| Summary: | Membrane-embedded transporters are crucial for the stability and performance of microbial production strains. Apart from engineering known transporters derived from model systems, it is equally important to identify transporters from nonconventional organisms that confer advantageous traits for biotechnological applications. Here, we transferred genes encoding fluoride exporter (FEX) proteins from three strains of early-branching anaerobic fungi (Neocallimastigomycota) to Saccharomyces cerevisiae. The heterologous transporters are localized to the plasma membrane and complement a fluoride-sensitive yeast strain that is lacking endogenous fluoride transporters up to 10.24 mM fluoride. Furthermore, we show that fusing an amino-terminal leader sequence to FEX proteins in yeast elevates protein yields, yet inadvertently causes a loss of transporter function. Adaptive laboratory evolution of FEX proteins restores fluoride tolerance of these strains, in one case exceeding the solute tolerance observed in wild type S. cerevisiae; however, the underlying molecular mechanisms and cause for the increased tolerance in the evolved strains remain elusive. Our results suggest that microbial cultures can achieve solvent tolerance through different adaptive trajectories, and the study is a promising step towards the identification, production, and biotechnological application of membrane proteins from nonconventional fungi. Keywords: Neocallimastigomycota, Anaerobic gut fungi, Membrane proteins, Microbial engineering, Fluoride export proteins
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