Abstract
Lignocellulosic ethanol offers a promising alternative to conventional fossil fuels. One among the major limitations in the lignocellulosic biomass hydrolysis is unavailability of efficient and environmentally biomass degrading technologies. Plant-based production of these enzymes on large scale offers a cost-effective solution. Cellulases, hemicellulases including mannanases and other accessory enzymes are required for conversion of lignocellulosic biomass into fermentable sugars. β-mannanase catalyzes endo-hydrolysis of the mannan backbone, a major constituent of woody biomass. In this study, the man1 gene encoding β-mannanase was isolated from Trichoderma reesei and expressed via the chloroplast genome. PCR and Southern hybridization analysis confirmed site-specific transgene integration into the tobacco chloroplast genomes and homoplasmy. Transplastomic plants were fertile and set viable seeds. Germination of seeds in the selection medium showed inheritance of transgenes into the progeny without any Mendelian segregation. Expression of endo-β-mannanase for the first time in plants facilitated its characterization for use in enhanced lignocellulosic biomass hydrolysis. Gel diffusion assay for endo-β-mannanase showed the zone of clearance confirming functionality of chloroplast-derived mannanase. Endo-β-mannanase expression levels reached up to 25 units per gram of leaf (fresh weight). Chloroplast-derived mannanase had higher temperature stability (40°C to 70°C) and wider pH optima (pH 3.0 to 7.0) than E.coli enzyme extracts. Plant crude extracts showed 6–7 fold higher enzyme activity than E.coli extracts due to the formation of disulfide bonds in chloroplasts, thereby facilitating their direct utilization in enzyme cocktails without any purification. Chloroplast-derived mannanase when added to the enzyme cocktail containing a combination of different plant-derived enzymes yielded 20% more glucose equivalents from pinewood than the cocktail without mannanase. Our results demonstrate that chloroplast-derived mannanase is an important component of enzymatic cocktail for woody biomass hydrolysis and should provide a cost-effective solution for its diverse applications in the biofuel, paper, oil, pharmaceutical, coffee and detergent industries.
Highlights
The world’s energy demands are ever increasing and cannot be sustained by conventional fuel sources alone
This is the first report of expression of fungal mannanase in plants and their direct utilization in enzyme cocktails, for lignocellulosic biomass hydrolysis without any need of purification
Tobacco chloroplast transformation vector pLD-man1 (Figure 1B) was constructed with man1 coding sequence based on the universal chloroplast vector that targets the transgene expression cassette into the transcriptionally active spacer region between the trnI and trnA genes (Figure 1A) of the chloroplast genome for integration via homologous recombination [44]
Summary
The world’s energy demands are ever increasing and cannot be sustained by conventional fuel sources alone. For breakdown of complex biomass, chemical and physical pretreatments of these materials are necessary These treatments are expensive, have serious environmental consequences and decrease enzymatic hydrolysis [3]. As the cellulosic biomass is composed of complex cellulose, hemicellulose and various entangled fibers, concurrent action of different enzyme classes such as cellulases, glucosidases, hemicellulases and accessory enzymes including esterases, lipases, pectate lyases are required, in large quantities [3]. Simultaneous action of these enzymes can increase the access of each enzyme to the cellulosic biomass
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