Abstract
During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO2 to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.
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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
Bro C, Regenberg B, Forster J, Nielsen J (2006) In silico aided metabolic engineering of Saccharomyces cerevisiae for improved bioethanol production. Metab Eng 8:102–111
Bruinenberg PM (1986) The NADP(H) redox couple in yeast metabolism. Antonie Van Leeuwenhoek 52:411–429
Bruinenberg PM, de Bot PHM, van Dijken JP, WA S (1983) The role of redox balances in the anaerobic fermentation of xylose by yeasts. Eur J Appl Microbiol Biotechnol 18:287–292
Eliasson A, Christensson C, Wahlbom CF, Hahn-Hägerdal B (2000) Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying XYL1, XYL2, and XKS1 in mineral medium chemostat cultures. Appl Environ Microbiol 66:3381–3386
Grotkjaer T, Christakopoulos P, Nielsen J, Olsson L (2005) Comparative metabolic network analysis of two xylose fermenting recombinant Saccharomyces cerevisiae strains. Metab Eng 7:437–444
Hahn-Hägerdal B, Karhumaa K, Jeppsson M, Gorwa-Grauslund MF (2007) Metabolic engineering for pentose utilization in Saccharomyces cerevisiae. Adv Biochem Eng Biotechnol 108:147–177
Heux S, Cadiere A, Dequin S (2008) Glucose utilization of strains lacking PGI1 and expressing a transhydrogenase suggests differences in the pentose phosphate capacity among Saccharomyces cerevisiae strains. FEMS Yeast Res 8:217–224
Ho NW, Chen Z, Brainard AP (1998) Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose. Appl Environ Microbiol 64:1852–1859
Hou J, Shen Y, Li XP, Bao XM (2007) Effect of the reversal of coenzyme specificity by expression of mutated Pichia stipitis xylitol dehydrogenase in recombinant Saccharomyces cerevisiae. Lett Appl Microbiol 45:184–189
Jeffries TW (2006) Engineering yeasts for xylose metabolism. Curr Opin Biotechnol 17:320–326
Jeffries TW, Jin YS (2004) Metabolic engineering for improved fermentation of pentoses by yeasts. Appl Microbiol Biotechnol 63:495–509
Jeppsson M, Johansson B, Hahn-Hägerdal B, Gorwa-Grauslund MF (2002) Reduced oxidative pentose phosphate pathway flux in recombinant xylose-utilizing Saccharomyces cerevisiae strains improves the ethanol yield from xylose. Appl Environ Microbiol 68:1604–1609
Jeppsson M, Johansson B, Jensen PR, Hahn-Hägerdal B, Gorwa-Grauslund MF (2003) The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains. Yeast 20:1263–1272
Matsushika A, Watanabe S, Kodaki T, Makino K, Inoue H, Murakami K, Takimura O, Sawayama S (2008) Expression of protein engineered NADP+-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant Saccharomyces cerevisiae. Appl Microbiol Biotechnol 81:243–255
Minard KI, McAlister-Henn L (2005) Sources of NADPH in yeast vary with carbon source. J Biol Chem 280:39890–39896
Moreira dos Santos M, Raghevendran V, Kotter P, Olsson L, Nielsen J (2004) Manipulation of malic enzyme in Saccharomyces cerevisiae for increasing NADPH production capacity aerobically in different cellular compartments. Metab Eng 6:352–363
Møller K, Bro C, Piskur J, Nielsen J, Olsson L (2002) Steady-state and transient-state analyses of aerobic fermentation in Saccharomyces kluyveri. FEMS Yeast Research 2:233–244
Nielsen J, Villadsen J, Liden G (2003) Bioreaction Engineering Principles. Kluwer Academic, New York
Nissen TL, Anderlund M, Nielsen J, Villadsen J, Kielland-Brandt MC (2001) Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2-oxoglutarate due to depletion of the NADPH pool. Yeast 18:19–32
Otero JM, Panagiotou G, Olsson L (2007) Fueling industrial biotechnology growth with bioethanol. Adv Biochem Eng Biotechnol 108:1–40
Outten CE, Culotta VC (2003) A novel NADH kinase is the mitochondrial source of NADPH in Saccharomyces cerevisiae. Embo J 22:2015–2024
Panagiotou G, Grotkjaer T, Hofmann G, Bapat PM, Olsson L (2009) Overexpression of a novel endogenous NADH kinase in Aspergillus nidulans enhances growth. Metab Eng 11:31–39
Petschacher B, Nidetzky B (2008) Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of Saccharomyces cerevisiae. Microb Cell Fact 7:9
Postma E, Verduyn C, Scheffers WA, Van Dijken JP (1989) Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae. Appl Environ Microbiol 55:468–477
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
Shi F, Kawai S, Mori S, Kono E, Murata K (2005) Identification of ATP-NADH kinase isozymes and their contribution to supply of NADP(H) in Saccharomyces cerevisiae. Febs J 272:3337–3349
Shianna KV, Marchuk DA, Strand MK (2006) Genomic characterization of POS5, the Saccharomyces cerevisiae mitochondrial NADH kinase. Mitochondrion 6:94–101
Sonderegger M, Jeppsson M, Hahn-Hägerdal B, Sauer U (2004) Molecular basis for anaerobic growth of Saccharomyces cerevisiae on xylose, investigated by global gene expression and metabolic flux analysis. Appl Environ Microbiol 70:2307–2317
Strand MK, Stuart GR, Longley MJ, Graziewicz MA, Dominick OC, Copeland WC (2003) POS5 gene of Saccharomyces cerevisiae encodes a mitochondrial NADH kinase required for stability of mitochondrial DNA. Eukaryot Cell 2:809–820
Turner WL, Waller JC, Snedden WA (2005) Identification, molecular cloning and functional characterization of a novel NADH kinase from Arabidopsis thaliana (thale cress). Biochem J 385:217–223
Vemuri GN, Eiteman MA, McEwen JE, Olsson L, Nielsen J (2007) Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 104:2402–2407
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
Verho R, Londesborough J, Penttila M, Richard P (2003) Engineering redox cofactor regeneration for improved pentose fermentation in Saccharomyces cerevisiae. Appl Environ Microbiol 69:5892–5897
Walfridsson M, Anderlund M, Bao X, Hahn-Hägerdal B (1997) Expression of different levels of enzymes from the Pichia stipitis XYL1 and XYL2 genes in Saccharomyces cerevisiae and its effects on product formation during xylose utilisation. Appl Microbiol Biotechnol 48:218–224
Watanabe S, Abu Saleh A, Pack SP, Annaluru N, Kodaki T, Makino K (2007) Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis. Microbiology 153:3044–3054
Wattanachaisaereekul S, Lantz AE, Nielsen ML, Nielsen J (2008) Production of the polyketide 6-MSA in yeast engineered for increased malonyl-CoA supply. Metab Eng 10:246–254
Zaldivar J, Nielsen J, Olsson L (2001) Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl Microbiol Biotechnol 56:17–34
Acknowledgments
We thank Tina Johanssen, Jette Mortensen, and Martin Nielsen for technical assistance with the research. This work was partly financially supported by the National High Technology Research and Development Program of China (No. 2007AA05Z402), Natural Science Foundation of China (No. 50273019), and the National Basic Research Program of China (No. 2007CB707803). Jin Hou acknowledges a fellowship from China scholarship council.
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Hou, J., Vemuri, G.N., Bao, X. et al. Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing Saccharomyces cerevisiae . Appl Microbiol Biotechnol 82, 909–919 (2009). https://doi.org/10.1007/s00253-009-1900-4
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DOI: https://doi.org/10.1007/s00253-009-1900-4