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

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.