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Stable transformants of the azaphilone pigment-producing Monascus purpureus obtained by protoplast transformation and Agrobacterium-mediated DNA transfer

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Abstract

The high-level pigment-producing Monascus strain IBCC1 was characterized by random amplification of polymorphic DNA as M. purpureus. This technique allowed us to distinguish between M. purpureus and M. ruber strains. Transformation of Monascus species has not been previously reported. Protoplast formation and regeneration from M. purpureus IBCC1 was optimized by modification of growth media, lytic enzyme mixture, osmotic stabilizer and regeneration media. Of the Monascus transformants, 60% were found to be mitotically stable and retained the plasmid inserted in the chromosome after repeated sporulation cycles. Additionally, an Agrobacterium-mediated DNA transfer system was developed. The transformants obtained by Agrobacterium-mediated DNA transfer remained fully stable (98%) after four sporulation rounds and showed bands of hybridization corresponding to integration of the plasmid in different sites of the genome. The green fluorescent protein marker was well expressed in the M. purpureus transformants. The development of transformation systems is a basic tool for advanced genetic manipulation of the natural pigment producers, M. purpureus and M. ruber.

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Acknowledgements

This work was supported by the Spanish MYCT grant 1FD1997-0224-CO3. We thank R. Fouces and R. Godio for providing plasmid pBG7.1. We acknowledge the technical assistance of Josefina Merino, Bernabé Martín and the collaboration of Inés Sánchez in the initial Agrobacterium transformation experiments. We thank Prof. A.L. Demain for critical reading of the manuscript.

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Correspondence to Paloma Liras.

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Communicated by U. Kück

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Campoy, S., Pérez, F., Martín, J.F. et al. Stable transformants of the azaphilone pigment-producing Monascus purpureus obtained by protoplast transformation and Agrobacterium-mediated DNA transfer. Curr Genet 43, 447–452 (2003). https://doi.org/10.1007/s00294-003-0417-0

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  • DOI: https://doi.org/10.1007/s00294-003-0417-0

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