Background:
Neocallimastix patriciarum is one of the common anaerobic fungi in the digestive tract of ruminants that can actively digest cellulosic materials and its cellulases are of 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 on N. patriciarum to accelerate gene identification, enzyme classification, and application in rice straw degradation. By complementary studies of transcriptome (Roche 454 GS and Illumina GA IIx) and secretome (ESI-Trap LC-MS/MS), we identified 219 putative glycosyl hydrolase (GH) contigs and classified them into 25 GH families. The secretome analysis identified four major enzymes involved in rice straw degradation: beta-glucosidase, endo-1,4-beta-xylanase, xylanase B and Cel48A exo-glucanase. From the sequences of assembled contigs we cloned 19 putative cellulase genes, including 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 beta-glucosidases (W5-16143) and one exo-cellulase (W5-CAT26) showed strong activities and could potentially be developed into commercial enzymes.Keywordsanaerobic fungi, biomass, rice straw, sugarcane, napiergrass, GH, next-generation sequencingSource:
http://www.biotechnologyforbiofuels.com/rss/