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A method for genetic transformation of Botryococcus braunii using a cellulase pretreatment

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Abstract

Botryococcus braunii race A is a green microalga that produces alkadienes and alkatrienes, fatty acids, and other compounds with commercial applications. A simple, effective, and reliable genetic transformation method for B. braunii would accelerate the development of a large-scale production method. We propose here a transformation method based on the treatment of B. braunii with a cellulolytic enzyme that transiently weakens its cell wall, facilitating its ability to incorporate foreign DNA. To test the method’s effectiveness, B. braunii cells were treated with cellulase. Weakening of the cell wall was confirmed using confocal and epifluorescence microscopy. Treated cells were transformed with plasmid pSI103, which contains the selective marker AphVIII, a gene that confers resistance to the antibiotic paromomycin (Par). Transformants were obtained on a selection medium containing Par, with a transformation efficiency of 80 transformants μg−1 of plasmid DNA. A PCR analysis showed the presence of the Par gene in the resistant transformants and its maintenance through the sixth generation, after 6 months with antibiotic selection. The availability of an efficient and simple transformation method for B. braunii will make its use more feasible in a wide range of biotechnological applications.

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Acknowledgments

We thank Dr. Carlos Riquelme for his help in the realization of this study. We are also especially grateful to Dr. Rosa León-Bañares of the Universidad de Huelva (Spain), who kindly gave us the pSI103 plasmid and polyclonal antibodies anti-AphVIII. This study was supported, in part, by research grants from Centro de Investigación Científico Tecnológico para la Minería (CICITEM), Project number R10C1004, and the Regional Government of Antofagasta.

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Authors and Affiliations

  1. Centro de Investigación Científico Tecnológico para la Minería CICITEM, Av. José Miguel Carrera 1701, Antofagasta, Chile

    Manuel Zapata & Mariella Rivas

  2. Laboratorio de Biotecnología Algal y Sustentabilidad, Facultad de Ciencias del Mar y Recursos Biológicos, Universidad de Antofagasta, Avda. Angamos 601, Antofagasta, Chile

    Héctor Berrios, Manuel Zapata & Mariella Rivas

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  1. Héctor Berrios
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  2. Manuel Zapata
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Correspondence to Mariella Rivas.

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Berrios, H., Zapata, M. & Rivas, M. A method for genetic transformation of Botryococcus braunii using a cellulase pretreatment. J Appl Phycol 28, 201–208 (2016). https://doi.org/10.1007/s10811-015-0596-3

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  • DOI: https://doi.org/10.1007/s10811-015-0596-3

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