Adaptive Evolution Reveals Mechanisms of Isobutanol Tolerance in Zymomonas mobilis

Isobutanol production has been demonstrated in E. coli and Z. mobilis using heterologous pathways, with titers reaching up to 60% of the theoretical yield under optimized conditions. However, these results often depend on impractical setups, such as continuous product extraction, oxygenation, or nutrient supplementation, and still produce poorly growing strains with unstable pathway expression. In Z. mobilis, carbon flux remains strongly biased toward ethanol, which competes directly with isobutanol for pyruvate and reducing power.
Here, to address this challenge, we conducted an adaptive evolution experiment to improve isobutanol resistance in Z. mobilis. The bacterium was subjected to prolonged growth under gradually increasing isobutanol concentrations, leading to the isolation of evolved strains with significantly enhanced tolerance. Whole-genome sequencing revealed conserved adaptive mechanisms, with genetic alterations affecting a small set of target genes shared across resistant strains. By linking these mutations to improved growth kinetics and resistance profiles, we provide insights into the genetic and physiological basis of isobutanol tolerance, guiding future metabolic engineering efforts.