Mannosylerythritol lipids (MELs) are biosurfactants produced from feedstocks by basidiomycetous yeasts. MELs exhibit different properties depending on their structures, such as the degree of acetylation or acylation and the chirality of the mannosylerythritol moiety. Pseudozyma tsukubaensis produces a diastereomer type of MEL-B (mono-acetylated MEL); therefore, deletion of an acetyltransferase could yield a diastereomer type of MEL-D (deacetylated MEL), which has only been produced in in vitro reactions of lipase using MEL-B as a substrate. Here, we deleted the gene PtMAT1 in P. tsukubaensis 1E5 encoding an acetyltransferase related to MEL biosynthesis via targeted gene deletion and generated a producer of the diastereomer type of MEL-D. The uracil auxotrophic mutant of P. tsukubaensis 1E5 (PtURA5-mutant) was used as a host strain for gene deletion. The gene PtMAT1 was replaced with a PtURA5 cassette by homologous recombination using uracil auxotrophy as a selectable marker. According to thin-layer chromatography and nuclear magnetic resonation spectroscopy, we identified the strain APtMAT1 as a producer of the diastereomer type of MEL-D instead of MEL-B. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.