Lipidomic profiling of Saccharomyces cerevisiae and Zygosaccharomyces bailii reveals critical changes in lipid composition in response to acetic acid stress

Abstract

When using microorganisms as cell factories in the production of bio-based fuels or chemicals from lignocellulosic hydrolysate, inhibitory concentrations of acetic acid, released from the biomass, reduce the production rate. The undissociated form of acetic acid enters the cell by passive diffusion across the lipid bilayer, mediating toxic effects inside the cell. In order to elucidate a possible link between lipid composition and acetic acid stress, the present study presents detailed lipidomic profiling of the major lipid species found in the plasma membrane, including glycerophospholipids, sphingolipids and sterols, in Saccharomyces cerevisiae (CEN.PK 113_7D) and Zygosaccharomyces bailii (CBS7555) cultured with acetic acid. Detailed physiological characterization of the response of the two yeasts to acetic acid has also been performed in aerobic batch cultivations using bioreactors. Physiological characterization revealed, as expected, that Z. bailii is more tolerant to acetic acid than S. cerevisiae. Z. bailii grew at acetic acid concentrations above 24 g L(-1), while limited growth of S. cerevisiae was observed after 11 h when cultured with only 12 g L(-1) acetic acid. Detailed lipidomic profiling using electrospray ionization, multiple-reaction-monitoring mass spectrometry (ESI-MRM-MS) showed remarkable changes in the glycerophospholipid composition of Z. bailii, including an increase in saturated glycerophospholipids and considerable increases in complex sphingolipids in both S. cerevisiae (IPC 6.2×, MIPC 9.1×, M(IP)2C 2.2×) and Z. bailii (IPC 4.9×, MIPC 2.7×, M(IP)2C 2.7×), when cultured with acetic acid. In addition, the basal level of complex sphingolipids was significantly higher in Z. bailii than in S. cerevisiae, further emphasizing the proposed link between lipid saturation, high sphingolipid levels and acetic acid tolerance. The results also suggest that acetic acid tolerance is associated with the ability of a given strain to generate large rearrangements in its lipid profile.

Figure 1. Fermentation profiles of S. cerevisiae and Z. bailii cultured with and without acetic acid.

The graphs show data until all the carbon sources had been utilized. A. S. cerevisiae cultured in minimal medium. B. S. cerevisiae cultured in minimal medium with 9 g L⁻¹ acetic acid. C. Z. bailii cultured in minimal medium. D. Z. bailii cultured in minimal medium with 24 g L⁻¹ acetic acid. The graphs represent the mean of n≥3 biological replicates with error bars indicating standard deviation. For the sake of clarity, error bars are omitted from the insert in figure 1B.

Figure 2. Glycerophospholipid profiles of S. cerevisiae and Z. bailii in response to acetic acid.

Sc: S. cerevisiae cultured in minimal medium. ScAA: S. cerevisiae cultured in minimal medium with 9 g L⁻¹ acetic acid. Zb: Z. bailii cultured in minimal medium. ZbAA: Z. bailii cultured in minimal medium with 24 g L⁻¹ acetic acid. Apparent quantities were calculated relative to the appropriate internal standard, and normalized to the total amount of phosphate in each sample (see Materials and Methods). A. Total glycerophospolipids (GPL) analyzed (PC, PE, PI, PS). B. Glycerophospholipid classes. C. Amount of unsaturations in total glycerophospholipids, presented per lipid, containing two fatty acyl chains. D. Total glycerophospholipid chain length, presented per lipid, containing two fatty acyl chains. *Significant difference compared with control condition, obtained by t-tests (P<0.05). The results were calculated from biological replicates (n = 4) and are given as the mean ± standard deviation. For lipid nomenclature, see Table 1.

Figure 3. Sphingolipid and sterol profiles of S. cerevisiae and Z. bailii in response to acetic acid.

Sc: S. cerevisiae cultured in minimal medium. ScAA: S. cerevisiae cultured in minimal medium with 9 g L⁻¹ acetic acid. Zb: Z. bailii cultured in minimal medium. ZbAA: Z. bailii cultured in minimal medium with 24 g L⁻¹ acetic acid. Apparent quantities were calculated relative to the appropriate internal standard, and normalized to the total amount of phosphate in each sample (see Materials and Methods). A. Head group classes of complex sphingolipids. B. Total sphingolipid chain length, presented per lipid, containing two fatty acyl chains. C. Total ceramides and ceramide sub-classes. Abbreviations: DHS: dihydrosphingosine, PHS: phytosphingosine. D. Ergosterol, ergosterol esters and total sterols. *Significant differences, obtained by t-test (P<0.05) compared with control condition (Sc or Zb). The results were calculated from biological replicates (n = 4) and are given as the mean ± standard deviation. For lipid nomenclature, see Table 1.

Figure 4. Overall lipidome response of S. cerevisiae and Z bailii cultured with and without acetic acid.

S. cerevisiae was cultured with 9 g L⁻¹ acetic acid and Z. bailii with 24g L⁻¹ acetic acid. A. Response of S. cerevisiae and Z. bailii to acetic acid. B. Comparison of S. cerevisiae to Z. bailii under control conditions and acetic acid stress. Total sphingolipids is an approximation as absolute quantities are not available (see Materials and Methods). The results were calculated from biological replicates (n = 4) and are given as the mean ± standard deviation.

Figure 5. Lipid metabolism in S. cerevisiae.

Simplified illustration. It is speculated that this illustration is applicable also to Z. bailii, due to the high degree of evolutionary conservation of the lipid metabolism. Boxes in bold indicate lipid classes analyzed in this study. Green/red arrows indicate a suggested higher/lower flux in Z. bailii resulting from acetic acid stress. Abbreviations: LCFA: long-chain fatty acids, LCB: long-chain base, VLCFA: very-long-chain fatty acids, G-3-P: glycerol-3-phosphate, Etn: ethanolamine. Illustration modified from based on information from the Saccharomyces Genome Database (www.yeastgenome.org). For lipid nomenclature, see Table 1.