Abstract
The yeast Dekkera bruxellensis is well-known for its adaptation to industrial ethanol fermentation processes, which can be further improved if nitrate is present in the substrate. To date, the assimilation of nitrate has been considered inefficient because of the apparent energy cost imposed on cell metabolism. Recent research, however, has shown that nitrate promotes growth rate and ethanol yield when oxygen is absent from the environment. Given this, the present work aimed to identify the biological mechanisms behind this physiological behaviour. Proteomic analyses comparing four contrasting growth conditions gave some clues on how nitrate could be used as primary nitrogen source by D. bruxellensis GDB 248 (URM 8346) cells in anaerobiosis. The superior anaerobic growth in nitrate seems to be a consequence of increased cell metabolism (glycolytic pathway, production of ATP and NADPH and anaplerotic reactions providing metabolic intermediates) regulated by balanced activation of TORC1 and NCR de-repression mechanisms. On the other hand, the poor growth observed in aerobiosis is likely due to an oxidative stress triggered by nitrate when oxygen is present. These results represent a milestone regarding the knowledge about nitrate metabolism and might be explored for future use of D. bruxellensis as an industrial yeast.
Key points
• Nitrate can be regarded as preferential nitrogen source for D. bruxellensis.
• Oxidative stress limits the growth of D. bruxellensis in nitrate in aerobiosis.
• Nitrate is a nutrient for novel industrial bioprocesses using D. bruxellensis.
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Data availability statement
All data generated or analysed during this study are included in this published article (and its supplementary information files) and further information are available from the corresponding author on reasonable request.
References
Aronova S, Wedaman K, Anderson S, Yates Y, Powers T (2017) Probing the membrane environment of the TOR kinases reveals functional interactions between TORC1, actin, and membrane trafficking in Saccharomyces cerevisiae. Mol Biol Cell 18:2779–2794. https://doi.org/10.1091/mbc.e07-03-0274
Bertram PG, Choi JH, Carvalho J, Ai W, Zeng C, Chan TF, Zheng XF (2000) Tripartite regulation of Gln3p by TOR, Ure2p, and phosphatases. J Biol Chem 46:5727–5733. https://doi.org/10.1074/jbc.M004235200
Blomqvist J, Eberhard T, Schnürer J, Passoth V (2010) Fermentation characteristics of Dekkera bruxellensis strains. Appl Microbiol Biotechnol 87:1487–1497. https://doi.org/10.1007/s00253-010-2619-y
Blomqvist J, Nogue VS, Gorwa-Grauslund M, Passoth V (2012) Physiological requirements for growth and competitiveness of Dekkera bruxellensis under oxygen-limited or anaerobic conditions. Yeast 29:265–274. https://doi.org/10.1002/yea.2904
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. https://doi.org/10.1006/abio.1976.9999
Bradshaw PC (2019) Cytoplasmic and mitochondrial NADPH-coupled redox systems in the regulation of aging. Nutrients 3:504. https://doi.org/10.3390/nu11030504
Butow RA, Avadhani NG (2004) Mitochondrial signaling: the retrograde response. Mol Cell 14:1–15. https://doi.org/10.1016/S1097-2765(04)00179-0
Cajueiro DBB, Parente DC, Leite FCB, de Morais Jr MA, de Barros PW (2017) Glutamine: a major player in nitrogen catabolite repression in the yeast Dekkera bruxellensis. Antonie Van Leeuwenhoek 110:1157–1168. https://doi.org/10.1007/s10482-017-0888-5
Carmona L, Varela J, Godoy L, Ganga MA (2016) Comparative proteome analysis of Brettanomyces bruxellensis under hydroxycinnamic acid growth. Electron J Biotechnol 23:37–43. https://doi.org/10.1016/j.ejbt.2016.07.005
Conrad M, Schothorst J, Kankipati HN, Zeebroeck GV, Rubio-Texeira M, Thevelein JM (2013) Nutrient sensing and signaling in the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev 38:254–299. https://doi.org/10.1111/1574-6976.12065
Corpas FJ, Palma JM (2020) Assessing nitric oxide (NO) in higher plants: an outline. Nitrogen 1:12–20. https://doi.org/10.3390/nitrogen1010003
da Silva JM, Silva GHTG, Parente DC, Leite FCB, Silva CS, Valente P, Ganga AM, Simões DA, de Morais MA Jr (2019) Biological diversity of carbon assimilation among isolates of the yeast Dekkera bruxellensis from wine and fuel-ethanol industrial processes. FEMS Yeast Res 19:foz022. https://doi.org/10.1093/femsyr/foz022
de Barros Pita W, Leite FCB, de Souza Liberal AT, Simões DA, de Morais MA Jr (2011) The ability to use nitrate confers advantage to Dekkera bruxellensis over S. cerevisiae and can explain its adaptation to industrial fermentation processes. Antonie van Leeuwenhoek 1:1–9. https://doi.org/10.1007/s10482-011-9568-z
de Barros Pita W, Leite FC, de Souza Liberal AT, Pereira LF, Carazzolle MF, Pereira GA, de Morais MA Jr (2012) A new set of reference genes for RT-qPCR assays in the yeast Dekkera bruxellensis. Can J Microbiol 12:1362–1367. https://doi.org/10.1139/cjm-2012-0457
de Barros Pita W, Castro-Silva D, Simões-Ardaillon D, Volkmar P, de Morais MA Jr (2013) Physiology and gene expression profiles of Dekkera bruxellensis in response to carbon and nitrogen availability. Antonie van Leeuwenhoek 5:855–868. https://doi.org/10.1007/s10482-013-9998-x
de Barros Pita W, Teles GH, Peña-Moreno IC, da Silva JM, Ribeiro KC, de Morais Junior MA (2019) The biotechnological potential of the yeast Dekkera bruxellensis. World J Microbiol Biotechnol 35: 103. https://doi.org/10.1007/s11274-019-2678-x
de Groot MLJ, Daran-Lapujade P, van Breukelen B, Knijnenburg TA, de Hulster EAF, Reinders MJT, Pronk JT, Heck AJR, Slijper M (2007) Quantitative proteomics and transcriptomics of anaerobic and aerobic yeast cultures reveals posttranscriptional regulation of key cellular processes. Microbiol 153:3864–3878. https://doi.org/10.1099/mic.0.2007/009969-0
de Souza RB, dos Santos BM, de Fátima Rodrigues de Souza R, da Silva PF, Lucena BT, de Morais MA Jr (2012) The consequences of Lactobacillus vini and Dekkera bruxellensis as contaminants of the sugarcane-based ethanol fermentation. Ind Microbiol Biotechnol 11:1645–1650. https://doi.org/10.1007/s10295-012-1167-0
Denis V, Daignan-Fornier B (1998) Synthesis of glutamine, glycine and 10-formyl tetrahydrofolate is coregulated with purine biosynthesis in Saccharomyces cerevisiae. Mol Gen Genet 259:246–255. https://doi.org/10.1007/s004380050810
Galafassi S, Capusoni S, Moktaduzzaman M, Compagno C (2013) Utilization of nitrate abolishes the “Custers effect” in Dekkera bruxellensis and determines a different pattern of fermentation products. J Ind Microbiol Biotechnol 34:297–303. https://doi.org/10.1007/s10295-012-1229-3
Gardner PR (2012) Hemoglobin: a nitric-oxide dioxygenase. Scientifica 683729:1–34. https://doi.org/10.6064/2012/683729
Grawert T, Fischer M, Bacher A (2013) Structures and reaction mechanisms of GTP cyclohydrolases. IUBMB Life 4:310–322. https://doi.org/10.1002/iub.1153
Haugen AC, Kelley R, Collins JB, Tucker CJ, Deng C, Afshari CA, Brown JM, Ideker T, Van Houten B (2004) Integrating phenotypic and expression profiles to map arsenic-response networks. Genome Biol 5:R95. https://doi.org/10.1186/gb-2004-5-12-r95
Jouhten P, Rintala E, Huuskonen A, Tamminen A, Toivari M, Wiebe M, Ruohonen L, Penttila M, Maaheimo H (2008) Oxygen dependence of metabolic fluxes and energy generation of Saccharomyces cerevisiae. BMC Syst Biol 2:60. https://doi.org/10.1186/1752-0509-2-60
Leite FCB, Basso TO, de Pita BW, Gombert AK, Simoes DA, de Morais MA Jr (2013) Quantitative aerobic physiology of the yeast Dekkera bruxellensis, a major contaminant in bioethanol production plants. FEMS Yeast Res 1:34–43.
Leite FCB, Leite DVR, Pereira LF, de Barros PW, De Morais Jr MA (2016) High intracellular trehalase activity prevents the storage of trehalose in the yeast Dekkera bruxellensis. Lett Appl Microbiol 63:210–214. https://doi.org/10.1111/lam.12609
Lisacek F (2006) Web-based MS/MS data analysis. Proteomics 6(Suppl 2):22–32. https://doi.org/10.1002/pmic.200600524
Lu AL, Li X, Gu Y, Wright PM, Chang DY (2001) Repair of oxidative DNA damage: mechanisms and functions. Cell Biochem Biophys 35:141–170. https://doi.org/10.1385/CBB:35:2:141
Mukai M, Mills SE, Poole RK, Yeh SR (2001) Flavohemoglobin, a globin with a peroxidase-like catalytic site. J Biol Chem 10:7272–7277. https://doi.org/10.1074/jbc.M009280200
Neto AG, Pestana-Calsa MC, de Morais Jr MA, Calsa T (2014) Proteome responses to nitrate in bioethanol production contaminant Dekkera bruxellensis. J Proteomics 104:104–111. https://doi.org/10.1016/j.jprot.2014.03.014
Pacheco CM, Pestana-Calsa MC, Gozzo FC, Mansur Custodio Nogueira RJ, Menossi M, Calsa T Jr (2013) Differentially delayed root proteome responses to salt stress in sugar cane varieties. J Proteome Res 12:5681–5695. https://doi.org/10.1021/pr400654a
Parente DC, Vidal EE, Leite FC, de Barros PW, de Morais MA Jr (2015) Production of sensory compounds by means of the yeast Dekkera bruxellensis in different nitrogen sources with the prospect of producing cachaca. Yeast 32:77–87. https://doi.org/10.1002/yea.3051
Parente DC, Cajueiro DBB, Moreno ICP, Leite FCB, de Barros PW, de Morais Jr MA (2017) On the catabolism of amino acids in the yeast Dekkera bruxellensis and the implications for industrial fermentation processes. Yeast 3:299–309. https://doi.org/10.1002/yea.3290
Passoth V, Blomqvist J, Schnürer J (2007) Dekkera bruxellensis and Lactobacillus vini form a stable ethanol-producing consortium in a commercial alcohol production process. Appl Environ Microbiol 73:4354–4356. https://doi.org/10.1128/AEM.00437-07
Peña-Moreno IC, Castro Parente D, da Silva JM, Andrade Mendonça A, Rojas LAV, de Morais Jr MA, de Barros PW (2019) Nitrate boosts anaerobic ethanol production in an acetate-dependent manner in the yeast Dekkera bruxellensis. J Ind Microbiol Biotechnol 46:209–220. https://doi.org/10.1007/s10295-018-2118-1
Pereira LF, Bassi AFG, Avansini SH, Neto AGB, Brasileiro BTRV, Ceccato-Antonini SR, de Morais Jr MA (2012) The physiological characteristics of the yeast Dekkera bruxellensis in fully fermentative conditions with cell recycling in mixed cultures with Saccharomyces cerevisiae. Antonie Van Leeuwenhoek 101:529–539. https://doi.org/10.1007/s10482-011-9662-2
Pereira LF, Lucatti E, Basso LC, de Morais Jr MA (2014) The fermentation of sugarcane molasses by Dekkera bruxellensis and the mobilization of reserve carbohydrates. Antonie Van Leeuwenhoek 105:481–489. https://doi.org/10.1007/s10482-013-0100-5
Reis ALS, Damilano LD, Menezes RSC, de Morais Jr MA (2016) Second-generation ethanol from sugarcane and sweet sorghum bagasses using the yeast Dekkera bruxellensis. Ind Crops Prod 92:255–262. https://doi.org/10.1016/j.indcrop.2016.08.007
Rodríguez A, De La Cera T, Herrero P, Moreno F (2001) The hexokinase 2 protein regulates the expression of the GLK1, HXK1 and HXK2 genes of Saccharomyces cerevisiae. Biochem J 355:625–631. https://doi.org/10.1042/bj3550625
Ronne H, Carlberg M, Hu GZ, Nehlin JO (1991) Protein phosphatase 2A in Saccharomyces cerevisiae: effects on cell growth and bud morphogenesis. Mol Cel Biol 10:4876–4884. https://doi.org/10.1128/mcb.11.10.4876
Siverio JM (2002) Assimilation of nitrate by yeasts. FEMS Microbiol Rev 3:277–284. https://doi.org/10.1111/j.1574-6976.2002.tb00615.x
Szklarczyk D, Morris JH, Cook H, Kuhn M, Wyder S, Simonovic M, Santos A, Doncheva NT, Roth A, Bork P, Jensen LJ, von Mering C (2017) The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res 45:D362–D368. https://doi.org/10.1093/nar/gkw937
Steensels J, Daenen L, Malcorps P, Derdelinckx G, Verachtert H, Verstrepen K (2015) Brettanomyces yeasts—from spoilage organisms to valuable contributors to industrial fermentations. Int J Food Microbiol 206:24–38. https://doi.org/10.1016/j.ijfoodmicro.2015.04.005
Teles GH, Da Silva JM, Mendonça AA, de Morais Jr MA, de Barros PW (2018) First aspects on acetate metabolism in the yeast Dekkera bruxellensis: a 2 few keys for improving ethanol fermentation. Yeast 10:577–584. https://doi.org/10.1002/yea.3348
Tiukova IA, Petterson ME, Tellgren-Roth C, Bunikis I, Eberhard T, Pettersson OV, Passoth V (2013) Transcriptome of the alternative ethanol production strain Dekkera bruxellensis CBS 11270 in sugar limited, low oxygen cultivation. PloS One 3:e58455. https://doi.org/10.1371/journal.pone.0058455
van Dijken JP, Scheffers WA (1986) Redox balances in the metabolism of sugars by yeasts. FEMS Microbiol Letters 32:199–224. https://doi.org/10.1016/0378-1097(86)90291-0
Van Rossum HM, Kozak BU, Pronk JT, van Marris AJA (2016) Engineering cytosolic acetyl-coenzyme A supply in Saccharomyces cerevisiae: pathway stoichiometry, free-energy conservation and redox-cofactor balancing. Metabol Eng 36:99–115. https://doi.org/10.1016/j.ymben.2016.03.006
Vaudel M, Barsnes H, Berven FS, Sickmann A, Martens L (2011) SearchGUI: an open-source graphical user interface for simultaneous OMSSA and X!Tandem searches. Proteomics 11:996–999. https://doi.org/10.1002/pmic.201000595
Vigentini I, Lucy Joseph CM, Picozzi C, Foschino R, Bisson LF (2013) Assessment of the Brettanomyces bruxellensis metabolome during sulphur dioxide exposure. FEMS Yeast Res 13:597–608. https://doi.org/10.1111/1567-1364.12060
Zhao XJ, Raitt D, Burke VP, Clewell AS, Kwast KE, Poyton RO (1996) Function and expression of flavohemoglobin in Saccharomyces cerevisiae. Evidence for a role in the oxidative stress response. J Biol Chem 41:25131–25138. https://doi.org/10.1074/jbc.271.41.25131
Acknowledgements
The authors gratefully thank the technical staff of the Northern Centre for Technological Strategies CETENE (Recife, Pernambuco, Brazil at https://www.cetene.gov.br/) for their kind help with MALDI-ToF and MS analyses and Dr. Jimmy Eng of the Centre for Advanced Proteomics of the University of Washington (Seattle, WA, USA at http://proteomicsresource.washington.edu/) for the use of MASCOT with the specific D. bruxellensis database. The English text of this paper has been revised by Sidney Pratt, Canadian, MAT (The Johns Hopkins University), RSAdip - TESL (Cambridge University).
Funding
This work was sponsored by grants of the National Council of Science and Technology (CNPq/process 409767/2018-2 and CNPq/process 303551/2017-8) and by the Bioethanol Research Network of the State of Pernambuco (CNPq-FACEPE/PRONEM APQ-1452-2.01/10).
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MAMJ, WBP and TCJ conceived and designed the research. ICPM, DCP, KMS and EPNP conducted the experiments. ICPM, DCP, EPNP and FACS performed the proteome annotation and GO analyses. ICPM, WBP, TCJ and MAMJ prepared the manuscript. All authors read and approved the submitted manuscript.
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Peña-Moreno, I.C., Parente, D.C., da Silva, K.M. et al. Comparative proteomic analyses reveal the metabolic aspects and biotechnological potential of nitrate assimilation in the yeast Dekkera bruxellensis. Appl Microbiol Biotechnol 105, 1585–1600 (2021). https://doi.org/10.1007/s00253-021-11117-0
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DOI: https://doi.org/10.1007/s00253-021-11117-0