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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 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.
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pone.0119917.g002: 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.

Mentions: We selected three genes, i.e., glyceraldehyde-3-phosphate dehydrogenase (gapA), lactate dehydrogenase (ldhA) and pyruvate-formate lyase (pflB), and tested the strength of their promoters by integrating them into the bacterial genome upstream of a reporter gene, lacZ. The native promoter and the RBS of lacZ were replaced with the test promoters and their corresponding RBSs. The promoter strengths were studied using the lacZ expression under both aerobic and anaerobic conditions. The expression of lacZ under the control of its native promoter was tested in the presence of IPTG under a de-repressed condition and was found to be 443 Miller units under the aerobic growth condition (Fig. 1A). The expression of lacZ while under the control of the gapA, ldhA and pflB promoters was 1.8-fold higher, 1.5-fold higher and 4.9-fold lower, respectively, compared with that of the native lacZ promoter (Fig. 1A). Under the anaerobic condition, the gapA, ldhA and pflB promoters showed 3.5-, 2.6- and 2.5-fold higher expression of lacZ, respectively, compared with that of the native lacZ promoter (Fig. 1B). To validate the findings in a plasmid-based system, we cloned the lacZ gene in pET28a(+) vector under the control of its native promoter as well as under the promoters for gapA, ldhA and pflB genes. All the promoters showed enhanced expression of β-galactosidase in the plasmid-based system as compared to genome integration-based system (Fig. 2). Because the gapA promoter yielded the highest expression of lacZ under both the aerobic and anaerobic condition, it was selected for further study.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0119917.g002: 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.
Mentions: We selected three genes, i.e., glyceraldehyde-3-phosphate dehydrogenase (gapA), lactate dehydrogenase (ldhA) and pyruvate-formate lyase (pflB), and tested the strength of their promoters by integrating them into the bacterial genome upstream of a reporter gene, lacZ. The native promoter and the RBS of lacZ were replaced with the test promoters and their corresponding RBSs. The promoter strengths were studied using the lacZ expression under both aerobic and anaerobic conditions. The expression of lacZ under the control of its native promoter was tested in the presence of IPTG under a de-repressed condition and was found to be 443 Miller units under the aerobic growth condition (Fig. 1A). The expression of lacZ while under the control of the gapA, ldhA and pflB promoters was 1.8-fold higher, 1.5-fold higher and 4.9-fold lower, respectively, compared with that of the native lacZ promoter (Fig. 1A). Under the anaerobic condition, the gapA, ldhA and pflB promoters showed 3.5-, 2.6- and 2.5-fold higher expression of lacZ, respectively, compared with that of the native lacZ promoter (Fig. 1B). To validate the findings in a plasmid-based system, we cloned the lacZ gene in pET28a(+) vector under the control of its native promoter as well as under the promoters for gapA, ldhA and pflB genes. All the promoters showed enhanced expression of β-galactosidase in the plasmid-based system as compared to genome integration-based system (Fig. 2). Because the gapA promoter yielded the highest expression of lacZ under both the aerobic and anaerobic condition, it was selected for further study.

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