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

Munjal N, Jawed K, Wajid S, Yazdani SS - PLoS ONE (2015)

Bottom Line: 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.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.An ethanologenic strain that constitutively secretes a cellulolytic enzyme is a promising platform for producing lignocellulosic ethanol.

View Article: PubMed Central - PubMed

Affiliation: Synthetic Biology and Biofuels Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0119917.g003: 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.

Mentions: In our previous study, we used a T7 promoter-based expression system, pET28a(+), for the secretion of endoglucanase and β-glucosidase [24]. Here, we replaced the T7 promoter of this vector with the constitutive gapA promoter as mentioned in Materials and Methods section, and analyzed the expression and secretion of the enzymes that were aerobically grown in E. coli BLR(DE3) cells. The total activities of the endoglucanase and β-glucosidase that were expressed under the gapA promoter were 0.76 and 2 μmol min-1 ml-1, the values that were 1.3 and 1.5-fold lower than those under the T7 promoter, respectively (Fig. 3A and 3B). We also found that these enzymes were secreted in the extracellular medium, and the extent of secretion was higher for β-glucosidase than for endoglucanase (Fig. 3A and 3B).


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

Munjal N, Jawed K, Wajid S, Yazdani SS - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4358894&req=5

pone.0119917.g003: 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.
Mentions: In our previous study, we used a T7 promoter-based expression system, pET28a(+), for the secretion of endoglucanase and β-glucosidase [24]. Here, we replaced the T7 promoter of this vector with the constitutive gapA promoter as mentioned in Materials and Methods section, and analyzed the expression and secretion of the enzymes that were aerobically grown in E. coli BLR(DE3) cells. The total activities of the endoglucanase and β-glucosidase that were expressed under the gapA promoter were 0.76 and 2 μmol min-1 ml-1, the values that were 1.3 and 1.5-fold lower than those under the T7 promoter, respectively (Fig. 3A and 3B). We also found that these enzymes were secreted in the extracellular medium, and the extent of secretion was higher for β-glucosidase than for endoglucanase (Fig. 3A and 3B).

Bottom Line: 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.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.An ethanologenic strain that constitutively secretes a cellulolytic enzyme is a promising platform for producing lignocellulosic ethanol.

View Article: PubMed Central - PubMed

Affiliation: Synthetic Biology and Biofuels Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.

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.

No MeSH data available.


Related in: MedlinePlus