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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

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.
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pone.0119917.g006: 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.

Mentions: The above studies confirmed that SSY12 secreted functional β-glucosidase into the extracellular medium, and this β-glucosidase hydrolyzed cellobiose into glucose units for further uptake and fermentation by the cells. We tested the fermentation of a wheat straw hydrolysate, which contained 53 mM glucose, 57 mM xylose and 10 mM cellobiose, by the SSY12 strain. 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).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0119917.g006: 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.
Mentions: The above studies confirmed that SSY12 secreted functional β-glucosidase into the extracellular medium, and this β-glucosidase hydrolyzed cellobiose into glucose units for further uptake and fermentation by the cells. We tested the fermentation of a wheat straw hydrolysate, which contained 53 mM glucose, 57 mM xylose and 10 mM cellobiose, by the SSY12 strain. 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).

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