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Microbial Carotenoids pp 239–256Cite as

Expression Vectors and Gene Fusions for the Directed Modification of the Carotenoid Biosynthesis Pathway in Mucor circinelloides

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1852))

Abstract

Several fungal species, particularly some included in the Mucoromycotina, have been used to develop fermentation processes for the production of β-carotene. Oxygenated derivatives of β-carotene (xanthophylls) are desirable value-added products, and the preference by the market of carotenoids from biological sources has increased the research in different carotenoid-producing organisms. We currently use Mucor circinelloides f. lusitanicus as a model organism to develop strains with an increased content of new and more valuable carotenoids. The main carotenoid accumulated by M. circinelloides is β-carotene, although it has some hydroxylase activity and produces low amounts of zeaxanthin. On the other hand, in astaxanthin-producing organisms two enzymatic activities are required for the production of astaxanthin from β-carotene: a hydroxylase and a ketolase. In this chapter, we delineate part of our efforts to construct genetically modified strains that could advance in the improvement of carotenoid accumulation by this fungus and the diversification of its carotenoid content. Accordingly, we describe detailed and empirically tested protocols for the construction of functional expression vectors and gene fusions.

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References

  1. Nishikawa Y et al (2005) Effects of astaxanthin and vitamin C on the prevention of gastric ulcerations in stressed rats. J Nutr Sci Vitaminol 51:135–141

    Article  CAS  PubMed  Google Scholar 

  2. Chew BP, Park JS (2004) Carotenoid action on the immune response. J Nutr 134:257–261

    Article  Google Scholar 

  3. Vainio H, Rautalahti M (1998) An international evaluation of the cancer preventive potential of carotenoids. Cancer Epidemiol Biomark Prev 7:725–728

    CAS  Google Scholar 

  4. Johnson EA, Schroeder WA (1995) Microbial carotenoids. In: Fiechter A (ed) Advances in biochemical engineering and biotechnology. Springer, Berlin

    Google Scholar 

  5. Misawa N, Shimada H (1997) Metabolic engineering for the production of carotenoids in non-carotenogenic bacteria and yeasts. J Biotechnol 59:169–181

    Article  CAS  PubMed  Google Scholar 

  6. Schmidt-Dannert C, Umeno D, Arnold FH (2000) Molecular breeding of carotenoid biosynthesis pathways. Nat Biotechnol 18:750–753

    Article  CAS  PubMed  Google Scholar 

  7. Umeno D, Tobias AV, Arnold FH (2005) Diversifying carotenoid biosynthetic pathways by directed evolution. Microbiol Mol Biol Rev 69:51–78

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ukibe K et al (2009) Metabolic engineering of Saccharomyces cerevisiae for astaxanthin production and oxidative stress tolerance. Appl Environ Microbiol 75:7205–7211

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. de la Fuente JL et al (2010) High-titer production of astaxanthin by the semi-industrial fermentation of Xanthophyllomyces dendrorhous. J Biotechnol 148:144–146

    Article  CAS  PubMed  Google Scholar 

  10. Domínguez-Bocanegra AR, Ponce-Noyola T, Torres-Muñoz JA (2007) Astaxanthin production by Phaffia rhodozyma and Haematococcus pluvialis: a comparative study. Appl Microbiol Biotechnol 75:783–791

    Article  CAS  PubMed  Google Scholar 

  11. Iturriaga EA, Velayos A, Eslava AP (2000) The structure and function of the genes involved in the biosynthesis of carotenoids in the Mucorales. Biotechnol Bioprocess Eng 5:263–274

    Article  CAS  Google Scholar 

  12. Velayos A, Eslava AP, Iturriaga EA (2000) A bifunctional enzyme with lycopene cyclase and phytoene synthase activities is encoded by the carRP gene of Mucor circinelloides. Eur J Biochem 267:5509–5519

    Article  CAS  PubMed  Google Scholar 

  13. Iturriaga EA et al (2001) The genetics and molecular biology of carotenoid biosynthesis in Mucor. In: Pandalai SG (ed) Recent research developments in current genetics. Research Signpost, Trivandrum

    Google Scholar 

  14. Velayos A et al (2003) Expression of the carG gene, encoding geranylgeranyl pyrophosphate synthase, is up-regulated by blue light in Mucor circinelloides. Curr Genet 43:112–120

    PubMed  CAS  Google Scholar 

  15. Iturriaga EA et al (2005) Strain and culture conditions improvement for β-carotene production with Mucor. In: Barredo JL (ed) Microbial processes and products. Humana Press, Totowa, NJ

    Google Scholar 

  16. Papp T et al (2006) Heterologous expression of astaxanthin biosynthesis genes in Mucor circinelloides. Appl Microbiol Biotechnol 69:526–531

    Article  CAS  PubMed  Google Scholar 

  17. Wolff AM, Arnau J (2002) Cloning of glyceraldehyde-3-phosphate dehydrogenase-encoding genes in Mucor circinelloides (Syn. racemosus) and use of the gpd1 promoter for recombinant protein production. Fungal Genet Biol 35:21–29

    Article  CAS  PubMed  Google Scholar 

  18. Larsen GG et al (2004) Characterization of the Mucor circinelloides regulated promoter gpd1P. Curr Genet 45:225–234

    Article  CAS  PubMed  Google Scholar 

  19. Tao L et al (2006) Engineering a β-carotene ketolase for astaxanthin production. Metab Eng 8:523–531

    Article  CAS  PubMed  Google Scholar 

  20. Fraser PD, Miura Y, Misawa N (1997) In vitro characterization of astaxanthin biosynthetic enzymes. J Biol Chem 272:6128–6135

    Article  CAS  PubMed  Google Scholar 

  21. Domenech C et al (1996) Separate compartments for the production of sterols, carotenoids and gibberellins in Gibberella fujikuroi. Eur J Biochem 239:720–725

    Article  CAS  PubMed  Google Scholar 

  22. Kuzina V, Domenech C, Cerdá-Olmedo E (2006) Relationships among the biosynthesis of ubiquinone, carotene, sterols, and triacylglycerols in Zygomycetes. Arch Microbiol 186:485–493

    Article  CAS  PubMed  Google Scholar 

  23. Csernetics A et al (2011) Expression of three isoprenoid biosynthesis genes and their effects on the carotenoid production of the zygomycete Mucor circinelloides. Fungal Genet Biol 48:696–703

    Article  CAS  PubMed  Google Scholar 

  24. Álvarez V et al (2006) The crtS gene of Xanthophyllomyces dendrorhous encodes a novel cytochrome-P450 hydroxylase involved in the conversion of β-carotene into astaxanthin and other xanthophylls. Fungal Genet Biol 43:261–272

    Article  CAS  PubMed  Google Scholar 

  25. Roncero MIG et al (1989) Characterization of a leuA gene and an ARS element from Mucor circinelloides. Gene 84:335–343

    Article  CAS  PubMed  Google Scholar 

  26. Benito EP et al (1995) Isolation, characterization and transformation, by autonomous replication, of Mucor circinelloides OMPdecase-deficient mutants. Mol Gen Genet 248:126–135

    Article  CAS  PubMed  Google Scholar 

  27. Misawa N et al (1995) Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level. J Bacteriol 177:6575–6584

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Garre V, Barredo JL, Iturriaga EA (2015) Transformation of Mucor circinelloides f. lusitanicus protoplasts. In: van den Berg MA, Maruthachalam K (eds) Genetic transformation systems in fungi, Fungal biology, vol 1. Springer International Publishing, Cham

    Google Scholar 

  29. Schipper MAA (1976) On Mucor circinelloides, Mucor racemosus and related species. Stud Mycol 12:1–40

    Google Scholar 

  30. Díaz-Mínguez JM, López-Matas MA, Eslava AP (1999) Complementary mating types of Mucor circinelloides show electrophoretic karyotype heterogeneity. Curr Genet 36:383–389

    Article  PubMed  Google Scholar 

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Acknowledgments

This work was supported by different grants of the Junta de Castilla y León (Spain) (GR64) and the Hungarian Scientific Research Fund and the National Office for Research and Technology (OTKA CK80188).

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Authors and Affiliations

  1. Área de Genética, Departamento de Microbiología y Genética, University of Salamanca, Salamanca, Spain

    Enrique A. Iturriaga, María Isabel Alvarez & Arturo P. Eslava

  2. Department of Microbiology, University of Szeged, Szeged, Hungary

    Tamás Papp

Authors
  1. Enrique A. Iturriaga
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  2. María Isabel Alvarez
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  3. Arturo P. Eslava
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  4. Tamás Papp
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Corresponding author

Correspondence to Enrique A. Iturriaga .

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Editors and Affiliations

  1. Instituto de Biotecnología de León (INBIOTEC), Parque Científico de León, León, Spain

    Carlos Barreiro

  2. Department of Biotechnology, Crystal Pharma, A Subsidiary of Albany, Molecular Research Inc. (AMRI), Parque Tecnológico de León, León, Spain

    José-Luis Barredo

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Iturriaga, E.A., Alvarez, M.I., Eslava, A.P., Papp, T. (2018). Expression Vectors and Gene Fusions for the Directed Modification of the Carotenoid Biosynthesis Pathway in Mucor circinelloides. In: Barreiro, C., Barredo, JL. (eds) Microbial Carotenoids. Methods in Molecular Biology, vol 1852. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8742-9_14

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  • DOI: https://doi.org/10.1007/978-1-4939-8742-9_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8741-2

  • Online ISBN: 978-1-4939-8742-9

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