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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 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.
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pone.0119917.g004: 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.

Mentions: We further tested expression of the endoglucanase and β-glucosidase under the anaerobic condition for their use in SSF or CBP. Since expression of cellulolytic enzymes under anaerobic condition will need to be sustainable for longer duration of time to match with biofuel productivity of the host [25], we tested the expression of the enzymes at different time points under anaerobic condition. The time kinetics data revealed that the extracellular expression of endoglucanase under the control of the gapA promoter was lower during the early period of growth, but reached a higher level in the later phase. The intracellular endoglucanase level maintained a steady increase for both types of promoters (Fig. 4A). In the case of β-glucosidase, a maximum jump in activity was observed in first 24 hr (Fig. 4B). There was a marginal increase in the β-glucosidase activity in all the samples after 24 hr, except for the extracellular enzyme activity of the T7 based promoter, which declined during the last 24 hr of growth (Fig. 4B). These growth and activity patterns resulted in higher specific activities for endoglucanase and β-glucosidase at 72 hr as compared to 24 hr under the anaerobic condition (Table 2).


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0119917.g004: 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.
Mentions: We further tested expression of the endoglucanase and β-glucosidase under the anaerobic condition for their use in SSF or CBP. Since expression of cellulolytic enzymes under anaerobic condition will need to be sustainable for longer duration of time to match with biofuel productivity of the host [25], we tested the expression of the enzymes at different time points under anaerobic condition. The time kinetics data revealed that the extracellular expression of endoglucanase under the control of the gapA promoter was lower during the early period of growth, but reached a higher level in the later phase. The intracellular endoglucanase level maintained a steady increase for both types of promoters (Fig. 4A). In the case of β-glucosidase, a maximum jump in activity was observed in first 24 hr (Fig. 4B). There was a marginal increase in the β-glucosidase activity in all the samples after 24 hr, except for the extracellular enzyme activity of the T7 based promoter, which declined during the last 24 hr of growth (Fig. 4B). These growth and activity patterns resulted in higher specific activities for endoglucanase and β-glucosidase at 72 hr as compared to 24 hr under the anaerobic condition (Table 2).

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