S-Space College of Engineering/Engineering Practice School (공과대학/대학원) Program in Bioengineering (협동과정-바이오엔지니어링전공) Theses (Ph.D. / Sc.D._협동과정-바이오엔지니어링전공)
Regulation of Hsf1 and Msn2 activity by CK2 kinase in Saccharomyces cerevisiae
- 공과대학 협동과정 바이오엔지니어링전공
- Issue Date
- 서울대학교 대학원
- CK2 kinase; Ppt1 phosphatase; Hsf1; Msn2; Msn4; Phosphorylation; Stress transcription factor; Stress tolerance; Saccharomyces cerevisiae
- 학위논문 (박사)-- 서울대학교 대학원 : 바이오엔지니어링전공, 2017. 2. 한지숙.
- Saccharomyces cerevisiae is one of the best-studied useful eukaryotic model organisms for basic biological research. In addition, S. cerevisiae is also very attractive microorganism as an industrial cell factory due to several advantages such as assured safety, well-established genetic and metabolic engineering methods, excellent fermentation ability, and rapid growth under anaerobic condition. Especially, it has great tolerance to a wide range of harsh environmental challenges not only a high concentration of substrate and product but also facing the fermentation processes and cell culture. In response to unfavorable environmental changes, cells rapidly adjust global gene expression programs with inducing many stress-related genes such as heat shock proteins (HSPs) for maintaining cellular homeostasis and cell survival. Stress-induced transcriptional activation of HSP genes is mainly regulated by two stress transcription factors, Hsf1 and Msn2/4.
In this dissertation, regulation mechanisms of Hsf1 and Msn2 by CK2 in response to ethanol and several environmental stress were elucidated and applied to stress-tolerant yeast strain.
Firstly, we found that CK2-dependent phosphorylation on S608 is an ethanol stress-specific repression mechanism of Hsf1, which does not affect the basal or heat-induced activity of Hsf1. This repression is relieved by dephosphorylation by Ppt1 which directly interacts with Hsf1 via its tetratricopeptide repeat (TPR) domain. In response to ethanol stress, PPT1 deletion and CK2 overexpression exert synergistic inhibitory effects on Hsf1 activation, whereas Hsf1S608A mutant shows enhanced activation. Therefore, regulation of the Hsf1 S608 phosphorylation status by reciprocal actions of CK2 and Ppt1 might play an important role to determine Hsf1 sensitivity towards ethanol stress.
Secondly, the reciprocal regulation mechanism of Hsf1 by CK2 and Ppt1 was applied to improve alcohols tolerance for industrial yeast strain. Improving yeast tolerance to alcohols is an important stage toward enabling high titer production. To enhance the ethanol stress-specific activation of Hsf1, CK2-dependent phosphorylation site S608 in Hsf1 was substituted with alanine. The Hsf1S608A strain was showed the improved tolerance to alcohols not just ethanol but also a variety of alcohols, such as methanol, 2,3-butandiol, 2-phenylethanol, 3-methyl-1-butanol, isobutanol, and hexanol. Therefore, CK2-dependent repression mechanism of Hsf1 is applicable for improving yeast tolerance to various alcohols.
Thirdly, we demonstrate that CK2-dependent phosphorylation positively regulates Msn2/4, the general stress response transcriptional activators in Saccharomyces cerevisiae, in response to various types of environmental stress conditions. CK2 overexpression elicits hyperactivation of Msn2/4, whereas deletion of one of the CK2 catalytic subunits, especially CKA2, leads to reduced transcriptional activity of Msn2/4 in response to glucose starvation, H2O2, and lactic acid. The CKA2 deletion mutant also shows increased stress sensitivity. CK2 phosphorylates Ser194 and Ser638 in Msn2 and replacement of these residues with alanine leads to reduced Msn2 activity upon stress and reduced tolerance to H2O2 and lactic acid. CKA2 deletion mutant shows shorter nuclear retention time of Msn2 upon lactic acid stress, suggesting that CK2 might regulate nuclear localization of Msn2. However, Msn2S194A, S638A mutant shows normal nuclear import and export patterns upon stress, suggesting that CK2 might positively regulate the general stress response not only by direct phosphorylation of Msn2/4, but also by regulating cellular translocation machinery.