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Title: Quantitative Transcription Dynamic Analysis Reveals Candidate Genes and Key Regulators for Ethanol Tolerance in Saccharomyces cerevisiae

item MA, MENGGEN - New Mexico State University
item Liu, Zonglin

Submitted to: BMC Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/21/2010
Publication Date: 6/10/2010
Citation: Ma, M., Liu, Z. 2010. Quantitative Transcription Dynamic Analysis Reveals Candidate Genes and Key Regulators for Ethanol Tolerance in Saccharomyces cerevisiae. Biomed Central (BMC) Genomics. 10:169.

Interpretive Summary: Yeast is a classic ethanol producing agent yet it is vulnerable to high concentrations of ethanol. For cost-efficient ethanol production, this higher ethanol concentration is desirable since it reduces energy costs involved in downstream distillation and waste treatment. The development of ethanol tolerant yeasts is needed in order to achieve this. At the Agricultural Research Service, we recently developed an ethanol-tolerant yeast with an inhibitor-tolerant background through evolutionary engineering. Using comparative studies, this research described mechanisms of the ethanol tolerance displayed by the tolerant yeast based on comparative gene expression analysis applying validated pathway-based qRT-PCR array assays. We found that many genes are necessary for yeast to survive and function against ethanol challenge including genes encoding numerous heat shock proteins, biosynthetic, and central metabolic pathways. Well integrated and reprogrammed glucose metabolic pathways including pentose phosphate pathways were vital to maintain yeast function and ethanol production. Our findings in this study add insight into ethanol tolerance mechanisms and will guide metabolic engineering efforts for more tolerant strain development. The currently developed ethanol-tolerant yeast has potential application for high gravity fermentation to produce higher titer of ethanol.

Technical Abstract: Derived from our lignocellulosic conversion inhibitor-tolerant yeast, we generated an ethanol-tolerant strain Saccharomyces cerevisiae NRRL Y-50316 by enforced evolutionary adaptation. Using a newly developed robust mRNA reference and a master equation unifying gene expression data analyses, we investigated comparative quantitative transcription dynamics of 175 genes selected from previous studies for an ethanol-tolerant yeast and its closely related parental strain. A highly fitted master equation was established and applied for quantitative gene expression analyses using pathway-based qRT-PCR array assays. The ethanol-tolerant Y-50316 displayed a significantly enriched background of mRNA abundance for at least 35 genes without ethanol challenge compared with its parental strain Y-50049. Under the ethanol challenge, the tolerant Y-50316 responded in consistent expressions over time for numerous genes belonging to groups of heat shock proteins, trehalose metabolism, glycolysis, pentose phosphate pathway, fatty acid metabolism, amino acid biosynthesis, pleiotropic drug resistance gene family, and transcription factors. The parental strain showed a brief induction response for many genes but turned repressed afterward. It was unable to withstand the ethanol stress and establish a viable culture and fermentation. The distinct expression dynamics between the two strains and their close association with cell growth, viability and ethanol fermentation profiles distinguished the tolerance-response from the stress-response in yeast under the ethanol challenge. At least 82 genes were identified as candidates and key genes for ethanol-tolerance and subsequent fermentation under the stress. Among which, 36 genes were newly recognized by the present study. Most of the ethanol-tolerance candidate genes were found to share protein binding motifs of transcription factors Msn4p/Msn2p, Yap1p, Hsf1p, and Pdr1p. Enriched background of transcription abundance and continuously enhanced expressions of ethanol-tolerance genes associated with heat shock proteins, trehalose-glycolysis-pentose phosphate pathways and PDR gene family are accountable for the tolerant yeast to withstand the ethanol stress, maintain active metabolisms, and complete ethanol fermentation under the ethanol stress. Transcription factor Msn4p appeared to be a key regulator of gene interactions for ethanol-tolerance in the tolerant yeast Y-50316.