1,721,103 research outputs found
A roadmap for interpreting 13C metabolite labeling patterns from cells
No full textMeasuring intracellular metabolism has increasingly led to important insights in biomedical research. [superscript 13]C tracer analysis, although less information-rich than quantitative [superscript 13]C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrientcontributions. Here, we review selected key issues in interpreting [superscript 13]C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiment
Downstream reactions and engineering in the microbially reconstituted pathway for Taxol
Full text linkTaxol (a trademarked product of Bristol-Myers Squibb) is a complex isoprenoid natural product which has displayed potent anticancer activity. Originally isolated from the Pacific yew tree (Taxus brevifolia), Taxol has been mass-produced through processes reliant on plant-derived biosynthesis. Recently, there have been alternative efforts to reconstitute the biosynthetic process through technically convenient microbial hosts, which offer unmatched growth kinetics and engineering potential. Such an approach is made challenging by the need to successfully introduce the significantly foreign enzymatic steps responsible for eventual biosynthesis. Doing so, however, offers the potential to engineer more efficient and economical production processes and the opportunity to design and produce tailored analog compounds with enhanced properties. This mini review will specifically focus on heterologous biosynthesis as it applies to Taxol with an emphasis on the challenges associated with introducing and reconstituting the downstream reaction steps needed for final bioactivity.National Institutes of Health (U.S.) (GM085323)Milheim Foundation (2006-2017
Kinetic isotope effects significantly influence intracellular metabolite [superscript 13]C labeling patterns and flux determination
No full textRigorous mathematical modeling of carbon-labeling experiments allows estimation of fluxes through the pathways of central carbon metabolism, yielding powerful information for basic scientific studies as well as for a wide range of applications. However, the mathematical models that have been developed for flux determination from [superscript 13]C labeling data have commonly neglected the influence of kinetic isotope effects on the distribution of [superscript 13]C label in intracellular metabolites, as these effects have often been assumed to be inconsequential. We have used measurements of the [superscript 13]C isotope effects on the pyruvate dehydrogenase enzyme from the literature to model isotopic fractionation at the pyruvate node and quantify the modeling errors expected to result from the assumption that isotope effects are negligible. We show that under some conditions kinetic isotope effects have a significant impact on the [superscript 13]C labeling patterns of intracellular metabolites, and the errors associated with neglecting isotope effects in [superscript 13]C-metabolic flux analysis models can be comparable in size to measurement errors associated with GC–MS. Thus, kinetic isotope effects must be considered in any rigorous assessment of errors in [superscript 13]C labeling data, goodness-of-fit between model and data, confidence intervals of estimated metabolic fluxes, and statistical significance of differences between estimated metabolic flux distributions.United States. Dept. of Energy (Grant DE-SC0008744)MIT-National Institute of General Medical Sciences (U.S.) (Biotechnology Training Program
Review of methods to probe single cell metabolism and bioenergetics
No full textSingle cell investigations have enabled unexpected discoveries, such as the existence of biological noise and phenotypic switching in infection, metabolism and treatment. Herein, we review methods that enable such single cell investigations specific to metabolism and bioenergetics. Firstly, we discuss how to isolate and immobilize individuals from a cell suspension, including both permanent and reversible approaches. We also highlight specific advances in microbiology for its implications in metabolic engineering. Methods for probing single cell physiology and metabolism are subsequently reviewed. The primary focus therein is on dynamic and high-content profiling strategies based on label-free and fluorescence microspectroscopy and microscopy. Non-dynamic approaches, such as mass spectrometry and nuclear magnetic resonance, are also briefly discussed.United States. Dept. of Energy. Genomic Sciences Program (Grant DE-SC0008744
Improving fatty acids production by engineering dynamic pathway regulation and metabolic control
Full text linkGlobal energy demand and environmental concerns have stimulated increasing efforts to produce carbon-neutral fuels directly from renewable resources. Microbially derived aliphatic hydrocarbons, the petroleum-replica fuels, have emerged as promising alternatives to meet this goal. However, engineering metabolic pathways with high productivity and yield requires dynamic redistribution of cellular resources and optimal control of pathway expression. Here we report a genetically encoded metabolic switch that enables dynamic regulation of fatty acids (FA) biosynthesis in Escherichia coli. The engineered strains were able to dynamically compensate the critical enzymes involved in the supply and consumption of malonyl-CoA and efficiently redirect carbon flux toward FA biosynthesis. Implementation of this metabolic control resulted in an oscillatory malonyl-CoA pattern and a balanced metabolism between cell growth and product formation, yielding 15.7- and 2.1-fold improvement in FA titer compared with the wild-type strain and the strain carrying the uncontrolled metabolic pathway. This study provides a new paradigm in metabolic engineering to control and optimize metabolic pathways facilitating the high-yield production of other malonyl-CoA–derived compounds.National Science Foundation (U.S.) (Award CBET1144226)National Science Foundation (U.S.) (Award CBET0836513)Rensselaer Polytechnic Institute. Biocatalysis and Metabolic Engineering Constellatio
Engineering Yarrowia lipolytica for poly-3-hydroxybutyrate production
No full textStrains of Yarrowia lipolytica were engineered to express the poly-3-hydroxybutyrate (PHB) biosynthetic pathway. The genes for β-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase, and PHB synthase were cloned and inserted into the chromosome of Y. lipolytica. In shake flasks, the engineered strain accumulated PHB to 1.50 and 3.84% of cell dry weight in complex medium supplemented with glucose and acetate as carbon source, respectively. In fed-batch fermentation using acetate as sole carbon source, 7.35 g/l PHB (10.2% of cell dry weight) was produced. Selection of Y. lipolytica as host for PHB synthesis was motivated by the fact that this organism is a good lipids producer, which suggests robust acetyl-CoA supply also the precursor of the PHB pathway. Acetic acid could be supplied by gas fermentation, anaerobic digestion, and other low-cost supply route.Tsinghua University (Beijing, China). School of Life SciencesUnited States. Department of Energy (DE-SC0008744)National Natural Science Foundation (China) (21476014 and 31100025
Origins of Cell-to-Cell Bioprocessing Diversity and Implications of the Extracellular Environment Revealed at the Single-Cell Level
No full textBioprocess limitations imposed by microbial cell-to-cell phenotypic diversity remain poorly understood. To address this, we investigated the origins of such culture diversity during lipid production and assessed the impact of the fermentation microenvironment. We measured the single-cell lipid production dynamics in a time-invariant microfluidic environment and discovered that production is not monotonic, but rather sporadic with time. To characterize this, we introduce bioprocessing noise and identify its epigenetic origins. We linked such intracellular production fluctuations with cell-to-cell productivity diversity in culture. This unmasked the phenotypic diversity amplification by the culture microenvironment, a critical parameter in strain engineering as well as metabolic disease treatment.United States. Dept. of Energy (Grant SC 0008744)National Institute of General Medical Sciences (U.S.) (Institutional Development Award P20 GM103408)Pacific Northwest National Laboratory (U.S.) (Linus Pauling Fellowship PN12005/2406
Ensemble Kinetic Modeling of Metabolic Networks from Dynamic Metabolic Profiles
No full textKinetic modeling of metabolic pathways has important applications in metabolic engineering, but significant challenges still remain. The difficulties faced vary from finding best-fit parameters in a highly multidimensional search space to incomplete parameter identifiability. To meet some of these challenges, an ensemble modeling method is developed for characterizing a subset of kinetic parameters that give statistically equivalent goodness-of-fit to time series concentration data. The method is based on the incremental identification approach, where the parameter estimation is done in a step-wise manner. Numerical efficacy is achieved by reducing the dimensionality of parameter space and using efficient random parameter exploration algorithms. The shift toward using model ensembles, instead of the traditional “best-fit” models, is necessary to directly account for model uncertainty during the application of such models. The performance of the ensemble modeling approach has been demonstrated in the modeling of a generic branched pathway and the trehalose pathway in Saccharomyces cerevisiae using generalized mass action (GMA) kinetics
Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production
Full text linkArtemisinin is a potent antimalarial drug; however, it suffers from unstable and insufficient supply from plant source. Here, we established a novel multivariate-modular approach based on experimental design for systematic pathway optimization that succeeded in improving the production of amorphadiene (AD), the precursor of artemisinin, in Escherichia coli. It was initially found that the AD production was limited by the imbalance of glyceraldehyde 3-phosphate (GAP) and pyruvate (PYR), the two precursors of the 1-deoxy-d-xylulose-5-phosphate (DXP) pathway. Furthermore, it was identified that GAP and PYR could be balanced by replacing the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) with the ATP-dependent galactose permease and glucose kinase system (GGS) and this resulted in fivefold increase in AD titer (11 to 60 mg/L). Subsequently, the experimental design-aided systematic pathway optimization (EDASPO) method was applied to systematically optimize the transcriptional expressions of eight critical genes in the glucose uptake and the DXP and AD synthesis pathways. These genes were classified into four modules and simultaneously controlled by T7 promoter or its variants. A regression model was generated using the four-module experimental data and predicted the optimal expression ratios among these modules, resulting in another threefold increase in AD titer (60 to 201 mg/L). This EDASPO method may be useful for the optimization of other pathways and products beyond the scope of this study.Singapore-MIT Alliance for Research and Technology (SMART
Metabolomic and 13C-metabolic flux analysis of a xylose-consuming Saccharomyces cerevisiae strain expressing xylose isomerase
Full text linkOver the past two decades, significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative Pentose Phosphate Pathway (PPP) is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis.Shell Oil CompanyNational Institute of General Medical Sciences (U.S.) Biotechnology Training Progra
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