Identification of an acetate-tolerant strain of Saccharomyces cerevisiae and characterization by gene expression analysis

bDevelopment of acetic acid-resistant Saccharomyces cerevisiae is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge due to limited information on effective genetic perturbation targets for improving acetic acid resistance in the yeast. This study employed a genomic-library-based inverse metabolic engineering approach to successfully identify a novel gene target, WHI2 (encoding a cytoplasmatic globular scaffold protein), which elicited improved acetic acid resistance in S. cerevisiae. Overexpression of WHI2 significantly improved glucose and/or xylose fermentation under acetic acid stress in engineered yeast. The WHI2-overexpressing strain had 5-times-higher specific ethanol productivity than the control in glucose fermentation with acetic acid. Analysis of the expression of WHI2 gene products (including protein and transcript) determined that acetic acid induced endogenous expression of Whi2 in S. cerevisiae. Meanwhile, the whi2⌬ mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting the important role of Whi2 in the acetic acid response in S. cerevisiae. Additionally, overexpression of WHI2 and of a cognate phosphatase gene, PSR1, had a synergistic effect in improving acetic acid resistance, suggesting that Whi2 might function in combination with Psr1 to elicit the acetic acid resistance mechanism. These results improve our understanding of the yeast response to acetic acid stress and provide a new strategy to breed acetic acid-resistant yeast strains for renewable biofuel production. Lignocellulosic biomass from nonfood stocks such as agricultural and forestry residues has been identified as the prime source for production of renewable biofuels to substitute for conventional fossil fuels in the face of growing demand for energy and rising concerns about greenhouse gas emissions (1-4). Bioconversion of plant cell wall materials by microbial fermentation is typically preceded by harsh (physico)chemical hydrolysis designed to release sugars; this hydrolysis treatment also generates by-products that are toxic to fermenting microorganisms (5, 6). Since hemicellulose and lignin in the plant cell wall are ubiquitously acetylated (7,8), the typical acidic pretreatment of lignocellulosic biomass generates substantial amounts of acetic acid (with concentrations ranging from 1 g/liter to 15 g/liter) in the resulting hydrolysates (9, 10). Acetic acid severely inhibits cell growth and fermentation activity in Saccharomyces cerevisiae (5, 6, 11-13), the predominant microorganism used in industrial fermentation (14, 15). Therefore, improvement in S. cerevisiae resistance to acetic acid is highly desired and critical for achieving efficient and economically viable bioconversion of cellulosic sugars to biofuels.The toxic effects of acetic acid in S. cerevisiae have been intensively characterized, and toxicity mechanisms have been proposed (10,11,(16)(17)(18)(19)(20). When the external pH is lower than the...

Section: Discussionsupporting

confidence: 54%

mentioning

confidence: 99%

Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of the WHI2 Gene Identified through Inverse Metabolic Engineering

Chen

Stabryla

Wei

2016

“…Transcriptional activation of Haa1 target genes occurs under conditions of lactic or acetic acid stress, and some of these genes are required to provide resistance to acetic acid (13,15,33). To examine whether overexpression of HAA1 enhanced the expression of its target genes, transcriptional induction of YGP1, GPG1, and SPI1, encoding a secretory glycoprotein, a gamma subunit of the heterotrimeric G protein, and a glycosylphosphatidylinositol (GPI)-anchored cell wall protein, respectively (16), was analyzed in the presence of lactic acid (Fig.…”

Section: Resultsmentioning

confidence: 99%

Nuclear Localization of Haa1, Which Is Linked to Its Phosphorylation Status, Mediates Lactic Acid Tolerance in Saccharomyces cerevisiae

Sugiyama

Akase

Nakanishi

et al. 2014

Improvement of the lactic acid resistance of the yeast Saccharomyces cerevisiae is important for the application of the yeast in industrial production of lactic acid from renewable resources. However, we still do not know the precise mechanisms of the lactic acid adaptation response in yeast and, consequently, lack effective approaches for improving its lactic acid tolerance. To enhance our understanding of the adaptation response, we screened for S. cerevisiae genes that confer enhanced lactic acid resistance when present in multiple copies and identified the transcriptional factor Haa1 as conferring resistance to toxic levels of lactic acid when overexpressed. The enhanced tolerance probably results from increased expression of its target genes. When cells that expressed Haa1 only from the endogenous promoter were exposed to lactic acid stress, the main subcellular localization of Haa1 changed from the cytoplasm to the nucleus within 5 min. This nuclear accumulation induced upregulation of the Haa1 target genes YGP1, GPG1, and SPI1, while the degree of Haa1 phosphorylation observed under lactic acid-free conditions decreased. Disruption of the exportin gene MSN5 led to accumulation of Haa1 in the nucleus even when no lactic acid was present. Since Msn5 was reported to interact with Haa1 and preferentially exports phosphorylated cargo proteins, our results suggest that regulation of the subcellular localization of Haa1, together with alteration of its phosphorylation status, mediates the adaptation to lactic acid stress in yeast.

“…Transcriptome analysis revealed that AFT1-and HAA1-regulated genes are clearly upregulated in this strain (4). Aft1 is a transcription factor involved in iron utilization and is activated by treatment with weak organic acids (12).…”

mentioning

confidence: 93%

“…We therefore propose a novel strategy to employ acid-resistant yeast strains for bioethanol fermentation under acidic conditions (14,18). To date, we have reported several acidtolerant yeasts, including an acetic acid-tolerant strain of Saccharomyces cerevisiae (4), an acetic acid-tolerant strain of Schizosaccharomyces pombe (14), and the lactic acid-tolerant yeast Candida glabrata (18).…”

mentioning

confidence: 99%

Enhancement of Acetic Acid Tolerance in Saccharomyces cerevisiae by Overexpression of the HAA1 Gene, Encoding a Transcriptional Activator

Tanaka

Ishii

Ogawa

et al. 2012

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104