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

Abstract

BackgroundGenetically 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.ResultsTcEG1 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0918-6) contains supplementary material, which is available to authorized users.

De novo transcriptome assembly of the bamboo snout beetle Cyrtotrachelus buqueti reveals ability to degrade lignocellulose of bamboo feedstock

“…Insect cellulase was identified in the intestines of termites and cockroaches [ 47 , 48 ] and subsequently detected in other insects [ 49 – 59 ]. Endogenous cellulase activity exists in insects and at least seven orders, comprising 28 species, have been found to contain a cellulase gene [ 6 , 60 ].…”

Expression Profiling of Plant Cell Wall-Degrading Enzyme Genes in Eucryptorrhynchus scrobiculatus Midgut

“…The potential of cellulase application in RNAi-medicated pest control strategies has been demonstrated by silencing a termite endogenous cellulase (Zhou et al, 2008). Plant cellulose can be digested by cellulase to produce the bioenergy source cellulosic ethanol (Willis et al, 2017). With the development of genetic engineering of bioenergy, elucidation of the functions of the major PCWDEs will be highly valuable.…”

Expression of a hyperthermophilic endoglucanase in hybrid poplar modifies the plant cell wall and enhances digestibility

“…3 F). Interestingly, reduced lignin contents have also been reported from transgenic switchgrass expressing beetle-derived endoglucanase [ 58 ]. Genes involved in cellulose, monolignol, and hemicellulose synthesis did not display altered expression in the Tn CelB-overexpressing transgenic plants, indicating that the alteration of cell wall composition in transgenic events does not appear to be due to changes in expression of cell wall component biosynthesis genes.…”