Identification and Characterization of a Novel Issatchenkia orientalis GPI-Anchored Protein, IoGas1, Required for Resistance to Low pH and Salt Stress.

Akinori Matsushika,Tamotsu Hoshino,Toshihiro Suzuki,Tetsuya Goshima,Kanako Negi,Shihui Yang

doi:10.1371/journal.pone.0161888

Abstract

The use of yeasts tolerant to acid (low pH) and salt stress is of industrial importance for several bioproduction processes. To identify new candidate genes having potential roles in low-pH tolerance, we screened an expression genomic DNA library of a multiple-stress-tolerant yeast, Issatchenkia orientalis (Pichia kudriavzevii), for clones that allowed Saccharomyces cerevisiae cells to grow under highly acidic conditions (pH 2.0). A genomic DNA clone containing two putative open reading frames was obtained, of which the putative protein-coding gene comprising 1629 bp was retransformed into the host. This transformant grew significantly at pH 2.0, and at pH 2.5 in the presence of 7.5% Na2SO4. The predicted amino acid sequence of this new gene, named I. orientalis GAS1 (IoGAS1), was 60% identical to the S. cerevisiae Gas1 protein, a glycosylphosphatidylinositol-anchored protein essential for maintaining cell wall integrity, and 58–59% identical to Candida albicans Phr1 and Phr2, pH-responsive proteins implicated in cell wall assembly and virulence. Northern hybridization analyses indicated that, as for the C. albicans homologs, IoGAS1 expression was pH-dependent, with expression increasing with decreasing pH (from 4.0 to 2.0) of the medium. These results suggest that IoGAS1 represents a novel pH-regulated system required for the adaptation of I. orientalis to environments of diverse pH. Heterologous expression of IoGAS1 complemented the growth and morphological defects of a S. cerevisiae gas1Δ mutant, demonstrating that IoGAS1 and the corresponding S. cerevisiae gene play similar roles in cell wall biosynthesis. Site-directed mutagenesis experiments revealed that two conserved glutamate residues (E161 and E262) in the IoGas1 protein play a crucial role in yeast morphogenesis and tolerance to low pH and salt stress. Furthermore, overexpression of IoGAS1 in S. cerevisiae remarkably improved the ethanol fermentation ability at pH 2.5, and at pH 2.0 in the presence of salt (5% Na2SO4), compared to that of a reference strain. Our results strongly suggest that constitutive expression of the IoGAS1 gene in S. cerevisiae could be advantageous for several fermentation processes under these stress conditions.

Highlights

To date, the global consumption of energy has been primarily dependent on finite non-renewable fossil fuels
To identify and isolate novel proteins involved in low-pH tolerance, we screened a genomic DNA library from I. orientalis NBRC1279 constructed with a multicopy plasmid, pPGK [46], containing the promoter and terminator of the phosphoglycerate kinase (PGK) -encoding locus
The longer predicted open reading frames (ORFs)-2 protein displayed 58–60% identity with both Gas1 of S. cerevisiae [44,45], which is necessary for cell wall construction, and the Phr1 and Phr2 proteins of the pathogenic fungus Candida albicans [60,61], which are involved in cell wall assembly and required for virulence in that species

Summary

Introduction

The global consumption of energy has been primarily dependent on finite non-renewable fossil fuels (petroleum). It is believed that the continued use of fossil fuels at the current rate will lead to an increase in global warming and cause more severe climate change; biofuels and bioproducts produced from biomass are increasingly viewed as a viable approach to prevent this scenario [1,2]. Bioethanol, an eco-friendly renewable liquid fuel, is currently the most widely used biofuel [3] but is generated from sugar-containing and starchy biomass, such as corn grain and sugarcane, which are edible agricultural products. The budding yeast Saccharomyces cerevisiae, which efficiently produces ethanol from hexose sugars, including sucrose and glucose, is the most commonly used microorganism in industrial ethanol production. Biomass pretreatment produces hydrolysates that are saline, have high osmotic pressure [7], and contain high concentrations of inhibitors [8], all of which negatively influence fermentation by yeast. The development of robust strains with high stress tolerance is crucial for reducing cooling and ethanol recovery costs, and for minimizing the risk of contamination during commercial ethanol production [15,16,17]

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Results

Conclusion