Abstract
The halo-, xero-, and osmotolerant oleaginous yeast Debaryomyces hansenii can grow in extreme environments, with some isolates tolerating up to 4 M NaCl. As such, it could serve as a robust microbial chassis for non-sterile biotechnological processes based on seawater or saline side streams. However, its biotechnological toolbox lacks key genetic tools, particularly for inducible expression, secretion, and covalent anchoring of proteins to the outer cell surface. To address this gap, we characterised how growth in a medium supplemented with 1 M NaCl remodels the D. hansenii cell wall. We also developed strong constitutive, stationary-phase, and NaCl-inducible expression vectors, and designed protein tags for C- and N-terminal cell wall anchoring. These tools enabled functional surface display of β-lactamase, acetylxylan esterase, and lipase A via native GPI-dependent and heterologous Pir-based anchors, while immunoblotting detected the reporters in both covalently and non-covalently bound cell wall fractions. Activities of surface-displayed enzymes increased by more than 80 % under hypersaline conditions, while DhCcw12-based C-terminal anchoring outperformed Pir-based N-terminal anchoring. Thus, we retooled the D. hansenii cell wall into a catalytically active biointerface and established this yeast as an osmotolerant chassis for hypersaline surface display.