Glucose-methanol co-utilization in Pichia pastoris studied by metabolomics and instationary 13C flux analysis

BMC Systems Biology

Aljoscha Wahl¹  Joan Albiol²  Pau Ferrer²  Walter van Gulik¹  Joseph J Heijnen¹  Angela ten Pierick¹  Marc Carnicer³  Camilo Suarez¹  Joel Jordಠ

[1] Department of Biotechnology, Kluyver Centre for Genomics of Industrial Fermentation, Delft University of Technology, Delft, The Netherlands;Department of Chemical Engineering, Escola d’Enginyeria, Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallès, Spain;Present address: Laboratoire Ingénierie des Systèmes Biologiques et des procédés, INSA, Toulouse, France

关键词: LC-MS;    GC-MS;    Instationary ¹³C-metabolic flux analysis;    Pichia pastoris;   

Others  :  1143040
DOI  :  10.1186/1752-0509-7-17

 received in 2012-08-01, accepted in 2013-02-15,  发布年份 2013

【 摘 要 】

Background

Several studies have shown that the utilization of mixed carbon feeds instead of methanol as sole carbon source is beneficial for protein production with the methylotrophic yeast Pichia pastoris. In particular, growth under mixed feed conditions appears to alleviate the metabolic burden related to stress responses triggered by protein overproduction and secretion. Yet, detailed analysis of the metabolome and fluxome under mixed carbon source metabolizing conditions are missing. To obtain a detailed flux distribution of central carbon metabolism, including the pentose phosphate pathway under methanol-glucose conditions, we have applied metabolomics and instationary ¹³C flux analysis in chemostat cultivations.

Results

Instationary ¹³C-based metabolic flux analysis using GC-MS and LC-MS measurements in time allowed for an accurate mapping of metabolic fluxes of glycolysis, pentose phosphate and methanol assimilation pathways. Compared to previous results from NMR-derived stationary state labelling data (proteinogenic amino acids, METAFoR) more fluxes could be determined with higher accuracy. Furthermore, using a thermodynamic metabolic network analysis the metabolite measurements and metabolic flux directions were validated. Notably, the concentration of several metabolites of the upper glycolysis and pentose phosphate pathway increased under glucose-methanol feeding compared to the reference glucose conditions, indicating a shift in the thermodynamic driving forces. Conversely, the extracellular concentrations of all measured metabolites were lower compared with the corresponding exometabolome of glucose-grown P. pastoris cells.

The instationary ¹³C flux analysis resulted in fluxes comparable to previously obtained from NMR datasets of proteinogenic amino acids, but allowed several additional insights. Specifically, i) in vivo metabolic flux estimations were expanded to a larger metabolic network e.g. by including trehalose recycling, which accounted for about 1.5% of the glucose uptake rate; ii) the reversibility of glycolytic/gluconeogenesis, TCA cycle and pentose phosphate pathways reactions was estimated, revealing a significant gluconeogenic flux from the dihydroxyacetone phosphate/glyceraldehydes phosphate pool to glucose-6P. The origin of this finding could be carbon recycling from the methanol assimilatory pathway to the pentose phosphate pool. Additionally, high exchange fluxes of oxaloacetate with aspartate as well as malate indicated amino acid pool buffering and the activity of the malate/Asp shuttle; iii) the ratio of methanol oxidation vs utilization appeared to be lower (54 vs 79% assimilated methanol directly oxidized to CO₂).

Conclusions

In summary, the application of instationary ¹³C-based metabolic flux analysis to P. pastoris provides an experimental framework with improved capabilities to explore the regulation of the carbon and energy metabolism of this yeast, particularly for the case of methanol and multicarbon source metabolism.

【 授权许可】

   
2013 Jordà et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 2.

Figure 4.

【 参考文献 】

• [1]Bollok M, Resina D, Valero F, Ferrer P: Recent patents on the pichia pastoris expression system, expanding the toolbox for recombinant protein production. Recent Pat Biotechnol 2009, 3:192-201.
• [2]Damasceno LM, Huang C-J, Batt C: Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol 2012, 93:31-39.
• [3]Mattanovich D, Branduardi P, Dato L, Gasser B, Sauer M, Porro D: Recombinant protein production in yeasts. Methods Mol Biol 2012, 824:329-358.
• [4]Abad S, Nahalka J, Bergler G, Arnold SA, Speight R, Fotheringham I, Nidetzky B, Glieder A: Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst. Microb Cell Fact 2010, 9:24-35. BioMed Central Full Text
• [5]Schroer K, Luef KP, Hartner FS, Glieder A, Pscheidt B: Engineering the Pichia pastoris methanol oxidation pathway for improved NADH regeneration during whole-cell biotransformation. Metab Eng 2010, 12:8-17.
• [6]Solà A, Maaheimo H, Ylönen K, Ferrer P, Szyperski T: Amino acid biosynthesis and metabolic flux profiling of Pichia pastoris. Eur J Biochem 2004, 271:2462-2470.
• [7]Solà A, Jouhten P, Maaheimo H, Szyperski T, Ferrer P: Metabolic flux profiling of Pichia pastoris grown on glycerol/methanol mixtures in chemostat cultures at low and high dilution rates. Microbiology 2007, 153:281-290.
• [8]Chung BKS, Selvarasu S, Camattari A, Ryu J, Lee H, Ahn J, Lee H, Lee D: Genome-scale metabolic reconstruction an in silico analysis of methylotrophic yeast Pichia pastoris for strain improvement. Microb Cell Fact 2010, 9:50. BioMed Central Full Text
• [9]Dragosits M, Stadlmann J, Albiol J, Baumann K, Maurer M, Gasser B, Sauer M, Altmann F, Ferrer P, Mattanovich D: The effect of temperature on the proteome of recombinant Pichia pastoris research articles. J Proteome Res 2009, 8:1380-1392.
• [10]Mattanovich D, Graf AB, Stadlmann J, Dragosits M, Redl A, Maurer M, Kleinheinz M, Sauer M, Altmann F, Gasser B: Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris. Microb Cell Fact 2009, 8:29. BioMed Central Full Text
• [11]Carnicer M, Canelas AB, Pierick A, Zeng Z, Dam J, Albiol J, Ferrer P, Heijnen JJ, Gulik W: Development of quantitative metabolomics for Pichia pastoris. Metabolomics 2011, 8:284-298.
• [12]Sohn SB, Graf AB, Kim TY, Gasser B, Maurer M, Ferrer P, Mattanovich D, Lee SY: Genome-scale metabolic model of methylotrophic yeast Pichia pastoris and its use for in silico analysis of heterologous protein production. Biotechnol J 2010, 5:701-715.
• [13]Harder M, Veenhuis M: Metabolism of one-carbon compounds. In The Yeasts. Volume 3 (Metabolism and physiology of Yeast). 2nd edition. Edited by Harrison JS, Rose AH. New York: Academic; 1989:289-316.
• [14]Cregg JM, Vedvick TS, Rasehke WC: Recent advances in the expression of foreign genes in Pichia pastoris. Biothecnology 1993, 2:905-910.
• [15]Cos O, Ramón R, Montesinos JL, Valero F: Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters, a review. Microb Cell Fact 2006, 5:17. BioMed Central Full Text
• [16]Inan M, Meagher MM: Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris. J Biosci Bioeng 2001, 92:585-589.
• [17]Jungo C, Schenk J, Pasquier M, Marison IW, von Stockar U: A quantitative analysis of the benefits of mixed feeds of sorbitol and methanol for the production of recombinant avidin with Pichia pastoris. J Biotechnol 2007, 131:57-66.
• [18]Ramón R, Ferrer P, Valero F: Sorbitol co-feeding reduces metabolic burden caused by the overexpression of a Rhizopus oryzae lipase in Pichia pastoris. J Biotechnol 2007, 130:39-46.
• [19]Arnau C, Ramón R, Casas C, Valero F: Optimization of the heterologous production of a Rhizopus oryzae lipase in Pichia pastoris system using mixed substrates on controlled fed-batch bioprocess. Enzyme Microb Technol 2010, 46:494-500.
• [20]Heyland J, Fu J, Blank LM, Schmid A: Quantitative physiology of Pichia pastoris during glucose-limited high-cell density fed-batch cultivation for recombinant protein production. Biotechnol Bioeng 2010, 107:357-368.
• [21]Driouch H, Melzer G, Wittmann C: Integration of in vivo and in silico metabolic fluxes for improvement of recombinant protein production. Metab Eng 2012, 14:47-58.
• [22]Jordà J, Jouhten P, Cámara E, Maaheimo H, Albiol J, Ferrer P: Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures. Microb Cell Fact 2012, 11:57. BioMed Central Full Text
• [23]Szyperski T: Biosynthetically directed fractional 13C-labeling of proteinogenic amino acids. An efficient analytical tool to investigate intermediary metabolism. Eur J Biochem 1995, 232:433-448.
• [24]Marx A, de Graaf AA, Wiechert W, Eggeling L: Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing. Biotechnol Bioeng 1996, 49:111-129.
• [25]Schmidt K, Nørregaard LC, Pedersen B, Meissner A, Duus JO, Nielsen JO, Villadsen J: Quantification of intracellular metabolic fluxes from fractional enrichment and 13C-13C coupling constraints on the isotopomer distribution in labeled biomass components. Metab Eng 1999, 1:166-179.
• [26]Fischer E, Sauer U: Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS. Eur J Biochem 2003, 270:880-891.
• [27]Wiechert W, Wurzel M: Metabolic isotopomer labeling systems Part I, global dynamic behavior. Math Biosci 2001, 169:173-205.
• [28]Sauer U: Metabolic networks in motion, 13C-based flux analysis. Mol Syst Biol 2006, 2:62.
• [29]Noack S, Nöh K, Moch M, Oldiges M, Wiechert W: Stationary versus non-stationary (13)C-MFA, a comparison using a consistent dataset. J Biotechnol 2011, 154:179-190.
• [30]Nasution U, van Gulik WM, Kleijn RJ, van Winden WA: Measurement of intracellular metabolites of primary metabolism and adenine nucleotides in chemostat cultivated Penicillium chrysogenum. InterScience 2006, 94:166.
• [31]Carnicer M, Baumann K, Töplitz I, Sánchez-Ferrando F, Mattanovich D, Ferrer P, Albiol J: Macromolecular and elemental composition analysis and extracellular metabolite balances of Pichia pastoris growing at different oxygen levels. Microb Cell Fact 2009, 8:65. BioMed Central Full Text
• [32]Baumann K, Maurer M, Dragosits M, Cos O, Ferrer P, Mattanovich D: Hypoxic fed-batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins. Biotechnol Bioeng 2008, 100:177-183.
• [33]Nöh K, Wiechert W: Experimental design principles for isotopically instationary C labeling experiments. Biotechnol Bioeng 2006, 94:234-251.
• [34]Lange HC, Eman MR, van Zuijlen G, Visser D, van Dam JC, Frank J, de Mattos MJ, Heijnen JJ: Improved rapid sampling for in vivo kinetics of intracellular metabolites in Saccharomyces cerevisiae. Biotechnol Bioeng 2001, 75:406-415.
• [35]Canales M, Buxadó JA, Heynngnezz L, Enriquez A: Mechanical disruption of Pichia pastoris yeast to recover the recombinant glycoprotein Bm86. Enzyme Microb Technol 1998, 23:58-63.
• [36]Mashego MR, Wu L, van Dam JC, Ras C, Vinke JL, van Winden WA, van Gulik WM, Heijnen JJ: MIRACLE, mass isotopomer ratio analysis of U-13C-labeled extracts. A new method for accurate quantification of changes in concentrations of intracellular metabolites. Biotechnol Bioeng 2004, 85:620-628.
• [37]Wu L, Mashego MR, Dam JCV, Proell AM, Vinke JL, Ras C, van Winden WA, van Gulik WM, Heijnen JJ: Quantitative analysis of the microbial metabolome by isotope dilution mass spectrometry using uniformly 13 C-labeled cell extracts as internal standards. Anal Biochem 2005, 336:164-171.
• [38]Nöh K, Wahl A, Wiechert W: Computational tools for isotopically instationary 13C labeling experiments under metabolic steady state conditions. Metab Eng 2006, 8:554-577.
• [39]Douma RD, de Jonge LP, Jonker CTH, Seifar RM, Heijnen JJ, van Gulik WM: Intracellular metabolite determination in the presence of extracellular abundance, Application to the penicillin biosynthesis pathway in Penicillium chrysogenum. Biotechnol Bioeng 2010, 107:105-115.
• [40]Canelas AB, ten Pierick A, Ras C, Seifar RM, van Dam JC, van Gulik WM, Heijnen JJ: Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics. Anal Chem 2009, 81:7379-7389.
• [41]Wahl SA, Dauner M, Wiechert W: New tools for mass isotopomer data evaluation in 13C flux analysis, Mass isotope correction, data consistency checking, and precursor relationships. Biotechnol Bioeng 2004, 3:259-268.
• [42]Kleijn RJ, van Winden WA, van Gulik WM, Heijnen JJ: Revisiting the 13C-label distribution of the non-oxidative branch of the pentose phosphate pathway based upon kinetic and genetic evidence. FEBS J 2005, 272:4970-4982.
• [43]Kümmel A, Panke S, Heinemann M: Putative regulatory sites unraveled by network-embedded thermodynamic analysis of metabolome data. Mol Syst Biol 2006, 2:1-10.
• [44]Zamboni N, Kümmel A, Heinemann M: anNET, a tool for network-embedded thermodynamic analysis of quantitative metabolome data. BMC Bioinforma 2008, 9:199. BioMed Central Full Text
• [45]Canelas AB, Ras C, ten Pierick A, van Gulik WM, Heijnen JJ: An in vivo data-driven framework for classification and quantification of enzyme kinetics and determination of apparent thermodynamic data. Metab Eng 2011, 13:294-306.
• [46]Kümmel A, Panke S, Heinemann M: Systematic assignment of thermodynamic constraints in metabolic network models. BMC Bioinforma 2006, 7:512. BioMed Central Full Text
• [47]Orij R, Brul S, Smits GJ: Intracellular pH is a tightly controlled signal in yeast. Biochim Biophys Acta 1810, 2011:933-944.
• [48]Wang NS, Stephanopoulos G: Application of macroscopic balances to the identification of gross measurement errors. Biotechnol Bioeng 1983, 25:2177-2208.
• [49]Heijden VD: Linear constraint relations in biochemical reaction systems, I. Classification of the calculability and the balanceability of conversion rates. Biotechnol Bioeng 1994, 43:3-10.
• [50]Verheijen PJT: Data reconciliation and error detection. In The Metabolic Pathway Engineering Handbook. 8th edition. Edited by Smolke C. Boca Raton: CRC Press; 2010:8.1-8.13.
• [51]Wisselink HW, Cipollina C, Oud B, Crimi B, Heijnen JJ, Pronk JT, van Maris AJA: Metabolome, transcriptome and metabolic flux analysis of arabinose fermentation by engineered Saccharomyces cerevisiae. Metab Eng 2010, 12:537-551.
• [52]Klimacek M, Krahulec S, Sauer U, Nidetzky B: Limitations in xylose-fermenting Saccharomyces cerevisiae, made evident through comprehensive metabolite profiling and thermodynamic analysis. Appl Environ Microbiol 2010, 76:7566-7574.
• [53]Carnicer M: Systematic metabolic analysis of recombinant Pichia pastoris under diferent oxygen conditions. Barcelona, Spain: Universitat Autònoma de Barcelona: PhD Thesis; 2012.
• [54]Canelas AB, van Gulik WM, Heijnen JJ: Determination of the cytosolic free NAD/NADH ratio in Saccharomyces cerevisiae under steady-state and highly dynamic conditions. Biotechnol Bioeng 2008, 100:734-743.
• [55]Aboka FO, Heijnen JJ, van Winden WA: Dynamic 13C-tracer study of storage carbohydrate pools in aerobic glucose-limited Saccharomyces cerevisiae confirms a rapid steady-state turnover and fast mobilization during a modest stepup in the glucose uptake rate. FEMS Yeast Res 2009, 9:191-201.
• [56]Zhao Z, ten Pierick A, de Jonge L, Heijnen JJ, Wahl A: Substrate cycles in Penicillium chrysogenum quantified by isotopic non-stationary flux analysis. Microb Cell Fact 2012, 11:140. BioMed Central Full Text
• [57]Bakker BM, Overkamp KM, van Maris AJ, Kötter P, Luttik MA, van Dijken JP, Pronk JT: Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae. FEMS Microbiol Rev 2001, 25:15-37.
• [58]Strijbis K, van Roermund CWT, Hardy GP, van den Burg J, Bloem K, de Haan J, van Vlies N, Wanders RJA, Vaz FM, Distel B: Identification and characterization of a complete carnitine biosynthesis pathway in Candida albicans. FASEB J 2009, 23:2349-2359.
• [59]Schutter KD, Lin Y, Tiels P, Hecke AV, Glinka S, Peer YVD, Callewaert N, Weber-Lehmann J, Rouze P: Genome sequence of the recombinant protein production host Pichia pastoris. Nat Biotechnol 2009, 27:561-566.
• [60]Baumann K, Carnicer M, Dragosits M, Graf AB, Stadlmann J, Jouhten P, Maaheimo H, Gasser B, Albiol J, Mattanovich D, Ferrer P: A multi-level study of recombinant Pichia pastoris in different oxygen conditions. BMC Syst Biol 2010, 4:141. BioMed Central Full Text
• [61]Grotkjaer T, Akesson M, Christensen B, Gombert AK, Nielsen J: Impact of transamination reactions and protein turnover on labeling dynamics in (13)C-labeling experiments. Biotechnol Bioeng 2004, 86:209-216.
• [62]Jones JG, Bellion E: Methanol oxidation and assimilation in Hansenula polymorpha. Biochem J 1991, 280:475-481.
• [63]Hartner FS, Glieder A: Regulation of methanol utilisation pathway genes in yeasts. Microb Cell Fact 2006, 5:39. BioMed Central Full Text
• [64]Blank LM, Lehmbeck F, Sauer U: Metabolic-flux and network analysis in fourteen hemiascomycetous yeasts. FEMS Yeast Res 2005, 5:545-558.
• [65]González-Siso MI, García-Leiro A, Tarrío N, Cerdán ME: Sugar metabolism, redox balance and oxidative stress response in the respiratory yeast Kluyveromyces lactis. Microb Cell Fact 2009, 8:46. BioMed Central Full Text
• [66]Verduyn C: Physiology of yeast in relation to biomass yields. Biomedical and Life Science 1991, 60:325-353.
• [67]Caspeta L, Shoaie S, Agren R, Nookaew I, Nielsen J: Genome-scale metabolic reconstructions of Pichia stipitis and Pichia pastoris and in-silico evaluation of their potentials. BMC Syst Biol 2012, 6:24. BioMed Central Full Text
• [68]Wiechert W, de Graaf AA: In vivo stationary flux analysis by 13C labeling experiments. Adv Biochem Eng Biotechnol 1996, 54:109-154.