Acetic acid inhibits nutrient uptake in Saccharomyces cerevisiae: auxotrophy confounds the use of yeast deletion libraries for strain improvement

Abstract

Acetic acid inhibition of yeast fermentation has a negative impact in several industrial processes. As an initial step in the construction of a Saccharomyces cerevisiae strain with increased tolerance for acetic acid, mutations conferring resistance were identified by screening a library of deletion mutants in a multiply auxotrophic genetic background. Of the 23 identified mutations, 11 were then introduced into a prototrophic laboratory strain for further evaluation. Because none of the 11 mutations was found to increase resistance in the prototrophic strain, potential interference by the auxotrophic mutations themselves was investigated. Mutants carrying single auxotrophic mutations were constructed and found to be more sensitive to growth inhibition by acetic acid than an otherwise isogenic prototrophic strain. At a concentration of 80 mM acetic acid at pH 4.8, the initial uptake of uracil, leucine, lysine, histidine, tryptophan, phosphate, and glucose was lower in the prototrophic strain than in a non-acetic acid-treated control. These findings are consistent with two mechanisms by which nutrient uptake may be inhibited. Intracellular adenosine triphosphate (ATP) levels were severely decreased upon acetic acid treatment, which likely slowed ATP-dependent proton symport, the major form of transport in yeast for nutrients other than glucose. In addition, the expression of genes encoding some nutrient transporters was repressed by acetic acid, including HXT1 and HXT3 that encode glucose transporters that operate by facilitated diffusion. These results illustrate how commonly used genetic markers in yeast deletion libraries complicate the effort to isolate strains with increased acetic acid resistance.

Adaptation and tolerance of bacteria against acetic acid

“…Once in the near-neutral environment, acetic acid dissociates causing a decrease in internal pH which, like in bacteria, leads to a dissipation of the electrochemical gradient maintained across the plasma membrane and inhibits the activity of metabolic enzymes (Booth and Statford 2003;Holyoak et al 1996;Pampulha and Loureiro-Dias 1990). Accumulation of acetate has been found to increase turgor pressure of yeast cells, to induce oxidative stress, to inhibit cell wall glucans' synthesis, and to deplete cells of ATP and amino acids, among other deleterious effects (Almeida et al 2009;Ding et al 2013;Semchyshyn et al 2011;Ullah et al 2013). Yeast cells counteract the internal acidification imposed by acetic acid stress essentially by up-regulating the activity of two proton pumps, one located in the plasma membrane (the PM-H + -ATPase) and the other in the vacuolar membrane (the V-ATPase), which excrete the exceeding protons to the exterior or to the vacuole lumen, respectively (Carmelo et al 1997;Kawahata et al 2006;Mira et al 2010b).…”

“…The large-scale phenotypic screenings carried out in the presence of stressful concentrations of acetic acid have expanded the range of biological functions required for maximal yeast tolerance to acetic acid being demonstrated, in some cases for the first time, such as for the crucial role of genes involved in intracellular trafficking and in the uptake of amino acids and K + (Ding et al 2013;Kawahata et al 2006;Mira et al 2010b). The identification of acetic acid resistance genes and/or of the biological functions maximizing tolerance to this acid provides a valuable asset for the rational design of acetic acid-tolerant yeast strains.…”

“…These studies have been boosted by the availability of an experimental system for this yeast, which allowed the development of a huge panoply of resources and tools that are not available for less genetically accessible systems. In particular, phenotypic screenings of large collections of mutants devoid of all non-essential yeast genes had been conducted using different experimental setups and exploring different genetic backgrounds to identify acetic acid resistance genes in yeast (Ding et al 2013;Kawahata et al 2006;Mira et al 2010b). Putting together the results obtained in these chemogenomic screenings with those resulting from other studies performed at a genomic scale or on a gene-by-gene basis, comprehensive models on the adaptive responses of Saccharomyces cerevisiae to acetic acid toxicity have been established, and these are well described in previous reviews (Mira et al 2010c;Mollapour et al 2008;Piper et al 2001).…”

Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress

“…Several previous reports have called for caution when using auxotrophic parent strains in genetic experiments (15,16,(57)(58)(59). We have now shown that in rich medium, the auxotrophic mutations do not seem to interfere with tolerance to any of the other stress factors tested, i.e., acetic acid at pH 5, hydrogen peroxide, and high temperature, except for osmotic stress caused by either high glucose or NaCl concentrations.…”

PEP3 overexpression shortens lag phase but does not alter growth rate in Saccharomyces cerevisiae exposed to acetic acid stress

“…Because of limitations inherent in the widely used S. cerevisiae gene deletion libraries that restrict the extent of acetic acid resistance that can be obtained in the multiply auxotrophic library mutants (Ding et al 2013), we sought acetic acid-resistant mutants by screening a library of overexpressed yeast genes (Jones et al 2008). Here, we report that overexpression of PEP3 increases yeast tolerance for acetic acid by shortening lag phase.…”