Mining of Novel Thermo-Stable Cellulolytic Genes from a Thermophilic Cellulose-Degrading Consortium by Metagenomics

In this study, metagenomics was applied to characterize the microbial community and to discover carbohydrate-active genes of an enriched thermophilic cellulose-degrading sludge. The 16S analysis showed that the sludge microbiome was dominated by genus of cellulolytic Clostridium and methanogenesis Methanothermobacter. In order to retrieve genes from the metagenome, de novo assembly of the 11,930,760 Illumina 100 bp paired-end reads (totally 1.2 Gb) was carried out. 75% of all reads was utilized in the de novo assembly. 31,499 ORFs (Open Reading Frame) with an average length of 852 bp were predicted from the assembly; and 64% of these ORFs were predicted to present full-length genes. Based on the Hidden Markol Model, 253 of the predicted thermo-stable genes were identified as putatively carbohydrate-active. Among them the relative dominance of GH9 (Glycoside Hydrolase) and corresponding CBM3 (Carbohydrate Binding Module) revealed a cellulosome-based attached metabolism of polysaccharide in the thermophilic sludge. The putative carbohydrate-active genes ranged from 20% to 100% amino acid sequence identity to known proteins in NCBI nr database, with half of them showed less than 50% similarity. In addition, the coverage of the genes (in terms of ORFs) identified in the sludge were developed into three clear trends (112×, 29× and 8×) in which 85% of the high coverage trend (112×) mainly consisted of phylum of Firmicutes while 49.3% of the 29× trend was affiliated to the phylum of Chloroflexi.本研究采用宏基因组学（metagenomics）技术，对富集得到的嗜热纤维素降解污泥的微生物群落进行表征，并挖掘其中的碳水化合物活性基因。16S分析结果显示，该污泥微生物组以解纤维素梭菌属（cellulolytic Clostridium）和产甲烷甲烷嗜热杆菌属（Methanothermobacter）为优势类群。为从宏基因组中获取基因，本研究对11930760条Illumina 100 bp双端测序读段（总数据量1.2 Gb）进行从头组装（de novo assembly），其中75%的读段被用于该组装过程。从组装结果中共预测得到31499个开放阅读框（Open Reading Frame, ORF），平均长度为852 bp；其中64%的开放阅读框被预测为完整长度基因。基于隐马尔可夫模型（Hidden Markov Model, HMM），本研究从预测得到的热稳定基因中鉴定出253个疑似碳水化合物活性基因。其中，糖苷水解酶家族9（Glycoside Hydrolase, GH9）及其对应的碳水化合物结合模块3（Carbohydrate Binding Module, CBM3）的相对丰度较高，表明该嗜热污泥中存在基于纤维小体的多糖附着代谢机制。这些疑似碳水化合物活性基因与NCBI非冗余蛋白数据库（NCBI nr）中已知蛋白的氨基酸序列一致性介于20%至100%之间，其中半数的序列相似性低于50%。此外，基于该污泥中鉴定得到的基因（以开放阅读框计）的覆盖度，可将其划分为3个显著的聚类趋势：112×、29×和8×。其中，112×高覆盖度聚类中85%的类群隶属于厚壁菌门（Firmicutes），而29×聚类中49.3%的类群隶属于绿弯菌门（Chloroflexi）。