肠道卵形拟杆菌基因组中的多糖合成酶挖掘与验证

Mining and verification of polysaccharide synthases in Bacteroides ovatus Microbial MAGs

摘要

目的 多糖是由单糖通过糖苷键连接而成的高分子碳水化合物,可通过促进有益菌增殖、调节菌群结构及促进肠道菌群代谢生成短链脂肪酸等机制发挥显著的益生元功能。卵形拟杆菌属是动物肠道微生物基因组中挖掘高效碳水化合物活性酶(CAZyme)的重要研究对象。方法 为深入探究该菌属的酶系特征,本研究分析了1 780个不同来源的卵形拟杆菌宏基因组组装基因组(MAG),发现其是CAZyme多样性的丰富储库,共预测到糖苷水解酶(GH)编码基因506 389条和糖基转移酶(GT)编码基因103 975条。后续实验结果表明,卵形拟杆菌不仅有降解多糖的功能,还有合成多糖的能力。通过多糖利用位点(PUL)注释,筛选得位点最多为119个的卵形拟杆菌基因组序列,利用分子对接与分子动力学模拟技术,预测鉴定出一个潜在的 CAZyme,该酶编码452个氨基酸,命名为V6248。结果 体外酶活验证结果表明,V6248是一种金属酶(Zn²⁺可提高其合成效率),能够催化纤维二糖、麦芽三糖、α-乳糖、麦芽糖、葡萄糖和木糖合成多糖,为多糖益生元的合成提供了新途径。结论 本研究不仅为新型CAZyme的高效挖掘提供了思路,也为开发新型生物催化剂提供了宝贵资源。

Abstract

Objective To polysaccharides are high-molecular-weight carbohydrates formed by monosaccharides connected via glycosidic bonds, which exert significant prebiotic functions through multiple mechanisms, including promoting the proliferation of beneficial bacteria, regulating the structure of intestinal flora, and facilitating the metabolism of intestinal flora to produce short-chain fatty acids. Bacteroides ovatus is a critical research target for mining efficient carbohydrate-active enzymes (CAZymes) from the animal intestinal microbial genome. Methods To deeply explore the enzymatic characteristics of this genus, this study analyzed 1 780 metagenome-assembled genomes (MAGs) of Bacteroides ovatus from different sources, and revealed that Bacteroides ovatus is a rich reservoir of CAZyme diversity. In total, 506 389 glycoside hydrolase (GH) coding genes and 103 975 glycosyltransferase (GT) coding genes were predicted. Subsequent experimental results show that Bacteroides ovatus not only possesses the function of degrading polysaccharides, but also has the capacity to synthesize polysaccharides. Through polysaccharide utilization loci (PUL) annotation, the genomic sequence of Bacteroides ovatus with the maximum number of PULs (119) was screened. Using molecular docking and molecular dynamics simulation, a potential CAZyme was predicted and identified. This enzyme encodes 452 amino acids and is named V6248. Results In vitro enzyme activity verification demonstrates that V6248 is a metalloenzyme, and Zn²⁺ can improve its synthetic efficiency. It can catalyze the synthesis of polysaccharides from cellobiose, maltotriose, α-lactose, maltose, glucose and xylose, which provides a novel approach for the synthesis of polysaccharide prebiotics. Conclusion This study not only provides insights for the efficient mining of novel CAZymes, but also offers valuable resources for the development of new biocatalysts.

关键词

多糖 / 卵形拟杆菌 / 碳水化合物活性酶 / 糖基转移酶 / 分子对接

Key words

polysaccharides / Bacteroides ovatus / carbohydrate-active enzymes / glycosyltransferase / molecular docking

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基金资助

国家自然基金项目(32560017);新疆维吾尔自治区天池英才工程项目