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Halophilic and osmotolerant yeast Debaryomyces hansenii has a high potential for cell factory applications due to its resistance to harsh environmental factors and compatibility with a wide substrate range. However, currently available genetic techniques do not allow the full potential of D. hansenii as a cell factory to be harnessed. Moreover, most of the currently available tools rely on the use of auxotrophic markers that are not suitable in wild-type prototrophic strains. In addition, the preferred non-homologous end-joining (NHEJ) DNA damage repair mechanism poses further challenges when precise gene targeting is required. In this study, we present a novel plasmid-based CRISPRCUG/Cas9 method for easy and efficient gene editing of the prototrophic strains of D. hansenii. Our toolset design is based on a dominant marker and facilitates quick assembly of the vectors expressing Cas9 and single or multiple single-guide RNAs (sgRNAs) that provide the possibility for multiplex gene engineering even in prototrophic strains. Moreover, we have constructed NHEJ-deficient D. hansenii that enable our CRISPRCUG/Cas9 tools to support the highly efficient introduction of point mutations and single/double gene deletions. Importantly, we also demonstrate that 90-nt single-stranded DNA oligonucleotides are sufficient for direct repair of DNA breaks induced by sgRNA-Cas9, resulting in precise edits reaching 100% efficiencies. In conclusion, tools developed in this study will greatly advance basic and applied research in D. hansenii. In addition, we envision that our tools can be rapidly adapted for gene editing of other non-conventional yeast species including the ones belonging to the CUG clade.

Feasible bioprocessing of lignocellulosic biomass requires the use of microbial strains with tolerance to inhibitor compounds and osmotic pressure, able to provide high product yield and productivity. In this sense, this study evaluated the potential of two non-conventional yeasts, Hansenula polymorpha CBS 4732 and Debaryomyces hansenii CBS 767, for use on biomass conversion in a biorefinery perspective. The ability of the strains to consume pentose and hexose sugars, to resist the toxic compounds present in hydrolysates, as well as to produce sugar alcohols and ethanol, was investigated. H. polymorpha showed highlighted resistance to toxic compounds and relevant ability to consume xylose and produce xylitol and ethanol under these conditions, at 37 °C. D. hansenii was a great producer of arabitol from glucose. The implications for sustainability due to the use of these yeasts in biorefineries was discussed. These results open up new perspectives for the development of the biorefinery sector.