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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 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.
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pone.0119917.g005: 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.

Mentions: We showed in our earlier report that the β-glucosidase expressed in this study under the control of a constitutive promoter can hydrolyze cellobiose and the cello-oligosaccharides that have a chain length of up to five glucose [10]. In our earlier study, we also constructed an ethanologenic strain via modulation of the endogenous pathway for the fermentation of glucose and xylose into ethanol [25]. We wanted to determine whether this ethanologenic strain SSY09(pZSack) (Table 1) could ferment cellobiose after the secretion of β-glucosidase via the plasmid pPgap-OsmY-Gluc1C. The FRT-kan-FRT cassette from the genome of this strain was removed because it had a selection marker similar to that in the plasmid pPgap-OsmY-Gluc1C, and the resultant transformant, SSY12, was grown in minimal medium containing cellobiose under the microaerobic condition. No significant cell growth or cellobiose utilization was observed during first 20 hr of cultivation (Fig. 5A). Cellobiose, however, was fermented beyond 20 hr and was completely utilized by 120 hr of cultivation. In addition to the cell growth, two major products were observed in the cell-free culture medium, i.e., ethanol and acetic acid, with the yields of 1.44 and 1.92 mM per mM cellobiose, respectively. We showed in our previous report that the ethanol yield and productivity using the SSY09(pZSack) strain were higher when the cells were grown in complex medium [25]. We therefore cultivated the SSY12 strain in LB medium containing cellobiose and observed an ethanol production with a yield of 3.2 mM per mM cellobiose (80% efficiency of biotransformation) (Fig. 5B). Production of significant amount of acetate indicated that the carbon present in the complex medium used for cultivation is also contributing to fermentative product formation.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0119917.g005: 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.
Mentions: We showed in our earlier report that the β-glucosidase expressed in this study under the control of a constitutive promoter can hydrolyze cellobiose and the cello-oligosaccharides that have a chain length of up to five glucose [10]. In our earlier study, we also constructed an ethanologenic strain via modulation of the endogenous pathway for the fermentation of glucose and xylose into ethanol [25]. We wanted to determine whether this ethanologenic strain SSY09(pZSack) (Table 1) could ferment cellobiose after the secretion of β-glucosidase via the plasmid pPgap-OsmY-Gluc1C. The FRT-kan-FRT cassette from the genome of this strain was removed because it had a selection marker similar to that in the plasmid pPgap-OsmY-Gluc1C, and the resultant transformant, SSY12, was grown in minimal medium containing cellobiose under the microaerobic condition. No significant cell growth or cellobiose utilization was observed during first 20 hr of cultivation (Fig. 5A). Cellobiose, however, was fermented beyond 20 hr and was completely utilized by 120 hr of cultivation. In addition to the cell growth, two major products were observed in the cell-free culture medium, i.e., ethanol and acetic acid, with the yields of 1.44 and 1.92 mM per mM cellobiose, respectively. We showed in our previous report that the ethanol yield and productivity using the SSY09(pZSack) strain were higher when the cells were grown in complex medium [25]. We therefore cultivated the SSY12 strain in LB medium containing cellobiose and observed an ethanol production with a yield of 3.2 mM per mM cellobiose (80% efficiency of biotransformation) (Fig. 5B). Production of significant amount of acetate indicated that the carbon present in the complex medium used for cultivation is also contributing to fermentative product formation.

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