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Application of 13C-[2] - and 13C-[1,2] acetate in metabolic labelling studies of yeast and insect cells

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Abstract

The advantage of using 13C-labelled glucose in metabolic studies is that it is an important carbon and energy source for almost all biotechnologically and medically important organisms. On the other hand, the disadvantage is its relatively high cost in the labelling experiments. Looking for cheaper alternatives we found that 13C-[2] acetate or 13C-[1,2] acetate is a prospective compound for such experiments. Acetate is well incorporated by many organisms, including mammalian and insect cell cultures as preferred source of acetyl-CoA. Our experimental results using 13C NMR demonstrated that acetate was efficiently incorporated into glutamate and alanine secreted by the insect cell culture. Using D-stat culture of Saccharomyces uvarum on glucose/13C-acetate mineral media we demonstrated that the labelling patterns of proteinogenic amino acids can be well predicted on the basis of specific substrate consumption rates using the modified scheme of yeast metabolism and stoichiometric modelling. According to this scheme aspartate and alanine in S. uvarum under the experimental conditions used is synthesised in the mitochondria. Synthesis of alanine in the mitochondria was also demonstrated for Spodoptera frugiperda. For both organisms malic enzyme was also operative. For S. uvarum it was shown that the activity of malic enzyme is sufficient for supporting the mitochondrial biosynthetic reactions with NADPH.

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Abbreviations

BDF:

biosynthetically directed fractional labelling

FCS:

foetal calf serum

IEAM:

yeast extract free medium

MEM:

minimal essential medium

MFA:

metabolic flux analysis

oxac :

cytosolic oxaloacetate

oxam :

mitochondrial oxaloacetate

PP pathway:

pentose phosphate pathway

Pyrc :

cytosolic pyruvate

Pyrm :

mitochondrial pyruvate

TCA:

tricarboxylic acid

References

  • Adamberg K., Kask S., Laht T.M. and Paalme T. (2003). The effect of temperature and pH on the growth of lactic acid bacteria: a pH-auxostat study. Int. J. Food Microbiol. 85:171–183

    Article  PubMed  CAS  Google Scholar 

  • Barton J.K., Den Hollander J.A., Hopfield J.J. and Shulman R.G. (1982). 13C Nuclear magnetic resonance study of trehalose mobilization in yeast spores. J. Bacteriol. 151:177–185

    PubMed  CAS  Google Scholar 

  • Bedard C., Tom R. and Kamen A. (1993). Growth, nutrient consumption, and end-product accumulation in Sf-9 and BTI-EAA insect cell cultures: insights into growth limitation and metabolism. Biotechnol. Prog. 9:615–624

    Article  PubMed  CAS  Google Scholar 

  • Cannizzaro C., Christensen B., Nielsen J. and Von Stockar U.(2004). Metabolic network analysis on Phaffia rhodozyma yeast using 13C-labeled glucose and gas chromatography-mass spectrometry. Metab. Eng. 6:340–351

    Article  PubMed  CAS  Google Scholar 

  • Chance E., Seeholzer S., Kobayashi K. and Williamson J. (1983). Mathematical analysis of isotope labeling in the citric acid cycle with applications to 13C NMR studies in perfused rat harts. J. Biol. Chem. 258:13785–13794

    PubMed  CAS  Google Scholar 

  • Christensen B. and Nielsen J. (1999). Metabolic network analysis. A powerful tool in metabolic engineering. Adv. Biochem. Eng. Biotechnol. 66:210–231

    Google Scholar 

  • Christensen B., Gombert A.K. and Nielsen J. (2002). Analysis of flux estimates based on 13C-labelling experiments. Eur. J. Biochem. 269:2795–2800

    Article  PubMed  CAS  Google Scholar 

  • Cortassa S., Aon J.C. and Aon M.A. (1995). Fluxes of carbon, phosphorylation, and redox intermediates during growth of Saccharomyces cerevisiae on different carbon sources. Biotechnol. Bioeng. 47:193–208

    Article  CAS  PubMed  Google Scholar 

  • Dallies N., François J. and Paquet V. (1998). A New method for quantitative determination of polysaccharides in the yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae. Yeast 14:1297–1306

    Article  PubMed  CAS  Google Scholar 

  • Dauner M. and Sauer U. (2001). Stoichiometric growth model for riboflavin-producing Bacillus subtilis. Biotechnol. Bioeng. 76:132–143

    Article  PubMed  CAS  Google Scholar 

  • DeLuna A., Avendaño A., Riego L. and González A. (2001). NADP-Glutamate Dehydrogenase Isoenzymes of Saccharomyces cerevisiae. J. Biol. Chem. 276(47):43775–43783

    Article  PubMed  CAS  Google Scholar 

  • DeLuna A., Qezada H., Gómez-Puyou A., González A. (2005). Asparaginyl deamidation in two glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae. Biochem. Biophys. Res. Comm. 328:1083–1090

    Article  PubMed  CAS  Google Scholar 

  • Dickinson J.R., Dawes I.W., Boyd A.S. and Baxter R.L. (1983). 13C NMR studies of acetate metabolism during sporulation of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 80(19):5847–51

    Article  PubMed  CAS  ADS  Google Scholar 

  • Dos Santos M.M., Gombert A.K., Christensen B., Olsson L. and Nielsen J. (2003). Identification of in vivo enzyme activities in the cometabolism of glucose and acetate by Saccharomyces cerevisiae by using 13C-labeled substrates. Eucaryotic Cell 2(3):599–608

    Article  CAS  Google Scholar 

  • Dos Santos M.M., Raghevendran V., Kötter P., Olsson L. and Nielsen J. (2004). Manipulation of malic enzyme in Saccharomyces cerevisiae for increasing production capacity aerobically in different cellular compartments. Metab. Eng. 6:352–363

    Article  CAS  Google Scholar 

  • Doverskog M., Jacobsson U., Chapman B.E., Kuchel P. and Häggström L. (2000). Determination of NADH-dependent glutamate synthase (GOGAT) in Spodoptera frugiperda (Sf9) insect cells by a selective 1H/15N NMR in vitro assay. J. Biotechnol. Apr 14;79(1):87–97

    Article  PubMed  CAS  Google Scholar 

  • Drews M., Doverskog M., Öhman L., Chapman B.E., Jacobsson U., Kuchel P.W. and Häggström L. (2000). Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by 1H/15N NMR. J. Biotechnol. 78:23–37

    Article  PubMed  CAS  Google Scholar 

  • Drews, M., Nisamedtinov, I. and Paalme, T. 2003. Application of quasi-steady-state cultures. In: Proceedings of the 1st International Congress on Bioreactor Technology in Cell, Tissue Culture and Biomedical Applications. BioBien Innovations, Piikiö, Finland, pp. 218–225

  • Falco S.C., Dumas K.S. and Livak K.J. (1985). Nucleotide sequence of the yeast ILV2 gene which encodes acetolactate synthase. Nucleic Acids Res. 13(11):4011–4027

    PubMed  CAS  Google Scholar 

  • Ferreira J.C., Thevelein J.M., Hohmann S., Paschoalin V.M.F., Trugo L.C. and Panek A.D. (1997). Biochim. Biophys. Acta 1335:40–50

    PubMed  CAS  Google Scholar 

  • Gerhardt P., Murray R.G.E., Wood W.A. and Krieg N.R. (1994). Methods for General and Molecular Bacteriology. American Society for Mirobiology, Washington, D.C

    Google Scholar 

  • Haselbeck R.J. and McAlister-Henn L. (1991). Isolation, nucleotide sequence, and disruption of the Saccharomyces cerevisiae gene encoding mitochondrial NADP(H)-specific isocitrate dehydrogenase. J. Biol. Chem. 266(4):2339–2345

    PubMed  CAS  Google Scholar 

  • Huh W.K., Falvo J.V., Gerke L.C., Carroll A.S., Howson R.W., Weissman J.S. and O’Shea E.K. (2003). Global analysis of protein localization in budding yeast. Nature 425(6959):686–691

    Article  PubMed  CAS  ADS  Google Scholar 

  • Kasemets K., Drews M., Nisamedtinov I., Adamberg K. and Paalme T. (2003). Modification of A-stat for the characterization of microorganisms. J. Microbiol. Methods 55(1):187–200

    Article  PubMed  CAS  Google Scholar 

  • Kumar A., Agarwal S., Heyman J.A., Matson S., Heidtman M., Piccirillo S., Umansky L., Drawid A., Jansen R., Liu Y., Cheung K.H., Miller P., Gerstein M., Roeder G.S. and Snyder M. (2002). Subcellular localization of the yeast proteome. Genes Dev. 16(6):707–719

    Article  PubMed  CAS  Google Scholar 

  • Kylie F., Mackenzie K., Singh K. and Brown A.D. (1987). Water stress plating hypersensitivity of yeasts: protective role of trehalose in Saccharomyces cerevisae. J. Gen. Microbiol. 134:1661–1666

    Google Scholar 

  • Kyoto Encyclopedia of Genes and Genomes (http://www.kegg. com/)

  • Maaheimo H., Fiaux J., Çakar Z.P., Bailey J. E., Sauer U. and Szyperski T. (2001). Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic 13C labeling of common amino acids. Eur. J. Biochem. 268:2464–2479

    Article  PubMed  CAS  Google Scholar 

  • Magasanik, B. 2003. Ammonia Assimilation by Saccharomyces cerevisiae. Eukaryotic cell. Oct.: 827–829

  • Michal G. (1998). Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology. Wiley, New York

    Google Scholar 

  • Paalme T., Olivson A. and Vilu R. (1982). 13C-NMR study of CO2-fixation during the heterotrophic growth in Chlorobium thiosulfatophilum. Biochim. Biophys. Acta 782:311–319

    Google Scholar 

  • Paalme T., Olivson A. and Vilu R. (1982). 13C-NMR study of the glucose synthesis pathways in the bacterium Chlorobium thiosulfatophilum. Biochim. Biophys. Acta 720:303–310

    Article  CAS  Google Scholar 

  • Paalme T., Elken R., Vilu R. and Korhola M. (1997). The growth efficiency of Saccharomyces cerevisiae on glucose/ethanol media with smooth change in the dilution rate (A-stat). Enzyme Microb. Technol. 20:174–181

    Article  CAS  Google Scholar 

  • Pang S.S. and Duggleby R.G. (1999). Expression, purification, characterization, and reconstitution of the large and small subunits of yeast acetohydroxyacid synthase. Biochemistry 38(16):5222–5231

    Article  PubMed  CAS  Google Scholar 

  • Radin N.S. (1981). Extraction of tissue lipids with a solvent of low toxicity. Methods Enzymol. 72:5–7

    Article  PubMed  CAS  Google Scholar 

  • Saccharomyces Genome Database (http://www.yeastgenome.-org/)

  • Sickmann A., Reinders J., Wagner Y., Joppich C., Zahedi R., Meyer H.E., Schonfisch B., Perschil I., Chacinska A., Guiard B., Rehling P., Pfanner N. and Meisinger C. (2003). The proteome of Saccharomyces cerevisiae mitochondria. Proc. Natl. Acad. Sci. USA 100(23):13207–13212

    Article  PubMed  CAS  ADS  Google Scholar 

  • Szyperski T., Bailey J.E. and Wütrich K. (1996). Detecting and dissecting metabolic fluxes using biosynthetic fractional 13C labeling and two-dimensional NMR spectroscopy. Tibtech. 14:453–458

    Google Scholar 

  • Tran-Dinh S., Fermendjian S., Sala E., Mermet-Bouvier R., Cohen M. and Fromageot P. (1973). 13C-Enriched amino acids. Chemical shifts, coupling constants J c–c and conformation. J. Am. Chem. Soc. 96(5):1484–1492

    Article  Google Scholar 

  • Tran-Dinh S., Bouet F., Huynh Q. and Herve M. (1996). Mathematical models for determining metabolic fluxes through the citric acid and glyoxylate cycles in Saccharomyces cerevisiae by 13C-NMR spectroscopy. Eur. J. Biochem. 242:770–778

    Article  PubMed  CAS  Google Scholar 

  • Vallino J.J. and Stephanopoulos G. (1993). Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction. Biotechnol. Bioeng. 41:633–646

    Article  CAS  PubMed  Google Scholar 

  • Varma A. and Palsson P.O. (1994). Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110. Appl. Environ. Microbiol. 60(10):3724–3731

    PubMed  CAS  Google Scholar 

  • Vilu, R., Paalme, T. and Vanatalu, K. 1990. In: Directed Design of Cells-Producers (in Russian). Microbial Conversion, Riga, pp. 14–21

  • Walker T.E., Han C.H., Kollman V.H., London R.E. and Matwiyoff N.A. (1982). 13C Nuclear magnetic resonance studies of the biosynthesis by Microbacterium ammoniaphilum of L-glutamate selectively enriched with carbon-13. J. Biol. Chem. 3:1189–1195

    Google Scholar 

  • Weiss S.A., Smith G.C., Kalter S.S. and Vaughn J.L. (1981). Improved method for the production of insect cell cultures in large volume. In Vitro. 17 (6):495–502

    CAS  Google Scholar 

  • Wiechert W. and De Graaf A. (1996). Bidirectional reaction steps in metabolic networks: I. Modeling and simulation of carbon isotope labeling experiments. Biotechnol. Bioeng. 55:101–117

    Article  Google Scholar 

  • Wiechert W. (2001). 13C metabolic flux analysis. Metab. Eng. 3:195–206

    Article  PubMed  CAS  Google Scholar 

  • Wütrich K. (1976). NMR in Biological Research: Peptides and Proteins. North-Holland, American Elsevier

    Google Scholar 

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Acknowledgements

This work was founded through Estonian Science Foundation (Grant Nos. 5129 and 5160).

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Authors and Affiliations

  1. Tallinn University of Technology, Ehitajate tee 5, 19086, Tallinn, Estonia

    Toomas Paalme, Ildar Nisamedtinov, Kristo Abner, Tiiu-Mai Laht &  Monika Drews

  2. National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618, Tallinn, Estonia

    Tõnis Pehk

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  1. Toomas Paalme
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  2. Ildar Nisamedtinov
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  4. Tiiu-Mai Laht
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  6. Tõnis Pehk
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Corresponding author

Correspondence to Ildar Nisamedtinov.

Appendices

Appendix A

Table 1 Flux equations for the key reactions according to the scheme shown in Figure 1 in S. uvarum 1.
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Appendix B

Table 2 Labeling equations of the key metabolic intermediates according to the model in S. uvarum 1.
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Paalme, T., Nisamedtinov, I., Abner, K. et al. Application of 13C-[2] - and 13C-[1,2] acetate in metabolic labelling studies of yeast and insect cells. Antonie Van Leeuwenhoek 89, 443–457 (2006). https://doi.org/10.1007/s10482-005-9053-7

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