浒苔多糖降解微生物的筛选及其降解机理的研究

Enteromorpha prolifera could form "green tides" and produce a large number of polysaccharides. The decomposition, utilization and transformation of these polysaccharides are of great significance to marine carbon cycle and the utilization of alga resources. Having polysaccharide utilization sites (PULs) and carbohydrate enzymes (CAZymes) in the genome, the Bacteroides, especially Flavobacteria, are good decomposers of macromolecular polysaccharides. Early study showed that the Flavobacteria increased significantly during the middle and later stages of the green tide of Enteromorpha proliferata. We speculated that the Flavobacteria might have PULS and CAZymes which can degrade Enteromorpha proliferata polysaccharides. In this project, techniques of microbial isolation, pure culture and substrates testing were used to screen polysaccharide-degrading microorganisms isolated from the green tide of Enteromorpha prolifera. The structures and functions of PULs and CAZymes were analyzed by whole-genome sequencing, bioinformatic analysis, proteomic analysis, gene knockout and complementation techniques. And the possible Enteromorpha prolifera PULs and their degradation pathways were predicted and verified. This project aimed to elucidate the mechanism of microbial degradation of Enteromorpha prolifera polysaccharides and screen out the microorganisms and their key CAZymes that can efficiently degrade Enteromorpha prolifera polysaccharides. This study will provide theoretical basis for the mechanism of Enteromorpha prolifera polysaccharides decomposition by marine microorganisms and CAZymes, and provide new ideas for industrial improvement of the utilization efficiency of Enteromorpha prolifera biomass.

浒苔绿潮产生大量的浒苔多糖，这些多糖的分解、利用及转化对海洋碳循环以及藻类资源利用都意义重大。拟杆菌，尤其是黄杆菌，基因组中有多糖利用位点（PULs）及碳水化合物酶（CAZymes），能分解利用大分子多糖。研究表明黄杆菌在浒苔绿潮中后期大量增多，我们推测其可能具有能降解浒苔多糖的PULs及CAZymes，能分解利用浒苔多糖。本项目利用微生物分离，纯培养以及基质测试技术从浒苔绿潮中筛选出浒苔多糖降解微生物，以黄杆菌为重点研究对象，利用全基因组测序，生物信息学，蛋白质组学，基因敲除及回补等技术分析其PULs和CAZymes的结构和功能，预测并验证其可能的浒苔多糖PULs及其代谢途径。以期阐明微生物降解浒苔多糖的机理，同时筛选出能高效降解浒苔多糖的微生物及关键性的CAZymes，为海洋微生物及CAZymes对浒苔多糖的分解代谢提供理论基础，为工业上提高浒苔生物量的回收利用效率提供新思路。

浒苔绿潮产生大量的浒苔多糖，这些多糖的分解、利用及转化对海洋碳循环以及藻类资源利用都意义重大。加快多糖物质的降解，是提高浒苔生物质的利用效率、实现资源转化的关键，也是浒苔绿潮污染治理中重要的一环。因此，针对浒苔绿潮爆发增加海洋碳负荷，以及浒苔生物量资源的回收利用问题，筛选出能高效降解浒苔多糖的微生物及其关键酶类是非常必要且迫切的。但是目前对浒苔多糖降解菌以及能高效分解浒苔多糖的酶知之甚少。本研究以浒苔绿潮为背景，以擅长分解多糖的海洋拟杆菌（尤其是黄杆菌）为研究对象，在广西北海及山东青岛的浒苔绿潮爆发地分离筛选出2株浒苔多糖降解菌：黄杆菌Algibacter wandonensis S7和及交替单胞菌Alteromonas confluentis A1-6。利用全基因组分析找到2株菌中都含有针对浒苔多糖的GH78、GH106、GH5、PL24、PL40以及硫酸酯酶等基因组成的浒苔多糖PUL。用转录组分析验证了黄杆菌Algibacter wandonensis S7 的浒苔多糖PUL中GH78、GH106、GH5、PL24、PL40以及硫酸酯酶等相关基因的表达显著上调，进一步验证了S7菌株为浒苔多糖降解菌，其含有针对浒苔多糖降解的PUL，其中GH78很可能是可以高效降解浒苔多糖的酶。. 本研究成功筛选出能高效降解浒苔多糖菌株黄杆菌Algibacter wandonensis S7和及交替单胞菌Alteromonas confluentis A1-6，找到了的降解浒苔多糖的关键性CAZymes（GH78），为海洋微生物及CAZymes对浒苔多糖的分解代谢机理提供了理论基础，为工业上提高浒苔生物量回收利用效率提供新思路。