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Munjal N.,Synthetic Biology and Biofuels Group | Jawed K.,Synthetic Biology and Biofuels Group | Wajid S.,Jamia Hamdard University | Yazdani S.S.,Synthetic Biology and Biofuels Group | Yazdani S.S.,Advanced BioEnergy
PLoS ONE | Year: 2015

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. © 2015 Munjal et al. Source


Fatma Z.,Synthetic Biology and Biofuels Group | Jawed K.,Synthetic Biology and Biofuels Group | Mattam A.J.,Synthetic Biology and Biofuels Group | Yazdani S.S.,Synthetic Biology and Biofuels Group | Yazdani S.S.,Advanced BioEnergy
Metabolic Engineering | Year: 2016

Long chain fatty alcohols have wide application in chemical industries and transportation sector. There is no direct natural reservoir for long chain fatty alcohol production, thus many groups explored metabolic engineering approaches for its microbial production. Escherichia coli has been the major microbial platform for this effort, however, terminal endogenous enzyme responsible for converting fatty aldehydes of chain length C14-C18 to corresponding fatty alcohols is still been elusive. Through our in silico analysis we selected 35 endogenous enzymes of E. coli having potential of converting long chain fatty aldehydes to fatty alcohols and studied their role under in vivo condition. We found that deletion of ybbO gene, which encodes NADP+ dependent aldehyde reductase, led to >90% reduction in long chain fatty alcohol production. This feature was found to be strain transcending and reinstalling ybbO gene via plasmid retained the ability of mutant to produce long chain fatty alcohols. Enzyme kinetic study revealed that YbbO has wide substrate specificity ranging from C6 to C18 aldehyde, with maximum affinity and efficiency for C18 and C16 chain length aldehyde, respectively. Along with endogenous production of fatty aldehyde via optimized heterologous expression of cyanobaterial acyl-ACP reductase (AAR), YbbO overexpression resulted in 169 mg/L of long chain fatty alcohols. Further engineering involving modulation of fatty acid as well as of phospholipid biosynthesis pathway improved fatty alcohol production by 60%. Finally, the engineered strain produced 1989 mg/L of long chain fatty alcohol in bioreactor under fed-batch cultivation condition. Our study shows for the first time a predominant role of a single enzyme in production of long chain fatty alcohols from fatty aldehydes as well as of modulation of phospholipid pathway in increasing the fatty alcohol production. © 2016 International Metabolic Engineering Society. Source


Milne C.B.,Urbana University | Eddy J.A.,Urbana University | Raju R.,University of Minnesota | Ardekani S.,Urbana University | And 7 more authors.
BMC Systems Biology | Year: 2011

Background: Solventogenic clostridia offer a sustainable alternative to petroleum-based production of butanol--an important chemical feedstock and potential fuel additive or replacement. C. beijerinckii is an attractive microorganism for strain design to improve butanol production because it (i) naturally produces the highest recorded butanol concentrations as a byproduct of fermentation; and (ii) can co-ferment pentose and hexose sugars (the primary products from lignocellulosic hydrolysis). Interrogating C. beijerinckii metabolism from a systems viewpoint using constraint-based modeling allows for simulation of the global effect of genetic modifications.Results: We present the first genome-scale metabolic model (iCM925) for C. beijerinckii, containing 925 genes, 938 reactions, and 881 metabolites. To build the model we employed a semi-automated procedure that integrated genome annotation information from KEGG, BioCyc, and The SEED, and utilized computational algorithms with manual curation to improve model completeness. Interestingly, we found only a 34% overlap in reactions collected from the three databases--highlighting the importance of evaluating the predictive accuracy of the resulting genome-scale model. To validate iCM925, we conducted fermentation experiments using the NCIMB 8052 strain, and evaluated the ability of the model to simulate measured substrate uptake and product production rates. Experimentally observed fermentation profiles were found to lie within the solution space of the model; however, under an optimal growth objective, additional constraints were needed to reproduce the observed profiles--suggesting the existence of selective pressures other than optimal growth. Notably, a significantly enriched fraction of actively utilized reactions in simulations--constrained to reflect experimental rates--originated from the set of reactions that overlapped between all three databases (P = 3.52 × 10-9, Fisher's exact test). Inhibition of the hydrogenase reaction was found to have a strong effect on butanol formation--as experimentally observed.Conclusions: Microbial production of butanol by C. beijerinckii offers a promising, sustainable, method for generation of this important chemical and potential biofuel. iCM925 is a predictive model that can accurately reproduce physiological behavior and provide insight into the underlying mechanisms of microbial butanol production. As such, the model will be instrumental in efforts to better understand, and metabolically engineer, this microorganism for improved butanol production. © 2011 Milne et al; licensee BioMed Central Ltd. Source


Agrawal R.,Govind Ballabh Pant University of Agriculture & Technology | Agrawal R.,Advanced BioEnergy | Verma A.K.,Govind Ballabh Pant University of Agriculture & Technology | Satlewal A.,Govind Ballabh Pant University of Agriculture & Technology
Innovative Food Science and Emerging Technologies | Year: 2015

β-glucosidases are among the key enzymes for juice and beverage industries. They are responsible for the release of aromatic compounds in fruits and fermentation products. In this study, β-glucosidase was isolated, purified, and characterized from an indigenously developed Bacillus subtilis mutant PS-5CM-UM3. It is a 56kDa protein monomer (isoelectric point of 5.6) belonging to 1 glycosyl hydrolase family. The purified β-glucosidase was immobilized on SiO2 nanoparticles (with 52% efficiency and 14.1% yield) to improve the thermostability and Michaelis constant (Km) value of β-glucosidase from 0.9 to 1.1mM. The immobilized enzyme showed improved storage stability and was reusable for up to 10cycles with 70% residual activity. β-glucosidase treatment in sugarcane juice elevated the phenolics content with about 2.6 folds and 2.4 folds increase in p-hydroxy benzoic acid (PHBA) and gallic acid, respectively. The results show that recyclable immobilized enzyme system is a novel green approach for improving the sugarcane juice properties. Industrial relevance: In this study, β-glucosidase originally isolated and purified from an indigenously developed Bacillus subtilis mutant was immobilized on SiO2 nanoparticles. The immobilization has improved the thermostability, storage stability, and Michaelis constant (Km) value of the β-glucosidase. The immobilized β-glucosidase is now reusable for 10cycles with 70% residual activity. Further, β-glucosidase treatment in sugarcane juice elevated the phenolics content with about 2.6 folds and 2.4 folds increase in p-hydroxy benzoic acid (PHBA) and gallic acid, respectively. Hence, this study provides a green and sustainable approach for the food industry to efficiently enhance the juice properties. © 2015 Elsevier Ltd. Source


Agrawal R.,Advanced BioEnergy | Verma A.K.,Govind Ballabh Pant University of Agriculture & Technology | Satlewal A.,Govind Ballabh Pant University of Agriculture & Technology
Innovative Food Science and Emerging Technologies | Year: 2016

Β-glucosidases are among the key enzymes for juice and beverage industries. They are responsible for the release of aromatic compounds in fruits and fermentation products. In this study, β-glucosidase was isolated, purified, and characterized from an indigenously developed Bacillus subtilis mutant PS-5CM-UM3. It is a 56 kDa protein monomer (isoelectric point of 5.6) belonging to 1 glycosyl hydrolase family. The purified β-glucosidase was immobilized on SiO2 nanoparticles (with 52% efficiency and 14.1% yield) to improve the thermostability and Michaelis constant (Km) value of β-glucosidase from 0.9 to 1.1mM. The immobilized enzyme showed improved storage stability and was reusable for up to 10 cycles with 70% residual activity. β-glucosidase treatment in sugarcane juice elevated the phenolics content with about 2.6 folds and 2.4 folds increase in p-hydroxy benzoic acid (PHBA) and gallic acid, respectively. The results show that recyclable immobilized enzyme system is a novel green approach for improving the sugarcane juice properties. Industrial relevance: In this study, β-glucosidase originally isolated and purified from an indigenously developed Bacillus subtilismutantwas immobilized on SiO2 nanoparticles. The immobilization has improved the thermostability, storage stability, and Michaelis constant (Km) value of the β-glucosidase. The immobilized β-glucosidase is nowreusable for 10 cycles with 70% residual activity. Further, β-glucosidase treatment in sugarcane juice elevated the phenolics content with about 2.6 folds and 2.4 folds increase in p-hydroxy benzoic acid (PHBA) and gallic acid, respectively. Hence, this study provides a green and sustainable approach for the food industry to efficiently enhance the juice properties. © 2016 Elsevier Ltd. All rights reserved. Source

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