Prediction-based protein engineering of domain I of Cry2A entomocidal toxin of Bacillus thuringiensis for the enhancement of toxicity against lepidopteran insects

Issues relating to sustenance of the usefulness of genetically modified first generation Bt crop plants in the farmer’s field are of great concern for crop scientists. Additional biotechnological strategies need to be in place to safeguard the possibility for yield loss of Bt crop by other lepidopteran insects that are insensitive to the Cry1A toxin, and also against the possibility for emergence of resistant insects. In this respect, Cry2A toxin has figured as a prospective candidate to be the second toxin to offer the required protection along with Cry1A. In the present study, the entomocidal potency of Cry2A toxin was enhanced through knowledge-based protein engineering of the toxin molecule. Deletion of 42 amino acid residues from the N-terminal end of the peptide followed by the replacement of Lys residues by nonpolar amino acids in the putative transmembrane region including the introduction of Pro resulted in a 4.1–6.6-fold increase in the toxicity of the peptide against three major lepidopteran insect pests of crop plants

Fathead minnow steroidogenesis: in silico analyses reveals tradeoffs between nominal target efficacy and robustness to cross-talk

<p>Abstract</p> <p>Background</p> <p>Interpreting proteomic and genomic data is a major challenge in predictive ecotoxicology that can be addressed by a systems biology approach. Mathematical modeling provides an organizational platform to consolidate protein dynamics with possible genomic regulation. Here, a model of ovarian steroidogenesis in the fathead minnow, <it>Pimephales promelas</it>, (FHM) is developed to evaluate possible transcriptional regulation of steroid production observed in microarray studies.</p> <p>Results</p> <p>The model was developed from literature sources, integrating key signaling components (G-protein and PKA activation) with their ensuing effect on steroid production. The model properly predicted trajectory behavior of estradiol and testosterone when fish were exposed to fadrozole, a specific aromatase inhibitor, but failed to predict the steroid hormone behavior occurring one week post-exposure as well as the increase in steroid levels when the stressor was removed. In vivo microarray data implicated three modes of regulation which may account for over-production of steroids during a depuration phase (when the stressor is removed): P450 enzyme up-regulation, inhibin down-regulation, and luteinizing hormone receptor up-regulation. Simulation studies and sensitivity analysis were used to evaluate each case as possible source of compensation to endocrine stress.</p> <p>Conclusions</p> <p>Simulation studies of the testosterone and estradiol response to regulation observed in microarray data supported the hypothesis that the FHM steroidogenesis network compensated for endocrine stress by modulating the sensitivity of the ovarian network to global cues coming from the hypothalamus and pituitary. Model predictions of luteinizing hormone receptor regulation were consistent with depuration and in vitro data. These results challenge the traditional approach to network elucidation in systems biology. Generally, the most sensitive interactions in a network are targeted for further elucidation but microarray evidence shows that homeostatic regulation of the steroidogenic network is likely maintained by a mildly sensitive interaction. We hypothesize that effective network elucidation must consider both the sensitivity of the target as well as the target's robustness to biological noise (in this case, to cross-talk) when identifying possible points of regulation.</p

Analysis of optimal phenotypic space using elementary modes as applied to Corynebacterium glutamicum

BACKGROUND: Quantification of the metabolic network of an organism offers insights into possible ways of developing mutant strain for better productivity of an extracellular metabolite. The first step in this quantification is the enumeration of stoichiometries of all reactions occurring in a metabolic network. The structural details of the network in combination with experimentally observed accumulation rates of external metabolites can yield flux distribution at steady state. One such methodology for quantification is the use of elementary modes, which are minimal set of enzymes connecting external metabolites. Here, we have used a linear objective function subject to elementary modes as constraint to determine the fluxes in the metabolic network of Corynebacterium glutamicum. The feasible phenotypic space was evaluated at various combinations of oxygen and ammonia uptake rates. RESULTS: Quantification of the fluxes of the elementary modes in the metabolism of C. glutamicum was formulated as linear programming. The analysis demonstrated that the solution was dependent on the criteria of objective function when less than four accumulation rates of the external metabolites were considered. The analysis yielded feasible ranges of fluxes of elementary modes that satisfy the experimental accumulation rates. In C. glutamicum, the elementary modes relating to biomass synthesis through glycolysis and TCA cycle were predominantly operational in the initial growth phase. At a later time, the elementary modes contributing to lysine synthesis became active. The oxygen and ammonia uptake rates were shown to be bounded in the phenotypic space due to the stoichiometric constraint of the elementary modes. CONCLUSION: We have demonstrated the use of elementary modes and the linear programming to quantify a metabolic network. We have used the methodology to quantify the network of C. glutamicum, which evaluates the set of operational elementary modes at different phases of fermentation. The methodology was also used to determine the feasible solution space for a given set of substrate uptake rates under specific optimization criteria. Such an approach can be used to determine the optimality of the accumulation rates of any metabolite in a given network

In Sitico quantification of optimal lysine synthesis during growth of Corynebacterium glutamicum on mixed substrates (Glucose and Lactate)

by Kalyan Gayen et al.

Metabolic network analysis of biobutanol production using Clostridium acetobutylicum

Biofuel production from renewable resources is being demanded due to petroleum dependency and continuous depletion of petroleum oil reservoirs. After knowing the applications of biobutanol as biofuel, its economical production is the challenge for researchers. There is two phased (acidogenesis and solventogenesis) metabolism network of Clostridium acetobutylicum has been studied. It is believed that solventogenesis phase of Clostridium acetobutylicum metabolism is key phase in biobutanol production. During solventogenesis, butanol, ethanol, and acetone are produced in acetone-butanol-ethanol (ABE) fermentation and bacteria uptake the acids (butyrate and acetate) produced in acidogenesis phase. Additionally, ethanol can be produced separately from butanol and acetone and with lowest yield. The uptake of acids and the formation of acetone are coupled and there cannot be any uptake of acetate and butyrate without the formation of an equivalent amount of acetone. We have performed metabolic network analysis of this organism using elementary mode analysis. Metabolic network analysis suggests that butanol synthesis is always coupled with acetone production. The gene, namely acetoacetate decarboxylase (adc) is responsible for the synthesis of acetone producing enzymes which can be engineered to enhance butanol production. We also studied the optimal solution space for biobutanol production which will help to understand the optimal feeding strategy semi-batch fermentation process.by M. Kumar and Kalyan Gaye

Biobutanol: The Future Biofuel

Emerging interest of economic biobutanol production at industrial level is being stimulated owing to flourishing environmental issues and hiking of price for petroleum-based liquid fuels due to continuous depletion of oil reserves. Moreover, biobutanol also demonstrated various significant properties over bioethanol (commercialized biofuel) such as high calorific value, low freezing point, high hydrophobicity, low heat of vaporization, no need of modification in exiting car engines, less corrosive, no blending limit (can be used up to 100%), its dibutyl ether derivative has potential for diesel fuel, etc. Unfortunately, economic feasibility of biobutanol fermentation is suffering due to low butanol titer as butanol itself acts as inhibitor during fermentation. To overcome this problem several genetic and metabolic engineering strategies are being tried. Still, none of the attempts are successful efficiently as butanol disrupts the cytoplasmic membrane and its functions, which are essential for survival of organism. Therefore, online product recovery technologies with continuous fermentation are being optimized to enhance the butanol productivity. However, studies based on economic evaluation of biobutanol production illustrated that production cost of biobutanol primarily depends on cost of raw material. In this direction, conversion of cheaper lignocellulosic biomass (agriculture waste and wood residue) to biobutanol is promising the great potential towards the economic feasibility of this liquid fuel.by Manish Kumar and Kalyan Gaye

Developments in biobutanol production: New insights

Biobutanol will become an attractive, economic and sustainable fuel as petroleum oil leads towards expensive fuel due to diminishing oil reserves and an increase of green house gases in the atmosphere. The major challenges in biobutanol production are low butanol titer, availability of compatible feedstocks, and product inhibition. These hurdles are being resolved using several genetic engineering techniques, metabolic engineering strategies, and promising integrated continuous fermentation processes with efficient product recovery techniques (like gas stripping). Adequate success in utilizing renewable and cost-effective cellulosic materials as feedstocks has opened up novel grounds for the advancement in economic biobutanol production. In this direction, Clostridium beijerinckii is being explored as promising strain to produce biobutanol from cellulosic materials. Moreover, high biobutanol titer is being focused through genetic modifications of Clostridia and non-Clostridia organisms (e.g., Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, and Bacillus subtilis) in both aerobic and anaerobic fermentation. Further, application of various novel genetic tools and genome sequencing of hyper-butanol-producing Clostridial organism will enhance the scope of genetic engineering for biobutanol production. Therefore, consolidation of academic and industrial research towards economic synthesis of biobutanol illustrates the possibility of substantial breakthrough in future. In this review, we focus on (i) selection of suitable bacterial strain (ii) availability of cheaper biomass to produce butanol (iii) metabolic engineering strategies of various microorganisms (iv) attempts at process development and (v) biobutanol recovery techniques that provide future direction of economical biobutanol fermentation.by Manish Kumar and Kalyan Gaye

Quantification of cell size distribution as applied to the growth of Corynebacterium glutamicum

It is known that the cell size is related to the physiological state of a cell. Therefore, cell size distribution directly reflects the average physiological properties of the cell culture. Cell size distribution can be enumerated by image analysis, flow cytometry and coulter counter. In this study, image analysis was used to characterize the cell size distribution during the growth of Corynebacterium glutamicum and was further analyzed by a distribution function. The parameters of the distribution function indicate the mean value and spread of the distribution. Analysis demonstrated that the maximum specific growth rate was higher (0.67 h−1) for the growth obtained through serial dilution of seed as compared to growth from a normal seed culture (0.53 h−1). This was due to a greater percentage of the cell population being in the state of division for the growth through serial dilution in the mid-log phase. The measurement of the cell size distribution demonstrated that the average cell size decreased during the course of growth. The distribution function was also used to enumerate the average specific growth rate of both the conditions of the culture. The demonstrated methodology can be used to predict an average growth property of a cell culture.© Elsevie

Developments in biobutanol production: New insights

Biobutanol will become an attractive, economic and sustainable fuel as petroleum oil leads towards expensive fuel due to diminishing oil reserves and an increase of green house gases in the atmosphere. The major challenges in biobutanol production are low butanol titer, availability of compatible feedstocks, and product inhibition. These hurdles are being resolved using several genetic engineering techniques, metabolic engineering strategies, and promising integrated continuous fermentation processes with efficient product recovery techniques (like gas stripping). Adequate success in utilizing renewable and cost-effective cellulosic materials as feedstocks has opened up novel grounds for the advancement in economic biobutanol production. In this direction, Clostridium beijerinckii is being explored as promising strain to produce biobutanol from cellulosic materials. Moreover, high biobutanol titer is being focused through genetic modifications of Clostridia and non-Clostridia organisms (e.g., Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, and Bacillus subtilis) in both aerobic and anaerobic fermentation. Further, application of various novel genetic tools and genome sequencing of hyper-butanol-producing Clostridial organism will enhance the scope of genetic engineering for biobutanol production. Therefore, consolidation of academic and industrial research towards economic synthesis of biobutanol illustrates the possibility of substantial breakthrough in future. In this review, we focus on (i) selection of suitable bacterial strain (ii) availability of cheaper biomass to produce butanol (iii) metabolic engineering strategies of various microorganisms (iv) attempts at process development and (v) biobutanol recovery techniques that provide future direction of economical biobutanol fermentation.Biobutanol Lignocellulosic material Process development Metabolic engineering Economic feasibility