Functional characterization of cellulases identified from the cow rumen fungus Neocallimastix patriciarum W5 by transcriptomic and secretomic analyses
- Equal contributors
1 Biodiversity Research Center, Academia Sinica, Taipei 115, Taiwan
2 Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
3 Graduate Institute of Biotechnology, National Pingtung University of Science & Technology, Neipu Hsiang, Pingtung 91201, Taiwan
4 Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan
5 Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung-Hsing University - Academia Sinica, Taipei 115, Taiwan
6 Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung 402, Taiwan
7 Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan
8 PhD Program in Microbial Genomics, National Chung Hsing University, Taichung 402, Taiwan
9 Department of Life Sciences, National Taiwan University, Taipei 106, Taiwan
10 Institute of Information Science, Academia Sinica, Taipei 115, Taiwan
11 Department of Computer Science and Information Engineering, National Taiwan Normal University, Taipei 116, Taiwan
12 Biotechnology Center, National Chung-Hsing University, Taichung 402, Taiwan
13 Department of Ecology and Evolution, University of Chicago, Chicago, IL 60637, USA
Biotechnology for Biofuels 2011, 4:24 doi:10.1186/1754-6834-4-24Published: 17 August 2011
Neocallimastix patriciarum is one of the common anaerobic fungi in the digestive tracts of ruminants that can actively digest cellulosic materials, and its cellulases have great potential for hydrolyzing cellulosic feedstocks. Due to the difficulty in culture and lack of a genome database, it is not easy to gain a global understanding of the glycosyl hydrolases (GHs) produced by this anaerobic fungus.
We have developed an efficient platform that uses a combination of transcriptomic and proteomic approaches to N. patriciarum to accelerate gene identification, enzyme classification and application in rice straw degradation. By conducting complementary studies of transcriptome (Roche 454 GS and Illumina GA IIx) and secretome (ESI-Trap LC-MS/MS), we identified 219 putative GH contigs and classified them into 25 GH families. The secretome analysis identified four major enzymes involved in rice straw degradation: β-glucosidase, endo-1,4-β-xylanase, xylanase B and Cel48A exoglucanase. From the sequences of assembled contigs, we cloned 19 putative cellulase genes, including the GH1, GH3, GH5, GH6, GH9, GH18, GH43 and GH48 gene families, which were highly expressed in N. patriciarum cultures grown on different feedstocks.
These GH genes were expressed in Pichia pastoris and/or Saccharomyces cerevisiae for functional characterization. At least five novel cellulases displayed cellulytic activity for glucose production. One β-glucosidase (W5-16143) and one exocellulase (W5-CAT26) showed strong activities and could potentially be developed into commercial enzymes.