Molecular Insights into Zymomonas mobilis Membrane Interactions with Lignocellulosic Inhibitors

Biomass from crop residues is an abundant feedstock for biorefining through both chemical or biological processes. After breaking down intact biomass through physical or chemical means, the resulting pretreatment product contains a wide variety of small molecules that can be funneled into engineered microbes that convert these building blocks into preferred products. Zymomonas mobilis is one such biorefinery chassis, which is resistant to ethanol stress but may be inhibited by organic acids, aldehydes, alcohols, ketones, and amides found in biomass hydrolysate. One hypothesis is that these molecules interact with or disrupt the bacterial membrane, triggering stress responses and hindering growth. To test this hypothesis at the molecular level, we employ all-atom molecular dynamics (MD) simulations to investigate the interactions of small molecules with a biologically relevant Z. mobilis membrane model. Simulations were conducted across a range of inhibitor concentrations from 0–2.5 mol%, analyzing key membrane properties such as area per lipid (APL), membrane thickness, lipid order parameter, lateral diffusion coefficient, and permeability coefficient. From simulation, we observe altered membrane structure and dynamics at high small molecule concentrations. Generally, the membranes become thinner, with a higher area per lipid and lower order parameter as the small molecule concentration increases. These trends are stronger for more hydrophobic molecules with greater hydrophobic bulk, as isobutanol, propanol, and propanoic acid showed greater membrane perturbations as the concentration increased compared with other small molecules. Tracking small molecule distributions directly in our equilibrium simulations allows us to determine concentration-dependent free energy profiles for these molecules. While the trends are noisy, generally the barriers to crossing the membrane decrease as the concentration increases, indicating that the membranes become leakier as small molecule concentrations rise. Comparing between native Z. mobilis membranes with hopanoids and membranes sharing the same phospholipid composition but without hopanoids, hopanoids stabilize and order the membrane for smaller molecules to maintain membrane structure, but appear insufficient for larger hydrophobic molecules like isobutanol. These findings provide a mechanistic understanding of how small molecules found in biomass degradation streams interact with the Z. mobilis membrane, offering valuable insights for future strain engineering efforts to optimize biofuel and bioproduct synthesis from biomass feedstocks. Modifying membrane composition could enhance microbial robustness, improving microbial survival and thus yields in industrial contexts.