability toward HMW PAHs, and would expand our understanding of the metabolic potential of marine bacteria for bioremediation applications.

以环境毒性高、难生物降解的高分子量PAHs为研究对象，以一株已经完成全基因组测序的海洋新鞘氨醇菌Novosphingobium pentaromativorans US6-1为模式菌株，该菌株是目前报道的第一株能以高分子量PAHs-苯并芘（Benzo[a]Pyrene）为唯一碳源和能源生长的海洋细菌。在前期基因组和表达蛋白质组研究的基础上，引入比较转录组的研究方法，分析该菌株在没有PAHs诱导和有不同PAHs诱导下的转录组，通过基因表达差异、基因间序列、共表达网络、代谢通路预测等生物信息分析，结合qPCR、基因敲除和回补等生物学验证，剖析该菌株捕获、转运及分解代谢5环PAHs-苯并芘通路上的基因结构、功能、表达调控方式，揭示基因表达过程中的信息传输规律，从整体框架上研究海洋细菌转运和降解高分子量PAHs的基因功能及基因调控网络，推测PAHs分子转运和分解代谢等重要生理过程的分子机制。

以环境毒性高、难生物降解的高分子量PAHs为研究对象，以一株能降解高分子量PAHs-苯并芘（Benzo[a]Pyrene）的海洋新鞘氨醇菌Novosphingobium pentaromativorans US6-1为模式菌株，在前期基因组和表达蛋白质组研究的基础上，引入比较转录组的研究方法，并结合目标基因qPCR、基因敲除与回补等分子生物学技术，对该菌株捕获、转运及分解5环PAHs-苯并芘等代谢通路上的基因结构、功能以及表达过程中的信息传输规律展开研究，主要研究结果如下：研究表明，US6-1具有完整的趋化通路，且对菲、芘和苯并芘具有趋化活性。通过对甲基趋化受体蛋白MCP18870基因以及组氨酸激酶基因cheA的表达活性、基因敲除与回补以及配体结合域的荧光光谱实验证明MCP18870能触发US6-1对邻苯二酚和龙胆酸的趋化，组氨酸激酶基因cheA对PAHs的降解具有调节作用；在BaP压力下US6-1的TonB转运系统中的多个TDBT基因和tonB基因活跃表达，通过对这些基因的表达活性、敲除与回补以及过表达等实验证明，TonB转运系统在PAHs的跨膜转运与降解调控过程中具有重要的作用；通过基因簇分析、异源表达、蛋白功能验证、代谢产物分析、基因敲除与回补等实验证明，细胞色素P450在BaP降解的起始步起关键作用，同时发现US6-1具有多条中心代谢路径（原儿茶酸、邻苯二酚以及龙胆酸）进入TCA循环以快速获得能量。以上研究结果对解析新鞘氨醇菌捕获、转运及分解高分子量PAHs-苯并芘的分子机制具有重要意义。