Abstract
The production of biofuels from lignocellulosic biomass appears to be attractive and viable due to the abundance and availability of this biomass. The hydrolysis of this biomass, however, is challenging because of the complex lignocellulosic structure. The ability to produce hydrolytic cellulase enzymes in a cost-effective manner will certainly accelerate the process of making lignocellulosic ethanol production a commercial reality. These cellulases may need to be produced aerobically to generate large amounts of protein in a short time or anaerobically to produce biofuels from cellulose via consolidated bioprocessing. Therefore, it is important to identify a promoter that can constitutively drive the expression of cellulases under both aerobic and anaerobic conditions without the need for an inducer. Using lacZ as reporter gene, we analyzed the strength of the promoters of four genes, namely lacZ, gapA, ldhA and pflB, and found that the gapA promoter yielded the maximum expression of the β-galactosidase enzyme under both aerobic and anaerobic conditions. We further cloned the genes for two cellulolytic enzymes, β-1,4-endoglucanase and β-1,4-glucosidase, under the control of the gapA promoter, and we expressed these genes in Escherichia coli, which secreted the products into the extracellular medium. An ethanologenic E. colistrain transformed with the secretory β-glucosidase gene construct fermented cellobiose in both defined and complex medium. This recombinant strain also fermented wheat straw hydrolysate containing glucose, xylose and cellobiose into ethanol with an 85% efficiency of biotransformation. An ethanologenic strain that constitutively secretes a cellulolytic enzyme is a promising platform for producing lignocellulosic ethanol.
Highlights
The abundance of lignocellulosic biomass and its renewable nature makes it an ideal feedstock for biofuel production [1, 2]
Because the glyceraldehyde-3-phosphate dehydrogenase (gapA) promoter yielded the highest expression of lacZ under both the aerobic and anaerobic condition, it was selected for further study
All the three sugars were utilized by SSY12 in 38 hr and we obtained 204 mM of ethanol at the end of fermentation with 85% of efficiency of biotransformation (Fig. 6). Industrial enzymes such as cellulases are used in variety of applications, the most important being the production of second-generation biofuels
Summary
The abundance of lignocellulosic biomass and its renewable nature makes it an ideal feedstock for biofuel production [1, 2]. Often commercially available inducible expression systems based on T5 and T7 promoters are used for overexpression of enzymes in the heterologous hosts [11, 12]. The expression of enzymes such as βglucosidase under anaerobic conditions will help in the hydrolysis and consumption of partially hydrolyzed soluble cellulosic materials, such as cellobiose and cello-oligosaccharides [21]. For this to occur, the cellobiose either needs to be transported inside the cells through various transporters or the enzyme must be secreted to the extracellular medium to hydrolyze its substrate. A β-glucosidase from Paenibacillus polymyxa has been expressed in E. coli and secreted into the extracellular medium with the help of an OsmY (a hyperosmotically inducible periplasmic protein) tag for the hydrolysis of cellobiose [24]
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