Biotechnology for Biofuels (Aug 2011)

Functional characterization of cellulases identified from the cow rumen fungus <b><it>Neocallimastix patriciarum </it></b>W5 by transcriptomic and secretomic analyses

  • Wang Tzi-Yuan,
  • Chen Hsin-Liang,
  • Lu Mei-Yeh J,
  • Chen Yo-Chia,
  • Sung Huang-Mo,
  • Mao Chi-Tang,
  • Cho Hsing-Yi,
  • Ke Huei-Mien,
  • Hwa Teh-Yang,
  • Ruan Sz-Kai,
  • Hung Kuo-Yen,
  • Chen Chih-Kuan,
  • Li Jeng-Yi,
  • Wu Yueh-Chin,
  • Chen Yu-Hsiang,
  • Chou Shao-Pei,
  • Tsai Ya-Wen,
  • Chu Te-Chin,
  • Shih Chun-Chieh A,
  • Li Wen-Hsiung,
  • Shih Ming-Che

DOI
https://doi.org/10.1186/1754-6834-4-24
Journal volume & issue
Vol. 4, no. 1
p. 24

Abstract

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Abstract Background 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. Results 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. Conclusions 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.

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