Protoplast Generation from the Ascofuranone-Producing Fungus Acremonium sclerotigenum

Abstract

Acremonium sclerotigenum (Bionectriaceae, Hypocreales, Sordariomycetes, Ascomycota) is a soil fungus found worldwide as an air and food contaminant. This fungus produces the antibiotic ascofuranone, a promising drug candidate against African trypanosomiasis; however, the details of ascofuranone biosynthesis have not yet been elucidated. Genetic manipulation techniques would greatly facilitate identification of the genes from ascofuranone biosynthetic pathway, but these techniques have not yet been established for this organism. Protoplast generation is a required step of the polyethylene glycol-mediated transformation of fungi. In this study, we aimed to establish a protocol for the isolation of protoplasts from A. sclerotigenum strain F-1392. The cell wall was digested with a commercial enzyme, Yatalase, to promote protoplast release. Spherical cells that were sensitive to osmotic shock were recovered only when the mycelia were pretreated with dithiothreitol. As a result, 2.0×10⁷ protoplasts were obtained from a 40-mL liquid culture of A. sclerotigenum; the regeneration efficiency was estimated as 17%. This study comprises the first step toward development of genetic manipulation techniques in A. sclerotigenum, allowing future genetic dissection of this important microorganism.