title: Functional characterization of cellulases identified from the cow rumen fungus Neocallimastix patriciarum W5 by transcriptomic and secretomic analyses.
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.
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Tzi-Yuan
authors.initials: TY
authors.lastname: Wang
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Hsin-Liang
authors.initials: HL
authors.lastname: Chen
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei 115, Taiwan.|Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Mei-Yeh J
authors.initials: MJ
authors.lastname: Lu
authors.affiliation: Graduate Institute of Biotechnology, National Pingtung University of Science & Technology, Neipu Hsiang, Pingtung 91201, Taiwan.
authors.forename: Yo-Chia
authors.initials: YC
authors.lastname: Chen
authors.affiliation: Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan.
authors.forename: Huang-Mo
authors.initials: HM
authors.lastname: Sung
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei 115, Taiwan.|Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung-Hsing University - Academia Sinica, Taipei 115, Taiwan.|Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung 402, Taiwan.
authors.forename: Chi-Tang
authors.initials: CT
authors.lastname: Mao
authors.affiliation: Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung-Hsing University - Academia Sinica, Taipei 115, Taiwan.|Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung 402, Taiwan.|Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Hsing-Yi
authors.initials: HY
authors.lastname: Cho
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei 115, Taiwan.|PhD Program in Microbial Genomics, National Chung Hsing University, Taichung 402, Taiwan.
authors.forename: Huei-Mien
authors.initials: HM
authors.lastname: Ke
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Teh-Yang
authors.initials: TY
authors.lastname: Hwa
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Sz-Kai
authors.initials: SK
authors.lastname: Ruan
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Kuo-Yen
authors.initials: KY
authors.lastname: Hung
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.|Department of Life Sciences, National Taiwan University, Taipei 106, Taiwan.
authors.forename: Chih-Kuan
authors.initials: CK
authors.lastname: Chen
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Jeng-Yi
authors.initials: JY
authors.lastname: Li
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Yueh-Chin
authors.initials: YC
authors.lastname: Wu
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Yu-Hsiang
authors.initials: YH
authors.lastname: Chen
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Shao-Pei
authors.initials: SP
authors.lastname: Chou
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Ya-Wen
authors.initials: YW
authors.lastname: Tsai
authors.affiliation: Institute of Information Science, Academia Sinica, Taipei 115, Taiwan.|Department of Computer Science and Information Engineering, National Taiwan Normal University, Taipei 116, Taiwan.
authors.forename: Te-Chin
authors.initials: TC
authors.lastname: Chu
authors.affiliation: Institute of Information Science, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Chun-Chieh A
authors.initials: CA
authors.lastname: Shih
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei 115, Taiwan.|Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung-Hsing University - Academia Sinica, Taipei 115, Taiwan.|Biotechnology Center, National Chung-Hsing University, Taichung 402, Taiwan.|Department of Ecology and Evolution, University of Chicago, Chicago, IL 60637, USA.
authors.forename: Wen-Hsiung
authors.initials: WH
authors.lastname: Li
authors.affiliation: Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung-Hsing University - Academia Sinica, Taipei 115, Taiwan.|Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan.|Biotechnology Center, National Chung-Hsing University, Taichung 402, Taiwan.
authors.forename: Ming-Che
authors.initials: MC
authors.lastname: Shih