High demand of green and sustainable way for producing chemicals lead to usage of microorganisms for bio-based production of various types of products including biosurfactants. One of the most promising biosurfactants are mannosylerythritol lipids, which have been gaining a huge interest due to its high yield and mild production, various application. Non-conventional dimorphic yeast Moesziomyces antarcticus was used as a host organism for genetic manipulations and production of MELs. The MELs, excreted by M. antarcticus consist of three main forms of MEL-A, MEL-B and MEL-C and only very low amount of non-acetylated MEL-D is being produced. For homogenous production of MEL-D, knock-out of the MAT1 gene, acetyltransferase responsible for acetylation of MEL-D was attempted. Different gene engineering techniques were applied for disruption of MAT1 gene such as gene replacement with homologous recombination using a PCR amplified fragment with upstream and downstream flanking regions of MAT1 embedding NatMX4 selection cassette; split-marker approach using truncated fragments with homologous regions for recombination within the NatMX4 cassette; RNP mediation for better gene targeting and increasement of homologous recombination by supplying with dDNA. Transformation with PCR amplified NatMX_MaMat1 fragment resulted 2-fold lower efficiency of NatMX4 cassette insertion into the chromosome compared to split-marker with 70 bp overlap. RNP mediated transformation with 100x dilution of RNP obtained couple of mutants. However, MAT1 gene was not successfully targeted by any of the cases resulting in random event integration and inability to disrupt the gene of interest. Construction of 231 bp overlap for a split-marker approach resulted in slight increase of transformation efficiency. Optimization of transformation was performed and pre-treatment of the cells before electroporation transformation increased the efficiency by 3-folds. Two transformation techniques were applied, where electroporation resulted in 26% higher transformation efficiency compared with PEG/LiAc/ssDNA mediated transformation and was used for further experiments with RNP.