Enhancement of carotenoid production by disrupting the C22-sterol desaturase gene (CYP61) in Xanthophyllomyces dendrorhous

BMC Microbiology

Jennifer Alcaíno¹  Víctor Cifuentes¹  Marcelo Baeza¹  Pilar Martínez-Moya¹  Dionisia Sepúlveda¹  Salvador Barahona¹  María Soledad Gutiérrez¹  Iris Loto¹ 

[1] Laboratorio de Genética. Departamento de Ciencias Ecológicas y Centro de Biotecnología, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Santiago Casilla 653, Chile

关键词: Cytochrome P450;    Sterol C22-sterol desaturase;    Ergosterol;    Astaxanthin;    Xanthophyllomyces dendrorhous;   

Others  :  1221707
DOI  :  10.1186/1471-2180-12-235

 received in 2012-08-03, accepted in 2012-10-12,  发布年份 2012

【 摘 要 】

Background

Xanthophyllomyces dendrorhous is a basidiomycetous yeast that synthesizes astaxanthin, which is a carotenoid with a great biotechnological impact. The ergosterol and carotenoid synthesis pathways are derived from the mevalonate pathway, and in both pathways, cytochrome P450 enzymes are involved.

Results

In this study, we isolated and described the X. dendrorhous CYP61 gene, which encodes a cytochrome P450 involved in ergosterol biosynthesis. This gene is composed of nine exons and encodes a 526 amino acid polypeptide that shares significant percentages of identity and similitude with the C22-sterol desaturase, CYP61, from other fungi. Mutants derived from different parental strains were obtained by disrupting the CYP61 gene with an antibiotic selection marker. These mutants were not able to produce ergosterol and accumulated ergosta-5,8,22-trien-3-ol and ergosta-5,8-dien-3-ol. Interestingly, all of the mutants had a more intense red color phenotype than their respective parental strains. The carotenoid composition was qualitatively and quantitatively analyzed by RP-HPLC, revealing that the carotenoid content was higher in the mutant strains without major changes in their composition. The expression of the HMGR gene, which encodes an enzyme involved in the mevalonate pathway (3-hydroxy-3-methylglutaryl-CoA reductase), was analyzed by RT-qPCR showing that its transcript levels are higher in the CYP61 mutants.

Conclusions

These results suggest that in X. dendrorhous, ergosterol regulates HMGR gene expression by a negative feedback mechanism and in this way; it contributes in the regulation of the carotenoid biosynthesis.

【 授权许可】

   
2012 Loto et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Golubev WI: Perfect state of Rhodomyces dendrorhous (Phaffia rhodozyma). Yeast 1995, 11:101-110.
• [2]Johnson EA: Phaffia rhodozyma: colorful odyssey. Int Microbiol 2003, 6:169-174.
• [3]Guerin M, Huntley ME, Olaizola M: Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol 2003, 21:210-216.
• [4]Schroeder WA, Johnson EA: Antioxidant role of carotenoids in Phaffia rhodozyma. J Gen Microbiol 1993, 139:907-912.
• [5]Schroeder WA, Johnson EA: Singlet oxygen and peroxyl radicals regulate carotenoid biosynthesis in Phaffia rhodozyma. J Biol Chem 1995, 270:18374-18379.
• [6]Schroeder WA, Johnson EA: Carotenoids protect Phaffia rhodozyma against singlet oxygen damage. J Ind Microbiol Biotechnol 1995, 14:502-507.
• [7]Fassett RG, Coombes JS: Astaxanthin: a potential therapeutic agent in cardiovascular disease. Mar Drugs 2011, 9:447-465.
• [8]Higuera-Ciapara I, Felix-Valenzuela L, Goycoolea FM: Astaxanthin: a review of its chemistry and applications. Crit Rev Food Sci Nutr 2006, 46:185-196.
• [9]Britton G, Liaaen-Jensen S, Pfander H: Carotenoids handbook. Switzerland: Birkhäuser Verlag; 2004.
• [10]Miziorko HM: Enzymes of the mevalonate pathway of isoprenoid biosynthesis. Arch Biochem Biophys 2011, 505:131-143.
• [11]Goldstein JL, Brown MS: Regulation of the mevalonate pathway. Nature 1990, 343:425-430.
• [12]Merkulov S, van Assema F, Springer J, Fernandez del Carmen A, Mooibroek H: Loning and characterization of the Yarrowia lipolytica squalene synthase (SQS1) gene and functional complementation of theSaccharomyces cerevisiae erg9 mutation. Yeast 2000, 16:197-206.
• [13]Verdoes JC, Krubasik P, Sandmann G, Van Ooyen AJJ: Isolation and functional characterization of a novel type of carotenoid biosynthetic gene from Xanthophyllomyces dendrorhous. Mol Gen Genet 1999, 262:453-461.
• [14]Verdoes JC, Misawa N, van Ooyen AJJ: Cloning and characterization of the astaxanthin biosynthetic gene encoding phytoene desaturase of Xanthophyllomyces dendrorhous. Biotechnol Bioeng 1999, 63:750-755.
• [15]Ojima K, Breitenbach J, Visser H, Setoguchi Y, Tabata K, Hoshino T, van den Berg J, Sandmann G: 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 2006, 275:148-158.
• [16]Álvarez V, Rodríguez-Sáiz M, de la Fuente JL, Gudiña EJ, Godio RP, Martín JF, Barredo JL: 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 2006, 43:261-272.
• [17]Zhang H, Im SC, Waskell L: Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4. J Biol Chem 2007, 282:29766-29776.
• [18]Degtyarenko KN, Archakov AI: Molecular evolution of P450 superfamily and P450-containing monooxygenase systems. FEBS Lett 1993, 332:1-8.
• [19]Kimmich N, Das A, Sevrioukova I, Meharenna Y, Sligar SG, Poulos TL: Electron transfer between cytochrome P450cin and its FMN-containing redox partner, cindoxin. J Biol Chem 2007, 282:27006-27011.
• [20]McLean KJ, Sabri M, Marshall KR, Lawson RJ, Lewis DG, Clift D, Balding PR, Dunford AJ, Warman AJ, McVey JP: Biodiversity of cytochrome P450 redox systems. Biochem Soc Trans 2005, 33:796-801.
• [21]Alcaíno J, Barahona S, Carmona M, Lozano C, Marcoleta A, Niklitschek M, Sepúlveda D, Baeza M, Cifuentes V: Cloning of the cytochrome p450 reductase (crtR) gene and its involvement in the astaxanthin biosynthesis of Xanthophyllomyces dendrorhous. BMC Microbiol 2008, 8:169. BioMed Central Full Text
• [22]Masamoto K, Misawa N, Kaneko T, Kikuno R, Toh H: Beta-carotene hydroxylase gene from the cyanobacterium Synechocystis sp. PCC6803. Plant Cell Physiol 1998, 39:560-564.
• [23]Zhang YQ, Rao R: Beyond ergosterol: linking pH to antifungal mechanisms. Virulence 2010, 1:551-554.
• [24]Kelly SL, Lamb DC, Baldwin BC, Corran AJ, Kelly DE: Characterization of Saccharomyces cerevisiae CYP61, sterol Δ22-desaturase, and inhibition by azole antifungal agents. J Biol Chem 1997, 272:9986-9988.
• [25]Skaggs BA, Alexander JF, Pierson CA, Schweitzer KS, Chun KT, Koegel C, Barbuch R, Bard M: Cloning and characterization of the Saccharomyces cerevisiae C-22 sterol desaturase gene, encoding a second cytochrome P-450 involved in ergosterol biosynthesis. Gene 1996, 169:105-109.
• [26]Veen M, Lang C: Production of lipid compounds in the yeast Saccharomyces cerevisiae. Appl Environ Microbiol 2004, 63:635-646.
• [27]Werck-Reichhart D, Feyereisen R: Cytochromes P450: a success story. Genome Biol 2000, 1:3003.1-3003.9.
• [28]Sirim D, Wagner F, Lisitsa A, Pleiss J: The cytochrome P450 engineering database: integration of biochemical properties. BMC Biochem 2009, 10:27. BioMed Central Full Text
• [29]van den Brink H, van Gorcom RFM, van den Hondel CA, Punt PJ: Cytochrome P450 enzyme systems in fungi. Fungal Genet Biol 1998, 23:1-17.
• [30]Hermosilla G, Martínez C, Retamales P, León R, Cifuentes V: Genetic determination of ploidy level in Xanthophyllomyces dendrorhous. Antonie Van Leeuwenhoek 2003, 84:279-287.
• [31]Niklitschek M, Alcaíno J, Barahona S, Sepúlveda D, Lozano C, Carmona M, Marcoleta A, Martínez C, Lodato P, Baeza M: Genomic organization of the structural genes controlling the astaxanthin biosynthesis pathway of Xanthophyllomyces dendrorhous. Biol Res 2008, 41:93-108.
• [32]Chang YC, Bien CM, Lee H, Espenshade PJ, Kwong-Chung KJ: Sre1p, A regulator of oxygen sensing and sterol homeostasis, is required for virulence in Cryptococcus neoformans. Molec Microbiol 2007, 64:614-629.
• [33]Hughes AL, Todd BL, Espenshade PJ: SREBP pathway responds to sterols and functions as an oxygen sensor in fission yeast. Cell 2005, 120:831-842.
• [34]Pearson WR, Wood T, Zhang Z, Miller W: Comparison of DNA sequences with protein sequences. Genomics 1997, 46:24-36.
• [35]Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2-[Delta][Delta] CT method. Methods 2001, 25:402-408.
• [36]Cresnar B, Petric S: Cytochrome P450 enzymes in the fungal kingdom. Biochim Biophys Acta 2011, 1814:29-35.
• [37]Lamb DC, Maspahy S, Kelly DE, Manning NJ, Geber A, Bennett JE, Kelly SL: Purification, reconstitution, and inhibition of cytochrome P-450 sterol Δ22-desaturase from the pathogenic fungus Candida glabrata. Antimicrob Agents Chemother 1999, 43:1725-1728.
• [38]Kristan K, Rizner TL: Steroid-transforming enzymes in fungi. J Steroid Biochem Mol Biol 2012, 129:79-91.
• [39]Nes WD, Zhou W, Ganapathy K, Liu JL, Vatsyayan R, Chamala S, Hernandez K, Miranda M: Sterol 24-C-methyltransferase: an enzymatic target for the disruption of ergosterol biosynthesis and homeostasis in Cryptococcus neoformans. Arch Biochem Biophys 2009, 481:210-218.
• [40]Morris DC, Safe S, Subden RE: Detection of the ergosterol and episterol isomers lichesterol and fecosterol in nystatin-resistant mutants of Neurospora crassa. Biochem Genet 1974, 12:459-466.
• [41]Kanafani ZA, Perfect JR: Antimicrobial resitance: resistance to antifungal agents: mechanisms and clinical impact. Clin Infect Dis 2008, 46:120-128.
• [42]Shingo H, Yoshihisa ODA, Nishino T, Katsuki H, Aoyama Y, Yoshtoa Y, Nagai J: Characterization of a Saccharomyces cerevisiae mutant, N22, defective in ergosterol synthesis and preparation of [28-14C] ergosta-5, 7-dien-3β-ol with the mutant. J Biochem 1983, 94:501-510.
• [43]Ziogas BN, Sisler HD, Lusby WR: Sterol content and other characteristics of pimaricin-resistant mutants of Aspergillus nidulans. Pestic Biochem Physiol 1983, 20:320-329.
• [44]Wozniak A, Lozano C, Barahona S, Niklitschek M, Marcoleta A, Alcaíno J, Sepulveda D, Baeza M, Cifuentes V: Differential carotenoid production and gene expression in Xanthophyllomyces dendrorhous grown in a nonfermentable carbon source. FEMS Yeast Res 2011, 11:252-262.
• [45]Lodato P, Alcaíno J, Barahona S, Niklitschek M, Carmona M, Wozniak A, Baeza M, Jiménez A, Cifuentes V: Expression of the carotenoid biosynthesis genes in Xanthophyllomyces dendrorhous. Biol Res 2007, 40:73-84.
• [46]Miao L, Chi S, Tang Y, Su Z, Yin T, Guan G, Li Y: Astaxanthin biosynthesis is enhanced by high carotenogenic gene expression and decrease of fatty acids and ergosterol in a Phaffia rhodozyma mutant strain. FEMS Yeast Res 2011, 11:192-201.
• [47]Calo P, Miguel T, Velázquez JB, Villa TG: Mevalonic acid increases trans-astaxanthin and carotenoid biosynthesis in Phaffia rhodozyma. Biotechnol Lett 1995, 17:575-578.
• [48]Shimada H, Kondo K, Fraser PD, Miura Y, Saito T, Misawa N: Increased carotenoid production by the food yeast Candida utilis through metabolic engineering of the isoprenoid pathway. Appl Environ Microbiol 1998, 64:2676-2680.
• [49]Parks LW, Casey WM: Physiological implications of sterol biosynthesis in yeast. Annu Rev Microbiol 1995, 49:95-116.
• [50]Tang Q, Li Y, Yuan QP: Effects of an ergosterol synthesis inhibitor on gene transcription of terpenoid biosynthesis in Blakeslea trispora. Curr Microbiol 2008, 57:527-531.
• [51]Yan G, Wen K, Duan C: Enhancement of β-carotene production by over-expression of HMG-CoA reductase coupled with addition of ergosterol biosynthesis inhibitors in recombinant Saccharomyces cerevisiae. Curr Microbiol 2012, 64:1-5.
• [52]Sambrook J, Russell DW: Molecular cloning. A laboratory manual. 3rd edition. Cold Spring Harbor NY: Cold Spring Harbor Laboratory Press; 2001.
• [53]Drocourt D, Calmels T, Reynes JP, Baron M, Tiraby G: Cassettes of the Streptoalloteichus hindustanus ble gene for transformation of lower and higher eukaryotes to phleomycin resistance. Nucleic Acids Res 1990, 18:4009-4009.
• [54]Calmels T, Parriche M, Durand H, Tiraby G: High efficiency transformation of Tolypocladium geodes conidiospores to phleomycin resistance. Curr Genet 1991, 20:309-314.
• [55]Boyle JS, Lew AM: An inexpensive alternative to glassmilk for DNA purification. TIG 1995, 11:8.
• [56]Hofmann K, Stoffel W: TMbase-A database of membrane spanning protein segments. Biol Chem Hoppe Seyler 1993, 374:166.
• [57]Zdobnov EM, Apweiler R: InterProScan–an integration platform for the signature-recognition methods in InterPro. Bioinformatics 2001, 17:847-848.
• [58]Sirim D, Widmann M, Wagner F, Pleiss J: Prediction and analysis of the modular structure of cytochrome P450 monooxygenases. BMC Struct Biol 2010, 10:34. BioMed Central Full Text
• [59]Adrio JL, Veiga M: Transformation of the astaxanthin-producing yeast Phaffia rhodozyma. Biotechnol Tech 1995, 9:509-512.
• [60]Kim IG, Nam SK, Sohn JH, Rhee SK, An GH, Lee SH, Choi ES: Cloning of the ribosomal protein L41 gene of Phaffia rhodozyma and its use as a drug resistance marker for transformation. Appl Environ Microbiol 1998, 64:1947-1949.
• [61]Fell JW, Blatt GM: Separation of strains of the yeasts Xanthophyllomyces dendrorhous and Phaffia rhodozyma based on rDNA IGS and ITS sequence analysis. J Ind Microbiol Biotechnol 1999, 23:677-681.
• [62]An GH, Schuman DB, Johnson EA: Isolation of Phaffia rhodozyma mutants with increased astaxanthin content. Appl Environ Microbiol 1989, 55:116-124.
• [63]Shang F, Wen S, Wang X, Tan T: Effect of nitrogen limitation on the ergosterol production by fed-batch culture of Saccharomyces cerevisiae. J Biotechnol 2006, 122:285-292.
• [64]Cheng B, Yuan Q, Sun X, Li W: Enhanced production of coenzyme Q10 by overexpressing HMG-CoA reductase and induction with arachidonic acid in Schizosaccharomyces pombe. Appl Biochem Biotechnol 2010, 160:523-531.
• [65]Lamacka M, Sajbidor J: Ergosterol determination in Saccharomyces cerevisiae comparison of different methods. Biotechnol Tech 1997, 11:723-725.
• [66]Wery J, Dalderup MJM, Ter Linde J, Boekhout T, Van Ooyen AJJ: Structural and phylogenetic analysis of the actin gene from the yeast Phaffia rhodozyma. Yeast 1996, 12:641-651.