High level expression of Acidothermus cellulolyticusβ-1, 4-endoglucanase in transgenic rice enhances the hydrolysis of its straw by cultured cow gastric fluid

Abstract

BackgroundLarge-scale production of effective cellulose hydrolytic enzymes is the key to the bioconversion of agricultural residues to ethanol. The goal of this study was to develop a rice plant as a bioreactor for the large-scale production of cellulose hydrolytic enzymes via genetic transformation, and to simultaneously improve rice straw as an efficient biomass feedstock for conversion of cellulose to glucose.ResultsIn this study, the cellulose hydrolytic enzyme β-1, 4-endoglucanase (E1) gene, from the thermophilic bacterium Acidothermus cellulolyticus, was overexpressed in rice through Agrobacterium-mediated transformation. The expression of the bacterial E1 gene in rice was driven by the constitutive Mac promoter, a hybrid promoter of Ti plasmid mannopine synthetase promoter and cauliflower mosaic virus 35S promoter enhancer, with the signal peptide of tobacco pathogenesis-related protein for targeting the E1 protein to the apoplastic compartment for storage. A total of 52 transgenic rice plants from six independent lines expressing the bacterial E1 enzyme were obtained that expressed the gene at high levels without severely impairing plant growth and development. However, some transgenic plants exhibited a shorter stature and flowered earlier than the wild type plants. The E1 specific activities in the leaves of the highest expressing transgenic rice lines were about 20-fold higher than those of various transgenic plants obtained in previous studies and the protein amounts accounted for up to 6.1% of the total leaf soluble protein. A zymogram and temperature-dependent activity analyses demonstrated the thermostability of the E1 enzyme and its substrate specificity against cellulose, and a simple heat treatment can be used to purify the protein. In addition, hydrolysis of transgenic rice straw with cultured cow gastric fluid for one hour at 39°C and another hour at 81°C yielded 43% more reducing sugars than wild type rice straw.ConclusionTaken together, these data suggest that transgenic rice can effectively serve as a bioreactor for the large-scale production of active, thermostable cellulose hydrolytic enzymes. As a feedstock, direct expression of large amount of cellulases in transgenic rice may also facilitate saccharification of cellulose in rice straw and significantly reduce the costs for hydrolytic enzymes.

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.…”

“…Although in planta expression of thermophilic endoglucanases has been achieved in the range of 10-26% TSP without any detrimental effects on transgenic plants [37][38][39][40][41][42][43][44][45][46][47][48], yields of 0.1-5% TSP are more typical, and several studies have described negative phenotypes associated with in planta expression of recombinant mesophilic cellulolytic enzymes [32,49].…”

“…Additionally, 3 0 -untranslated sequences downstream of the stop codon playing a critical role in processing have been included along with signal sequences targeting and stabilizing the message for polyadenylation (Table S1 in the supplementary material online). Polyadenylation strongly enhances the heterologous expression of microbial genes in plants [33,34,[40][41][42]. Sequences that are located immediately around the translational start site, termed Kozak sequences [56], can also be modified to fit the consensus initiation sequence of plants in order to achieve higher rates of translation.…”

“…N-terminal or C-terminal transit peptides generally possess the necessary information for the correct targeting of proteins to subcellular organelles. Subcellular targeting of heterologous proteins generally yields higher accumulation levels in transgenic plants than expression in the cytosol [32][33][34][35][36][37][38][39][40][41][42]47,51]. When expressed in subcellular compartments, heterologous proteins typically display correct folding and activity, appropriate glycosylation, reduced degradation, and increased stability, as compared to expression and accumulation in the cytosol [8,44].…”

“…Development of hyperthermophilic fermentation microbes would confer an additional advantage when using the in planta approach. 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].…”