Towards membrane engineering as a tool in cell factory design: A case study on acetic acid tolerance in Saccharomyces cerevisiae

Doctoral thesis, 2017

The sustainable production of fuels, chemicals, and materials using renewable resources is a necessity if we are to reduce our ecological footprint and the rate of climate change. Lignocellulosic biomass, the major constituent of plant cell walls, is a renewable raw material with great potential due to its high abundance. The conversion of lignocellulosic material into desired products using micro­organisms is a promising option, although many microbial production processes fail to reach the titers required for process economy due to cellular inhibition. The inhibitory action of some compounds is related to the physiochemical properties of the cell membrane. Inhibitors may enter the cell by passive diffusion through the lipid bilayer of the cell membrane, or may inhibit cells by partition in the lipid bilayer, altering the membrane properties. The aim of the research described in this thesis was to evaluate the possibility of engineering the lipid composition of the cell membrane to create microbial cell factories with maintained production capacity when exposed to compounds whose mechanism of inhibition relates to the physio­chemical properties of the membrane. Attempts were made to increase the tolerance of Saccharomyces cerevisiae to the lignocellulose-derived inhibitor acetic acid, by engineering the cell membrane in order to reduce the rate of acetic acid diffusion. Studies of the acetic-acid-tolerant yeast Zygosaccharomyces bailii revealed that its high tolerance relies on its ability to remodel the cell membrane lipid composition so as to greatly increase the fraction of sphingolipids. Further evidence that sphingolipids reduce the rate of acetic acid diffusion was obtained by molecular dynamics simulations of model membranes with increasing fraction of sphingolipids. The lipid metabolism of S. cerevisiae was then engineered in an attempt to increase the fraction of sphingolipids in the cell membrane. However, sphingolipid synthesis was unchanged or decreased in these strains. The effect of sphingolipids on acetic acid tolerance in S. cerevisiae could therefore not be elucidated, but insight was gained into sphingolipid regulation. To understand the variation in membrane permeation, in particular the extent to which compounds partitioning in the cell membrane change the rate of acetic acid diffusion, the effects of ethanol and n-butanol were investigated. It was found that target titers in ethanol and n‑butanol production significantly increased the rate of acetic acid diffusion; n-butanol having a stronger effect than ethanol. Molecular dynamics simulations were then used to suggest mechanisms for the experimental observations.