Cultivation-independent methods, including metagenomics, are tools for the exploration and discovery of biotechnological compounds produced by microbes in natural environments. Glycoside hydrolases (GHs) enzymes are extremely desired and important in the industry of production for goods and biofuel and removal of problematic biofilms and exopolysaccharide (EPS). Biofilms and EPS are complex, requiring a wide range of enzymes for a complete degradation. The aim of this study was to identify potential GH microbial producers and GH genes with biotechnological potential, using EPS-complex structure (WH15EPS) of Acidobacteria Granulicella sp. strain WH15 as an enrichment factor, in cultivation-independent and cultivation-dependent methods. We performed stable isotope probing (SIP) combined with metagenomics on topsoil litter amended with WH15EPS and coupled solid culture-EPS amended medium with metagenomics. SIP metagenome analysis of the soil litter demonstrated that phyla Proteobacteria, Actinobacteria, Acidobacteria, and Planctomycetes were the most abundant in WH15EPS amended and unamended treatments. The enrichment cultures in solid culture medium coupled to metagenomics demonstrated an enrichment in Proteobacteria, and the metagenome assembly of this enrichment cultures resulted in 4 metagenome-assembled genomes (MAGs) of microbes with low identity (42–86%) to known microorganisms. Among all carbohydrate-active enzymes (CAZymes) retrieved genes, glycoside transferase (GT) was the most abundant family, either in culture-independent or culture-based metagenome datasets. Within the glycoside hydrolases (GHs), GH13 was the most abundant family in both metagenome datasets. In the “heavy” fraction of the culture-independent metagenome SIP dataset, GH109 (α-N-acetylgalactosaminidases), GH117 (agarases), GH50 (agarases), GH32 (invertases and inulinases), GH17 (endoglucanases), and GH71 (mutanases) families were more abundant in comparison with the controls. Those GH families are affiliated to microorganism that are probably capable to degrade WH15EPS and potentially applicable for biofilm deconstruction. Subsequent in culture-based metagenome, the assembled 4 MAGs (unclassified Proteobacteria) also contained GH families of interest, involving mannosidases, lysozymes, galactosidases, and chitinases. We demonstrated that functional diversity induced by the presence of WH15EPS in both culture-independent and culture-dependent approaches was enriched in GHs, such as amylases and endoglucanases that could be applied in chemical, pharmaceutical, and food industrial sectors. Furthermore, WH15EPS may be used for the investigation and isolation of yet unknown taxa, such as unclassified Proteobacteria and Planctomycetes, increasing the number of current cultured bacterial representatives with potential biotechnological traits.

中文翻译：

---

不依赖培养和依赖培养的基因组揭示了参与复杂微生物聚合物降解的微生物群落的遗传和酶促潜力。

与耕种无关的方法，包括宏基因组学，是探索和发现自然环境中微生物产生的生物技术化合物的工具。糖苷水解酶（GHs）酶在商品和生物燃料生产以及去除有问题的生物膜和胞外多糖（EPS）的生产行业中极为需要并且很重要。生物膜和EPS是复杂的，需要多种酶才能完全降解。这项研究的目的是使用酸性细菌Granulicella sp。的EPS复合结构（WH15EPS）来确定潜在的GH微生物生产者和具有生物技术潜力的GH基因。不依赖于培养和依赖于培养的方法中，将菌株WH15作为富集因子。我们对表土垫料进行了稳定的同位素探测（SIP），结合了宏基因组学，并改良了WH15EPS，并改良了带有宏基因组学的固体培养物EPS改良培养基。对土壤垫料的SIP元基因组分析表明，在WH15EPS改良和未经修正的处理中，门菌属，放线菌，嗜酸菌和轮生菌最丰富。固体培养基中的富集培养与宏基因组学相结合，证明了变形杆菌的富集，这种富集培养的元基因组组装产生了4个由基因组组装的微生物基因组（MAGs），与已知微生物的同源性较低（42–86％）。在所有与糖有关的活性酶（CAZymes）检索到的基因中，糖苷转移酶（GT）是最丰富的家族，无论是与培养无关的还是基于培养的元基因组数据集。在糖苷水解酶（GHs）中，GH13是两个元基因组数据集中最丰富的家族。在与培养无关的基因组SIP数据集的“重度”部分中，GH109（α-N-乙酰半乳糖苷酶），GH117（琼脂糖），GH50（琼脂糖），GH32（蔗糖酶和菊粉酶），GH17（内切葡聚糖酶）和GH71（淀粉酶） ）家庭比对照组多。这些GH家族隶属于可能能够降解WH15EPS并可能适用于生物膜解构的微生物。随后在基于文化的基因组中，组装的4个MAG（未分类的变形杆菌）也包含感兴趣的GH家族，涉及甘露糖苷酶，溶菌酶，半乳糖苷酶和几丁质酶。我们证明，在不依赖文化的和依赖文化的方法中，由WH15EPS的存在引起的功能多样性都富含GH，例如淀粉酶和内切葡聚糖酶，可用于化学，制药和食品工业领域。此外，WH15EPS可用于调查和分离尚未分类的类群，如未分类的变形杆菌和扁平菌，从而增加了具有潜在生物技术特征的当前培养细菌代表的数量。