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

Willis, Jonathan D.; Grant, Joshua N.; Mazarei, Mitra; Kline, Lindsey M.; Rempe, Caroline S.; Collins, A. Grace; Turner, Geoffrey B.; Decker, Stephen R.; Sykes, Robert W.; Davis, Mark F.; Labbe, Nicole; Jurat-Fuentes, Juan L.; Stewart, Jr., C. Neal

doi:10.1186/s13068-017-0918-6

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

Journal Article · Thu Nov 30 00:00:00 EST 2017 · Biotechnology for Biofuels

DOI:https://doi.org/10.1186/s13068-017-0918-6· OSTI ID:1618687

Willis, Jonathan D.; Grant, Joshua N.; Mazarei, Mitra; Kline, Lindsey M.; Rempe, Caroline S.; Collins, A. Grace; Turner, Geoffrey B.; Decker, Stephen R.; Sykes, Robert W.; Davis, Mark F.; Labbe, Nicole; Jurat-Fuentes, Juan L.;

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 in 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.

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Research Organization:: Univ. of Tennessee, Knoxville, TN (United States); National Renewable Energy Laboratory (NREL), Golden, CO (United States); Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: PS02-06ER64304; AC36-08GO28308

OSTI ID:: 1618687

Alternate ID(s):: OSTI ID: 1430816

Report Number(s):: NREL/JA-2700-70636; 230; PII: 918

Journal Information:: Biotechnology for Biofuels, Journal Name: Biotechnology for Biofuels Vol. 10 Journal Issue: 1; ISSN 1754-6834

Publisher:: Springer Science + Business MediaCopyright Statement

Country of Publication:: Netherlands

Language:: English

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Cited by: 5 works

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Related Subjects

09 BIOMASS FUELS
switchgrass
auto-hydrolysis
glycosyl hydrolase
β-1
4-Endoglucanase
insect
cellulase
biofuel
Tribolium castaneum

MOESM1 of The TcEG1 beetle (Tribolium castaneum) cellulase produced in transgenic switchgrass is active at alkaline pH and auto-hydrolyzes biomass for increased cellobiose release Willis, Jonathan; Grant, Joshua; Mazarei, Mitra https://doi.org/10.6084/m9.figshare.5648803.v1 The current record is supplemented by this dataset	dataset	November 2017
The TcEG1 beetle (Tribolium castaneum) cellulase produced in transgenic switchgrass is active at alkaline pH and auto-hydrolyzes biomass for increased cellobiose release [Supplementary Data] Willis, Jonathan; Grant, Joshua; Mazarei, Mitra https://doi.org/10.6084/m9.figshare.c.3942325 The current record is supplemented by this collection	collection	November 2017
The TcEG1 beetle (Tribolium castaneum) cellulase produced in transgenic switchgrass is active at alkaline pH and auto-hydrolyzes biomass for increased cellobiose release [Supplemental Data] Willis, Jonathan; Grant, Joshua; Mazarei, Mitra https://doi.org/10.6084/m9.figshare.c.3942325.v1 The current record is supplemented by this dataset	dataset	November 2017