Preparation of protoplasts of Rhizopus niveus and their transformation with plasmid DNA.

Abstract

An efficient method for protoplast formation of Rhizopus niveus, a filamentous fungus with high capability to secrete enzymes out of the cells, was established using three lytic enzymes in combination, and a plasmid with a selection marker for transformation was constructed by transcriptional fusion of a Rhizopus promoter with the G418 resistance gene derived from Tn903. Then the protoplasts were mixed with the plasmid DNA in the presence of polyethylene glycol 4000 and CaCl2. G418-resistant colonies were obtained, indicating that transformation was successful. Southern blot analysis of DNA from a transformant shows that the introduced DNA was present not only integrated into the host chromosome but also replicating extrachromosomalh, and that it was rearranged with host DNA during the course of cell cultivation. This is the first report of a transformation system for Rhizopus. This system will open the possibility for breeding R. niveus at the molecular level.