Consolidated Pretreatment and Hydrolysis of Plant Biomass Expressing Cell Wall Degrading Enzymes

Abstract

Significant amounts of cell wall degrading (CWD) enzymes are required to degrade lignocellulosic biomass into its component sugars. One strategy for reducing exogenous enzyme production requirements is to produce the CWD enzymes in planta. For this work, various CWD enzymes were expressed in maize (Zea mays). Following growth and dry down of the plants, harvested maize stover was tested to determine the impact of the expressed enzymes on the production of glucose and xylose using different exogenous enzyme loadings. In this study, a consolidated pretreatment and hydrolysis process consisting of a moderate chemical pretreatment at temperatures below 75°C followed by enzymatic hydrolysis using an in-house enzyme cocktail was used to evaluate engineered transgenic feedstocks. The carbohydrate compositional analysis showed no significant difference in the amounts of glucan and xylan between the transgenic maize plants expressing CWD enzyme(s) and the control plants. Hydrolysis results demonstrated that transgenic plants expressing CWD enzymes achieved up to 141% higher glucose yield and 172% higher xylose yield over the control plants from enzymatic hydrolysis under the experimental conditions. The hydrolytic performance of a specific xylanase (XynA) expressing transgenic event (XynA.2015.05) was heritable in the next generation, and the improved properties can be achieved even with a 25% reduction in exogenous enzyme loading. Simultaneous saccharification and fermentation of biomass hydrolysates from two different transgenic maize lines with yeast (Saccharomyces cerevisiae D5A) converted 65% of the biomass glucan into ethanol, versus only a 42% ethanol yield with hydrolysates from control plants, corresponding to a 55% improvement in ethanol production.

Recombinant hyperthermophilic enzyme expression in plants: a novel approach for lignocellulose digestion

“…Heterologous expression of these enzymes in plants has several potential advantages over other expression systems (e.g., Escherichia coli and yeast). These include better post-translational processing, wellestablished production and harvesting methods, and the ability to scale up to meet market demands in an economically and environmentally sustainable manner [15,[40][41][42][43][44]. The greatest potential benefit offered by plants as expression systems for lignocellulose-digesting enzymes is that they can serve as the source of the cellulosic biomass required for sugar fermentation.…”

“…Comparison of a traditional pre-fermentation pipeline (purple boxes) and an approach based on in planta expression of hyperthermophilic lignocellulose digesting enzymes combining pretreatment and saccharification in a single reactor (green boxes). [15,38]. In planta expression of recombinant lignocellulosedigesting genes has involved a range of different promoters and other regulatory elements and targeting of the enzymes to the specific organelles via different transit peptides for accumulation and storage or targeting to the cell wall for immediate modification of the polysaccharides (Table S1 in the supplementary material online).…”

“…A recent systematic study on transgenic maize (Zea mays) expressing various mesophilic and thermophilic lignocellulose-digesting enzymes, where total plant biomass was harvested and used as a feedstock for sugar production, has demonstrated the potential of this approach [15]. Transgenic maize biomass produced up to 1.4-fold higher glucose and 1.7-fold higher xylose yields than control plants treated with different exogenous enzyme loadings during enzymatic hydrolysis at temperatures below 758C.…”

“…The major economic burden of biomass refineries include the pretreatment processing of the lignocellulosic biomass and the cost of production of hydrolytic enzymes [14,15]. In order to reduce these cost elements, researchers have focused on genetic engineering of plants for desirable lignin content or composition by partial removal or redistribution of lignin.…”

“…Plants are increasingly being investigated as potential 'green bioreactors' for the cost-effective and large-scale production of biologically active recombinant enzymes [31][32][33][34][35][36], but the production of cellulolytic enzymes using transgenic plants has mostly been restricted to mesophilic and thermotolerant enzymes [37][38][39][40][41][42][43][44][45]. To the best of our knowledge, the effects of such expression on the efficiency of biomass conversion have been reported in few instances [15].…”