A constitutive expression system for cellulase secretion in Escherichia coli and its use in bioethanol production

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.

Fig 1. Expression of β-galactosidase via its native and heterologous promoter in genome integration based system.

Cells were grown (A) aerobically and (B) anaerobically, harvested and used to monitor β-galactosidase activity. The data are presented as the average and standard deviation of two independent experiments.

Fig 2. Expression of β-galactosidase via its native and heterologous promoter in plasmid based system.

Cells were grown aerobically and anaerobically, harvested and used to monitor β-galactosidase activity. The data are presented as the average and standard deviation of two independent experiments.

Fig 3. Expression of cellulases under the constitutive gapA promoter and the inducible T7 promoter.

Cells were grown aerobically for 16 hr, harvested and used to monitor the endoglucanase (A) and β-glucosidase (B) and activity in both the extracellular and intracellular fractions. The data are presented as the average and standard deviation of two independent experiments.

Fig 4. Time profiles of anaerobic cellulase expression under the constitutive gapA promoter and the inducible T7 promoter.

Cells were grown anaerobically and used to monitor the (A) endoglucanase and (B) β-glucosidase activity in both the extracellular and intracellular fractions. The data are presented as the average and standard deviation of two independent experiments.

Fig 5. Time profiles of cellobiose fermentation by the engineered strain.

The engineered E. coli strain SSY12 bearing the plasmid pPgap-OsmY-Gluc1C was grown in minimal medium (A) or complex medium (B) containing cellobiose under a microaerobic condition, and the metabolites and cell growth were monitored throughout the cultivation period.

Fig 6. Time profiles of hydrolysate fermentation by the engineered strain.

The engineered E. coli strain SSY12 bearing the plasmid pPgap-OsmY-Gluc1C was grown in LB+biomass hydrolysate medium under a microaerobic condition and the metabolites were monitored throughout the cultivation period. The data are presented as an average and standard deviation of two bioreactor batches.