深海细菌参与D-氨基酸再循环利用的生命过程、机制及相关酶资源应用潜力评价

Most deep-sea bacteria can be infected and lysed by virus, releasing organic matters into deep-sea environments. The peptidoglycan of bacterial cell wall is composed by glycan and polypeptide, which occupies 10% (G-) or 50~80% (G+) dry weight of bacteria. D-amino acids are unique components of peptidoglycan. The concentration of D-amino acids in marine environments is very high. D-amino acids are important organic carbon and nitrogen sources, but they are toxic to bacteria. Accumulation of D-amino acids in marine could influence marine ecosystem. However, little is known about the molecular mechanisms of recycling and detoxication of D-amino acids driven by marine bacteria. In this project, we will first screen bacteria and enzymes from deep sea samples, which can degrade peptidoglycan. We will subsequently select typical bacteria and enzymes, and study the metabolic pathways and the molecular mechanisms of recycling and detoxication of D-amino acids. Then, we will analyze GOS and Tara Oceans metagenomics databases to elucidate the universality and the ecological significance of the metabolic pathways of D-amino acids. Based on these results, we will investigate the application potential of the identified enzymes to control the infection of antibiotic resistant bacteria, which will lay foundation to develop novel drugs.

绝大多数的深海细菌能够被病毒侵染、裂解，释放菌体有机质到深海环境。细菌细胞壁肽聚糖由聚糖和肽链组成，占菌体干重的10%（G-）或50-80%（G+）。D-氨基酸是肽聚糖中的独特组分，海洋中储量巨大，既是重要的有机碳、氮，又对细菌具有毒性，若在深海中积累，将严重影响深海生态系统运转。但有关深海细菌参与D-氨基酸再循环利用和解毒的生命过程和分子机制尚不完全清楚。本项目首先从深海获取可降解肽聚糖中肽链的菌株、蛋白酶及其基因资源，构建相关菌株、蛋白酶及基因资源库；选取可降解肽聚糖的新型蛋白酶和可再循环利用D-氨基酸并解除其毒性的典型菌株，揭示深海细菌对D-氨基酸再循环利用和解毒的生命过程与分子机制。分析GOS和Tara Oceans等全球海洋宏基因组数据库，阐明上述代谢途径的普遍性及生态意义。最后开展新型蛋白酶在防治抗药性细菌感染中应用研究，为开发抗感染新药奠定基础。

细菌肽聚糖和D-氨基酸都是海洋中难降解有机质，数量庞大，但目前海洋细菌如何推动细菌肽聚糖和D-氨基酸的降解代谢和再循环利用还不清楚。海洋中相关的细菌、 酶和基因资源尚有待于挖掘。项目首先挖掘了一批海洋细菌、酶和基因资源。在此基础上，揭示了深海沉积物细菌Pseudoalteromonas sp. CF6-2分泌的M23家族蛋白酶Pseudoalterin的诱导表达分泌机制、晶体结构及其吸附和降解革兰氏阳性菌肽聚糖的分子机制，以及菌株SMCF6-2以分泌的M23家族蛋白酶Pseudoalterin为武器捕食革兰氏阳性菌的机制。揭示了菌株SMCF6-2及其它海洋细菌利用D-Ala、D-Glu和其它D-氨基酸的代谢通路和机制。揭示了海洋细菌利用革兰氏阴性菌肽聚糖中的特征氨基酸-二氨基庚二酸（diaminopimelic acid, DAP）的代谢通路和机制。对蛋白酶Pseudoalterin的应用潜力进行了探索，建立了Pseudoalterin的低成本发酵技术，研发了该酶的稳定剂，评价了该酶对动物的安全性及其在治疗多抗金色葡萄球菌MASA引起的感染的效果。项目还揭示了海洋中富营养菌和寡营养菌的共存机制以及一个外切型肝素酶的结构、催化机制和应用潜力。本项目的研究对阐明海洋生态系统中细菌间的相互作用和物质循环具有重要意义，并为海洋资源的进一步开发应用奠定了很好的基础。项目还资助了其它相关的海洋微生物研究。相关研究成果已发表45篇SCI论文，申请并授权国家发明专利3项。培养博士后6名，博士6名，硕士2名。