Skip to main content

Integration of a Bacterial β-Carotene Ketolase Gene into the Mucor circinelloides Genome by the Agrobacterium tumefaciens-Mediated Transformation Method

  • Protocol
  • First Online:
Microbial Carotenoids From Fungi

Part of the book series: Methods in Molecular Biology ((MIMB,volume 898))

Abstract

Plasmids introduced in Mucor circinelloides (and most transformable Mucorales) tend to replicate autonomously, and hardly ever integrate in the genome. This is critical if we want to express exogenous genes, because plasmids are easily lost during vegetative growth, and the ratio of plasmid molecules/nuclei is invariably low. Linearized molecules of DNA have been used to get their genomic integration but the transformation efficiency drops extremely. We have developed and highly optimized an efficient Agrobacterium-mediated transformation system for M. circinelloides to facilitate the integration of transforming DNA in the genome of the recipient strain that could also be used for other Mucorales.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Iturriaga EA, Papp T, Breum J, Arnau J, Eslava AP (2005) Strain and culture conditions improvement for β-carotene production with Mucor. In: Barredo JL (ed) Microbial processes and products. Humana Press, Totowa

    Google Scholar 

  2. Papp T, Velayos A, Bartók T, Eslava AP, Vágvölgyi C, Iturriaga EA (2006) Heterologous expression of astaxanthin biosynthesis genes in Mucor circinelloides. Appl Microbiol Biotechnol 69:526–531

    Article  PubMed  CAS  Google Scholar 

  3. Monfort A, Cordero L, Maicas S, Polaina J (2003) Transformation of Mucor miehei results in plasmid deletion and phenotypic instability. FEMS Microbiol Lett 224:101–106

    Article  PubMed  CAS  Google Scholar 

  4. Michielse CB, Salim K, Ragas P, Ram AFJ, Kudla B, Jarry B, Punt J, van den Hondel CAMJJ (2004) Development of a system for integrative and stable transformation of the zygomycete Rhizopus oryzae by Agrobacterium-mediated DNA transfer. Mol Genet Genomics 271:499–510

    Article  PubMed  CAS  Google Scholar 

  5. Ibrahim AS, Skory CD (2007) Genetic manipulation of zygomycetes. In: Kavanagh K (ed) Medical mycology. Wiley, New York, pp 305–326

    Google Scholar 

  6. Nyilasi I, Ács K, Papp T, Vágvölgyi C (2005) Agrobacterium tumefaciens-mediated transformation of Mucor circinelloides. Folia Microbiol 50:415–420

    Article  CAS  Google Scholar 

  7. Nyilasi I, Papp T, Csernetics Á, Vágvölgyi C (2008) Agrobacterium tumefaciens-mediated transformation of the zygomycete fungus, Backusella lamprospora. J Basic Microbiol 48:59–64

    Article  PubMed  CAS  Google Scholar 

  8. Hamilton CM, Frary A, Lewis C, Tanksley S (1996) Stable transfer of intact high molecular weight DNA into plant chromosomes. Proc Natl Acad Sci USA 93:9975–9979

    Article  PubMed  CAS  Google Scholar 

  9. de Groot MJA, Bundock P, Hooykaas PJJ, Beijersbergen AGM (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol 16:839–842

    Article  PubMed  Google Scholar 

  10. Covert SF, Kapoor P, Lee M, Briley A, Nairn CJ (2001) Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol Res 105:259–264

    Article  CAS  Google Scholar 

  11. Velayos A, López-Matas MA, Ruiz-Hidalgo MJ, Eslava AP (1997) Complementation analysis of carotenogenic mutants of Mucor circinelloides. Fungal Genet Biol 22:19–27

    Article  PubMed  CAS  Google Scholar 

  12. Koncz C, Schell J (1986) The promoter of the TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396

    Article  CAS  Google Scholar 

  13. Bundock P, Hooykaas PJJ (1996) Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination. Proc Natl Acad Sci USA 93:15272–15275

    Article  PubMed  CAS  Google Scholar 

  14. McBride KE, Summerfelt KR (1990) Improved binary vectors for Agrobacterium-mediated plant transformation. Plant Mol Biol 14:269–276

    Article  PubMed  CAS  Google Scholar 

  15. Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Nat Biotechnol 9:963–967

    Article  CAS  Google Scholar 

  16. Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in the region pTiBo542 outside the T-DNA. J Bacteriol 168:1291–1301

    PubMed  CAS  Google Scholar 

  17. Mullins ED, Chen X, Romaine P, Raina R, Geiser DM, Kang S (2001) Agrobacterium-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer. Phytopathology 91:173–180

    Article  PubMed  CAS  Google Scholar 

  18. Iturriaga EA, Díaz-Mínguez JM, Benito EP, Álvarez MI, Eslava AP (1992) Heterologous transformation of Mucor circinelloides with the Phycomyces blakesleeanus leu1 gene. Curr Genet 21:215–223

    Article  PubMed  CAS  Google Scholar 

  19. Fullner KJ, Nester EW (1996) Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens. J Bacteriol 178:1498–1504

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

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

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Tamás Papp .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media New York

About this protocol

Cite this protocol

Papp, T., Csernetics, Á., Nyilasi, I., Vágvölgyi, C., Iturriaga, E.A. (2012). Integration of a Bacterial β-Carotene Ketolase Gene into the Mucor circinelloides Genome by the Agrobacterium tumefaciens-Mediated Transformation Method. 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_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-918-1_7

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-917-4

  • Online ISBN: 978-1-61779-918-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics