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.
Highlights
Large-scale production of effective cellulose hydrolytic enzymes is the key to the bioconversion of agricultural residues to ethanol
Rice transformation and transgene confirmation The A. cellulolyticus cellulose hydrolytic enzyme β-1 (E1) and hygromycin phosphotransferase II (HptII) genes in the p1500 binary vector were successfully introduced into rice genome by Agrobacterium-mediated transformation
High levels of A. cellulolyticus E1 protein in transgenic rice plants have been achieved via gene expression, driven by the strong Mac promoter, with accumulation in the apoplast compartment by a proper signal peptide
Summary
Large-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 largescale 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. Lignocellulose is the major polysaccharide component of global plant mass, which consists of hemicellulose, lignin and cellulose, a polymer of thousands of 1, 4-blinked unions of D-glucose [5,6]. The conversion of the polysaccharide component of lignocellulose into ethanol for use as an alternative transportation fuel and other useful chemicals requires a series of complete pretreatment and hydrolysis procedures [2,6,10]. The complete hydrolysis of cellulose (saccharification) requires at least three different hydrolytic enzymes, including b-1,4-endoglucanse (EC 3.2.1.4), b-1, 4-exoglucanse (EC 3.2.1.91), and b-D-glucosidase (EC 3.2.1.21) [2,10,11]
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