Glycerol uptake and synthesis systems contribute to the osmotic tolerance of Kluyveromyces marxianus

The accumulation of glycerol is essential for yeast viability upon hyperosmotic stress. In this study, the STL1 homolog KmSTL1, encoding a putative glycerol transporter contributing to cell osmo-tolerance, was identified in Kluyveromyces marxianus NBRC1777. We constructed the KmSTL1, KmGPD1, and KmFPS1 single-deletion mutants and the KmSTL1/KmGPD1 and KmSTL1/KmFPS1 double-deletion mutants of K. marxianus. Deletion of KmSTL1 or KmGPD1 resulted in K. marxianus cell sensitization to hyperosmotic stress, whereas deletion of KmFPS1 improved stress tolerance. The expression of KmSTL1 was osmotically induced, whereas that of KmFPS1 was osmotically inhibited. The expression of KmGPD1 was constitutive and continuous in the ΔKmSTL1 mutant strain but inhibited in the ΔKmFPS1 mutant strain due to feedback suppression by glycerol. In summary, our findings indicated that K. marxianus would increase glycerol synthesis by increasing GPD1 expression, increase glycerol import from the extracellular environment by increasing STL1 expression, and reduce glycerol efflux by reducing FPS1 expression under hyperosmotic stress.