Sphingolipids contribute to acetic acid resistance in Zygosaccharomyces bailii

Abstract

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.

Sphingolipid biosynthesis upregulation by TOR complex 2–Ypk1 signaling during yeast adaptive response to acetic acid stress

“…It was found that exponentially-growing yeast cells adapted to a concentration of acetic acid that reduced their doubling time by 50% exhibited an increase in their total content of complex sphingolipids and extensive changes in the profile of the complex sphingolipids present, compared to cells cultivated in the same way, but not exposed to acetic acid [46]. The conclusions of our study are also in agreement with a recent report [52] providing evidence that the intrinsically high sphingolipid content in the PM of Zygosaccharomyces bailii , a hemiascomycete distantly related to S. cerevisiae , is an important contributor to the high acetic acid tolerance of this organism. In this regard, we demonstrated that, at least in S. cerevisiae , it is IPC and MIPC, and not M(IP) 2 C, that are required for acetic acid tolerance.…”

“…In this same regard, a scs7Δ mutant, which lacks an enzyme necessary to alpha-hydroxylate the VLCFA in sphingolipids, contains an altered spectrum of IPC species and is more sensitive to acetic acid than the corresponding parental strain [20]. Given that TORC2, Ypk1 and the mechanism by which they regulate sphingolipid production appear to be largely conserved throughout the fungal clade [40], the response we have described here likely plays an important role in the tolerance to acetic acid of many other yeast species, such as Z. bailii [52]. …”

“…First, it is unclear why elevation of complex sphingolipids permits growth on medium containing inhibitory concentrations of acetic acid. One suggestion is that an increase in the content of VLCFA-containing sphingolipids leads to a thicker and more dense PM, thereby increasing the free energy barrier for permeation of acetic acid [52], or enhancing cohesion among the lipids in the outer leaflet, which would be important to counteract the hypotonic shock reported to be induced by this weak acid [55]. Moreover, inadequate sphingolipid biosynthesis is known to deleteriously influence the trafficking of integral membrane proteins from the Golgi body to the PM [56, 57], and several proteins required for this vesicle-mediated protein sorting (Sur2, Sur4 and Ypc1) have known roles in sphingolipid metabolism [58].…”

Engineering of membrane complex sphingolipids improves osmotic tolerance of Saccharomyces cerevisiae

“…Some attempts had been made to change sphingolipid content via engineering or the simulation of molecular dynamics (23-25). An increase in sphingolipids resulted in a Zygosaccharomyces bailii membrane, which was more packed and dense and increased acetic acid resistance (24). These findings indicated that the importance of developing novel strategies to improve stress resistance by engineering complex sphingolipid metabolism.…”

The Zygosaccharomyces bailii transcription factor Haa1 is required for acetic acid and copper stress responses suggesting subfunctionalization of the ancestral bifunctional protein Haa1/Cup2

“…Although the mechanisms underlying the response and extreme tolerance of Z. bailii to acetic acid are still poorly characterized, a number of relevant physiological strategies have been reported. These include the capacity of the yeast cells to tolerate short-term intracellular pH changes [11, 12], co-consume acetic acid and glucose [13–15] and exhibit high basal level of complex sphingolipids proposed to decrease plasma membrane permeability to this weak acid [16]. Also, a recent genome-wide study identified the Z. bailii transcription factor ZbMsn4 [17], homologous to the S. cerevisiae stress-responsive transcriptional activators Msn4 and Msn2 [18], as an acetic acid tolerance determinant.…”