Kinetic isotope effects significantly influence intracellular metabolite [superscript 13]C labeling patterns and flux determination

Rigorous 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 (13) C labeling data have commonly neglected the influence of kinetic isotope effects on the distribution of (13) C label in intracellular metabolites, as these effects have often been assumed to be inconsequential. We have used measurements of the (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 (13) C labeling patterns of intracellular metabolites, and the errors associated with neglecting isotope effects in (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 (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.

[1]  H. Schmidt,et al.  Carbon isotope effects on the pyruvate dehydrogenase reaction and their importance for relative carbon-13 depletion in lipids. , 1987, The Journal of biological chemistry.

[2]  W. Cleland,et al.  Use of multiple isotope effects to study the mechanism of 6-phosphogluconate dehydrogenase. , 1984, Biochemistry.

[3]  W. Wiechert,et al.  Bidirectional reaction steps in metabolic networks: I. Modeling and simulation of carbon isotope labeling experiments. , 1997, Biotechnology and bioengineering.

[4]  H Sahm,et al.  Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing , 1996, Biotechnology and bioengineering.

[5]  B. Christensen,et al.  Isotopomer analysis using GC-MS. , 1999, Metabolic engineering.

[6]  J. Hayes,et al.  Carbon isotopic fractionation in the biosynthesis of bacterial fatty acids. Ozonolysis of unsaturated fatty acids as a means of determining the intramolecular distribution of carbon isotopes , 1982 .

[7]  M. J. Deniro,et al.  Mechanism of carbon isotope fractionation associated with lipid synthesis. , 1977, Science.

[8]  J Villadsen,et al.  Quantification of intracellular metabolic fluxes from fractional enrichment and 13C-13C coupling constraints on the isotopomer distribution in labeled biomass components. , 1999, Metabolic engineering.

[9]  W Wiechert,et al.  Bidirectional reaction steps in metabolic networks: IV. Optimal design of isotopomer labeling experiments. , 1999, Biotechnology and bioengineering.

[10]  H. Schmidt.  Fundamentals and systematics of the non-statistical distributions of isotopes in natural compounds , 2003, Naturwissenschaften.

[11]  Jörg Schwender,et al.  Metabolic flux analysis as a tool in metabolic engineering of plants. , 2008, Current opinion in biotechnology.

[12]  W. Cleland,et al.  Oxidative decarboxylation of 6-phosphogluconate by 6-phosphogluconate dehydrogenase proceeds by a stepwise mechanism with NADP and APADP as oxidants. , 1998, Biochemistry.

[13]  C. R. Benedict,et al.  Carbon isotope discrimination in a plant possessing the C4 dicarboxylic acid pathway. , 1970, Biochemical and biophysical research communications.

[14]  P. Abelson,et al.  Carbon isotope fractionation in formation of amino acids by photosynthetic organisms. , 1961, Proceedings of the National Academy of Sciences of the United States of America.

[15]  U. Sauer,et al.  Metabolic fluxes in riboflavin-producing Bacillus subtilis , 1997, Nature Biotechnology.

[16]  U. Sauer,et al.  Large-scale 13C-flux analysis reveals mechanistic principles of metabolic network robustness to null mutations in yeast , 2005, Genome Biology.

[17]  Christoph Wittmann,et al.  Genealogy Profiling through Strain Improvement by Using Metabolic Network Analysis: Metabolic Flux Genealogy of Several Generations of Lysine-Producing Corynebacteria , 2002, Applied and Environmental Microbiology.

[18]  Gregory Stephanopoulos,et al.  Expanding the concepts and tools of metabolic engineering to elucidate cancer metabolism , 2012, Biotechnology progress.

[19]  Gregory Stephanopoulos,et al.  Determination of confidence intervals of metabolic fluxes estimated from stable isotope measurements. , 2006, Metabolic engineering.

[20]  Gregory Stephanopoulos,et al.  Evaluation of 13C isotopic tracers for metabolic flux analysis in mammalian cells. , 2009, Journal of biotechnology.

[21]  G. Gleixner,et al.  Carbon Isotope Effects on the Fructose-1,6-bisphosphate Aldolase Reaction, Origin for Non-statistical 13C Distributions in Carbohydrates* , 1997, The Journal of Biological Chemistry.

[22]  Gregory Stephanopoulos,et al.  Accurate assessment of amino acid mass isotopomer distributions for metabolic flux analysis. , 2007, Analytical chemistry.

[23]  G. Stephanopoulos,et al.  Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction , 2000, Biotechnology and bioengineering.

[24]  Christoph Wittmann,et al.  Analysis of 13C labeling enrichment in microbial culture applying metabolic tracer experiments using gas chromatography-combustion-isotope ratio mass spectrometry. , 2008, Analytical biochemistry.

[25]  W. Cleland,et al.  Use of multiple isotope effects to determine enzyme mechanisms and intrinsic isotope effects. Malic enzyme and glucose-6-phosphate dehydrogenase. , 1982, Biochemistry.

[26]  Scott B. Crown,et al.  Rational design of 13C-labeling experiments for metabolic flux analysis in mammalian cells , 2012, BMC Systems Biology.

[27]  U. Sauer,et al.  Article number: 62 REVIEW Metabolic networks in motion: 13 C-based flux analysis , 2022 .

[28]  Jens Nielsen,et al.  Analysis of flux estimates based on (13)C-labelling experiments. , 2002, European journal of biochemistry.

[29]  W. Sackett,et al.  Enzymatic fractionation of carbon isotopes by phosphoenolpyruvate carboxylase from c(4) plants. , 1973, Plant physiology.

[30]  T. Szyperski.  Biosynthetically Directed Fractional 13C‐labeling of Proteinogenic Amino Acids , 1995 .

[31]  G. Stephanopoulos,et al.  Metabolic flux analysis in a nonstationary system: fed-batch fermentation of a high yielding strain of E. coli producing 1,3-propanediol. , 2007, Metabolic engineering.

[32]  C. Hwang,et al.  Multiple isotope effects as a probe of proton and hydride transfer in the 6-phosphogluconate dehydrogenase reaction. , 1998, Biochemistry.

[33]  Elmar Heinzle,et al.  13C metabolic flux analysis for larger scale cultivation using gas chromatography-combustion-isotope ratio mass spectrometry. , 2010, Metabolic engineering.

[34]  E. Heinzle,et al.  Mass spectrometry for metabolic flux analysis. , 1999, Biotechnology and bioengineering.

[35]  W. Wiechert,et al.  Bidirectional reaction steps in metabolic networks: II. Flux estimation and statistical analysis. , 1997, Biotechnology and bioengineering.

[36]  J. Heijnen,et al.  Metabolic-flux analysis of Saccharomyces cerevisiae CEN.PK113-7D based on mass isotopomer measurements of (13)C-labeled primary metabolites. , 2005, FEMS yeast research.

[37]  J. Nielsen,et al.  It Is All about MetabolicFluxes , 2003, Journal of bacteriology.

[38]  W. Cleland,et al.  Isotope effect studies of the chemical mechanism of pig heart NADP isocitrate dehydrogenase. , 1988, Biochemistry.

[39]  U. Sauer,et al.  GC‐MS Analysis of Amino Acids Rapidly Provides Rich Information for Isotopomer Balancing , 2000, Biotechnology progress.

[40]  J. Nielsen,et al.  Quantitative analysis of metabolic fluxes in Escherichia coli, using two-dimensional NMR spectroscopy and complete isotopomer models. , 1999, Journal of biotechnology.

[41]  Uwe Sauer,et al.  Transcriptional regulation of respiration in yeast metabolizing differently repressive carbon substrates , 2010, BMC Systems Biology.

[42]  W. Wiechert,et al.  Bidirectional reaction steps in metabolic networks: III. Explicit solution and analysis of isotopomer labeling systems. , 1999, Biotechnology and bioengineering.

[43]  J. Nielsen,et al.  Network Identification and Flux Quantification in the Central Metabolism of Saccharomyces cerevisiae under Different Conditions of Glucose Repression , 2001, Journal of bacteriology.

[44]  U. Sauer,et al.  Large-scale in vivo flux analysis shows rigidity and suboptimal performance of Bacillus subtilis metabolism , 2005, Nature Genetics.

[45]  G. Stephanopoulos,et al.  Elementary metabolite units (EMU): a novel framework for modeling isotopic distributions. , 2007, Metabolic engineering.

[46]  U. Sauer,et al.  Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS. , 2003, European journal of biochemistry.

[47]  Xiao-Jiang Feng,et al.  Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy , 2008, Nature Biotechnology.

[48]  S. Epstein,et al.  Metabolic fractionation of C13 & C12 in plants , 1961 .

[49]  T. Szyperski.  Biosynthetically directed fractional 13C-labeling of proteinogenic amino acids. An efficient analytical tool to investigate intermediary metabolism. , 1995, European journal of biochemistry.

[50]  Y. Shachar-Hill,et al.  Metabolic flux analysis in plants: coping with complexity. , 2009, Plant, cell & environment.

[51]  P. Verheijen,et al.  Possible pitfalls of flux calculations based on (13)C-labeling. , 2001, Metabolic engineering.