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Kushwaha H.R.,Synthetic Biology and Biofuel Group | Singla-Pareek S.L.,International Center for Genetic Engineering and Biotechnology | Pareek A.,Jawaharlal Nehru University
Journal of Biomolecular Structure and Dynamics | Year: 2014

Prokaryotes and eukaryotes respond to various environmental stimuli using the two-component system (TCS). Essentially, it consists of membrane-bound histidine kinase (HK) which senses the stimuli and further transfers the signal to the response regulator, which in turn, regulates expression of various target genes. Recently, sequence-based genome wide analysis has been carried out in Arabidopsis and rice to identify all the putative members of TCS family. One of the members of this family i.e. AtHK1, (a putative osmosensor, hybrid-type sensory histidine kinase) is known to interact with AtHPt1 (phosphotransfer proteins) in Arabidopsis. Based on predicted rice interactome network (PRIN), the ortholog of AtHK1 in rice, OsHK3b, was found to be interacting with OsHPt2. The analysis of amino acid sequence of AtHK1 showed the presence of transmitter domain (TD) and receiver domain (RD), while OsHK3b showed presence of three conserved domains namely CHASE (signaling domain), TD, and RD. In order to elaborate on structural details of functional domains of hybrid-type HK and phosphotransfer proteins in both these genera, we have modeled them using homology modeling approach. The structural motifs present in various functional domains of the orthologous proteins were found to be highly conserved. Binding analysis of the RD domain of these sensory proteins in Arabidopsis and rice revealed the role of various residues such as histidine in HPt protein which are essential for their interaction. © 2013 © 2013 The Author(s). Published by Taylor & Francis.

Mattam A.J.,Synthetic Biology and Biofuel Group | Clomburg J.M.,Rice University | Gonzalez R.,Rice University | Yazdani S.S.,Synthetic Biology and Biofuel Group
Biotechnology Letters | Year: 2013

Glycerol has attracted the attention of scientific and industrial communities due to its generation in bulk quantities as a byproduct of biofuel industries. With the rapid growth of these industries in recent years, glycerol is frequently treated as a very low-value byproduct or even a waste product with a disposal cost associated to it. Glycerol is not only abundant and inexpensive but also can generate more reducing equivalents than glucose or xylose. This unique characteristic of glycerol offers a tremendous opportunity for its biological conversion to valuable products at higher yield. This review focuses on research efforts to utilize glycerol as a carbon source for the production of a variety of fuels and chemicals by both native and metabolically engineered microorganisms. © 2013 Springer Science+Business Media Dordrecht.

Turan S.,Synthetic Biology and Biofuel Group | Cornish K.,Ohio State University | Kumar S.,Synthetic Biology and Biofuel Group
Australian Journal of Crop Science | Year: 2012

Salinity stress limits crop yield affecting plant growth and restricting the use of land. As world population is increasing at alarming rate, agricultural land is shrinking due to industrialization and/or habitat use. Hence, there is a need to utilize salt affected land to meet the food requirement. Although some success has been achieved through conventional breeding but its use is limited due to reproductive barrier and scarcity of genetic variations among major crops. The genetic engineering has proven a revolutionary technique to generate salt tolerant plants as one can transfer desired gene from any genetic resource and/or alter the expression of existing gene(s). There are examples of improved salinity tolerance in various crop plants through the use of genetic engineering. However, there is a further need of improvement for successful release of salt tolerant cultivars at field level. In this review, we have given a detailed update on production of salt-tolerant plants through genetic engineering. Future prospects and concerns, along with the importance of novel techniques, as well as plant breeding are also discussed.

Gupta S.,Synthetic Biology and Biofuel Group | Adlakha N.,Synthetic Biology and Biofuel Group | Yazdani S.S.,Synthetic Biology and Biofuel Group
Protein Expression and Purification | Year: 2013

Escherichia coli is considered one of the most appropriate hosts for the production of recombinant proteins. However, its usage is undermined by its inability to efficiently secrete proteins into the extracellular medium. We selected two cellulolytic enzymes with potential biofuel applications, β-1,4-endoglucanase (Endo5A) and β-1,4-glucosidase (Gluc1C), and determined the genetic and environmental parameters for their optimal secretion into culture medium. Endo5A and Gluc1C were fused with the hyperosmotically inducible periplasmic protein of E. coli, OsmY, and their activities in the extracellular, periplasmic and cytoplasmic fractions were monitored. Most of the endoglucanase activity (0.15 μmol min-1 ml-1) and β-glucosidase activity (2.2 μmol min-1 ml-1) in the extracellular fraction was observed at 16 h post-induction. To reduce the overall cost, we expressed Endo5A and Gluc1C together either via a synthetic operon or through a bifunctional chimeric protein. Both systems efficiently secreted the enzymes, as evident from the functional activities and protein profiles on SDS-PAGE gels. The enzymes secreted via a synthetic operon showed higher activities (0.14 μmol min-1 ml-1 for endoglucanase and 2.4 μmol min-1 ml-1 for β-glucosidase) as compared to the activities shown by the- bifunctional chimera (0.075 μmol min-1 ml-1 for endoglucanase and 2.0 μmol min-1 ml-1 for β-glucosidase). The cellulase secretion system developed here has potential for use in the production of lignocellulosic biofuels. © 2012 Elsevier Inc. All rights reserved.

Adlakha N.,Synthetic Biology and Biofuel Group | Sawant S.,Institute of Chemical Technology | Anil A.,Institute of Chemical Technology | Lali A.,Institute of Chemical Technology | Yazdani S.S.,Synthetic Biology and Biofuel Group
Applied and Environmental Microbiology | Year: 2012

Identification and design of new cellulolytic enzymes with higher catalytic efficiency are a key factor in reducing the production cost of lignocellulosic bioalcohol. We report here identification of a novel β-glucosidase (Gluc1C) from Paenibacillus sp. strain MTCC 5639 and construction of bifunctional chimeric proteins based on Gluc1C and Endo5A, a β-1,4-endoglucanase isolated from MTCC 5639 earlier. The 448-amino-acid-long Gluc1C contained a GH superfamily 1 domain and hydrolyzed cellodextrin up to a five-sugar chain length, with highest efficiency toward cellobiose. Addition of Gluc1C improved the ability of Endo5A to release the reducing sugars from carboxymethyl cellulose. We therefore constructed six bifunctional chimeric proteins based on Endo5A and Gluc1C varying in the positions and sizes of linkers. One of the constructs, EG5, consisting of Endo5A-(G4S)3-Gluc1C, demonstrated 3.2- and 2-fold higher molar specific activities for β-glucosidase and endoglucanase, respectively, than Gluc1C and Endo5A alone. EG5 also showed 2-fold higher catalytic efficiency than individual recombinant enzymes. The thermal denaturation monitored by circular dichroism (CD) spectroscopy demonstrated that the fusion of Gluc1C with Endo5A resulted in increased thermostability of both domains by 5oC and 9oC, respectively. Comparative hydrolysis experiments done on alkali-treated rice straw and CMC indicated 2-fold higher release of product by EG5 than that by the physical mixture of Endo5A and Gluc1C, providing a rationale for channeling of intermediates. Addition of EG5 to a commercial enzyme preparation significantly enhanced release of reducing sugars from pretreated biomass, indicating its commercial applicability. © 2012, American Society for Microbiology.

Adlakha N.,Synthetic Biology and Biofuel Group | Rajagopa R.,Insect Resistance Group | Rajagopa R.,University of Delhi | Kumar S.,Plant Transformation Group | And 2 more authors.
Applied and Environmental Microbiology | Year: 2011

Insects living on wood and plants harbor a large variety of bacterial flora in their guts for degrading biomass. We isolated a Paenibacillus strain, designated ICGEB2008, from the gut of a cotton bollworm on the basis of its ability to secrete a variety of plant-hydrolyzing enzymes. In this study, we cloned, expressed, and characterized two enzymes, β-1,4-endoglucanase (Endo5A) and β-1,4-endoxylanase (Xyl11D), from the ICGEB2008 strain and synthesized recombinant bifunctional enzymes based on Endo5A and Xyl11D. The gene encoding Endo5A was obtained from the genome of the ICGEB2008 strain by shotgun cloning. The gene encoding Xyl11D was obtained using primers for conserved xylanase sequences, which were identified by aligning xylanase sequences in other species of Paenibacillus. Endo5A and Xyl11D were overexpressed in Escherichia coli, and their optimal activities were characterized. Both Endo5A and Xyl11D exhibited maximum specific activity at 50°C and pH 6 to 7. To take advantage of this feature, we constructed four bifunctional chimeric models of Endo5A and Xyl11D by fusing the encoding genes either end to end or through a glycine-serine (GS) linker. We predicted three-dimensional structures of the four models using the I-TASSER server and analyzed their secondary structures using circular dichroism (CD) spectroscopy. The chimeric model Endo5A-GS-Xyl11D, in which a linker separated the two enzymes, yielded the highest C-score on the I-TASSER server, exhibited secondary structure properties closest to the native enzymes, and demonstrated 1.6-fold and 2.3-fold higher enzyme activity than Endo5A and Xyl11D, respectively. This bifunctional enzyme could be effective for hydrolyzing plant biomass owing to its broad substrate range. © 2011, American Society for Microbiology.

Munjal N.,Synthetic Biology and Biofuel Group | Mattam A.J.,Synthetic Biology and Biofuel Group | Pramanik D.,Synthetic Biology and Biofuel Group | Srivastava P.S.,Jamia Hamdard University | Yazdani S.S.,Synthetic Biology and Biofuel Group
Microbial Cell Factories | Year: 2012

Background: E. coli is a robust host for various genetic manipulations and has been used commonly for bioconversion of hexose and pentose sugars into valuable products. One of the products that E. coli make under fermentative condition is ethanol. However, availability of limited reducing equivalence and generation of competing co-products undermine ethanol yield and productivity. Here, we have constructed an E. coli strain to produce high yield of ethanol from hexose and pentose sugars by modulating the expression of pyruvate dehydrogenase and acetate kinase and by deleting pathways for competing co-products.Results: The availability of reducing equivalence in E. coli was increased by inducing the expression of the pyruvate dehydrogenase (PDH) operon under anaerobic condition after replacement of its promoter with the promoters of ldhA, frdA, pflB, adhE and gapA. The SSY05 strain, where PDH operon was expressed under gapA promoter, demonstrated highest PDH activity and maximum improvement in ethanol yield. Deletion of genes responsible for competing products, such as lactate (ldhA), succinate (frdA), acetate (ack) and formate (pflB), led to significant reduction in growth rate under anaerobic condition. Modulation of acetate kinase expression in SSY09 strain regained cell growth rate and ethanol was produced at the maximum rate of 12 mmol/l/h from glucose. The resultant SSY09(pZSack) strain efficiently fermented xylose under microaerobic condition and produced 25 g/l ethanol at the maximum rate of 6.84 mmol/l/h with 97% of the theoretical yield. More importantly, fermentation of mixture of glucose and xylose was achieved by SSY09(pZSack) strain under microaerobic condition and ethanol was produced at the maximum rate of 0.7 g/l/h (15 mmol/l/h), respectively, with greater than 85% of theoretical yield.Conclusions: The E. coli strain SSY09(pZSack) constructed via endogenous pathway engineering fermented glucose and xylose to ethanol with high yield and productivity. This strain lacking any foreign gene for ethanol fermentation is likely to be genetically more stable and therefore should be tested further for the fermentation of lignocellulosic hydrolysate at higher scale. © 2012 Munjal et al.; licensee BioMed Central Ltd.

Ahmad I.,Synthetic Biology and Biofuel Group | Fatma Z.,Synthetic Biology and Biofuel Group | Yazdani S.S.,Synthetic Biology and Biofuel Group | Kumar S.,Synthetic Biology and Biofuel Group
Algal Research | Year: 2013

The marine based algal biofuel may be the best possible options for future energy, particularly for India where freshwater and land resources are limited. We have characterized a novel marine microalga, isolated from an Indian Ocean using genotypic and biochemical studies, which contain high lipids (48%) on dry wt. basis, among marine-microalgae reported from the Indian subcontinent. The classification of new species based on DNA barcodes studies using 16S rRNA and 23S rRNA has indicated its genetic similarity to green algae Parachlorella kessleri. Subsequently, FAME analysis has showed that it contain about 43%, saturated fatty acids, which makes new algal species highly suitable for further exploration and commercial production of the biodiesel. © 2012 Elsevier B.V.

Gaur N.A.,U.S. National Institutes of Health | Gaur N.A.,Synthetic Biology and Biofuel Group | Hasek J.,Academy of Sciences of the Czech Republic | Brickner D.G.,Northwestern University | And 8 more authors.
Genetics | Year: 2013

There is increasing evidence that certain Vacuolar protein sorting (Vps) proteins, factors that mediate vesicular protein trafficking, have additional roles in regulating transcription factors at the endosome. We found that yeast mutants lacking the phosphatidylinositol 3-phosphate [PI(3)P] kinase Vps34 or its associated protein kinase Vps15 display multiple phenotypes indicating impaired transcription elongation. These phenotypes include reduced mRNA production from long or G+C-rich coding sequences (CDS) without affecting the associated GAL1 promoter activity, and a reduced rate of RNA polymerase II (Pol II) progression through lacZ CDS in vivo. Consistent with reported genetic interactions with mutations affecting the histone acetyltransferase complex NuA4, vps15Δ and vps34Δ mutations reduce NuA4 occupancy in certain transcribed CDS. vps15Δ and vps34Δ mutants also exhibit impaired localization of the induced GAL1 gene to the nuclear periphery. We found unexpectedly that, similar to known transcription elongation factors, these and several other Vps factors can be cross-linked to the CDS of genes induced by Gcn4 or Gal4 in a manner dependent on transcriptional induction and stimulated by Cdk7/Kin28-dependent phosphorylation of the Pol II C-terminal domain (CTD). We also observed colocalization of a fraction of Vps15-GFP and Vps34-GFP with nuclear pores at nucleus-vacuole (NV) junctions in live cells. These findings suggest that Vps factors enhance the efficiency of transcription elongation in a manner involving their physical proximity to nuclear pores and transcribed chromatin. © 2013 by the Genetics Society of America.

Yadav S.,National Institute of Plant Genome Research | Kushwaha H.R.,Jawaharlal Nehru University | Kushwaha H.R.,Synthetic Biology and Biofuel Group | Kumar K.,National Institute of Plant Genome Research | Verma P.K.,National Institute of Plant Genome Research
International Journal of Biological Macromolecules | Year: 2012

Glutaredoxins (GRXs) are small, ubiquitous, multifunctional, heat-stable and glutathione-dependent thiol-disulphide oxidoreductases, classified under thioredoxin-fold superfamily. In the green lineage, GRXs constitute a complex family of proteins. Based on their active site, GRXs are classified into two subfamilies: dithiol and monothiol. Monothiol GRXs contain 'CGFS' as a redox active motif and assist in maintaining redox state and iron homeostasis within the cell. Using RACE strategy, a full length cDNA of chickpea (Cicer arietinum) glutaredoxin 3 (CarGRX3) was cloned and sequenced. The cDNA contains open reading frame of 537bp encoding 178 amino acids and exhibits features of other known 'CGFS' type GRXs. Based on the multiple sequence alignment among CarGRX3 and monothiol GRXs of other photosynthetic organisms, the characteristic motif (KGX4PXCGFSX[29/30/32]KX4WPTXPQX4GX3GGXDI) with 18 invariant residues was observed. The proposed structure of CarGRX3 was compared with structurally resolved monothiol GRXs of other organisms. The CarGRX3 and nearest Arabidopsis homolog (AtGRXcp) shares 76% sequence identity which was reflected by their 3D-structure conservation. The structure of chickpea monothiol GRX (CarGRX3) coordinates glutathione ligated [2Fe-2S] cluster in a homodimeric form, highlighting the structural basis for iron-sulfur cluster (ISC) assembly and delivery to acceptor proteins. The present study on CarGRX3 model highlighted the utility of the theoretical approaches to understand complex biological phenomena such as glutathione docking and incorporation of GSH-ligated [2Fe-2S] cluster. © 2012 Elsevier B.V.

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