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Archives of Microbiology

, Volume 196, Issue 6, pp 411–421 | Cite as

Analysis of proteomic changes in colored mutants of Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

  • Alejandra Barbachano-Torres
  • Lina M. Castelblanco-Matiz
  • Ana C. Ramos-Valdivia
  • Carlos M. Cerda-García-Rojas
  • Luis M. Salgado
  • César M. Flores-Ortiz
  • Teresa Ponce-NoyolaEmail author
Original Paper

Abstract

The yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin as its most prevalent xanthophyll derivative. Comparisons between the protein profiles of mutant lines of this yeast can provide insight into the carotenogenic pathway. Differently colored mutants (red, orange, pink, yellow, and white) were obtained from this yeast species, and their protein profiles were determined using two-dimensional polyacrylamide gel electrophoresis (2DE). Individual proteins differentially expressed were identified using mass spectrometry. The red mutants hyperproduced total carotenoids (mainly astaxanthin), while in white and orange mutants, mutagenesis affected the phytoene dehydrogenase activity as indicated by the accumulation of phytoene. Inactivation of astaxanthin synthase after the mutagenic treatment was evident in β-carotene accumulating mutants. Differences in the proteomic profiles of wild-type X. dendrorhous and its colored mutants were demonstrated using 2DE. Of the total number of spots detected in each gel (297–417), 128 proteins were present in all strains. The red mutant showed the greatest number of matches with respect to the wild type (305 spots), while the white and yellow mutants, which had reduced concentrations of total carotenoids, presented the highest correlation coefficient (0.6) between each other. A number of differentially expressed proteins were sequenced, indicating that tricarboxylic acid cycle and stress response proteins are closely related to the carotenogenic process.

Keywords

Carotenoid biosynthesis Proteomic profile Secondary metabolism Stress response Tricarboxylic acid cycle 

Notes

Acknowledgments

We thank Professors E. Cerdá-Olmedo and J. Ávalos and their working groups from the Genetic Department at Seville University for their technical support in mutant isolation and characterization. A Barbachano-Torres and LM Castelblanco-Matiz thank CONACYT-México for doctoral (172824 and 219320, respectively) fellowships.

Supplementary material

203_2014_979_MOESM1_ESM.tiff (325 kb)
Fig. 1 Survival of X. dendrorhous after its exposition to several NTG concentrations and exposition time: □15 min, ♦30 min (TIFF 324 kb)

References

  1. Alcaíno J, Barahona S, Carmona M, Lozano C, Marcoleta A, Niklitschek M, Sepúlveda D, Marcelo B (2008) Cloning of the cytochrome P450 reductase (crtR) gene and its involvement in the astaxanthin biosynthesis of X. dendrhorhous. BMC Microbiol 8(189):1–13Google Scholar
  2. Álvarez V, Rodríguez-Sáiz M, de la Fuente JL, Gudiña EJ, Godio RP, Martín JF, Barredo JL (2006) The crtS gene of Xanthophyllomyces dendrorhous encodes a novel cytochrome-P450 hydroxylase involved in the conversion of beta-carotene into astaxanthin and other xanthophylls. Fungal Genet Biol 43:261–272PubMedCrossRefGoogle Scholar
  3. An GH (2001) Improved growth of the red yeast, Phaffia rhodozyma (Xanthophyllomyces dendrorhous), in the presence of tricarboxylic acid cycle intermediates. Biotechnol Lett 23:1005–1009CrossRefGoogle Scholar
  4. An GH, Schumau DB, Johnson EA (1989) Isolation of Phaffia rhodozyma mutants with increased astaxanthin content. Appl Environ Microbiol 55:116–124PubMedCentralPubMedGoogle Scholar
  5. Asadollahi MA, Maury J, Patil KR, Schalk M, Clark A, Nielsen J (2009) Enhancing sesquiterpene production in Saccharomyces cerevisiae through in silico driven metabolic engineering. Metabolic Eng 11:328–334CrossRefGoogle Scholar
  6. Barbachano-Torres A, Ramos-Valdivia AC, Cerda-García-Rojas CM, Salgado-Rodríguez LM, Flores-Ortiz C, Ponce-Noyola T (2012) Carotenogenesis induction with hydrogen peroxide in Xanthophyllomyces dendrorhous colored mutants. In: Mendez-Vilas A (ed) Microbes in applied research: current advances and challenges. World Scientific Publishing Co Pvt. Ltd., Singapore, pp 598–602CrossRefGoogle Scholar
  7. Becker J, Craig EA (1994) Heat-shock proteins as molecular chaperones. Eur J Biochem 219:11–23PubMedCrossRefGoogle Scholar
  8. Bhosale P, Bernstein PS (2005) Microbial xanthophylls. Appl Microbiol Biotechnol 68:445–455PubMedCrossRefGoogle Scholar
  9. Browning KS, Uhlinger DJ, Reed LJ (1988) Nucleotide sequence for yeast dihydrolipoamide dehydrogenase. PNAS 85:1831–1834PubMedCentralPubMedCrossRefGoogle Scholar
  10. Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, Carnemolla B, Orecchia P, Zardi L, Righetti PG (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25:1327–1333PubMedCrossRefGoogle Scholar
  11. Chávez-Cabrera C, Flores-Bustamante ZR, Marsch R, del Montes CM, Sánchez S, Cancino-Díaz JC, Flores-Cotera LB (2010) ATP-citrate lyase activity and carotenoid production in batch cultures of Phaffia rhodozyma under nitrogen-limited and nonlimited conditions. Appl Microbiol Biotechnol 85:1953–1960PubMedCrossRefGoogle Scholar
  12. Cifuentes V, Hermosilla G, Martínez C, León R, Pincheira G, Jiménez A (1997) Genetics and electrophoretic karyotyping of wild-type and astaxanthin mutant strains of Phaffia rhodozyma. Antonie Van Leeuwenhoek 72:111–117PubMedCrossRefGoogle Scholar
  13. DeLuna A, Avendano A, Riego L, González A (2001) NADP-glutamate dehydrogenase isoenzymes of Saccharomyces cerevisiae. Purification, kinetic properties, and physiological roles. J Biol Chem 276:43775–43783PubMedCrossRefGoogle Scholar
  14. Farah ME, Amberg DC (2007) Conserved actin cysteine residues are oxidative stress sensors that can regulate cell death in yeast. Mol Biol Cel 18:1359–1365CrossRefGoogle Scholar
  15. Flaherty KM, McKay DB, Kabsch W, Holmes KC (1991) Similarity of the three-dimensional structures of actin and the ATPase fragment of a 70-kDa heat shock cognate protein. PNAS 88:5041–5045PubMedCentralPubMedCrossRefGoogle Scholar
  16. Giuliano G, Pollock D, Scolnik PA (1986) The gene crtI mediates the conversion of phytoene into colored carotenoids in Phodopseudomonas capsulata. J Biol Chem 261:12925–12929PubMedGoogle Scholar
  17. González-Roncero MI, Zabala C, Cerdá Olmedo E (1984) Mutagenesis in multinucleate cells: the effects of N-methyl-N′-nitro-N-nitrosoguanidine on Phycomyces spores. Mutat Res 125:195–204CrossRefGoogle Scholar
  18. Griffith CL, Klutts JS, Zhang L, Levery SB, Doering TL (2004) UDP-glucose dehydrogenase plays multiple roles in the biology of the pathogenic fungus Cryptococcus neoformans. J Biol Chem 279:51669–51676PubMedCrossRefGoogle Scholar
  19. Hermosilla G, Martínez C, Retamales P, León R, Cifuentes V (2003) Genetic determination of ploidy level in Xanthophyllomyces dendrorhous. Antonie Van Leeuwenhoek 84:279–287PubMedCrossRefGoogle Scholar
  20. Hu ZC, Zheng YG, Wang Z, Shen YC (2006) pH control strategy in astaxanthin fermentation bioprocess by Xanthophyllomyces dendrorhous. Enzyme Microbial Technol 39:586–590CrossRefGoogle Scholar
  21. Johnson EA (2003) Phaffia rhodozyma: colorful odyssey. Int Microbiol 6:169–174PubMedCrossRefGoogle Scholar
  22. Johnson EA, An GH (1991) Astaxanthin from microbial sources. Crit Rev Biotech 11:297–326CrossRefGoogle Scholar
  23. Kampranis S, Makris AM (2012) Developing a yeast cell factory for the production of terpenoids. Comput Struct Biotechnol J 3:1–7. doi: 10.5936/csbj.201210006 Google Scholar
  24. Kocharin K (2013) Metabolic engineering of Saccharomyces cerevisiae for polyhydroxybutyrate production. Dissertation, Chalmers University of Technology, Göteborg, SwedenGoogle Scholar
  25. Kondo H, Nakamura Y, Dong YX, Nikawa JI, Sueda S (2004) Pyridoxine biosynthesis in yeast: participation of ribose 5-phosphate ketol-isomerase. Biochem J 379:65–70PubMedCentralPubMedCrossRefGoogle Scholar
  26. Kucsera J, Pfeiffer I, Ferenczy L (1998) Homothallic life cycle in the diploid red yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma). Antonie Van Leeuwenhoek 73:163–168PubMedCrossRefGoogle Scholar
  27. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  28. Lawrence CW (2002) Classical mutagenesis techniques. Methods Enzymol 350:189–199PubMedCrossRefGoogle Scholar
  29. Lee YJ, Kim KJ, Kang HY, Kim HR, Maeng PJ (2012) Involvement of GDH3-encoded NADP+-dependent glutamate dehydrogenase in yeast cell resistance to stress-induced apoptosis in stationary phase cells. J Biol Chem 287:44221–44233PubMedCentralPubMedCrossRefGoogle Scholar
  30. Liu YS, Wu JY (2006) Hydrogen peroxide-induced astaxanthin biosynthesis and catalase activity in Xanthophyllomyces dendrorhous. Appl Microbiol Biotechnol 73:663–668PubMedCrossRefGoogle Scholar
  31. Liu ZQ, Zhang JF, Zheng YG, Shen YC (2008) Improvement of astaxanthin production by a newly isolated Phaffia rhodozyma mutant with low-energy ion beam implantation. J Appl Microbiol 104:861–872PubMedCrossRefGoogle Scholar
  32. Long TV (2004) Process for production of carotenoids, xanthophylls and apo-carotenoids utilizing eukaryotic microorganisms. US PATENT 7563935 B2Google Scholar
  33. Lowry OH, Rosebrough NJ, Farr GL, Randall RS (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  34. Martinez-Moya P, Watt SA, Niehaus K, Alcaíno J, Baeza M, Cifuentes V (2011) Proteomic analysis of the carotenogenic yeast Xanthophyllomyces dendrorhous. BMC Microbiol 11:1–14CrossRefGoogle Scholar
  35. McNulty H, Jacob RF, Mason P (2008) Biologic activity of carotenoids related to distinct membrane physicochemical interactions. Am J Cardiol 101:20–29CrossRefGoogle Scholar
  36. Medwid RD (1998) Phaffia rhodozyma is polyploid. J Ind Microbiol Biotechnol 21:228–232CrossRefGoogle Scholar
  37. Miller SM, Magasanik B (1990) Role of NAD-linked glutamate dehydrogenase in nitrogen metabolism in Saccharomyces cerevisiae. J Bacteriol 172:4927–4935PubMedCentralPubMedGoogle Scholar
  38. Misawa N, Shimada H (1998) Metabolic engineering for the production of carotenoids in non-carotenogenic bacteria and yeasts. J Biotechnol 59:169–181CrossRefGoogle Scholar
  39. Mittenhuber G (2001) Phylogenetic analyses and comparative genomics of vitamin B6 (Pyridoxine) and pyridoxal phosphate biosynthesis pathways. J Mol Microbiol Biotechnol 3:1–20PubMedGoogle Scholar
  40. Molloy MP (2008) Isolation of bacterial cell membranes proteins using carbonate extraction. Methods Mol Biol 424:397–401PubMedCrossRefGoogle Scholar
  41. Ojima K, Breitenbach J, Visser H, Setoguchi Y, Tabata K, Hoshino T, van den Berg J, Sandmann G (2006) Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and its assignment as a β-carotene 3-hydroxylase/4-ketolase. Mol Genet Genomics 275:148–158PubMedCrossRefGoogle Scholar
  42. Olaizola M (2000) Commercial production of astaxanthin from Haematococcus pluvialis using 25,000-liter outdoor photobioreactors. J App Phycol 12:499–506CrossRefGoogle Scholar
  43. Qin G, Gu H, Ma L, Peng Y, Deng XW, Chen Z, Qu LJ (2007) Disruption of phytoene desaturase results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellins biosynthesis. Cell Res 17:471–482PubMedCrossRefGoogle Scholar
  44. Rodríguez-Sáiz M, Godio RP, Alvarez V, de la Fuente JL, Martín JF, Barredo JL (2009) The NADP-dependent glutamate dehydrogenase gene from the astaxanthin producer Xanthophyllomyces dendrorhous: use of Its promoter for controlled gene expression. Mol Biotechnol 41:165–172PubMedCrossRefGoogle Scholar
  45. Sandmann G, Albrecht M, Schnurr G, Knörzer O, Böger P (1999) The biotechnological potential and design of novel carotenoids by gene combination in Escherichia coli. Trends Biotechnol 17:233–237PubMedCrossRefGoogle Scholar
  46. Schroeder WA, Johnson EA (1993) Antioxidant role of carotenoids in Phaffia rhodozyma. J Gen Microbiol 139:907–912CrossRefGoogle Scholar
  47. Sedmak JJ, Weerasinghe DK, Jolly SO (1990) Extraction and quantification of astaxanthin from Phaffia rhodozyma. Biotechnol Tech 4:107–112CrossRefGoogle Scholar
  48. Shi Y, Thomas JO (1992) The transport of proteins into the nucleus requires the 70-kilodalton heat shock protein or its cytosolic cognate. Mol Cell Biol 12:2186–2192PubMedCentralPubMedGoogle Scholar
  49. Tahara EB, Barros MH, Oliveira GA, Netto LE, Kowaltowski AJ (2007) Dihydrolipoyl dehydrogenase as a source of reactive oxygen species inhibited by caloric restriction and involved in Saccharomyces cerevisiae aging. FASEB J 21:274–283PubMedCrossRefGoogle Scholar
  50. Tanaka T, Tateno Y, Gojobori T (2005) Evolution of vitamin B6 (pyridoxine) metabolism by gain and loss of genes. Mol Biol Evol 22:243–250PubMedCrossRefGoogle Scholar
  51. Tretter L, Adam-Vizi V (2005) Alpha-ketoglutarate dehydrogenase: a target and generator of oxidative stress. Philos Trans R Soc B 360:2335–2345CrossRefGoogle Scholar
  52. Verdoes JC, Wery J, Boekhout T, Van Ooyen AJ (1997) Molecular characterization of the glyceraldehyde-3-phosphate dehydrogenase gene of Phaffia rhodozyma. Yeast 13:1231–1242PubMedCrossRefGoogle Scholar
  53. Verdoes JC, Krubasik KP, Sandmann G, Van Ooyen AJ (1999) Isolation and functional characterisation of a novel type of carotenoid biosynthetic gene from Xanthophyllomyces dendrorhous. Mol Gen Genet 262:453–461PubMedCrossRefGoogle Scholar
  54. Verdoes JC, Sandmann G, Visser H, Diaz M, van Mossel M, van Ooyen A (2003) Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma). Appl Environ Microbiol 69:3728–3738PubMedCentralPubMedCrossRefGoogle Scholar
  55. Wang C, Long X, Mao X, Dong H, Xu L, Li Y (2010) SigN is responsible for differentiation and stress responses based on comparative proteomic analysis of Streptomyces coelicolor wild-type and sigN deletion strains. Microbiol Res 165:221–231PubMedCrossRefGoogle Scholar
  56. Yamane Y, Higashida K, Nakashimada Y, Kakizono T, Nishio N (1997) Influence of oxygen and glucose on primary metabolism and astaxanthin production by Phaffia rhodozyma in batch and fed-batch cultures: kinetic and stoichiometric analysis. Appl Environ Microbiol 63:4471–4478PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Alejandra Barbachano-Torres
    • 1
  • Lina M. Castelblanco-Matiz
    • 1
  • Ana C. Ramos-Valdivia
    • 1
  • Carlos M. Cerda-García-Rojas
    • 2
  • Luis M. Salgado
    • 3
  • César M. Flores-Ortiz
    • 4
  • Teresa Ponce-Noyola
    • 1
    Email author
  1. 1.Department of Biotechnology and BioengineeringCINVESTAV-IPNMexico CityMexico
  2. 2.Department of ChemistryCINVESTAV-IPNMexico CityMexico
  3. 3.CICATA-QroInstituto Politécnico NacionalMexico CityMexico
  4. 4.FES-Iztacala UNAMMexico CityMexico

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