Expressing intrinsically-disordered tardigrade proteins has positive effects on acute but not chronic stress tolerance in Saccharomyces cerevisiae

Abstract

The production of high value and commodity chemicals, biopharmaceuticals and biofuels using Saccharomyces cerevisiae is hindered by various stress factors that affect yield and efficiency. Tardigrades, known for their remarkable stress tolerance, express unique proteins responsible for their resilience. This study evaluates the impact of expressing the tardigrade proteins CAHS3, MAHS, and RvLEAM on stress tolerance in S. cerevisiae. Our results show that high yields of these proteins do not impede yeast growth, except for CAHS3, which reduces proliferation. Expression of MAHS enhances acute heat tolerance, while MAHS and RvLEAM confer increased tolerance to acute hyperosmotic stress. Both CAHS3 and RvLEAM improve desiccation survival. However, these proteins do not provide benefits under chronic stress conditions such as prolonged exposure to high temperature, hyperosmotic stress, or solvents. These findings highlight the potential utility of tardigrade proteins for transient stress protection in industrial bioprocesses and suggest future engineering approaches for improved stress tolerance in yeast.

Fig 1. Expression of tardigrade transgenes in S. cerevisiae.

(A) DNA constructs used in the expression and localization of GFP-fused tardigrade proteins. (B) Representative fluorescence microscopy images of S. cerevisiae expressing AcGFP1 as a cytosolic control, MAHS-AcGFP1, CAHS3-AcGFP1 and RvLEAM-AcGFP1 (green). Mitochondria are identified with TMRE (red), which stains live mitochondria. Images were taken using a 100x objective and merged images of green fluorescence, red fluorescence, and brightfield contrast (blue) are shown. Colocalization of red and green fluorescence shows in yellow.

Fig 2. Plasmid constructs used and the effect of the tardigrade proteins on cell growth.

(A) Graphic illustration of the transcriptional units assembled in the 2μ plasmid that were used for stress tolerance testing. (B) Growth curves of the yeast strains, three of them expressing tardigrade proteins (pRvLEAM, pMAHS, pCAHS3) and one carrying an empty vector (pControl).

Fig 3. Effect of the tardigrade proteins on cell viability after heat shock.

(A) Representative images of a 3-day growth of the different plasmid-carrying yeast strains spot-plated after 1 h heat stress in a 5-fold dilution starting at an OD₆₀₀ of 0.5. Showing pMAHS as the lone survivor at 50°C. (B) Live/Dead cytometry assay for pMAHS and pControl analyzed using two-way ANOVA with a Bonferroni correction (n = 3).

Fig 4. Effect of tardigrade proteins in desiccation and hyperosmolarity acute stress.

Yeast expressing CAHS3, MAHS or RvLEAM, as well as BY4741 containing an empty vector (pCAHS3, pMAHS, pRvLEAM and pControl respectively) were subjected to desiccation and hyperosmolarity stress prior to Live/Dead assay. Stress conditions were performed simultaneously and shared the control condition. Live/Dead assay showed significant stress tolerance improvement for pRvLEAM in both stress conditions and improvement for pMAHS under hyperosmolarity stress (n = 3).

Fig 5. Spot plate growth under chronic stress.

(A) Representative images of the different plasmid-carrying yeast strains spot-plated in a 5-fold serial dilution starting at an OD₆₀₀ of 0.5 after 2 days of growth at 30°C and 37°C. (B) Representative images of the different plasmid-carrying yeast strains spot-plated as in (A) after 6 days of growth in 7.5% NaCl, and after 9 days of growth in 10% NaCl. (C) Representative images of the yeast on ButOH after 5 days in 1% ButOH, and after 8 days in 1.5% ButOH exhibiting lack of better growth in the tardigrade protein expressing strains than the control pControl.