Variation among metschnikowia pulcherrima isolates for genetic modification and homologous recombination

Abstract

Metschnikowia pulcherrima is a non-conventional yeast with the potential to be used in bi-otechnological processes, especially involving low-cost feedstock exploitation. However, there are a lack of tools for researching it at a molecular level and for producing genetically modified strains. We tested the amenability to genetic modification of ten different strains, establishing a transformation protocol based on LiAc/PEG that allows us to introduce heterologous DNA. Non-homolo-gous integration was broadly successful and homologous recombination was successful in two strains. Chemical inhibition of non-homologous end joining recombination had a modest effect on the improvement of homologous recombination rates. Removal of selective markers via flippase recombinase was successful across integrated loci except for those targeted to the native URA3 lo-cus, suggesting that the genome sequence or structure alters the efficacy of this system.