肠道微生物褐藻胶多糖利用基因位点AlgPULs的功能与调控机制研究

Alginate has been widely used in food, pharmaceutical and industrial fields, the degradation products of which were reported to exhibit prebiotics, antioxidation, antitumor activity. There is no direct experimental evidence for that human intestinal microbiota is capable of metabolizing alginate so far. Functions of the genes involved in this process have not been reported either. By searching from human intestinal microbial genome database, 62 genes encoding alginate lyases in 34 intestinal strains were explored. Then up-and down-stream of the genes were analyzed and several Bacteroides strains including Bacteroides clarus were predicted to have potential alginate utilization loci (AlgPULs), in which proteins involved in alginate degradation, transport and transcription regulation were encoded. Based on the background, we are going to identify the alginate lyases by enzymatic characterization and structure analysis, reveal the transcription regulation mechanism of AlgPULs by EMSA and RT-PCR, and analyze functions of the genes involved in alginate utilization genetically. Finally the genes of AlgPULs involved in alginate utilization will be demonstrated and the functions of them are supposed to be elucidated. This study is expected to provide information for unravelling the physiological benefits of alginate degradation in vivo.

褐藻胶（Alginate）在食品、医药等领域具有广泛应用，其降解产物寡糖具有益生元、抗氧化、抗肿瘤等生物活性。但目前尚无人类肠道菌利用褐藻胶的实验证据，且参与该过程的相关基因也未见报道。前期我们从人类肠道微生物基因组数据库挖掘了34株肠道菌的62个褐藻胶裂解酶基因，对基因上下游分析发现，Bacteroides clarus等肠道拟杆菌具有潜在褐藻胶利用基因位点（AlgPULs），预测编码褐藻胶降解、转运、利用及基因调控相关的蛋白。基于此，本项目将通过酶学表征、结构解析鉴定褐藻胶裂解酶的功能；通过凝胶阻滞、RT-PCR等研究AlgPULs基因簇的转录调控机制；通过基因敲除、表型分析等遗传学手段验证AlgPULs各基因的功能。通过上述研究，明确AlgPULs基因簇中参与褐藻胶利用的基因，解析肠道菌降解、利用褐藻胶相关的基因功能，为揭示肠道菌利用褐藻胶的生理意义提供信息。

褐藻胶（Alginate）在食品、医药等领域具有广泛应用，其降解产物寡糖具有益生元、抗氧化、抗肿瘤等生物活性。但其在肠道环境中的代谢机制尚不明确。前期通过生物信息学分析发现，多株肠道拟杆菌具有潜在的褐藻胶利用基因位点（AlgPULs）。本项目以肠道菌Bacteroides clarus 为模型解析了肠道微生物AlgPUL的分子作用机制。该AlgPUL 受到转录因子BcGntR 的抑制调控，不饱和褐藻胶二糖能够解除转录抑制，激活AlgPUL的转录。该AlgPUL 含有的两个褐藻胶裂解酶AlyPL6和AlyPL17均为外切酶，且底物特异性互补。其中AlyPL6 偏好聚甘露糖醛酸古罗糖醛酸（PMG）和聚古罗糖醛酸（PG），而AlyPL17 偏好聚甘露糖醛酸（PM）。对AlyPL6晶体结构的解析发现其C 端结构域能够帮助底物处于正确的催化位置，而非参与底物结合。AlyPL6和AlyPL17在细胞周质空间协同作用，降解由BcSusCD 复合体转运的不饱和褐藻胶寡糖底物。这些特性决定了B. clarus 专一性代谢不饱和褐藻胶寡糖。获得的单糖最终通过Entner-Doudoroff (ED) 途径代谢。与葡萄糖代谢相比，ED途径导致细胞内还原力的缺乏从而改变了细胞内短链脂肪酸和氨基酸的代谢，使主要代谢产物由乳酸转变为乙酸。基于上述结果，本课题最终建立了B. clarus 代谢褐藻胶寡糖的模型，代表着第一个人肠道微生物褐藻胶代谢模型，为褐藻胶寡糖生物活性的研究提供了理论依据。.肠道微生物通过水平基因转移从食物中的海洋微生物获得褐藻胶PUL。除了肠道微生物AlgPUL的研究，我们还关注了海洋微生物的AlgPUL。与肠道菌B. clarus 不同，海洋菌Defluviitalea phaphyphila 能利用高聚合度的褐藻胶生长。在解析D. phaphyphila 褐藻降解系统的工作中，本项目鉴定了其中一个褐藻胶裂解酶AlgAT0。揭示了AlgAT0 通过提高钙离子亲和力来增强底物亲和力，从而适应高温环境的分子机制。该研究不仅从新的视角揭示了耐高温酶的高温适应性机制，还为耐高温酶的定向改造提供了新思路。