Heterologous Acidothermus cellulolyticus 1,4-β-endoglucanase E1 produced within the corn biomass converts corn stover into glucose

Abstract

Commercial conversion of lignocellulosic biomass to fermentable sugars requires inexpensive bulk production of biologically active cellulase enzymes, which might be achieved through direct production of these enzymes within the biomass crops. Transgenic corn plants containing the catalytic domain of Acidothermus cellulolyticus E1 endo-1,4-beta glucanase and the bar bialaphos resistance coding sequences were generated after Biolistic (BioRad Hercules, CA) bombardment of immature embryo-derived cells. E1 sequences were regulated under the control of the cauliflower mosaic virus 35S promoter and tobacco mosaic virus translational enhancer, and E1 protein was targeted to the apoplast using the signal peptide of tobacco pathogenesis-related protein to achieve accumulation of this enzyme. The integration, expression, and segregation of E1 and bar transgenes were demonstrated, respectively, through Southern and Western blotting, and progeny analyses. Accumulation of up to 1.13% of transgenic plant total soluble proteins was detected as biologically active E1 by enzymatic activity assay. The corn-produced heterologous E1 could successfully convert ammonia fiber explosion-pretreated corn stover polysaccharides into glucose as a fermentable sugar for ethanol production, confirming that the E1 enzyme is produced in its active form.

In planta differential targeting analysis of Thermotoga maritima Cel5A and CBM6-engineered Cel5A for autohydrolysis

“…Subcellular organelles provide the matrix for correct folding, activity, glycosylation, reduced degradation, and increased stability, with a large accumulation of the recombinant protein (Horn et al 2004;Bae et al 2006a;Sticklen 2006). Transgenic cellulases can autohydrolyze their cell walls once their cells are disrupted, and direct conversion of biomass to glucose has been examined in several crops (Kawazu et al 1999;Montalvo-Rodriguez et al 2000;Oraby et al 2007;Ransom et al 2007).…”

Cell Wall Engineering by Heterologous Expression of Cell Wall-Degrading Enzymes for Better Conversion of Lignocellulosic Biomass into Biofuels

“…Major drawbacks of heterologous expression of cellulases are dramatic yield penalty and detrimental effect on plant development. To avoid these negative effects on plant growth and development, cellulases have been targeted to different cellular compartments [63,[67][68][69][70][71][72][73][74][75][76][77][78]. For instance, Mahadevan et al [63] successfully generated tobacco plants expressing endoglucanases (CEL5) originated from Thermotoga maritima with an Arabidopsis rubisco activase signal peptide for chloroplast targeting.…”

Commercial cellulosic ethanol: The role of plant-expressed enzymes

“…Subsequently, A. cellulolyticus endoglucanase E1 was found in truncated but active form in tobacco and in duckweed, consistent with proteolytic removal of the cellulose binding domain (CBD), leaving just the catalytic domain (CD; Dai et al 2000a;Sun et al 2007). Expression of A. cellulolyticus endoglucanase CD had equal or higher activity than the fulllength protein when expressed in potato or tobacco (Dai et al 2000b;Ziegelhoffer et al 2001), and apoplast-targeted CD has also been successfully expressed in maize (Biswas et al 2006;Ransom et al 2007). Importantly, although the enzyme is not able to survive a mild AFEX pretreatment (Teymouri et al 2004), rice-and maize-expressed A. cellulolyticus CD has activity on pretreated corn stover and rice straw (Oraby et al 2007;Ransom et al 2007).…”