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Functional characterization of cellulases identified from the cow rumen fungus Neocallimastix patriciarum W5 by transcriptomic and secretomic analyses

DOI: 10.1186/1754-6834-4-24

Keywords: anaerobic fungi, biomass, rice straw, sugarcane, napiergrass, GH, next-generation sequencing

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Abstract:

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.Cellulosic ethanol produced by microbial fermentation from feedstocks has been proposed to replace fossil fuels in transportation. A key step in cellulosic ethanol production is to break down cellulose into glucose and hemicellulose into xylose, which can subsequently be converted into ethanol by fermentative microbes. Therefore, finding efficient cellulases is important to bioethanol production, as well as for hydrolyzing feedstocks into sugars in general. Neocallimastix species is one of the major anaerobic fungi in the rumen of water buffalo capable of efficiently digesting cellulosic biomass [1-4]. Such anaerobic fungi are potential sources for highly active cellulolytic enzymes that are useful for cellulose hydrolysis [5-7]. Plant cell wall degrading enzymes from rumen fungi such as Neocallimastix patriciarum may

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