Strategies for Manipulating Metabolic Fluxes in Biotechnology

Strategies for biotechnologically manipulating metabolic fluxes are critically examined in relation to a model system. The common idea of first identifying the rate-limiting enzyme in the biosynthetic pathway to a desired end-product, and then increasing its activity, is shown to be completely ineffective; such manipulation typically produces only trivial changes in flux. Manipulating the activities of all of the enzymes in a biosynthetic pathway by amounts calculated to increase a desired flux while leaving all other fluxes and all concentrations unchanged is potentially effective, and can be applied to any system without regard to its regulatory design. however, it requires accurate knowledge of the initial state of the system and the ability to make precise changes to numerous activities. The classical information about the regulatory mechanisms that exist in living organisms suggests that one can make much simpler manipulations, involving only the steps that remove the desired end-product, with almost equally satisfactory results.     

Estimating Relative Changes of Metabolic Fluxes

Fluxes are the central trait of metabolism and Kinetic Flux Profiling (KFP) is an effective method of measuring them. To generalize its applicability, we present an extension of the method that estimates the relative changes of fluxes using only relative quantitation of ¹³C-labeled metabolites. Such features are directly tailored to the more common experiment that performs only relative quantitation and compares fluxes between two conditions. We call our extension rKFP. Moreover, we examine the effects of common missing data and common modeling assumptions on (r)KFP, and provide practical suggestions. We also investigate the selection of measuring times for (r)KFP and provide a simple recipe. We then apply rKFP to ¹³C-labeled glucose time series data collected from cells under normal and glucose-deprived conditions, estimating the relative flux changes of glycolysis and its branching pathways. We identify an adaptive response in which de novo serine biosynthesis is compromised to maintain the glycolytic flux backbone. Together, these results greatly expand the capabilities of KFP and are suitable for broad biological applications.     

Metabolic Fluxes in an Illuminated Arabidopsis Rosette

Photosynthesis is the basis for life, and its optimization is a key biotechnological aim given the problems of population explosion and environmental deterioration. We describe a method to resolve intracellular fluxes in intact Arabidopsis thaliana rosettes based on time-dependent labeling patterns in the metabolome. Plants photosynthesizing under limiting irradiance and ambient CO₂ in a custom-built chamber were transferred into a ¹³CO₂-enriched environment. The isotope labeling patterns of 40 metabolites were obtained using liquid or gas chromatography coupled to mass spectrometry. Labeling kinetics revealed striking differences between metabolites. At a qualitative level, they matched expectations in terms of pathway topology and stoichiometry, but some unexpected features point to the complexity of subcellular and cellular compartmentation. To achieve quantitative insights, the data set was used for estimating fluxes in the framework of kinetic flux profiling. We benchmarked flux estimates to four classically determined flux signatures of photosynthesis and assessed the robustness of the estimates with respect to different features of the underlying metabolic model and the time-resolved data set.     

Metabolic Effects on Ion Fluxes in Tolypella intricata

The uptake of C1 ions by cells of Tolypella intricata is greatlyincreased by light, and must be an active process. K ions inthe cells (at 90–110 mM concentration) are in approximateelectrochemical equilibrium with the external solution, butthe K influx is affected (directly or indirectly) by cellularmetabolism. The K influx is increased by light, and the increaseis greater in the presence of C1 than when C1 is removed fromthe solution. K uptake is inhibited by chemicals which alsoinhibit the C1 pump.It is suggested that light increases thepermeability of the plasmalemma to K, but there must also belinks between K and C1 uptake. The possible nature of theselinks is discussed.The internal Na concentration (3–10mM) is considerably below the expected equilibrium concentration,but the Na influx is also very low (and is not increased bylight). The permeability of the plasmalemma to Na is thus verylow, and there can be little active extrusion of Na under normalconditions.     

森林生态系统生物能流的瞬态分析

森林生态系统生物能流的瞬态分析欧阳兵（中国科学院大气物理所大气数值模拟国家重点实验室,北京１０００８０）Ｔｒａｎｓｉｅｎｔ－ＳｔａｔｅＡｎａｌｙｓｉｓｏｎＢｉｏｔｉｃＥｎｅｒｇｙＦｌｏｗｉｎＦｏｒｅｓｔＥｃｏｓｙｓｔｅｍ．￥ＯｕｙａｎｇＢｉｎｇ（ＬＡ...     

Estimating Metabolic Fluxes Using a Maximum Network Flexibility Paradigm

MotivationGenome-scale metabolic networks can be modeled in a constraint-based fashion. Reaction stoichiometry combined with flux capacity constraints determine the space of allowable reaction rates. This space is often large and a central challenge in metabolic modeling is finding the biologically most relevant flux distributions. A widely used method is flux balance analysis (FBA), which optimizes a biologically relevant objective such as growth or ATP production. Although FBA has proven to be highly useful for predicting growth and byproduct secretion, it cannot predict the intracellular fluxes under all environmental conditions. Therefore, alternative strategies have been developed to select flux distributions that are in agreement with experimental “omics” data, or by incorporating experimental flux measurements. The latter, unfortunately can only be applied to a limited set of reactions and is currently not feasible at the genome-scale. On the other hand, it has been observed that micro-organisms favor a suboptimal growth rate, possibly in exchange for a more “flexible” metabolic network. Instead of dedicating the internal network state to an optimal growth rate in one condition, a suboptimal growth rate is used, that allows for an easier switch to other nutrient sources. A small decrease in growth rate is exchanged for a relatively large gain in metabolic capability to adapt to changing environmental conditions.ResultsHere, we propose Maximum Metabolic Flexibility (MMF) a computational method that utilizes this observation to find the most probable intracellular flux distributions. By mapping measured flux data from central metabolism to the genome-scale models of Escherichia coli and Saccharomyces cerevisiae we show that i) indeed, most of the measured fluxes agree with a high adaptability of the network, ii) this result can be used to further reduce the space of feasible solutions iii) this reduced space improves the quantitative predictions made by FBA and contains a significantly larger fraction of the measured fluxes compared to the flux space that was reduced by a uniform sampling approach and iv) MMF can be used to select reactions in the network that contribute most to the steady-state flux space. Constraining the selected reactions improves the quantitative predictions of FBA considerably more than adding an equal amount of flux constraints, selected using a more naïve approach. Our method can be applied to any cell type without requiring prior information.AvailabilityMMF is freely available as a MATLAB plugin at: http://cs.ru.nl/~wmegchel/mmf.     

Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, ¹³C metabolic flux analysis with parallel studies on [1-¹³C^Fru]sucrose, [1-¹³C^Glc]sucrose, and [¹³C₆^Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTS^Man or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.     

Principal-Component Analysis of the Characteristics Desirable in Baker's Yeasts

Twenty-seven properties considered to be required for good bakery products were examined in 56 industrial and 2 laboratory yeast strains. The data obtained were applied to principal-component analysis, one of the multivariate statistical analyses. The first and second principal components together were extracted, and these accounted for 77.7% of the variance. The first principal component was interpreted as the glycolytic activity of yeast in dough, and the second one was interpreted as the balance of leavening abilities in sweet and flour doughs from the factor loadings. The scattergram on the two principal components was effective in grouping the 58 yeast strains used.     

Quantitative Analysis of NAD Synthesis-Breakdown Fluxes

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Principal-Component Analysis of the Characteristics Desirable in Baker's Yeasts

Twenty-seven properties considered to be required for good bakery products were examined in 56 industrial and 2 laboratory yeast strains. The data obtained were applied to principal-component analysis, one of the multivariate statistical analyses. The first and second principal components together were extracted, and these accounted for 77.7% of the variance. The first principal component was interpreted as the glycolytic activity of yeast in dough, and the second one was interpreted as the balance of leavening abilities in sweet and flour doughs from the factor loadings. The scattergram on the two principal components was effective in grouping the 58 yeast strains used.     

Identification of Enzymes and Quantification of Metabolic Fluxes in the Wild Type and in a Recombinant Aspergillus oryzae Strain

Two α-amylase-producing strains of Aspergillus oryzae, a wild-type strain and a recombinant containing additional copies of the α-amylase gene, were characterized with respect to enzyme activities, localization of enzymes to the mitochondria or cytosol, macromolecular composition, and metabolic fluxes through the central metabolism during glucose-limited chemostat cultivations. Citrate synthase and isocitrate dehydrogenase (NAD) activities were found only in the mitochondria, glucose-6-phosphate dehydrogenase and glutamate dehydrogenase (NADP) activities were found only in the cytosol, and isocitrate dehydrogenase (NADP), glutamate oxaloacetate transaminase, malate dehydrogenase, and glutamate dehydrogenase (NAD) activities were found in both the mitochondria and the cytosol. The measured biomass components and ash could account for 95% (wt/wt) of the biomass. The protein and RNA contents increased linearly with increasing specific growth rate, but the carbohydrate and chitin contents decreased. A metabolic model consisting of 69 fluxes and 59 intracellular metabolites was used to calculate the metabolic fluxes through the central metabolism at several specific growth rates, with ammonia or nitrate as the nitrogen source. The flux through the pentose phosphate pathway increased with increasing specific growth rate. The fluxes through the pentose phosphate pathway were 15 to 26% higher for the recombinant strain than for the wild-type strain.     

Yeasts in Pucciniomycotina

Recent results in taxonomic, phylogenetic and ecological studies of basidiomycetous yeast research are remarkable. Here, Pucciniomycotina with yeast stages are reviewed. The phylogenetic origin of single-cell basidiomycetes still remains unsolved. But the massive occurrence of yeasts in basal basidiomycetous taxa indicates their early evolutionary presence. Yeasts in Cryptomycocolacomycetes, Mixiomycetes, Agaricostilbomycetes, Cystobasidiomycetes, Septobasidiales, Heterogastridiomycetes, and Microbotryomycetes will be discussed. The apparent loss of yeast stages in Tritirachiomycetes, Atractiellomycetes, Helicobasidiales, Platygloeales, Pucciniales, Pachnocybales, and Classiculomycetes will be mentioned briefly for comparative purposes with dimorphic sister taxa. Since most phylogenetic papers suffer considerably from the lack of adequate illustrations, plates for representative species of orders have been arranged. The structural qualities are the prerequisites for specific functions that cannot be expressed by phylogenetic dendrograms.     

The Carbon Assimilation Network in Escherichia coli Is Densely Connected and Largely Sign-Determined by Directions of Metabolic Fluxes

Gene regulatory networks consist of direct interactions but also include indirect interactions mediated by metabolites and signaling molecules. We describe how these indirect interactions can be derived from a model of the underlying biochemical reaction network, using weak time-scale assumptions in combination with sensitivity criteria from metabolic control analysis. We apply this approach to a model of the carbon assimilation network in Escherichia coli. Our results show that the derived gene regulatory network is densely connected, contrary to what is usually assumed. Moreover, the network is largely sign-determined, meaning that the signs of the indirect interactions are fixed by the flux directions of biochemical reactions, independently of specific parameter values and rate laws. An inversion of the fluxes following a change in growth conditions may affect the signs of the indirect interactions though. This leads to a feedback structure that is at the same time robust to changes in the kinetic properties of enzymes and that has the flexibility to accommodate radical changes in the environment.     

Predicting Growth Conditions from Internal Metabolic Fluxes in an In-Silico Model of E. coli

A widely studied problem in systems biology is to predict bacterial phenotype from growth conditions, using mechanistic models such as flux balance analysis (FBA). However, the inverse prediction of growth conditions from phenotype is rarely considered. Here we develop a computational framework to carry out this inverse prediction on a computational model of bacterial metabolism. We use FBA to calculate bacterial phenotypes from growth conditions in E. coli, and then we assess how accurately we can predict the original growth conditions from the phenotypes. Prediction is carried out via regularized multinomial regression. Our analysis provides several important physiological and statistical insights. First, we show that by analyzing metabolic end products we can consistently predict growth conditions. Second, prediction is reliable even in the presence of small amounts of impurities. Third, flux through a relatively small number of reactions per growth source (∼10) is sufficient for accurate prediction. Fourth, combining the predictions from two separate models, one trained only on carbon sources and one only on nitrogen sources, performs better than models trained to perform joint prediction. Finally, that separate predictions perform better than a more sophisticated joint prediction scheme suggests that carbon and nitrogen utilization pathways, despite jointly affecting cellular growth, may be fairly decoupled in terms of their dependence on specific assortments of molecular precursors.     

Occurrence and Growth of Yeasts in Yogurts

Yogurts purchased from retail outlets were examined for the presence of yeasts by being plated onto oxytetracycline malt extract agar. Of the 128 samples examined, 45% exhibited yeast counts above 10³ cells per g. A total of 73 yeast strains were isolated and identified as belonging to the genera Torulopsis, Kluyveromyces, Saccharomyces, Candida, Rhodotorula, Pichia, Debaryomyces, and Sporobolomyces. Torulopsis candida and Kluyveromyces fragilis were the most frequently isolated species, followed by Saccharomyces cerevisiae, Rhodotorula rubra, Kluyveromyces lactis, and Torulopsis versatilis. The growth of yeasts in yogurts was related to the ability of the yeasts to grow at refrigeration temperatures, to ferment lactose and sucrose, and to hydrolyze milk casein. Most yeast isolates grew in the presence of 100 μg of sorbate and benzoate preservatives per ml. Higher yeast counts from yogurts were obtained when the yogurts were plated onto oxytetracycline malt extract agar than when they were plated onto acidified malt extract agar.     

Yeasts in greenhouse environment

Summary The examination of a total of 98 yeast cultures collected from the buds, the open flowers and the host animals of 8 different species of plants and from the related soils of the vessels in a greenhouse, has demonstrated the presence of 13 different yeasts, including 3 sporogenous and 10 asporogenous species. The more frequent species were: Torulopsis bacillaris (Kr. et Krumbh.) Lodder (frequency 37.5%) and Candida reukaufii (Grüss) Diddens et Lodder (37.5%), on the open flowers. Rhodotorula mucilaginosa (Jörg.) Harrison (37.5%), and Rhodotorula glutinis (Fres.) Harrison var. rubescens (Saito) Lodder (25%), on the bud flowers. Torulopsis bacillaris (Kr. et Krumbh.) Lodder (50%) on the animals. Saccharomyces ellipsoideus Hansen (37.5%) and Pichia fermentans Lodder (37.5%), in the soils of the related vessels.The yeast like form Aureobasidium pullulans (De Bary) Arnaud, has 50% of frequency on the bud flowers and 12.5% on the open flowers.