Next-generation sequencing and computational biology have facilitated the implementation of new combinatorial screening approaches to discover novel enzymes of biotechnological interest. In this study, we describe the successful establishment of a multi-omic approach for the identification of thermostable hydrolase-encoding genes by determination of gene expression levels. We applied this combinatorial approach using an anaerobic enrichment culture from an Azorean hot spring sample grown on green coffee beans as recalcitrant substrate. An in-depth analysis of the microbial community resulted in microorganisms capable of metabolizing the selected substrate, such as the genera Caloramator, Dictyoglomus and Thermoanaerobacter as active and abundant microorganisms. To discover glycoside hydrolases, 90,342 annotated genes were screened for specific reaction types. A total number of 106 genes encoding cellulases (EC 3.2.1.4), beta-glucosidases (EC 3.2.1.21) and endo-1,4-beta-mannosidases (EC 3.2.1.78) were selected. Mapping of RNA-Seq reads to the related metagenome led to expression levels for each gene. Amongst those, 14 genes, encoding glycoside hydrolases, showed highest expression values, and were used for further cloning. Four proteins were biochemically characterized and were identified as thermoactive glycoside hydrolases with a broad substrate range. This work demonstrated that a combinatory omic approach is a suitable strategy identifying unique thermoactive enzymes from environmental samples.

下一代测序和计算生物学促进了新的组合筛选方法的实施，以发现具有生物技术意义的新型酶。在这项研究中，我们描述了通过确定基因表达水平成功建立多基因组方法来鉴定热稳定水解酶编码基因的方法。我们使用厌氧浓缩培养物，从以绿色咖啡豆作为顽固底物生长的Azorean温泉样品中进行厌氧浓缩培养，从而应用了这种组合方法。对微生物群落的深入分析产生了能够代谢选定底物的微生物，例如Caloramator，  Dictyoglomus和Thermoanaerobacter属。作为活跃而丰富的微生物。为了发现糖苷水解酶，针对特定的反应类型筛选了90342个带注释的基因。总共选择了106个编码纤维素酶（EC 3.2.1.4），β-葡萄糖苷酶（EC 3.2.1.21）和1,4-β-甘露糖苷酶（EC 3.2.1.78）的基因。RNA-Seq读图到相关的基因组的映射导致每个基因的表达水平。在这些基因中，编码糖苷水解酶的14个基因表现出最高的表达值，并被用于进一步克隆。四种蛋白质经过生物化学鉴定，被鉴定为具有广泛底物范围的热活性糖苷水解酶。这项工作表明组合的组学方法是一种从环境样品中鉴定独特的热活性酶的合适策略。