Abstract
BackgroundEnzymatic conversion of lignocellulosic biomass into soluble sugars is a major bottleneck in the plant biomass utilization. Several anaerobic organisms cope these issues via multiple-enzyme complex system so called ‘cellulosome’. Hence, we proposed a “biomimic operon” concept for making an artificial cellulosome which can be used as a promising tool for the expression of cellulosomal enzymes in Bacillus subtilis.ResultsAccording to the proteomic analysis of Clostridium thermocellum ATCC27405 induced by Avicel or cellobiose, we selected eight highly expressed cellulosomal genes including a scaffoldin protein gene (cipA), a cell-surface anchor gene (sdbA), two exoglucanase genes (celK and celS), two endoglucanase genes (celA and celR), and two xylanase genes (xynC and xynZ). Arranging these eight genes in two different orders, we constructed two different polycistronic operons using the ordered gene assembly in Bacillus method. This is the first study to express the whole CipA along with cellulolytic enzymes in B. subtilis. Each operon was successfully expressed in B. subtilis RM125, and the protein complex assembly, cellulose-binding ability, thermostability, and cellulolytic activity were demonstrated. The operon with a higher xylanase activity showed greater saccharification on complex cellulosic substrates such as Napier grass than the other operon.ConclusionsIn this study, a strategy for constructing an efficient cellulosome system was developed and two different artificial cellulosomal operons were constructed. Both operons could efficiently express the cellulosomal enzymes and exhibited cellulose saccharification. This strategy can be applied to different industries with cellulose-containing materials, such as papermaking, biofuel, agricultural compost, mushroom cultivation, and waste processing industries.
Highlights
Enzymatic conversion of lignocellulosic biomass into soluble sugars is a major bottleneck in the plant biomass utilization
The selected genes were amplified from the C. thermocellum ATCC 27405 genome
Each cellulosomal gene was amplified from the C. thermocellum genome along with 16-bp native ribosomal-binding site (RBS) and we have demonstrated the expression of each gene in B. subtilis, confirming that the RBS from C. thermocellum works in B. subtilis
Summary
Enzymatic conversion of lignocellulosic biomass into soluble sugars is a major bottleneck in the plant biomass utilization. The major bottleneck is the low cellulolytic efficiency in converting the recalcitrant crystalline cellulose [3]. The highly ordered crystalline cellulose is buried within the architecture of cross-linked hemicellulose matrix covered by lignin, making it inaccessible for cellulolytic enzymes [4, 5]. Various techniques have been used to reduce the recalcitrant nature of lignocellulosic biomass, including ammonia treatment, chemical hydrolysis, and steam explosion. These methods increase the costs and produce toxic byproducts, which can inhibit the microbial growth [6]. A large number of studies have focused on the pretreatment of biomass using the cellulase cocktail comprised of natural or tailor-made microbial enzymes [7]
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