In an effort to move towards a biobased economy and sustainable production of chemicals, we investigate strategies to improve the robustness of microbial cells by engineering the cell membrane to increase the tolerance to inhibitors. Our efforts have been focused on acetic acid, a major inhibitor of yeast cells used in the production of chemicals from lignocellulosic raw material. Acetic acid enters the cell mainly by passive diffusion across the plasma membrane and therefore our aim is to modify the lipid composition of this membrane to reduce the permeability of the acid. In the proposed project, we will i) study the effect of inclusion of bacterial lipids in yeast membranes and ii) the size effect of the partitioning of secondary solvents, such as alcohols.
In the first part, the aim is to use simulations to investigate changes to the physicochemical properties of the membrane when introducing bacterial lipids in yeast. These simulations will be used to guide metabolic engineering strategies with the aim to produce novel strains with improved tolerance to organic acids. The first step in this project is to develop new parameters for the lipid species; we need parameters for lipids with unusually long acyl chains and with cyclopropane moieties on the tails. A procedure to obtain these parameters has been established and this step will be straightforward: it entails running several test simulations to check the validity of the parameters. The novel lipids will then be used to assemble novel membranes that will be subjected to long simulations. We will measure basic membrane properties such as thickness and rigidity but also analyze clustering phenomena and the distribution of the novel lipids in the membrane. Finally, we will measure the permeation of organic acids and phenolic compounds through a few selected membranes by performing umbrella sampling.
In the second part, the aim is to investigate the effect of simulation size on the water–membrane partitioning of butanol, ethanol, acetone and DMSO. We have in an earlier study established that chemicals produced by the yeast cells, such as butanol and ethanol, affects the permeation of acetic acid. Therefore, it is essential to quantify the amount of membrane partitioning of these molecules and how they affect the properties of the membrane. We also observed a size effect of the partitioning in the simulation – more alcohols partitioned in the membrane when we increased the solvation level. This observation had to be investigated further and using a previous SNIC project we have produced preliminary data that a size effect exists for butanol, ethanol, acetone and DMSO. In the proposed project we will extend these simulation and run replicates to establish statistically converged and reliable results. The study entails modeling a small model membrane (64 lipids) solvated with different levels of water molecules (40, 80, 160, 320 and 640 water molecules per lipid). In this system, butanol, ethanol, acetone and DMSO will be inserted at various concentrations and subjected to 500 ns simulations to equilibrate the partitioning between water and membrane.