We report here that tobacco and maize expressing heterologous E1 endoglucanase from Acidothermus cellulolyticus were observed to become less recalcitrant compared to wild-type biomass when subjected to pretreatment and post-pretreatment enzymatic hydrolysis. This reduction in recalcitrance was manifest through lower severity requirements to achieve comparable levels of conversion to wild-type biomass. Our studies indicate that the decreased recalcitrance was not due to post-pretreatment residual E1cd activity and could not be reproduced by the addition of exogenous E1cd to the biomass prior to pretreatment, indicating that the expression of E1cd during cell wall construction altered the inherent recalcitrance of the cell wall. In our experiments, expression of E1cd in maize increased the digestibility of biomass cell walls after pretreatment at expression levels far below those normally required for efficient E1 hydrolysis of cellulose. It also enabled lower-severity pretreated corn stover to become as digestible as higher-severity pretreated stover with the same enzyme loadings. In addition to decreased pretreatment severity requirements, the expression of E1 in corn stover is likely to enable decreased enzyme loadings for hydrolysis, decreased inhibitor formation and decreased degradation product formation. This effect is also likely to extend to other endoglucanases, especially those from glycoside hydrolase family 5.
We observed a shared phenomenon when expressing an endoglucanase in both corn stover and tobacco; that is, E1-expressing plants are more easily saccharified by cellulase enzymes following various severity pretreatments. Our results indicate that E1 expression in both corn and tobacco enabled reduction of the required pretreatment severity to permit the same level of sugar conversion as was obtained with higher-temperature pretreatments of wild-type samples. The possibility of reducing pretreatment severity is highly relevant to the biofuels industry because of the potential cost savings from lower energy and enzyme usage, reduced inhibitor formation and sugar losses, decreased catalyst addition and lower costs of facility construction.
Throughout the digestion experiments, transgenic E1 biomass, whether stover or tobacco, was shown to be either more digestible than the wild type after identical pretreatment or as digestible as more severely pretreated wild-type biomass. For example, with the 100 mg of enzyme per gram of biomass digestions, the yield of glucose from E1-expressing transgenic corn stover pretreated at 140°C appeared to be comparable to the yield from nontransgenic corn stover pretreated at about 170°C (Figure 5). This effect was even more distinct at lower enzyme loadings (15 mg of protein per gram of biomass; Figure 4). Tobacco also showed that transgenic expression of E1 benefited enzymatic conversion, although this effect was more pronounced at lower severities (Figure 3). As incubation of the biomass after the addition of exogenous E1 before pretreatment did not have any discernible impact on enzymatic conversion, we conclude that the effect is not due to residual post-pretreatment E1 activity. Also, as the incubation time was fairly long, the exogenous E1 would be expected to cleave some of the cellulose before pretreatment, although presumably the accessibility would be limited to outer layers of cellulose.
Our work in extracting E1 from the cell walls and quantifying it by Western blot analysis indicated that the E1 protein is tightly associated with the cell wall. Our normal extraction protocol of 100% ethylene glycol (as used in the cel7a purification method) did not remove detectable amounts of E1 from the biomass even after increasing the extraction temperature to ~96°C. Treatment by boiling with NuPAGE LDS sample buffer did remove E1 from the cell walls, indicating that the transgenic E1, even though it lacks a carbohydrate binding module, was tightly associated with the cell wall.
Imaging of plant cell wall sections by anti-E1 antibody localization clearly showed the presence of E1 throughout the cell wall, specifically in the thicker sclerenchyma cells of the rind and vascular bundles. Stover samples showed much higher levels of intrawall E1 deposition than did the tobacco samples, which seemed to have more E1 localized to the inner layers of the cell wall (Figures 8 and 9). This may indicate that measured E1 levels were higher in tobacco simply because the E1 was easier to extract. It may also explain why the tobacco conversion enhancement due to E1 expression was not apparent at the highest pretreatment severity; that is, the innermost layers of the tobacco cell wall were not as disrupted by pretreatment as were those of stover, which had more broad distribution of E1 throughout the cell wall. It is unclear from this work whether the localization was due to a preference of E1 to bind to a particular wall type, to a difference in cell-type expression or to some other variable (such as cell wall-type permeability to antibody). Additional maize transformation vectors have been generated in which E1 is driven by other strong promoters and fused to alternative signal peptides to target accumulation in the vacuole and endoplasmic reticulum, and these constructs will be used to generate additional transgenic lines for study. Entrained E1 may enhance the conversion, but only to the extent that it is distributed throughout the cell wall. Broader distribution of E1 in the stover cell wall interior could have allowed more rapid and complete digestion, which is indicated by the increased extent of conversion of E1 stover compared to E1 tobacco under identical high-enzyme loadings. Without the increased pretreatment efficacy from intercalated E1, the tobacco digestions all may have simply reached their limit at the 170°C pretreatment condition. Localization of cellulases using immunocytochemistry followed by transmission electron microscopy as described by Donohoe et al. supports this idea, as cell wall disruption and enzyme penetration are directly proportional to pretreatment severity.
Our pretreatment, digestion, and imaging results lead us to believe that the cellulose in the E1 stover and tobacco has been modified by the expression of E1 during plant growth, biomass storage or both. On this basis, we conclude that the increased enzymatic conversion of the transgenic E1 biomass is due to the intrawall (and possibly intracellulose microfibril) localization of the E1 protein resulting from expression during cell wall formation. This conclusion is supported by the observation that wild-type corn stover treated with exogenous E1 prior to pretreatment was not as digestible as the transformed stover. We believe that this result was due to the ability of transgenic E1 to access more cell wall compartments containing cellulose and perhaps even the cell membrane-cell wall space occupied by the nascent (growing) cellulose microfibril during cellulose synthesis than was possible with the simple external addition of enzyme. This idea is supported by our imaging data showing that E1 expressed during plant growth is targeted to the cell wall, where it may work by nicking the cellulose chains as they are formed or laid down, resulting in either less crystalline cellulose material or simply more free chain ends for cellobiohydrolases, which could result in increased conversion. It is not known whether the reduced activity of this thermal tolerant enzyme at growth condition also benefited this effect. Alternatively, expression of the E1 enzyme in plant tissue may likely have led to increased conversion through a nonenzymatic effect, such as the ability to bind strongly to polysaccharides. By simply binding to the cellulose microfibril during plant growth and development, E1 may decrease the recalcitrance of the cell wall to chemical pretreatment. The comparable results between tobacco and corn expressing E1 suggest that low-level expression of E1 or other endoglucanases in plants may be a general phenomenon by which the conversion of biomass may be improved.