Measuring non-steady-state metabolic fluxes in starch-converting faecal microbiota in vitro
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This paper explores human gut bacterial metabolism of starch using a combined analytical and computational modelling approach for metabolite and flux analysis. Non-steady-state isotopic labelling experiments were performed with human faecal microbiota in a well-established in vitro model of the human colon. After culture stabilisation, [U-13C] starch was added and samples were taken at regular intervals. Metabolite concentrations and 13C isotopomeric distributions were measured amongst other things for acetate, propionate and butyrate by mass spectrometry and NMR. The vast majority of metabolic flux analysis methods based on isotopomer analysis published to date are not applicable to metabolic non-steady-state experiments. We therefore developed a new ordinary differential equation-based representation of a metabolic model of human faecal microbiota to determine eleven metabolic parameters that characterised the metabolic flux distribution in the isotope labelling experiment. The feasibility of the model parameter quantification was demonstrated on noisy in silico data using a downhill simplex optimisation, matching simulated labelling patterns of isotopically labelled metabolites with measured metabolite and isotope labelling data. Using the experimental data, we determined an increasing net label influx from starch during the experiment from 94±1 µmol/l/min to 133±3 µmol/l/min. Only about 12% of the total carbon flux from starch reached propionate. Propionate production mainly proceeded via succinate with a small contribution via acrylate. The remaining flux from starch yielded acetate (35%) and butyrate (53%). Interpretation of 13C NMR multiplet signals further revealed that butyrate, valerate and caproate were mainly synthesised via cross-feeding, using acetate as a co-substrate. This study demonstrates for the first time that the experimental design and the analysis of the results by computational modelling allows the determination of time-resolved effects of nutrition on the flux distribution within human faecal microbiota in metabolic non-steady-state.
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Antoniewicz, M.R., Kelleher, J.K. and Stephanopoulos, G., 2007. Elementary metabolite units (EMU): a novel framework for modeling isotopic distributions. Metabolic Engineering 9: 68-86.
'Elementary metabolite units (EMU): a novel framework for modeling isotopic distributions ' () 9 Metabolic Engineering : 68 -86.
-
Backhed, F., Ley, R.E., Sonnenburg, J.L., Peterson, D.A. and Gordon, J.I., 2005. Host-bacterial mutualism in the human intestine. Science 307: 1915-1920.
'Host-bacterial mutualism in the human intestine ' () 307 Science : 1915 -1920.
-
Baxter, C.J., Liu, J.L., Fernie, A.R. and Sweetlove, L.J., 2007. Determination of metabolic fluxes in a non-steady-state system. Phytochemistry 68: 2313-2319.
'Determination of metabolic fluxes in a non-steady-state system ' () 68 Phytochemistry : 2313 -2319.
-
Binsl, T.W., Alders, D.J.C., Heringa, J., Groeneveld, A.B.J. and Van Beek, J.H.G.M., 2010. Computational quantification of metabolic fluxes from a single isotope snapshot: application to an animal biopsy. Bioinformatics 26: 653-660.
'Computational quantification of metabolic fluxes from a single isotope snapshot: application to an animal biopsy ' () 26 Bioinformatics : 653 -660.
-
Carroll, J.J. and Mather, A.E., 1992. The system carbon dioxide-water and the Krichevsky-Kasarnovsky equation. Journal of Solution Chemistry 21: 607-621.
'The system carbon dioxide-water and the Krichevsky-Kasarnovsky equation ' () 21 Journal of Solution Chemistry : 607 -621.
-
Chance, E.M., Seeholzer, S.H., Kobayashi, K. and Williamson, J.R., 1983. Mathematical analysis of isotope labeling in the citric acid cycle with applications to 13C NMR studies in perfused rat hearts. Journal of Biological Chemistry 258: 13785-13794.
'Mathematical analysis of isotope labeling in the citric acid cycle with applications to 13C NMR studies in perfused rat hearts ' () 258 Journal of Biological Chemistry : 13785 -13794.
-
Counotte, G.H.M., Prins, R.A., Janssen, R.H.A.M. and De Bie, M.J.A., 1981. Role of Megasphaera elsdenii in the fermentation of DL-[2-13C] in the rumen of dairy cattle. Applied and Environmental Microbiology 42: 649-655.
-
De Graaf, A.A., 2006. Use of 13C labelling and NMR spectroscopy in metabolic flux analysis. In: Barbotin, J.-N. and Portais, J.-C. (eds.) NMR in microbiology: theory and application. Horizon Scientific Press, Norwich, UK, pp. 73-104.
-
De Graaf, A.A. and Venema, K., 2007. Gaining insight into microbial physiology in the large intestine: a special role for stable isotopes. Advances in Microbial Physiology 53: 73-168; 313-314.
-
De Graaf, A.A., Maathuis, A., De Waard, P., Deutz, N.E.P., Dijkema, C., De Vos, W.M. and Venema, K., 2010. Profiling human gut bacterial metabolism and its kinetics using [U-13C]glucose and NMR. NMR in Biomedicine 23: 2-12.
-
Egert, M., De Graaf, A.A., Smidt, H., De Vos, W.M. and Venema, K., 2006. Beyond diversity: functional microbiomics of the human colon. Trends in Microbiology 14: 86-91.
'Beyond diversity: functional microbiomics of the human colon ' () 14 Trends in Microbiology : 86 -91.
-
Leclerc, M., Bernalier, A., Lelait, M. and Grivet, J.P., 1996. 13C-NMR study of glucose and pyruvate catabolism in four acetogenic species isolated from the human colon. FEMS Microbiology Letters 146: 199-204.
'13C-NMR study of glucose and pyruvate catabolism in four acetogenic species isolated from the human colon ' () 146 FEMS Microbiology Letters : 199 -204.
-
McGarr, S.E., Ridlon, J.M. and Hylemon, P.B., 2005. Diet, anaerobic bacterial metabolism, and colon cancer: a review of the literature. Journal of Clinical Gastroenterology 39: 98-109.
'Diet, anaerobic bacterial metabolism, and colon cancer: a review of the literature ' () 39 Journal of Clinical Gastroenterology : 98 -109.
-
Meesters, R.J.W., Van Eijk, H.M.H., Ten Have, G.A.M., De Graaf, A.A., Venema, K., Van Rossum, B.E.J. and Deutz, N.E.P., 2007. Application of liquid chromatography-mass spectrometry to measure the concentrations and study the synthesis of short chain fatty acids following stable isotope infusions. Journal of Chromatography B 854: 57-62.
'Application of liquid chromatography-mass spectrometry to measure the concentrations and study the synthesis of short chain fatty acids following stable isotope infusions ' () 854 Journal of Chromatography B : 57 -62.
-
Minekus, M., Smeets-Peeters, M., Bernalier, A., Marol-Bonnin, S., Havenaar, R., Marteau, P., Alric, M., Fonty, G. and In 't Veld, J.H.H., 1999. A computer controlled system to simulate conditions of the large intestine with peristaltic mixing, water absorption and absorption of fermentation products. Applied Microbiology and Biotechnology 53: 108-114.
'A computer controlled system to simulate conditions of the large intestine with peristaltic mixing, water absorption and absorption of fermentation products ' () 53 Applied Microbiology and Biotechnology : 108 -114.
-
Nöh, K. and Wiechert, W., 2006. Experimental design principles for isotopically instationary 13C labeling experiments. Biotechnology and Bioengineering 10: 233-251.
'Experimental design principles for isotopically instationary 13C labeling experiments ' () 10 Biotechnology and Bioengineering : 233 -251.
-
Petersen, S., De Graaf, A.A., Eggeling, L., Möllney, M., Wiechert, W. and Sahm, H., 2000. In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum. Journal of Biological Chemistry 275: 35932-35941.
'In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum ' () 275 Journal of Biological Chemistry : 35932 -35941.
-
Pryde, E.S., Duncan, S.H., Hold, G.L., Stewart, C.S. and Flint, H.J., 2002. The microbiology of butyrate formation in the human colon. FEMS Microbiology Letters 217: 133-139.
'The microbiology of butyrate formation in the human colon ' () 217 FEMS Microbiology Letters : 133 -139.
-
Van Beek, J.H.G.M., Csont, T., De Kanter, F.J.J. and Bussemaker, J., 1998. Simple model analysis of 13C NMR spectra to measure oxygen consumption using frozen tissue samples. Advances in Experimental Medicine and Biology 454: 475-485.
'Simple model analysis of 13C NMR spectra to measure oxygen consumption using frozen tissue samples ' () 454 Advances in Experimental Medicine and Biology : 475 -485.
-
Van Beek, J.H.G.M., Van Mil, H., King, R., De Kanter, F., Alders, D.J.C. and Bussemaker, J., 1999. A 13C NMR double-labeling method to quantitate local myocardial O2 NMR consumption using frozen tissue samples. American Journal of Physiology -Heart and Circulatory Physiology 277: H1630-H1640.
'A 13C NMR double-labeling method to quantitate local myocardial O2 NMR consumption using frozen tissue samples ' () 277 American Journal of Physiology -Heart and Circulatory Physiology : H1630 -H1640.
-
Van Nuenen, M.H.M.C., Meyer, P.D. and Venema, K., 2003. The effect of various inulins and Clostridium difficile on the metabolic activity of the human colonic microbiota in vitro. Microbial Ecology in Health and Disease 15: 137-144.
'The effect of various inulins and Clostridium difficile on the metabolic activity of the human colonic microbiota in vitro ' () 15 Microbial Ecology in Health and Disease : 137 -144.
-
Van Winden, W.A., Heijnen, J.J. and Verheijen, P.J.T., 2002. Cumulative bondomers: a new concept in flux analysis from 2D [13C,1H] COSY NMR data. Biotechnology and Bioengineering 80: 731-745.
-
Venema, K., Van Nuenen, M.H.M.C., Van den Heuvel, E.G., Pool, W. and Van der Vossen, J.M.B.M., 2003. The effect of lactulose on the composition of the intestinal microbiota and short-chain fatty acid production in human volunteers and a computer-controlled model of the proximal large intestine. Microbial Ecology in Health and Disease 15: 94-105.
'The effect of lactulose on the composition of the intestinal microbiota and short-chain fatty acid production in human volunteers and a computer-controlled model of the proximal large intestine ' () 15 Microbial Ecology in Health and Disease : 94 -105.
-
Wiechert, W. and De Graaf, A.A., 1997. Bidirectional reaction steps in metabolic networks: I. modeling and simulation of carbon isotope labeling experiments. Biotechnology and Bioengineering 55: 101-117.
'Bidirectional reaction steps in metabolic networks: I ' () 55 modeling and simulation of carbon isotope labeling experiments. Biotechnology and Bioengineering : 101 -117.
-
Wiechert, W., Möllney, M., Isermann, N., Wurzel, M. and De Graaf, A.A., 1999. Bidirectional reaction steps in metabolic networks: III. explicit solution and analysis of isotopomer labeling systems. Biotechnology and Bioengineering 66: 71-85.
'Bidirectional reaction steps in metabolic networks: III ' () 66 explicit solution and analysis of isotopomer labeling systems. Biotechnology and Bioengineering : 71 -85.
Supplementary Materials
| å ¨é¨æé´ | è¿å»ä¸å¹´ | è¿å»30天 | |
|---|---|---|---|
| æè¦æµè§æ¬¡æ° | 466 | 219 | 20 |
| å ¨ææµè§æ¬¡æ° | 21 | 3 | 0 |
| PDFä¸è½½æ¬¡æ° | 24 | 0 | 0 |
Measuring non-steady-state metabolic fluxes in starch-converting faecal microbiota in vitro
äºBeneficial MicrobesSearch for other papers by T. Binsl in
Current site
Google Scholar
PubMed
Search for other papers by A. De Graaf in
Current site
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PubMed
Search for other papers by K. Venema in
Current site
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Search for other papers by J. Heringa in
Current site
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Search for other papers by A. Maathuis in
Current site
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PubMed
Search for other papers by P. De Waard in
Current site
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PubMed
Search for other papers by J.H. Van Beek in
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PubMed
This paper explores human gut bacterial metabolism of starch using a combined analytical and computational modelling approach for metabolite and flux analysis. Non-steady-state isotopic labelling experiments were performed with human faecal microbiota in a well-established in vitro model of the human colon. After culture stabilisation, [U-13C] starch was added and samples were taken at regular intervals. Metabolite concentrations and 13C isotopomeric distributions were measured amongst other things for acetate, propionate and butyrate by mass spectrometry and NMR. The vast majority of metabolic flux analysis methods based on isotopomer analysis published to date are not applicable to metabolic non-steady-state experiments. We therefore developed a new ordinary differential equation-based representation of a metabolic model of human faecal microbiota to determine eleven metabolic parameters that characterised the metabolic flux distribution in the isotope labelling experiment. The feasibility of the model parameter quantification was demonstrated on noisy in silico data using a downhill simplex optimisation, matching simulated labelling patterns of isotopically labelled metabolites with measured metabolite and isotope labelling data. Using the experimental data, we determined an increasing net label influx from starch during the experiment from 94±1 µmol/l/min to 133±3 µmol/l/min. Only about 12% of the total carbon flux from starch reached propionate. Propionate production mainly proceeded via succinate with a small contribution via acrylate. The remaining flux from starch yielded acetate (35%) and butyrate (53%). Interpretation of 13C NMR multiplet signals further revealed that butyrate, valerate and caproate were mainly synthesised via cross-feeding, using acetate as a co-substrate. This study demonstrates for the first time that the experimental design and the analysis of the results by computational modelling allows the determination of time-resolved effects of nutrition on the flux distribution within human faecal microbiota in metabolic non-steady-state.
| å ¨é¨æé´ | è¿å»ä¸å¹´ | è¿å»30天 | |
|---|---|---|---|
| æè¦æµè§æ¬¡æ° | 466 | 219 | 20 |
| å ¨ææµè§æ¬¡æ° | 21 | 3 | 0 |
| PDFä¸è½½æ¬¡æ° | 24 | 0 | 0 |
