Generation of high-lipid producing yeast cells using theendoplasmic reticulum-stress response
Yukio KIMATA
Graduate School of Science and Technology, Nara Institute of Science andTechnology
Research objective
In general, eukaryotic cells contain the endoplasmic reticulum (ER), which is a suc-shaped
organelle with a flat or tubular morphology and covered with a single phospholipid bilayer.
The role of the ER includes the formation of higher-order structures of secretory proteins andcell surface proteins, and the biosynthesis of lipids. Molecular chaperones and related proteins
reside in the ER lumen, and lipid metabolism/synthase family proteins are present on the ERsurface. mRNAs encoding secretory proteins and cell surface proteins are translated byribosomes attached to the ER surface, and nascent polypeptides are folded in the ER. The
failure of these processes is referred to as ER stress, and cells evoke an ER stress response tocope with serious cellular damage.
For the ER stress response in yeast cells, the ER transmembrane protein Ire1 acts as a sensor
that detects denatured proteins accumulated in the ER lumen and activates an intracellularsignal transduction pathway1). The cytosolic domain of Ire1 has a protein kinase region and anendoribonuclease region. In response to ER stress, Ire1 induces autophosphorylation, whichleads to its activation as an endoribonuclease. In Saccharomyces cerevisiae, the target of Ire1
as an endoribonuclease is the mRNA transcript of HAC1 gene. The HAC1 mRNA, namely theimmature HAC1 mRNA, has an intron and is not translated into a protein. In contrast, theactivation of Ire1 proteins induces splicing of the HAC1 mRNA and translation into thetranscription factor protein Hac1. The Hac1 protein t induces the transcription of ER-locatedmolecular chaperones and phospholipid biosynthetic enzymes, leading to the reactivation ofER functions and enlargement of the ER.
In the present study, we addressed the following issues, which are deeply related to ER stressand ER stress response in yeast cells.
(1) Biological significance of Ire1 regulation by its kinase region(2) Effect of molecular oxygen on ER stress and ER stress response in S. cerevisiae(3) ER stress response in the methanol-utilizing yeast Pichia pastoris (Komagataella phaffii).
(4) Application of the S. cerevisiae strain that constitutively expresses mature HAC1 mRNA.

Methods
The reverse transcriptase (RT)-PCR technique was used to monitor the splicing of HAC1mRNA, and PolyT primers were used in the RT reaction. For PCR, we used a HAC1-specificprimer set that amplifies the non-spliced and spliced sequences as different-sized DNA
products.
The genetic manipulation of S. cerevisiae and P. pastoris, and the analyses of nucleic acid,protein, and lipidic samples were performed as described in the literature2)-3).
Results(1) Even when Ire1 carried a mutation that abolished its requirement for the luminal domaindependent detection of ER-accumulated unfolded proteins for its activation, the HAC1-mRNA
splicing was not constitutive but was dependent on the nutrient condition. Contrarily, theHAC1-mRNA splicing was constitutively evoked when another mutation was introduced into
the kinase region of its cytosolic domain2).
(2) When S. cerevisiae cells were cultured under anaerobic conditions, the HAC1 mRNA
splicing level induced by the conventional ER stress inducers tunicamycin and dithiothreitol
was higher than that induced under aeration conditions. The same pattern was observed when
mitochondrial respiration was halted by the rho0 mitochondrial-gene deletion mutation4).
(3) Unlike S. cerevisiae, in P. pastoris, HAC1 mRNA splicing was observed at high levels
even under normal culture conditions. When the P. pastoris Ire1 gene was introduced into S.
cerevisiae, HAC1 mRNA splicing was not induced under normal conditions. Therefore, P.
pastoris cells were likely to be under ER stress even under normal culture conditions. Theprotein secretion level of P. pastoris was significantly higher than that of S. cerevisiae.
Moreover, ER stress response-target genes of IRE1 and HAC1-gene deletion mutants of P.
pastoris that were analyzed using RNAseq have been listed.
(4) The constitutive and high expression of the spliced HAC1 mRNA in S. cerevisiae induced
the expression of lipid biosynthetic enzymes, leading to the accumulation of phospholipids
and triglycerides. It also increased the secretion of free fatty acids. However, overexpressionof spliced HAC1 mRNA drastically retarded cellular growth. To cope with this problem, fastgrowing spliced HAC1 mRNA-expressing cells were screened. The mutant carried a loss-offunction mutation in the histone deacetylase gene HDA3.
Conclusion
Here, we showed that the protein kinase region of yeast Ire1 was involved in the regulation ofER stress responses by nutrient conditions. Additionally, in S. cerevisiae, molecular oxygen
attenuated ER stress independent of mitochondrial respiration. Unlike S. cerevisiae, P.
pastoris robustly secreted proteins, leading to potent ER stress, even under normal conditions.

By combining the constitutive and high expression of the spliced HAC1 mRNA and the lossof-function mutation of the HDA3 gene, S. cerevisiae strains that produced abundant lipidic
molecules without severe growth retardation (Fig. 1) were obtained.

Fig. 1 Production of lipidic molecules in yeast cells. Wild type and mutant (high expressionof the spliced HAC1 mRNA and HDA3 deletion (HAC1i/hda3) S. cerevisiae cells werecultured at 30 °C in SD medium and were checked for lipid contents.
References1) Le, Q.G., and Kimata, Y. (2021) Multiple ways for stress sensing and regulation of theendoplasmic reticulum-stress sensors. Cell Struct. Funct. 46: 37–49.
2) Le, Q.G., Ishiwata-Kimata, Y., Phuong, T.H., Fukunaka, S., Kohno, K., and Kimata, Y.
(2021) The ADP-binding kinase region of Ire1 directly contributes to its responsiveness toendoplasmic reticulum stress. Sci. Rep. 11: 4506.
3) Fauzee, Y.N.B.M., Taniguchi, N., Ishiwata-Kimata, Y., Takagi, H., and Kimata, Y. (2020)
The unfolded protein response in pichia pastoris without external stressing stimuli. FEMS
Yeast Res. 20: foaa053.
4) Phuong, H.T., Ishiwata-Kimata, Y., Nishi, Y., Oguchi, N., Takagi, H., and Kimata, Y.
(2021) Aeration mitigates endoplasmic reticulum stress in Saccharomyces cerevisiae even
without mitochondrial respiration. Microb. Cell 8: 77–86.