Protoplast Isolation and Cell Wall Regeneration of Cunninghamella Bainieri

Cunninghamella bainieri is as an oleaginous fungus capable of producing lipids at a high percentage of its cell dry weight. This fungus may serve as a model to elucidate the biosynthetic pathways for polyunsaturated fatty acids (PUFAs) and physiological properties of an oleaginous organism using genetic and biochemical analyses. Thus, establishment of a protoplast and regeneration system is crucial towards developing a transformation system for this important fungal species. Protoplast formation and regeneration were studied in the zygomycetous filamentous fungus, C. bainieri. In this study, a method was conducted for obtaining protoplasts from C. bainieri using a lytic enzymatic approach. The study also applies the use of protoplasts in the regeneration of protoplasts. The study was carried out by manipulating parameters such as mycelium age, concentration of lytic enzyme, duration of incubation in lytic mixture, concentration of osmotic stabilizers, pre-treatment with the thiol compound, dithiotreitol (DTT) and concentration of regeneration agar medium. The results showed that from lg wet weight of mycelial culture, the highest protoplast yield was 1.10 X 107 protoplasts/ml using an 18-hour mycelial culture in a lytic mixture of 7.5 mg/ml lytic enzyme incubated for 3 hours in 1.2 M magnesium sulphate osmotic buffer without pre-treatment with DTT. The highest percentage of protoplast regeneration obtained from this study was 86.5% using the Czapek-Dox regeneration medium at a concentration of 0.7% agar (w/v). To date, this is the first report of protoplast isolation and regeneration for this important oleaginous fungus