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Carbon fluxes of xylose-consuming Saccharomyces cerevisiae strains are affected differently by NADH and NADPH usage in HMF reduction

  • Applied Microbial and Cell Physiology
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Abstract

Industrial Saccharomyces cerevisiae strains able to utilize xylose have been constructed by overexpression of XYL1 and XYL2 genes encoding the NADPH-preferring xylose reductase (XR) and the NAD+-dependent xylitol dehydrogenase (XDH), respectively, from Pichia stipitis. However, the use of different co-factors by XR and XDH leads to NAD+ deficiency followed by xylitol excretion and reduced product yield. The furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural inhibit yeast metabolism, prolong the lag phase, and reduce the ethanol productivity. Recently, genes encoding furaldehyde reductases were identified and their overexpression was shown to improve S. cerevisiae growth and fermentation rate in HMF containing media and in lignocellulosic hydrolysate. In the current study, we constructed a xylose-consuming S. cerevisiae strain using the XR/XDH pathway from P. stipitis. Then, the genes encoding the NADH- and the NADPH-dependent HMF reductases, ADH1-S110P-Y295C and ADH6, respectively, were individually overexpressed in this background. The performance of these strains, which differed in their co-factor usage for HMF reduction, was evaluated under anaerobic conditions in batch fermentation in absence or in presence of HMF. In anaerobic continuous culture, carbon fluxes were obtained for simultaneous xylose consumption and HMF reduction. Our results show that the co-factor used for HMF reduction primarily influenced formation of products other than ethanol, and that NADH-dependent HMF reduction influenced product formation more than NADPH-dependent HMF reduction. In particular, NADH-dependent HMF reduction contributed to carbon conservation so that biomass was produced at the expense of xylitol and glycerol formation.

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References

  • Almeida JRM, Modig T, Petersson A, Hahn-Hägerdal B, Lidén G, Gorwa-Grauslund MF (2007) Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J Chem Technol Biotechnol 82:340–349

    Article  CAS  Google Scholar 

  • Almeida JRM, Modig T, Röder A, Lidén G, Gorwa-Grauslund MF (2008a) Pichia stipitis xylose reductase helps detoxifying lignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfural (HMF). Biotechnol Biofuels 1:12

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Almeida JRM, Röder A, Modig T, Laadan B, Lidén G, Gorwa-Grauslund MF (2008b) NADH- vs NADPH-coupled reduction of 5-hydroxymethyl furfural (HMF) and its implications on product distribution in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 78:939–945

    Article  PubMed  CAS  Google Scholar 

  • Almeida JRM, Bertilsson M, Gorwa-Grauslund MF, Gorsich S, Lidén G (2009) Metabolic effects of furaldehydes and impacts on biotechnological processes. Appl Microbiol Biotechnol 82:625–638

    Article  PubMed  CAS  Google Scholar 

  • Anderlund M, Nissen TL, Nielsen J, Villadsen J, Rydstrom J, Hahn-Hägerdal B, Kielland-Brandt MC (1999) Expression of the Escherichia coli pntA and pntB genes, encoding nicotinamide nucleotide transhydrogenase, in Saccharomyces cerevisiae and its effect on product formation during anaerobic glucose fermentation. Appl Environ Microbiol 65:2333–2340

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Bengtsson O, Hahn-Hägerdal B, Gorwa-Grauslund MF (2009) Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae. Biotechnol Biofuels 2:9

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Bruinenberg PM, Debot PHM, Vandijken JP, Scheffers WA (1983) The role of redox balances in the anaerobic fermentation of xylose by yeasts. Eur J Appl Microbiol 18:287–292

    Article  CAS  Google Scholar 

  • Chu BC, Lee H (2007) Genetic improvement of Saccharomyces cerevisiae for xylose fermentation. Biotechnol Adv 25:425–441

    Article  CAS  PubMed  Google Scholar 

  • Dunlop AP (1948) Furfural formation and behavior. Ind Eng Chem 40:204–209

    Article  CAS  Google Scholar 

  • Farrell AE, Plevin RJ, Turner BT, Jones AD, O'Hare M, Kammen DM (2006) Ethanol can contribute to energy and environmental goals. Science 311:506–508

    Article  PubMed  CAS  Google Scholar 

  • Hahn-Hägerdal B, Galbe M, Gorwa-Grauslund MF, Lidén G, Zacchi G (2006) Bio-ethanol—the fuel of tomorrow from the residues of today. Trends Biotechnol 24:549–556

    Article  PubMed  CAS  Google Scholar 

  • Hahn-Hägerdal B, Karhumaa K, Fonseca C, Spencer-Martins I, Gorwa-Grauslund MF (2007a) Towards industrial pentose-fermenting yeast strains. Appl Microbiol Biotechnol 74:937–953

    Article  PubMed  CAS  Google Scholar 

  • Hahn-Hägerdal B, Karhumaa K, Jeppsson M, Gorwa-Grauslund MF (2007b) Metabolic engineering for pentose utilization in Saccharomyces cerevisiae. Adv Biochem Eng Biotechnol 108:147–177

    PubMed  Google Scholar 

  • Hauf J, Zimmermann FK, Müller S (2000) Simultaneous genomic overexpression of seven glycolytic enzymes in the yeast Saccharomyces cerevisiae. Enzyme Microb Technol 26:688–698

    Article  PubMed  CAS  Google Scholar 

  • Jeffries TW (2006) Engineering yeasts for xylose metabolism. Curr Opin Biotechnol 17:320–326

    Article  PubMed  CAS  Google Scholar 

  • Karhumaa K, Hahn-Hägerdal B, Gorwa-Grauslund MF (2005) Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering. Yeast 22:359–368

    Article  PubMed  CAS  Google Scholar 

  • Karhumaa K, Garcia Sanchez R, Hahn-Hägerdal B, Gorwa-Grauslund MF (2007) Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae. Microb Cell Fact 6:5

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Klinke HB, Thomsen AB, Ahring BK (2004) Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl Microbiol Biotechnol 66:10–26

    Article  PubMed  CAS  Google Scholar 

  • Kotter P, Ciriacy M (1993) Xylose fermentation by Saccharomyces cerevisiae. Appl Microbiol Biotechnol 38:776–783

    Article  Google Scholar 

  • Laadan B (2008) Yeasts as cellular factories. PhD Thesis. Lund University

  • Laadan B, Almeida JRM, Rådström P, Hahn-Hägerdal B, Gorwa-Grauslund M (2008) Identification of an NADH-dependent 5-hydroxymethylfurfural-reducing alcohol dehydrogenase in Saccharomyces cerevisiae. Yeast 25:191–198

    Article  PubMed  CAS  Google Scholar 

  • Larroy C, Fernandez MR, Gonzalez E, Pares X, Biosca JA (2002) Characterization of the Saccharomyces cerevisiae YMR318C (ADH6) gene product as a broad specificity NADPH-dependent alcohol dehydrogenase: relevance in aldehyde reduction. Biochem J 361:163–172

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Larsson S, Cassland P, Jönsson LJ (2001a) Development of a Saccharomyces cerevisiae strain with enhanced resistance to phenolic fermentation inhibitors in lignocellulose hydrolysates by heterologous expression of laccase. Appl Environ Microbiol 67:1163–1170

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Larsson S, Nilvebrant NO, Jönsson LJ (2001b) Effect of overexpression of Saccharomyces cerevisiae Pad1p on the resistance to phenylacrylic acids and lignocellulose hydrolysates under aerobic and oxygen-limited conditions. Appl Microbiol Biotechnol 57:167–174

    Article  PubMed  CAS  Google Scholar 

  • Liu ZL, Slininger PJ, Dien BS, Berhow MA, Kurtzman CP, Gorsich SW (2004) Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2, 5-bis-hydroxymethylfuran. J Ind Microbiol Biotechnol 31:345–352

    Article  PubMed  CAS  Google Scholar 

  • Liu ZL, Moon J, Andersh BJ, Slininger PJ, Weber S (2008) Multiple gene-mediated NAD(P) H-dependent aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cerevisiae. Appl Microbiol Biotechnol 81:743–753

    Article  PubMed  CAS  Google Scholar 

  • Modig T, Almeida JRM, Gorwa-Grauslund MF, Lidén G (2008) Variability of the response of Saccharomyces cerevisiae strains to lignocellulose hydrolysate. Biotechnol Bioeng 100:423–429

    Article  PubMed  CAS  Google Scholar 

  • Nilsson A, Gorwa-Grauslund MF, Hahn-Hägerdal B, Lidén G (2005) Cofactor dependence in furan reduction by Saccharomyces cerevisiae in fermentation of acid-hydrolyzed lignocellulose. Appl Environ Microbiol 71:7866–7871

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Nissen TL, Kielland-Brandt MC, Nielsen J, Villadsen J (2000) Optimization of ethanol production in Saccharomyces cerevisiae by metabolic engineering of the ammonium assimilation. Metab Eng 2:69–77

    Article  PubMed  CAS  Google Scholar 

  • Oura E (1977) Reaction products of yeast fermentations. Process Biochem 12:19–21

    CAS  Google Scholar 

  • Palmqvist E, Hahn-Hägerdal B (2000) Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresource Technol 74:25–33

    Article  CAS  Google Scholar 

  • Petersson A, Almeida JRM, Modig T, Karhumaa K, Hahn-Hägerdal B, Gorwa-Grauslund MF, Lidén G (2006) A 5-hydroxymethyl furfural reducing enzyme encoded by the Saccharomyces cerevisiae ADH6 gene conveys HMF tolerance. Yeast 23:455–464

    Article  PubMed  CAS  Google Scholar 

  • Rizzi M, Erlemann P, Bui-Thanh N-A, Dellweg H (1988) Xylose fermentation by yeasts. 4. Purification and kinetic studies of xylose reductase from Pichia stipitis. Appl Microbiol Biotechnol 29:148–154

    Article  CAS  Google Scholar 

  • Rizzi M, Harwart K, Erlemann P, Buithanh NA, Dellweg H (1989) Xylose fermentation by yeasts. 6. Purification and properties of the Nad + -xylitol-dehydrogenase from the yeast Pichia stipitis. J Ferment Bioeng 67:20–24

    Article  CAS  Google Scholar 

  • Roca C, Nielsen J, Olsson L (2003) Metabolic engineering of ammonium assimilation in xylose-fermenting Saccharomyces cerevisiae improves ethanol production. Appl Environ Microbiol 69:4732–4736

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Saint-Prix F, Bonquist L, Dequin S (2004) Functional analysis of the ALD gene family of Saccharomyces cerevisiae during anaerobic growth on glucose: the NADP + -dependent Ald6p and Ald5p isoforms play a major role in acetate formation. Microbiology 150:2209–2220

    Article  PubMed  CAS  Google Scholar 

  • Smiley K, Bolen P (1982) Demonstration of d-xylose reductase and d-xylitol dehydrogenase in Pachysolen tannophilus. Biotechnol Lett 9:607–610

    Article  Google Scholar 

  • Sonderegger M, Schumperli M, Sauer U (2004) Metabolic engineering of a phosphoketolase pathway for pentose catabolism in Saccharomyces cerevisiae. Appl Environ Microbiol 70:2892–2897

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Taherzadeh MJ, Gustafsson L, Niklasson C, Lidén G (1999) Conversion of furfural in aerobic and anaerobic batch fermentation of glucose by Saccharomyces cerevisiae. J Biosci Bioeng 87:169–174

    Article  PubMed  CAS  Google Scholar 

  • Taherzadeh MJ, Gustafsson L, Niklasson C, Lidén G (2000) Physiological effects of 5-hydroxymethylfurfural on Saccharomyces cerevisiae. Appl Microbiol Biotechnol 53:701–708

    Article  PubMed  CAS  Google Scholar 

  • Ulbricht RJ, Northup SJ, Thomas JA (1984) A review of 5-hydroxymethylfurfural (HMF) in parenteral solutions. Fundam Appl Toxicol 4:843–853

    Article  PubMed  CAS  Google Scholar 

  • van Maris AJA, Winkler AA, Kuyper M, de Laat WTAM, van Dijken JP, Pronk JT (2007) Development of efficient xylose fermentation in Saccharomyces cerevisiae: xylose isomerase as a key component. Adv Biochem Eng Biot 108:179–204

    Google Scholar 

  • Verduyn C, Postma E, Scheffers WA, Van Dijken JP (1992) Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501–517

    Article  PubMed  CAS  Google Scholar 

  • Verho R, Londesborough J, Penttilä M, Richard P (2003) Engineering redox cofactor regeneration for improved pentose fermentation in Saccharomyces cerevisiae. Appl Environ Microbiol 69:5892–5897

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Wahlbom CF, Hahn-Hägerdal B (2002) Furfural, 5-hydroxymethyl furfural, and acetoin act as external electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae. Biotechnol Bioeng 78:172–178

    Article  PubMed  CAS  Google Scholar 

  • Wahlbom CF, Eliasson A, Hahn-Hägerdal B (2001) Intracellular fluxes in a recombinant xylose-utilizing Saccharomyces cerevisiae cultivated anaerobically at different dilution rates and feed concentrations. Biotechnol Bioeng 72:289–296

    Article  PubMed  CAS  Google Scholar 

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Acknowledgment

This work was supported by the Swedish Energy Agency.

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Correspondence to Marie-F. Gorwa-Grauslund.

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Almeida, J.R.M., Bertilsson, M., Hahn-Hägerdal, B. et al. Carbon fluxes of xylose-consuming Saccharomyces cerevisiae strains are affected differently by NADH and NADPH usage in HMF reduction. Appl Microbiol Biotechnol 84, 751–761 (2009). https://doi.org/10.1007/s00253-009-2053-1

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