Improvement of Transformation System in Filamentous Fungus Aspergillus oryzae

Lee, Jae Won; Yun, Chan Do; Hahm, Young Tae

Abstract

Aspergillus oryzae is a filamentous fungus classified in the group Aspergillaceae Ascomycetes. A. oryzae is an important microorganism for industrial production of enzymes and fermented food products. It secrets large quantities of proteins or enzymes into the culture medium which makes this organism appealing for the production of heterologous proteins. Recently Electric field-mediated transformation method, electroporation, has been applied to fungal transformation. It is fast, simple to handle, and avoids the use of some chemicals. The optimum conditions for A. oryzae were determined with pILJ-16 and ~0.2 x 105 protoplast cell at various field strength. The survived population of protoplasts in the electric field were ~80% of nonprotoplast cell population at 1.3 KV/cm to ~50% at 6.3 KV/cm. The electrotransformation efficiency, expressed as transformants/microgram of input DNA/population of protoplast cells, increased with the increment of the field strength up to 6.3 KV/cm. The highest value, 14.35%, was obtained at 6.3 KV/cm and 1540ohm. Some antibiotics for the dominant selectable makers were applied to A. oryzae and Tolypocladium inflatum. Whereas phleomycin was very effective on T. inflatum, hygromycin B and phleomycin were not effective on A. oryzae. Protoplasts were obtained with hemicellulase and celluclast, instead of novozyme234. More than 104 transformants/microgram of DNA with hemicellulase-treated protoplasts were obtained by using electroporation at the condition of 2,500 voltage, 1,540 ohm and 0.50 capacitance. Less than 102 transformants/microgram of DNA were obtained with Novozyme234- and celluclast-treated protoplasts.