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Lignocellulosic raw material plays a crucial role in the development of sustainable processes for the production of fuels and chemicals. Weak acids such as acetic acid and formic acid are troublesome inhibitors restricting efficient microbial conversion of the biomass to desired products. To improve our understanding of weak acid inhibition and to identify engineering strategies to reduce acetic acid toxicity, the highly acetic‐acid‐tolerant yeast  Zygosaccharomyces bailii  was studied. The impact of acetic acid membrane permeability on acetic acid tolerance in  Z. bailii  was investigated with particular focus on how the previously demonstrated high sphingolipid content in the plasma membrane influences acetic acid tolerance and membrane permeability. Through molecular dynamics simulations, we concluded that membranes with a high content of sphingolipids are thicker and more dense, increasing the free energy barrier for the permeation of acetic acid through the membrane.  Z. bailii  cultured with the drug myriocin, known to decrease cellular sphingolipid levels, exhibited significant growth inhibition in the presence of acetic acid, while growth in medium without acetic acid was unaffected by the myriocin addition. Furthermore, following an acetic acid pulse, the intracellular pH decreased more in myriocin‐treated cells than in control cells. This indicates a higher inflow rate of acetic acid and confirms that the reduction in growth of cells cultured with myriocin in the medium with acetic acid was due to an increase in membrane permeability, thereby demonstrating the importance of a high fraction of sphingolipids in the membrane of  Z. bailii  to facilitate acetic acid resistance; a property potentially transferable to desired production organisms suffering from weak acid stress. Biotechnol. Bioeng. 2016;113: 744–753. © 2015 The Authors.  Biotechnology and Bioengineering  Published by Wiley Periodicals, Inc.

Sphingolipids contribute to acetic acid resistance in Zygosaccharomyces bailii