EcoCyc: fusing model organism databases with systems biology.

EcoCyc (http://EcoCyc.org) is a model organism database built on the genome sequence of Escherichia coli K-12 MG1655. Expert manual curation of the functions of individual E. coli gene products in EcoCyc has been based on information found in the experimental literature for E. coli K-12-derived strains. Updates to EcoCyc content continue to improve the comprehensive picture of E. coli biology. The utility of EcoCyc is enhanced by new tools available on the EcoCyc web site, and the development of EcoCyc as a teaching tool is increasing the impact of the knowledge collected in EcoCyc

Metabolic modeling and analysis of the metabolic switch in Streptomyces coelicolor

Background The transition from exponential to stationary phase in Streptomyces coelicolor is accompanied by a major metabolic switch and results in a strong activation of secondary metabolism. Here we have explored the underlying reorganization of the metabolome by combining computational predictions based on constraint-based modeling and detailed transcriptomics time course observations. Results We reconstructed the stoichiometric matrix of S. coelicolor, including the major antibiotic biosynthesis pathways, and performed flux balance analysis to predict flux changes that occur when the cell switches from biomass to antibiotic production. We defined the model input based on observed fermenter culture data and used a dynamically varying objective function to represent the metabolic switch. The predicted fluxes of many genes show highly significant correlation to the time series of the corresponding gene expression data. Individual mispredictions identify novel links between antibiotic production and primary metabolism. Conclusion Our results show the usefulness of constraint-based modeling for providing a detailed interpretation of time course gene expression data

Robust Nonlinear Model Predictive Control of Biosystems described by Dynamic Metabolic Flux Models

The accuracy of the model used for prediction in Nonlinear Model Predictive Controller (NMPC) is one of the main factors affecting the closed loop performance. Since it is impossible to formulate a perfect model for a real process, there are always differences between the responses predicted by the model and the responses observed from the process. Hence, robustness to model error is an essential property that the controller must have to be adopted in industrial applications. Propagating the uncertainty in the model onto the variables used by the controller is one the key challenges for efficient implementation of a robust controller. Uncertainty propagation approaches such as Monte Carlo simulations and the Polynomial Chaos Expansions (PCE) has been found to suffer from exponentially increasing computational effort with the number of uncertain parameters. Accordingly, the main goal of this thesis is to develop a novel formulation of NMPC based on an uncertainty propagation approach that is more computationally efficient as compared to previously reported approaches. The proposed robust controller in this thesis is specifically targeted to biosystems that are modeled by Dynamic Metabolic Flux models. These models that are becoming increasingly popular for modelling bioprocesses are based on the premise that microorganisms have learned through natural evolution to optimally allocate their resources (nutrients) to maximize a biological objective such as growth or ATP production. Accordingly, these flux models are formulated by LP (Linear Programming) optimizations with constraints that are solved at each time interval and then can be used in conjunction with mass balances to predict the dynamic evolution of different metabolites. The uncertainty in these models is associated to inaccuracies of model parameters involved in the constraints. Thus, although the problem can be solved for particular model parameters by an LP, in the presence of uncertainty the problem becomes nonlinear since different active sets of constraints may become active for parameters’ values within their possible range of variation. Accordingly, the solution space of this nonlinear system can be divided into a set of polyhedrons where each point corresponds to a particular set of parameters within their range of uncertainty. The solution space is often referred in the thesis as the RHS (Right Hand Side) space since it is defined by the variations in the RHS of the constraints with respect to the uncertain parameters. To identify these polyhedrons a dividing procedure has been developed. Since all the polyhedrons can be proven to be convex cones based on a standard simplex form of LP, this dividing method is referred to as the Convex Cone Method (CCM). The regions found by the CCM method are then compared to regions calculated with 100 Percent Rule where the latter has been often used to find a region of existence of a particular tableau in the Simplex method. From this comparison it is found that the CCM can both identify all the possible tableaus with a given region of uncertain parameters and it can also be used the probability for occurrence of each one of the tableaus. These two facts make the CCM an attractive basis for uncertainty propagation in an LP problem instead of the 100 Percent Rule. After identifying the possible tableaus for a given region of model parameters, a novel method is developed for propagating uncertainty onto the controlled variables to be referred to as Tableau Based Tree (TBT) method. The TBT method is based on the concept of propagating uncertainty into the prediction horizon of the controlled by using a tree structure which branches correspond to different tableaus identified by the CCM approach. It is then shown that the conservativeness of the NMPC controller can be significantly reduced based on this tree structure as compared to a Monte Carlo approach for uncertainty propagation. After propagating the uncertainty onto the relevant variables, the control actions for each branch of the tree structure can be obtained by a simple linear calculation. An EMPC (Economic Model Predictive Controller) is adopted in this work as a special realization of an NMPC algorithm where the controller pursues the maximization of an economic objective function. A simple theoretical comparison with a Monte Carlo uncertainty propagation approach shows that the TBT method have a potential to save considerable computational effort as compared to Monte Carlo simulation and PCEs. Finally, the TBT-based robust EMPC is applied in a case study dealing with a fed-batch reactor which is described by dynamic metabolic flux model (DMFM)

Análise de fluxo metabólico de leveduras que naturalmente fazem a conversão de xilose em etanol

Tese (doutorado)—Universidade de Brasília, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2018.A demanda mundial por combustível continua crescendo. Entre as fontes alternativas de energia a produção de etanol tem lugar de destaque. No entanto, está produção ainda pode aumentar com o aproveitamento da xilose proveniente da biomassa lignocelulósica. Vários estudos estão sendo realizados para identificar os mecanismos moleculares envolvidos no metabolismo de xilose. Neste estudo é proposto a integração dados fisiológicas e moleculares para caracterizar o fluxo metabólico de leveduras. Foi avaliado a capacidade fermentativa entre diferentes leveduras naturalmente consumidoras de xilose: Scheffersomyces stipitis, Spathaspora passalidarum, Spathaspora arborariae e Candida tenuis. Para entender o metabolismo dessas leveduras foi construído um modelo de fluxo metabólico utilizando as taxas de produção para restringir o modelo e calcular a distribuição interna de carbono. Pela primeira vez, é estimado o fluxo metabólico nas leveduras Spathaspora. O modelo de fluxo metabólico de xilose até a formação de etanol foi inicialmente validado a partir do teste de correlação entre fluxos calculados e medidos. O modelo de fluxo metabólico foi útil para aumentar a acurácia de dados de metaboloma. 74% das taxas de fluxos calculados e medidos apresentaram semelhanças acima de 90%. O modelo caracterizou a S. stipitis e S. passalidarum como tendo as melhores propriedades naturais para fermentar xilose e produzir etanol.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).Global demand for fuel continues to grow. Among the alternative sources of energy, the production of ethanol has a prominent place. However, this production can yet increase with the use of xylose from the lignocellulosic biomass. Several studies are being carried out to identify the molecular mechanisms involved in the metabolism of xylose. In this study it is proposed the integration of physiological and molecular data to characterize the metabolic flux of yeasts. It was assessed the fermentative capacity among different yeasts naturally consuming xylose: Scheffersomyces stipitis, Spathaspora passalidarum, Spathaspora arborariae and Candida tenuis. To understand the metabolism of these yeasts a metabolic flux model was constructed using the production rates to constrain the model and to calculate the internal carbon distribution. For the first time, is estimated the metabolic flux into Spathaspora yeasts. The metabolic flux model was initially validated from a correlation test between calculated and measured fluxes. The metabolic flux model was useful to increase accuracy of metabolome data. 74% of calculated and measured fluxes rate shown similarity above 90%. The model characterized S. stipitis and S. passalidarum as having the best natural properties for fermenting xylose and producing ethanol

The differentiation of pluripotent stem cells to hepatic cells – Parallels between maturation status and metabolic state

Hepatocytes derived from human pluripotent stem cells (PSCs) hold great promise as an unlimited cell source for liver cell therapy and in vitro toxicity studies. Through the treatment of a series of cytokines and growth factors to mimic embryonic development, PSCs can be guided to differentiate through the endodermal and hepatic commitment stages to become hepatocytelike cells (HLCs). As PSCs differentiate toward endoderm, then to hepatic lineage, the glycolysis and amino acid metabolic rate decreased significantly. Flux analysis using a compartmentalized metabolic flux model that considers cytosolmitochondria interactions revealed that the progressive decline in glycolysis flux coincides with an increase in activities of oxidative phosphorylation (OxPhos) and TCA cycle. This increase in OxPhos activity was also accompanied by increased mitochondria activity. Transcriptome analysis showed that the expression of a number of enzymes and transporter in glucose metabolism decreased as PSCs differentiate toward HLCs. Using a kinetic model of energy metabolism, we showed that the decrease in the expression of those genes could account for the metabolic shift during the differentiation. Our results suggest that metabolic shift may play a role in in vitro PSC differentiation to HLC. Consistently, aborting the metabolic shift by culturing differentiating HLCs at a high glucose level showed a decreased degree of maturation. We then asked the question whether such metabolic shift occurred during embryonic liver development. Lacking fetal liver metabolism data, we examined the transcriptome data of developing liver in mouse embryo. We compiled the transcriptome data of human PSCs differentiation to HLCs and mouse embryonic liver development and performed cross-species in vivo vs. in vitro meta-analysis. After batch corrections on the combined data set cells at different stages of HLC differentiation and different embryonic days of mouse liver development aligned chronologically on a unified developmental “time” scale. The results show that in vitro HLC differentiation of human PSCs reached an equivalent period of E(Embryo day)13-E15 in mouse development, but lacked full maturity of hepatocytes. Furthermore, the enzymes of glucose metabolism behaved similarly in embryonic liver development and in HLC differentiation up to E15. In late stages of embryonic liver development, many of the metabolic enzymes subsequently switch their isoforms to those of the mature hepatocyte. The isoform switch of glycolytic enzymes may reflect the final switch to the mature metabolic nature of the liver. Although, we observe many similar trends in our differentiation, failure to switch isoforms in in vitro differentiation is a key contributor to the lack of maturity of HLCs. In conclusion, the energy metabolism undergoes significant changes over the course of in vitro differentiation from PSCs towards hepatocytes. The shift in energy metabolism is the result, but has also been proposed to be a possible driver, of the differentiation. To enhance the maturation of HLCs, correcting the expression of the genes that fail to progress concordantly as in mouse embryonic liver beyond E15 is a tempting proposition. However, this metabolic study also suggests that providing an appropriate environment to elicit a shift toward the metabolic state of mature hepatocytes may be equally important

Quantitative analysis of metabolic pathways in Catharanthus roseus hairy roots metabolically engineered for terpenoid indole alkaloid overproduction

The important anticancer pharmaceuticals, vinblastine and vincristine, are produced by Catharanthus roseus. Given their cytotoxicity, these valuable alkaloids are produced in very small quantities within the aerial parts of the plant. The high cost of isolating the drugs has led to research efforts to increase the alkaloid content of C. roseus cell cultures, tissue cultures, and seedlings. The metabolic engineering of C. roseus strives to overcome the strict regulation of the biosynthetic pathways. Seedlings of C. roseus were elicited with methyl jasmonate (MeJA) to induce expression of octadecanoid–responsive Catharanthus AP2–domain 3 (ORCA3), a transcription regulator of several biosynthetic genes. ORCA3 exhibited increases up to 25–fold observed 0.5 h after MeJA treatment with the transcript levels of biosynthetic genes following with variable timing. The amounts of certain terpenoid indole alkaloid (TIA) metabolites, including the important vinblastine precursors, catharanthine and vindoline, were increased significantly. Three hairy root cultures of C. roseus were investigated. The ASAB–1 line expressing a feedback–resistant anthranilate synthase (AS) α subunit from Arabidopsis under the control of a glucocorticoid–inducible promoter and an ASβ subunit from Arabidopsis under the control of the constitutive CaMV 35S promoter, the EHIDXS–4–1 line expressing 1–deoxy–D–xylulose 5–phosphate synthase (DXS) under the control of a glucocorticoid–inducible promoter, and the EHIT16H–34–1 line tabersonine 16–hydroxylase (T16H) under the control of a glucocorticoid–inducible promoter. These lines were used to investigate the regulatory nature of the biosynthetic network by quantifying the effect of light–adaptation, biosynthetic enzyme overexpression, and the combination of these two factors on the production of TIAs. Comprehensive metabolite profiling and a stoichiometric model were employed to reveal mechanisms of regulation. The results point towards controlling metabolite degradation as a potential focus for metabolic engineering efforts. A proof of concept of a method for the introduction of 13C–labeling at the time of gene induction and preliminary results are presented. This method allows for the creation of metabolic flux maps of central carbon metabolism before and after the gene has been induced. The flux maps will reveal limitations in central carbon metabolism that affect the production potential of secondary metabolism. The long term stability of a transgenic C. roseus hairy root line containing the inducible expression of a feedback–insensitive ASα is reported. After 5 years in liquid culture, the presence and inducible expression of the inserted AS gene was confirmed. This report also demonstrates that it may take as long as two years for the metabolite profile to stabilize. Transgenic C. roseus hairy root lines were created that individually overexpress DXS and geraniol 10–hydroxylase (G10H) under the control of a glucocorticoid–inducible promoter. Double overexpression lines that overexpress DXS and ASα subunit or DXS and G10H with both genes under control of a glucocorticoid–inducible promoter were also created. The double overexpression lines displayed pertinent increases in TIA levels, surpassing the single overexpression lines. The value of ultraviolet (UV) and mass spectra in identifying compounds in chromatographic methods is presented. The UV and mass spectra of important C. roseus secondary metabolites are included. A method for the isolation of important C. roseus alkaloids is presented. A biomass extraction and analytical HPLC protocol was adapted for semi–preparative scale in order to obtain tabersonine, lochnericine, and hyrhammericine standards. Previously unidentified tabersonine–like compounds were also isolated for future identification

Fermentation of mixed glucose-xylose substrates by engineered strains of Saccharomyces cerevisiae: role of the coenzyme specificity of xylose reductase, and effect of glucose on xylose utilization

<p>Abstract</p> <p>Background</p> <p>In spite of the substantial metabolic engineering effort previously devoted to the development of <it>Saccharomyces cerevisiae </it>strains capable of fermenting both the hexose and pentose sugars present in lignocellulose hydrolysates, the productivity of reported strains for conversion of the naturally most abundant pentose, xylose, is still a major issue of process efficiency. Protein engineering for targeted alteration of the nicotinamide cofactor specificity of enzymes catalyzing the first steps in the metabolic pathway for xylose was a successful approach of reducing xylitol by-product formation and improving ethanol yield from xylose. The previously reported yeast strain BP10001, which expresses heterologous xylose reductase from <it>Candida tenuis </it>in mutated (NADH-preferring) form, stands for a series of other yeast strains designed with similar rational. Using 20 g/L xylose as sole source of carbon, BP10001 displayed a low specific uptake rate <it>q</it>_xylose(g xylose/g dry cell weight/h) of 0.08. The study presented herein was performed with the aim of analysing (external) factors that limit <it>q</it>_xyloseof BP10001 under xylose-only and mixed glucose-xylose substrate conditions. We also carried out a comprehensive investigation on the currently unclear role of coenzyme utilization, NADPH compared to NADH, for xylose reduction during co-fermentation of glucose and xylose.</p> <p>Results</p> <p>BP10001 and BP000, expressing <it>C. tenuis </it>xylose reductase in NADPH-preferring wild-type form, were used. Glucose and xylose (each at 10 g/L) were converted sequentially, the corresponding <it>q</it>_substratevalues being similar for each strain (glucose: 3.0; xylose: 0.05). The distribution of fermentation products from glucose was identical for both strains whereas when using xylose, BP10001 showed enhanced ethanol yield (BP10001 0.30 g/g; BP000 0.23 g/g) and decreased yields of xylitol (BP10001 0.26 g/g; BP000 0.36 g/g) and glycerol (BP10001 0.023 g/g; BP000 0.072 g/g) as compared to BP000. Increase in xylose concentration from 10 to 50 g/L resulted in acceleration of substrate uptake by BP10001 (0.05 - 0.14 g/g CDW/h) and reduction of the xylitol yield (0.28 g/g - 0.15 g/g). In mixed substrate batches, xylose was taken up at low glucose concentrations (< 4 g/L) and up to fivefold enhanced xylose uptake rate was found towards glucose depletion. A fed-batch process designed to maintain a "stimulating" level of glucose throughout the course of xylose conversion provided a <it>q</it>_xylosethat had an initial value of 0.30 ± 0.04 g/g CDW/h and decreased gradually with time. It gave product yields of 0.38 g ethanol/g total sugar and 0.19 g xylitol/g xylose. The effect of glucose on xylose utilization appears to result from the enhanced flux of carbon through glycolysis and the pentose phosphate pathway under low-glucose reaction conditions.</p> <p>Conclusions</p> <p>Relative improvements in the distribution of fermentation products from xylose that can be directly related to a change in the coenzyme preference of xylose reductase from NADPH in BP000 to NADH in BP10001 increase in response to an increase in the initial concentration of the pentose substrate from 10 to 50 g/L. An inverse relationship between xylose uptake rate and xylitol yield for BP10001 implies that xylitol by-product formation is controlled not only by coenzyme regeneration during two-step oxidoreductive conversion of xylose into xylulose. Although xylose is not detectably utilized at glucose concentrations greater than 4 g/L, the presence of a low residual glucose concentration (< 2 g/L) promotes the uptake of xylose and its conversion into ethanol with only moderate xylitol by-product formation. A fed-batch reaction that maintains glucose in the useful concentration range and provides a constant <it>q</it>_glucosemay be useful for optimizing <it>q</it>_xylosein processes designed for co-fermentation of glucose and xylose.</p

A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581T

The halophilic γ-proteobacterium Halomonas elongata DSM 2581T thrives at high salinity by synthesizing and accumulating the compatible solute ectoine. Ectoine levels are highly regulated according to external salt levels but the overall picture of its metabolism and control is not well understood. Apart from its critical role in cell adaptation to halophilic environments, ectoine can be used as a stabilizer for enzymes and as a cell protectant in skin and health care applications and is thus produced annually on a scale of tons in an industrial process using H. elongata as producer strain. This paper presents the complete genome sequence of H. elongata (4 061 296 bp) and includes experiments and analysis identifying and characterizing the entire ectoine metabolism, including a newly discovered pathway for ectoine degradation and its cyclic connection to ectoine synthesis. The degradation of ectoine (doe) proceeds via hydrolysis of ectoine (DoeA) to Nα-acetyl-l-2,4-diaminobutyric acid, followed by deacetylation to diaminobutyric acid (DoeB). In H. elongata, diaminobutyric acid can either flow off to aspartate or re-enter the ectoine synthesis pathway, forming a cycle of ectoine synthesis and degradation. Genome comparison revealed that the ectoine degradation pathway exists predominantly in non-halophilic bacteria unable to synthesize ectoine. Based on the resulting genetic and biochemical data, a metabolic flux model of ectoine metabolism was derived that can be used to understand the way H. elongata survives under varying salt stresses and that provides a basis for a model-driven improvement of industrial ectoine production

MATHEMATICAL MODELING OF \u3ci\u3eCLOSTRIDIUM THERMOCELLUM’S\u3c/i\u3e METABOLIC RESPONSES TO ENVIRONMENTAL PERTURBATION

Clostridium thermocellum is a thermophilic anaerobe that is capable of producing ethanol directly from lignocellulosic compounds, however this organism suffers from low ethanol tolerance and low ethanol yields. In vivo mathematical modeling studies based on steady state traditional metabolic flux analysis, metabolic control analysis, transient and steady states’ flux spectrum analysis (FSA) were conducted on C. thermocellum’s central metabolism. The models were developed in Matrix Laboratory software ( MATLAB® (The Language of Technical Computing), R2008b, Version 7.7.0.471)) based on known stoichiometry from C. thermocellum pathway and known physical constraints. Growth on cellobiose from Metabolic flux analysis (MFA) and Metabolic control analysis (MCA) of wild type (WT) and ethanol adapted (EA) cells showed that, at lower than optimum exogenous ethanol levels, ethanol to acetate (E/A) ratios increased by approximately 29% in WT cells and 7% in EA cells. Sensitivity analyses of the MFA and MCA models indicated that the effects of variability in experimental data on model predictions were minimal (within ±5% differences in predictions if the experimental data varied up to ±20%). Steady state FSA model predictions showed that, an optimum hydrogen flux of ~5mM/hr in the presence of pressure equal to or above 7MPa inhibits ferrodoxin hydrogenase which causes NAD re-oxidation in the system to increase ethanol yields to about 3.5 mol ethanol/mol cellobiose

Bioeconomy Initiative at MBI International

Di-carboxylic acids have the potential to replace petrochemicals used in the polymer industry (Werpy and Petersen, 2004). MBI developed a process for the production of succinic acid using a proprietary organism. During this work MBI assessed the feasibility to produce other carboxylic acids either using A. succinogenes or other organisms. The development of recombinant A. succinogenes strain derivatives for a mono-carboxylic acid through over-expression of enzymatic activities was successful. Fermentations achieved titers of 58 g/L for this organic acid. Recombinant strains that produced the same acid, but a different stereoisomer, reached titers of 10 g/L. Attempts to increase the titers for this isomer as well as other organic acids were unsuccessful. MBI is looking for commercial partners to pursue the development of recombinant A. succinogenes strains for the production of other organic acids. Attempts to develop recombinant strains of A. succinogenes for fumaric acid production through introduction of various antisense RNA constructs were unsuccessful. Alternative suitable organisms were evaluated and Rhizopus oryzae, a natural fumaric acid producer with potential for process improvements, was selected. A novel fermentation and one-step recovery process was developed that allowed capture of IP, produced titers of &gt;80 g/L with a productivity of 1.8 g/L-h and 57% (g/g glucose) yield. The process was scaled to 2000 L pilot scale. The economic analysis projected a production cost of 72 c/lb. Recycling and re-use of the base was demonstrated and incorporated into the process. The ability of the organism to produce fumaric acid from other carbon sources and biomass hydrolysate was demonstrated. The production of other organic acids was evaluated and techno-economic de-risking roadmap documents were prepared