Construction of a ceramide production system using rescue
mechanism against impaired biosynthesis of sphingolipids
Motohiro TANI
Department of Chemistry, Faculty of Sciences, Kyushu University
Research objective
Sphingolipids are essential membrane lipids in eukaryotes ranging from fungi to humans.
Ceramides, which constitute the hydrophobic part of sphingolipids, exist in the human stratum
corneum and contribute to the barrier function and moisturization of the skin. In recent years,
attention has been focused on the use of ceramides in health foods, cosmetics, andpharmaceuticals. Budding yeast can be a good source of ceramides; however, ceramides arepresent only in trace amounts in natural resources, including yeast, because most of thesynthesized ceramides are converted into complex sphingolipids containing a hydrophilic headgroup. Thus, although the conversion of ceramides to complex sphingolipids is necessary toobtain a larger amount of ceramides from yeast, doing so would substantially impair the
metabolic balance of sphingolipids and can be lethal for the yeast. Therefore, the constructionof a ceramide mass production system using yeast is difficult. Recently, we found that theactivation of a specific signaling system (HOG pathway) rectified the growth defect causedby the repression of complex sphingolipid biosynthesis (1). In this study, we aim to establish
a mass production system for ceramides by analyzing this rescue mechanism in detail.
Methods
To suppress the conversion of ceramides to complex sphingolipids in budding yeast, AUR1,
which encodes for an IPC (the simplest structure of complex sphingolipids) synthase, wasexpressed under the control of a tetracycline-regulatable promoter (tet-AUR1 strain). The
expression of AUR1 was repressed by the addition of doxycycline (Dox). DNA microarray
analysis was used to perform a comprehensive search for genes whose transcription increased
in a HOG-pathway-dependent manner under AUR1-repressive conditions.
Results
Identification of the final target factor of the HOG pathway
Transcriptome analysis led to the identification of 59 genes whose expression increased in a
Hog1 (MAPK of the HOG pathway)-dependent manner under AUR1-repressive conditions.
These candidate genes were separately overexpressed in tet-AUR1 cells, and genes that wereable to rescue the impaired biosynthesis of complex sphingolipids were screened. As a result,FMP48, UIP4, GIP2, TPS2, and MGA1 genes were identified. From these, FMP48 and TPS2
were identified as important candidate genes, because the deletion of one of these genes
promoted growth defects due to AUR1 repression (Fig. 1A). FMP48 encodes a putative
Ser/Thr kinase, and it was found that the overexpression of FMP48 suppresses growth defects
and increases mitochondria-derived reactive oxygen species (ROS), which are produced byAUR1 repression (Fig. 1B). Comprehensive analysis of protein interactions showed thatFmp48 physically interacts with the transcriptional regulator Mks1 (2), and it was suggested
that the effects of FMP48 under AUR1-repressive conditions are mediated by MKS1.
TPS2 encodes trehalose-6-phosphate phosphatase involved in trehalose biosynthesis.
Deletion of TPS2 promoted growth inhibition caused by AUR1 repression; however, deletionof the trehalose-6-phosphate synthase gene TPS1 had no promoting effect. Thus, it is assumed
that the rescue effect of TPS2 is not mediated by the promotion of trehalose biosynthesis, but
is exerted by regulating the amount of trehalose-6-phosphate, a precursor of trehalose.
Trehalose-6-phosphate is a negative regulator of glycolysis. Therefore, glycolysis activity wasevaluated by examining the production of ethanol in a fermentable medium. As a result, it was
found that AUR1 repression reduced the amount of ethanol produced per living cell, and thisreduction was promoted by the deletion of TPS2 or HOG1. These results suggest that TPS2 isupregulated under AUR1 repression and contributes to the suppression of the growth defects
by regulating glycolysis.

Fig. 1. (A) Effect of deletion of candidate genes that rescue growth defects caused byrepression of complex sphingolipid biosynthesis (AUR1 repression). (B) Effect ofoverexpression of FMP48 (TEFp-FMP48) on increased ROS levels in AUR1-repressed cells.
Detection of ROS was performed by staining with 2’,7’-dichlorofruorescein diacetate, anindicator of ROS in cells.
Crosstalk of Protein kinase A and the HOG pathway under impaired biosynthesis ofcomplex sphingolipids
Previously, we reported that the deletion of RAS2, which encodes a component of thecAMP/protein kinase A (PKA) signaling pathway, confers resistance to growth inhibition
caused by AUR1 repression (1). In the present study, we found that gene mutations (pde2∆,ira2∆), which cause the activation of the cAMP/PKA pathway, enhance growth inhibition

caused by AUR1 repression (3) (Fig. 2A). To elucidate the function of the cAMP/PKA
pathway under AUR1-repressive conditions, we screened multicopy suppressor genes that can
suppress this enhancement of the growth inhibition. As a result, the PBS2 gene encoding
MAPKK of the HOG pathway was identified. We also found that the mutant strain withincreased cAMP/PKA pathway activity decreased the phosphorylation of Hog1 under AUR1repressive conditions (Fig. 2B). These results suggest that the cAMP/PKA pathway
contributes to the proper activation of the HOG pathway under impaired biosynthesis ofcomplex sphingolipids (3).

Fig. 2. (A) Effect of deletion of components of the cAMP/PKA pathway on growth defectcaused by repression AUR1. (B) Effect of deletion of PDE2 or IRA2 on phosphorylation ofHog1 caused by AUR1 repression.
Conclusion
This study revealed how yeast cells protect themselves against the impaired biosynthesis ofcomplex sphingolipids. The TORC2-Ypk1 pathway is involved in the repair of complex
sphingolipid biosynthetic pathways by activating the sphingolipid biosynthetic pathway;
however, activation of the HOG pathway is not itself involved in the repair of the complex
sphingolipid biosynthesis system. By combining these findings, we aim to develop mutant
strains exhibiting resistance to complex sphingolipid reduction and abnormal ceramide
accumulation in the future.
References
1) Yamaguchi, Y. et al. (2018) Protective role of the HOG pathway against the growth defectcaused by impaired biosynthesis of complex sphingolipids in yeast Saccharomyces
cerevisiae. Mol. Microbiol. 107: 363-386.
2) Breitkreutz, A. et al. (2010) A global protein kinase and phosphatase interaction networkin Yeast. Science 328: 1043-1046.
3) Urita, A., Ishibashi, Y., Kawaguchi, R., Yanase, Y., and Tani, M. (2022) Crosstalk between
protein kinase A and the HOG pathway under impaired biosynthesis of complex
sphingolipids in budding yeast. FEBS J. 289: 766-786.