Abstract
The carotene producer fungus Mucor circinelloides is the zygomycete more amenable to genetic manipulations by using molecular tools. Since the initial development of an effective procedure of genetic transformation, more than two decades ago, the availability of new molecular approaches such as gene replacement techniques and gene expression inactivation by RNA silencing, in addition to the sequencing of its genome, has made Mucor a valuable organism for the study of a number of processes. Here we describe in detail the main techniques and methods currently used to manipulate M. circinelloides, including transformation, gene replacement, gene silencing, RNAi, and immunoprecipitation.
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References
Silva F, Torres-Martínez S, Garre V (2006) Distinct white collar-1 genes control specific light responses in Mucor circinelloides. Mol Microbiol 61:1023–1037
Silva F, Navarro E, Peñaranda A, Murcia-Flores L, Torres-Martínez S, Garre V (2008) A RING-finger protein regulates carotenogenesis via proteolysis-independent ubiquitylation of a White Collar-1-like activator. Mol Microbiol 70:1026–1036
Idnurm A, Walton FJ, Floyd A, Heitman J (2008) Identification of the sex genes in an early diverged fungus. Nature 451:193–196
Lee SC, Corradi N, Byrnes EJ III, Torres-Martínez S, Dietrich FS, Keeling PJ, Heitman J (2008) Microsporidia evolved from ancestral sexual fungi. Curr Biol 18:1–5
Zemach A, McDaniel IE, Silva P, Zilberman D (2010) Genome-wide evolutionary analysis of eukaryotic DNA methylation. Science 328:916–919
Nicolás FE, Torres-Martínez S, Ruiz-Vázquez RM (2003) Two classes of small antisense RNAs in fungal RNA silencing triggered by non integrative transgenes. EMBO J 22:3983–3991
Nicolás FE, De Haro JP, Torres-Martínez S, Ruiz-Vázquez RM (2007) Mutants defective in a Mucor circinelloides dicer-like gene are not compromised in siRNA silencing but display developmental defects. Fungal Genet Biol 44:504–516
Nicolás FE, Torres-Martínez S, Ruiz-Vázquez RM (2009) Transcriptional activation increases RNA silencing efficiency and stability in the fungus Mucor circinelloides. J Biotechnol 142:123–126
De Haro JP, Calo S, Cervantes M, Nicolás FE, Torres-Martínez S, Ruiz-Vázquez RM (2009) A single gene required for efficient gene silencing associated with two classes of small antisense RNAs in Mucor circinelloides. Eukaryot Cell 8:1486–1497
Nicolás FE, Moxon S, De Haro JP, Calo S, Grigoriev IV, Torres-Martínez S, Moulton V, Ruiz-Vázquez RM, Dalmay T (2010) Endogenous short RNAs generated by Dicer 2 and RNA-dependent RNA polymerase 1 regulate mRNAs in the basal fungus Mucor circinelloides. Nucleic Acids Res 38:5535–5541
Rodríguez-Sáiz M, Paz B, De La Fuente JL, López-Nieto MJ, Cabri W, Barredo JL (2004) Blakeslea trispora genes for carotene biosynthesis. Appl Environ Microbiol 70:5589–5594
Ruiz-Hidalgo MJ, Benito EP, Sandmann G, Eslava AP (1997) The phytoene dehydrogenase gene of Phycomyces: regulation of its expression by blue light and vitamin A. Mol Gen Genet 253:734–744
Arrach N, Fernández-Martín R, Cerdá-Olmedo E, Ávalos J (2001) A single gene for lycopene cyclase, phytoene synthase and regulation of carotene biosynthesis in Phycomyces. Proc Natl Acad Sci USA 98:1687–1692
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
Velayos A, Blasco JL, Álvarez MI, Iturriaga EA, Eslava AP (2000) Blue-light regulation of the phytoene dehydrogenase (carB) gene expression in Mucor circinelloides. Planta 210:938–946
van Heeswijck R, Roncero MIG (1984) High frequency transformation of Mucor with recombinant plasmid DNA. Carlsberg Res Commun 49:691–702
Gutiérrez A, López-García S, Garre V (2011) High reliability transformation of the basal fungus Mucor circinelloides by electroporation. J Microbiol Methods 84:442–446
Navarro E, Lorca-Pascual JM, Quiles-Rosillo MD, Nicolás FE, Garre V, Torres-Martínez S, Ruiz-Vázquez RM (2001) A negative regulator of light-inducible carotenogenesis in Mucor circinelloides. Mol Gen Genet 266:463–470
Nicolás FE, Calo S, Murcia-Flores L, Garre V, Ruiz-Vázquez RM, Torres-Martínez S (2008) A RING-finger photocarotenogenic repressor involved in asexual sporulation in Mucor circinelloides. FEMS Microbiol Lett 280:81–88
Roncero MIG (1984) Enrichment method for the isolation of auxotrophic mutants of Mucor using the polyenic antibiotic N-glycosylpolyfungin. Carlsberg Res Commun 49:685–690
Wolf AM, Arnau J (2002) Cloning of glyceraldehyde-3-phosphate dehydrogenase-encoding genes in Mucor circinelloides (syn. racemosus) and use of the gpd1 promoter in recombinant protein production. Fungal Genet Biol 35:21–29
Roncero MIG, Jepsen LP, Strøman P, van Heeswijck R (1989) Characterization of a leuA gene and ARS element from Mucor circinelloides. Carlsberg Res Commun 84:335–343
Acknowledgments
The work in our laboratory is supplied by Fundacion Séneca de la Comunidad Autónoma de la Región de Murcia, Spain (project 08802/PI/08), by D.G. de Investigación y Política Científica (Comunidad Autónoma de la Región de Murcia, Spain; Project Bio-BMC 07/01-0005), and Spanish Ministerio de Ciencia e Innovación (BFU2006-02408, BFU2009-07220).
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Torres-Martínez, S., Ruiz-Vázquez, R.M., Garre, V., López-García, S., Navarro, E., Vila, A. (2012). Molecular Tools for Carotenogenesis Analysis in the Zygomycete Mucor circinelloides . In: Barredo, JL. (eds) Microbial Carotenoids From Fungi. Methods in Molecular Biology, vol 898. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-918-1_5
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DOI: https://doi.org/10.1007/978-1-61779-918-1_5
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