Kalyan Gayen

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

BackgroundQuantification 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.ResultsQuantification 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.ConclusionWe 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.

Metabolic engineering for enhanced hydrogen production: a review

Hydrogen gas exhibits potential as a sustainable fuel for the future. Therefore, many attempts have been made with the aim of producing high yields of hydrogen gas through renewable biological routes. Engineering of strains to enhance the production of hydrogen gas has been an active area of research for the past 2 decades. This includes overexpression of hydrogen-producing genes (native and heterologous), knockout of competitive pathways, creation of a new productive pathway, and creation of dual systems. Interestingly, genetic mutations in 2 different strains of the same species may not yield similar results. Similarly, 2 different studies on hydrogen productivities may differ largely for the same mutation and on the same species. Consequently, here we analyzed the effect of various genetic modifications on several species, considering a wide range of published data on hydrogen biosynthesis. This article includes a comprehensive metabolic engineering analysis of hydrogen-producing organisms, namely Escherichia coli, Clostridium, and Enterobacter species, and in addition, a short discussion on thermophilic and halophilic organisms. Also, apart from single-culture utilization, dual systems of various organisms and associated developments have been discussed, which are considered potential future targets for economical hydrogen production. Additionally, an indirect contribution towards hydrogen production has been reviewed for associated species.

Role of extracellular cues to trigger the metabolic phase shifting from acidogenesis to solventogenesis in Clostridium acetobutylicum.

Clostridium acetobutylicum exhibits a two-step metabolic pathway where substrates are first converted to organic acids accompanied by a decrease in pH. The acids are then assimilated to organic solvents. The transition from the acid-producing (acidogenesis) to the solvent-producing phase (solventogenesis) is controlled by integration of a number of cellular and environmental cues, whose precise mode of action are not well understood. In this study, a series of batch experiments were performed to understand the impact of extracellular cues in regulating the dynamics of acidogenesis and solventogenesis. It is demonstrated that the two phases operate independently of each other and the growth phase of the cell, i.e. the cues controlling a phase are not linked to the status of the other phase or the growth phase of the cell. Kinetic experiments demonstrated that there exist two previously uncharacterized negative feedback loops controlling the amounts of acids produced in the acidogenesis phase.

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

BackgroundInterpreting 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, Pimephales promelas, (FHM) is developed to evaluate possible transcriptional regulation of steroid production observed in microarray studies.ResultsThe 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.ConclusionsSimulation 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 targets robustness to biological noise (in this case, to cross-talk) when identifying possible points of regulation.

Production of biodiesel from microalgae through biological carbon capture: a review

Gradual increase in concentration of carbon dioxide (CO2) in the atmosphere due to the various anthropogenic interventions leading to significant alteration in the global carbon cycle has been a subject of worldwide attention and matter of potential research over the last few decades. In these alarming scenario microalgae seems to be an attractive medium for capturing the excess CO2 present in the atmosphere generated from different sources such as power plants, automobiles, volcanic eruption, decomposition of organic matters and forest fires. This captured CO2 through microalgae could be used as potential carbon source to produce lipids for the generation of biofuel for replacing petroleum-derived transport fuel without affecting the supply of food and crops. This comprehensive review strives to provide a systematic account of recent developments in the field of biological carbon capture through microalgae for its utilization towards the generation of biodiesel highlighting the significance of certain key parameters such as selection of efficient strain, microalgal metabolism, cultivation systems (open and closed) and biomass production along with the national and international biodiesel specifications and properties. The potential use of photobioreactors for biodiesel production under the influence of various factors viz., light intensity, pH, time, temperature, CO2 concentration and flow rate has been discussed. The review also provides an economic overview and future outlook on biodiesel production from microalgae.

A review on the production of fermentable sugars from lignocellulosic biomass through conventional and enzymatic route—a comparison

ABSTRACT The scarcity of fossil fuels has urged the economically developed countries to find the resources for an alternative energy sources. In apprehension to this, biofuels, like bioethanol and biobutanol, produced from lignocellulosic biomass were considered as potential alternative. There are several methods for the pretreatment of biomass before it is being used as a feedstock for the production of fermentable sugars. However, one of the crucial concerns here is to enumerate an economic pretreatment scheme that can be implemented in large scale for the production of mostly exposed cellulosic part from biomass. This will ensure an effective hydrolysis of cellulose for the production of fermentable sugars and the production of biobutanol from these derived sugars. Moreover, the keynote understanding of an effective fermentation is the production of less inhibitory compounds like furfural, hydroxymethyl furfural during the hydrolysis of cellulose. Enzymatic hydrolysis of cellulose was reported as the most efficient method is this aspect. Trichoderma sp. was found the mostly used resources for the enzyme called cellulase and Aspergillus sp. for hemicellulase enzymes. The most crucial part here is the isolation of proper enzyme that will increase the rate of hydrolysis. Moreover, selection of proper pretreatment process will be a key benefit to the production of fermentable sugars through enzymatic hydrolysis. Based on the biomass nature, the evaluated hot water pretreatment followed by enzymatic hydrolysis with a provision of enzyme reusability (like encapsulated or enzyme separation with membrane) seems to be promising for enhanced biofuel-production.

Acetone-butanol-ethanol fermentation analysis using only high performance liquid chromatography

Currently, sample analysis of Acetone-butanol-ethanol (ABE) fermentation in Clostridium acetobutylicum is performed through the simultaneous use of both High Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC). In this study, a novel method was developed for the quantification of substrate (glucose) and products (acetic acid, ethanol, butyric acid, acetone, and butanol) of ABE fermentation using only HPLC. The most favorable characterization of peaks of tested compounds were observed in a refractive index (RI) detector maintaining a flow rate of 0.5 ml min−1 with a column temperature of 30 °C. However, an overlap between butyric acid and acetone peaks was detected, and therefore a methodology was developed accounting for respective peak heights for the quantification of these compounds. During validation of the method, linear regression analysis of the calibration plot illustrated that there was a good linear relationship (correlation coefficient R2 > 0.9981) between peak area and concentration in the range of 0.31–5.0 mg ml−1. The quantitative recoveries of tested components were very close in range, 97.99–103.46%, and Relative Standard Deviation (RSD) values were lower than 4.90%. The results of statistical analysis proved that the method is precise, repeatable, reproducible, accurate, and sensitive, and hence can be employed for the quantification of components involved in ABE fermentation. This method was also used to quantify the products and substrates of fermentation samples using individual glucose and a combination of sugars.

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.

Lysine overproducing Corynebacterium glutamicum is characterized by a robust linear combination of two optimal phenotypic states.

A homoserine auxotroph strain of Corynebacterium glutamicum accumulates storage compound trehalose with lysine when limited by growth. Industrially lysine is produced from C. glutamicum through aspartate biosynthetic pathway, where enzymatic activity of aspartate kinase is allosterically controlled by the concerted feedback inhibition of threonine plus lysine. Ample threonine in the medium supports growth and inhibits lysine production (phenotype-I) and its complete absence leads to inhibition of growth in addition to accumulating lysine and trehalose (phenotype-II). In this work, we demonstrate that as threonine concentration becomes limiting, metabolic state of the cell shifts from maximizing growth (phenotype-I) to maximizing trehalose phenotype (phenotype-II) in a highly sensitive manner (with a Hill coefficient of 4). Trehalose formation was linked to lysine production through stoichiometry of the network. The study demonstrated that the net flux of the population was a linear combination of the two optimal phenotypic states, requiring only two experimental measurements to evaluate the flux distribution. The property of linear combination of two extreme phenotypes was robust for various medium conditions including varying batch time, initial glucose concentrations and medium osmolality.

Enzymatic hydrolysis of banana stems (Musa acuminata): Optimization of process parameters and inhibition characterization

ABSTRACT In current work, an optimum solid loading (solid: liquid = 1:20), pH (4.8), temperature (50°C), and enzyme dosing of 20 filter paper unit (amount of enzyme required to release 1 µmol of glucose as reducing sugar from filter paper in per mL per minute) were enumerated for enzymatic hydrolysis of banana stem using cellulase from Trichoderma reesei. Further, inhibition study on enzymatic hydrolysis of banana stem was investigated by the supplementation of monosaccharides (glucose, galactose, mannose, xylose, and arabinose), disaccharide (cellobiose), and inhibitors (acetic acid and furfural obtained from pre-enzymatic hydrolysis steps). Glucose and cellobiose showed inhibitory effect on enzymatic hydrolysis of pretreated banana stem at or above 8 g/L while galactose, mannose, and xylose showed a significant inhibitory effect at or above 4 g/L. Instead of inhibition, arabinose enhanced the enzymatic hydrolysis with increase in total reducing sugars. Acetic acid did not show any significant inhibition while furfural inhibited the system at a comparative low concentration of 2 g/L. Further, scanning electron microscopy analysis was performed to investigate the difference in ultra-structural morphology of raw biomass, pretreated biomass, and biomass obtained after enzymatic hydrolysis.