Metatranscriptomics of the Hu sheep rumen microbiome reveals novel cellulases

Abstract

Background: Cellulosic biomass has great potential as a renewable biofuel resource. Robust, high-performance enzymes are needed to effectively utilize this valuable resource. In this study, metatranscriptomics was used to explore the carbohydrate-active enzymes (CAZymes), especially glycoside hydrolases (GHs), present in the rumen microbiome of Hu sheep. Select CAZymes were experimentally verified and characterized after cloning and expression in E. coli. Results: The metatranscriptomes of six Hu sheep rumen microbiomes yielded 42.3 Gbp of quality-checked sequence data that represented in total 2,380,783 unigenes after de novo assembling using Trinity and clustered with CD-HIT-EST. Annotation using the CAZy database revealed that 2.65% of the unigenes encoded GHs, which were assigned to 111 different CAZymes families. Firmicutes (18.7%) and Bacteroidetes (13.8%) were the major phyla to which the unigenes were taxonomically assigned. In total, 14,489 unigenes were annotated to 15 cellulase-containing GH families, with GH3, GH5 and GH9 being the predominant. From these putative cellulase-encoding unigenes, 4225 open reading frames (ORFs) were predicted to contain 2151 potential cellulase catalytic modules. Additionally, 147 ORFs were found to encode proteins that contain carbohydrate-binding modules (CBMs). Heterogeneous expression of 30 candidate cDNAs from the GH5 family in E. coli BL21 showed that 17 of the tested proteins had endoglucanase activity, while 7 exhibited exoglucanase activity. Interestingly, two of the GH5 proteins (Cel5A-h28 and Cel5A-h11) showed high specific activity against carboxymethylcellulose (CMC) and p-nitrophenyl-β-d-cellobioside (pNPC) (222.2 and 142.8 U/mg), respectively. The optimal pH value for activity of Cel5A-h11 and Cel5A-h28 was 6.0 for both enzymes, and optimal temperatures were 40 and 50 °C, respectively. Both enzymes retained over 70 and 60%, respectively, of their original activities after incubation at 40 °C for 60 min. However, their activities were rapidly diminished upon exposure to higher temperatures. Cel5A-h11 and Cel5A-h28 retained more than 80 and 60% of their maximal enzymatic activities after incubation for 16 h in buffered solutions in the pH range from 4.0 to 9.0. Conclusion: The metatranscriptomic results revealed that the rumen microbiome of Hu sheep encoded a repertoire of new enzymes capable of cellulose degradation and metatranscriptomics was an effective method to discover novel cellulases for biotechnological applications.