Heterologous transporters from anaerobic fungi bolster fluoride tolerance in Saccharomyces cerevisiae

Abstract

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: Anaerobic gut fungi; Fluoride export proteins; Membrane proteins; Microbial engineering; Neocallimastigomycota.

Fig. 1

Fex proteins extrude fluoride from cells. (a) Cartoon depiction of a cell producing a fluoride transporter (FEX). (b) Anaerobic fungal FEX transporters restore fluoride tolerance in a yeast knockout (KO) strain. 0.15 ODU of induced yeast cultures were harvested and plated on SG-URA supplemented with 2% w/v galactose and 50 μM NaF. Streaked plates were incubated at 30 °C for approximately 48 h. KO = BJ5465 fex1:GSHU Δfex2 carrying the empty pYC vector. WT = BJ5465 carrying the empty pYC vector. N. c. FEX-GH = KO with pYC-N. californiae FEX-GH. A. r. FEX-GH = KO with pYC-A. robustus FEX-GH. P. f. FEX-GH = KO with pYC-P. finnis FEX-GH. S. c. Fex1p-GH = KO with pYC-S. cerevisiae Fex1p-GH.

Fig. 2

KO-strains harboring anaerobic fungal FEX proteins show attenuated growth in up to 10.24 mM NaF. OD₆₀₀values of indicated yeast strains were measured after ∼22 h growth in the presence of inducer (2% w/v galactose) and increasing concentrations of NaF. Wildtype (WT) and a KO strain expressing S. cerevisiae Fex1p-GH exhibit growth in up to 40.96 ​mM NaF. In contrast, KO strains expressing gut fungal homologs show reduced fluoride tolerance exhibiting growth in up to 10.24 ​mM NaF. Indicated is the standard error of the mean of three biological replicates. Statistically significant differences in growth between FEX-producing strains and the KO control strain were determined using a one-tailed Student's t-test: *** ​= ​p-value<0.001, ** ​= ​0.001<p-value<0.01, * ​= ​0.01<p-value<0.05. KO = BJ5465 fex1:GSHU Δfex2 carrying the empty pYC vector. WT = BJ5465 carrying the empty pYC vector. S. c. Fex1p-GH = KO with pYC S. cerevisiae Fex1p-GH. P. f. FEX-GH = KO with pYC P. finnis FEX-GH. A. r. FEX-GH = KO with pYC A. robustus FEX-GH. N. c. FEX-GH = KO with pYC N. californiae FEX-GH.

Fig. 3

Confocal micrographs show localization of GFP-tagged FEX proteins produced in a S. cerevisiae knockout strain. Yeast knockout strains producing the indicated FEX homologs exhibit localization of fluorescent protein to the endoplasmic reticulum and plasma membrane signifying proper membrane protein folding and targeting. GFP (top) and transmission (bottom) micrographs were obtained in the absence of fluoride after ∼22–24 h induction of FEX gene expression in raffinose- and galactose-based medium. Microscopy was performed using an Olympus Fluoview 1000 Spectral confocal microscope equipped with a 60x/NA 1.3 objective. Laser intensities were adjusted for each strain to bring fluorescence intensities near saturating levels. All scale bars represent 5 μm.

Fig. 4

Knockout strains producing gut fungal FEX-GH proteins exhibit decreased total protein yield compared to a strain producing S. cerevisiae Fex1p-GH. Analysis of strains using fluorescence-activated cell sorting (FACS) provides a proxy measurement of total protein yield. Compared to a knockout strain expressing the native S. cerevisiae Fex1p-GH from a low copy vector, strains expressing gut fungal homologs from the same vector backbone display lower cellular mean fluorescence intensities (MFI). Strains were cultured and gene expression was induced in the absence of fluoride. Indicated is the standard deviation from the mean of three biological replicates. For each replicate, fluorescence data was collected for ∼40,000 single cells.

Fig. 5

Addition of Prepro leader peptide to FEX-GH proteins leads to improved total protein yields and reduced cellular fluoride tolerance. (a) Knockout strains producing Prepro-FEX-GH homologs in the absence of fluoride exhibit cellular MFI values significantly greater than the strain producing S. cerevisiae Fex1p-GH. The increase in cellular MFI relative to S. cerevisiae Fex1p-GH is indicative of improved protein yields, suggesting a beneficial effect arises due to addition of Prepro. (b) The strains producing Prepro-FEX-GH homologs exhibit low tolerances to NaF suggesting that the Prepro peptide is deleterious to protein function. Indicated is the standard deviation from the mean of three biological replicates. For each replicate in panel a, fluorescence data was collected for ∼40,000 single cells. In panel b, OD₆₀₀values of KO strains expressing Prepro-FEX-GH were measured after ∼22–24 ​h growth in the presence of inducer (2% w/v galactose) and increasing concentrations of NaF. Statistically significant differences in growth between FEX-producing strains and the KO control strain were determined using a one-tailed Student's t-test: *** ​= ​p-value<0.001, ** ​= ​0.001<p-value<0.01, * ​= ​0.01<p-value<0.05. KO = BJ5465 fex1:GSHU Δfex2 carrying the empty pYC vector. P. f. FEX-GH = KO with pYC P. finnis FEX-GH. A. r. FEX-GH = KO with pYC A. robustus FEX-GH. N. c. FEX-GH = KO with pYC N. californiae FEX-GH.

Fig. 6

Evolved Prepro-FEX-producing strains exhibit distinct changes in fluoride tolerance. Following adaptive evolution, the OD₆₀₀ values of evolved strains were measured and normalized to the OD₆₀₀ of a wildtype BJ5465 empty vector control grown at each NaF concentration. A KO strain producing S. cerevisiae Fex1p-GH provides a reference controlling for promoter strength and a closer comparison with respect to transporter gene copy number. This strain exhibits comparable or greater growth compared to WT yeast at all but the highest NaF concentrations tested. Similar to S. cerevisiae Fex1p-GH, A. robustus Prepro-FEX-GH 40.96 grows to cell densities comparable to, or greater than, WT at all but the highest NaF concentrations. In contrast, P. finnis Prepro-FEX-GH 5.12 grows to a lower concentration than WT yeast in all cases. OD₆₀₀ values of evolved KO strains were measured after ∼24 h growth in the presence of inducer (2% w/v galactose) and increasing concentrations of NaF. Indicated is the standard error of the mean of three biological replicates. KO = BJ5465 fex1:GSHU Δfex2 carrying the empty pYC vector. S. c. Fex1p-GH = KO with pYC S. cerevisiae Fex1p-GH.P.f. FEX-GH = KO with pYC P. finnis FEX-GH. A. r. FEX-GH = KO with pYC A. robustus FEX-GH.

Fig. 7

Evolved Prepro-FEX-producing strains exhibit divergent fluorescence phenotypes. (a) P. finnis Prepro-FEX-GH 5.12 exhibits significantly improved MFI relative to its parent strain indicative of increased total protein yield. In contrast, the MFI of A. robustus Prepro-FEX-GH 40.96 is similar to that of the unevolved strain. (b) Histograms reveal phenotypic heterogeneity at the population level. While P. finnis Prepro-FEX-GH 5.12 exhibits complete homogeneity in the presence of NaF, A. robustus Prepro-FEX-GH 40.96 displays increased heterogeneity. Fluorescence data represents the mean fluorescence intensity corresponding to ∼40,000 single cells. KO = BJ5465 fex1:GSHU Δfex2 carrying the empty pYC vector. P. f. FEX-GH = KO with pYC P. finnis FEX-GH. A. r. FEX-GH = KO with pYC A. robustus FEX-GH.