Institute of Technical Microbiology, University of Technology Hamburg, Kasernenstrasse 12, Hamburg, Germany.
Department of Evolutionary Biology and Environmental Studies, University of Zurich, Zurich, Switzerland.
Extremophiles. 2021 Mar;25(2):101-114. doi: 10.1007/s00792-020-01214-9. Epub 2021 Jan 8.
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兴趣的新酶。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组学方法来鉴定耐热水解酶编码基因的方法。我们使用来自亚速尔群岛温泉样本的厌氧富集培养物，以绿咖啡豆为抗性底物，应用这种组合方法。对微生物群落的深入分析导致能够代谢所选底物的微生物，如 Caloramator、Dictyoglomus 和 Thermoanaerobacter 等活跃且丰富的微生物。为了发现糖苷水解酶，筛选了 90,342 个注释基因，以寻找特定的反应类型。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）和内切 1,4-β-甘露糖苷酶（EC 3.2.1.78）的基因。将 RNA-Seq 读序列映射到相关的宏基因组中，得到每个基因的表达水平。在这些基因中，有 14 个编码糖苷水解酶的基因表达水平最高，被用于进一步克隆。四种蛋白质进行了生化特性鉴定，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，组合组学方法是从环境样品中鉴定独特热活性酶的合适策略。
