Physiological and Transcriptional Responses of Saccharomyces cerevisiae to d -Limonene Show Changes to the Cell Wall but Not to the Plasma Membrane

Abstract

bMonoterpenes can, upon hydrogenation, be used as light-fraction components of sustainable aviation fuels. Fermentative production of monoterpenes in engineered microorganisms, such as Saccharomyces cerevisiae, has gained attention as a potential route to deliver these next-generation fuels from renewable biomass. However, end product toxicity presents a formidable problem for microbial synthesis. Due to their hydrophobicity, monoterpene inhibition has long been attributed to membrane interference, but the molecular mechanism remains largely unsolved. In order to gain a better understanding of the mode of action, we analyzed the composition and structural integrity of the cell envelope as well as the transcriptional response of yeast cells treated with an inhibitory amount of d-limonene (107 mg/liter). We found no alterations in membrane fluidity, structural membrane integrity, or fatty acid composition after the solvent challenge. A 4-fold increase in the mean fluorescence intensity per cell (using calcofluor white stain) and increased sensitivity to cell wall-degrading enzymes demonstrated that limonene disrupts cell wall properties. Global transcript measurements confirmed the membrane integrity observations by showing no upregulation of ergosterol or fatty acid biosynthesis pathways, which are commonly overexpressed in yeast to reinforce membrane rigidity during ethanol exposure. Limonene shock did cause a compensatory response to cell wall damage through overexpression of several genes (ROM1, RLM1, PIR3, CTT1, YGP1, MLP1, PST1, and CWP1) involved with the cell wall integrity signaling pathway. This is the first report demonstrating that cell wall, rather than plasma membrane, deterioration is the main source of monoterpene inhibition. We show that limonene can alter the structure and function of the cell wall, which has a clear effect on cytokinesis.

Evolutionary Engineering Improves Tolerance for Replacement Jet Fuels in Saccharomyces cerevisiae

“…Phase toxicity occurs beyond a solvent's solubility and is defined as inhibition due to a distinct second phase (2). The molecular mechanisms of phase toxicity remain poorly understood, but we have demonstrated that limonene interferes with cell wall integrity (CWI) rather than membranes in S. cerevisiae (10). Accordingly, rational strategies for alleviating toxicity, such as manipulation of membrane fluidity or expression of membranebound solvent pumps, have failed to resolve the problem of phase toxicity (11).…”

“…Molecular toxicity is caused by water-soluble compounds partitioning into the plasma membrane (PM) and interfering with membrane properties (9). Molecular toxicity is proportional to a solvent's aqueous concentration and reaches its maximum at a solvent's solubility point (i.e., at membrane and water saturation) (10). Phase toxicity occurs beyond a solvent's solubility and is defined as inhibition due to a distinct second phase (2).…”

“…Total RNA was isolated from S288C and tTcb3p cultures during the mid-exponential-growth phase described previously (10). For control (no limonene) and limonene-challenged (107 mg/liter) cultures, approximately 10 ml of culture was quickly pelleted (RCF, 17,572 ϫ g) for 2 min at 4°C, immediately resuspended in 2 ml of RNAlater solution (Life Technologies, Carlsbad, CA), and stored at 4°C.…”

Modelling sponge-symbiont metabolism

“…A fatty acid methyl ester (FAME) analysis was performed on the extracted lipids from section 3.3.6 as previously described (Brennan et al, 2013). The lipid solid was dissolved in hexane and 5.1 μg of nondecanoic acid (Sigma-Aldrich, St. Louis, MO, USA) was used as an internal standard (ISTD) to a final concentration of 3.6 μg / mL.…”

“…The lipid solid was dissolved in hexane and 5.1 μg of nondecanoic acid (Sigma-Aldrich, St. Louis, MO, USA) was used as an internal standard (ISTD) to a final concentration of 3.6 μg / mL. Saponification, methylation and GC/MS analysis of the lipid mass fraction followed the protocol described in (Brennan et al, 2013). The software program AMDIS (version 2.64, National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA) was used for the analysis of the fatty acid and sterols.…”