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Title: The TcEG1 beetle (Tribolium castaneum) cellulase produced in transgenic switchgrass is active at alkaline pH and auto-hydrolyzes biomass for increased cellobiose release

Genetically engineered biofuel crops, such as switchgrass (Panicum virgatum L.), that produce their own cell wall-digesting cellulase enzymes would reduce costs of cellulosic biofuel production. To date, non-bioenergy plant models have been used in nearly all studies assessing the synthesis and activity of plant-produced fungal and bacterial cellulases. One potential source for cellulolytic enzyme genes is herbivorous insects adapted to digest plant cell walls. Here we examine the potential of transgenic switchgrass-produced TcEG1 cellulase from Tribolium castaneum (red flour beetle). This enzyme, when overproduced in Escherichia coli and Saccharomyces cerevisiae, efficiently digests cellulose at optima of 50 °C and pH 12.0. TcEG1 that was produced in green transgenic switchgrass tissue had a range of endoglucanase activity of 0.16–0.05 units (µM glucose release/min/mg) at 50 °C and pH 12.0. TcEG1 activity from air-dried leaves was unchanged from that from green tissue, but when tissue was dried in a desiccant oven (46 °C), specific enzyme activity decreased by 60%. When transgenic biomass was “dropped-in” into an alkaline buffer (pH 12.0) and allowed to incubate at 50 °C, cellobiose release was increased up to 77% over non-transgenic biomass. Saccharification was increased in one transgenic event by 28%, which had a concurrent decrease inmore » lignin content of 9%. Histological analysis revealed an increase in cell wall thickness with no change to cell area or perimeter. Transgenic plants produced more, albeit narrower, tillers with equivalent dry biomass as the control. This work describes the first study in which an insect cellulase has been produced in transgenic plants; in this case, the dedicated bioenergy crop switchgrass. Switchgrass overexpressing the TcEG1 gene appeared to be morphologically similar to its non-transgenic control and produced equivalent dry biomass. Therefore, we propose TcEG1 transgenics could be bred with other transgenic germplasm (e.g., low-lignin lines) to yield new switchgrass with synergistically reduced recalcitrance to biofuel production. In addition, transgenes for other cell wall degrading enzymes may be stacked with TcEG1 in switchgrass to yield complementary cell wall digestion features and complete auto-hydrolysis.« less
Authors:
 [1] ;  [2] ;  [1] ;  [3] ;  [4] ;  [2] ;  [5] ;  [5] ;  [5] ;  [5] ;  [3] ;  [6] ; ORCiD logo [7]
  1. Univ. of Tennessee, Knoxville, TN (United States). Dept. of Plant Sciences; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center
  2. Univ. of Tennessee, Knoxville, TN (United States). Dept. of Plant Sciences
  3. Univ. of Tennessee, Knoxville, TN (United States). Center for Renewable Carbon
  4. Univ. of Tennessee, Knoxville, TN (United States). Dept. of Plant Sciences. UT‑ORNL Graduate School of Genome Science and Technology
  5. National Renewable Energy Lab. (NREL), Golden, CO (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center
  6. Univ. of Tennessee, Knoxville, TN (United States). Dept. of Entomology and Plant Pathology
  7. Univ. of Tennessee, Knoxville, TN (United States). Dept. of Plant Sciences. UT‑ORNL Graduate School of Genome Science and Technology; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center
Publication Date:
Report Number(s):
NREL/JA-2700-70636
Journal ID: ISSN 1754-6834
Grant/Contract Number:
AC36-08GO28308
Type:
Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 10; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Research Org:
Univ. of Tennessee, Knoxville, TN (United States); National Renewable Energy Lab. (NREL), Golden, CO (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; switchgrass; auto-hydrolysis; glycosyl hydrolase; β-1,4-Endoglucanase; insect; cellulase; biofuel; Tribolium castaneum
OSTI Identifier:
1430816