Leucyl-tRNA synthetase as a molecular target of isobutanol-mediated growth inhibition in Saccharomyces cerevisiae.

Our dependence on fossil fuels and ongoing environmental degradation urgently require sustainable energy solutions. Biofuels that do not rely on fossil resources have attracted significant scientific interest. Among these, isobutanol produced by yeast is a promising next-generation biofuel owing to its high-energy density and compatibility with existing fuel infrastructure. However, the inherently low tolerance of yeast to isobutanol constrains its production, posing a major barrier to the large-scale implementation of bio-based isobutanol. Here, we provide compelling evidence that supplementation with excess branched-chain amino acids (BCAAs), particularly leucine, alleviates isobutanol-induced growth inhibition in yeast. Using saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy and structural modeling, we showed that isobutanol directly interacts with cytoplasmic leucyl-tRNA synthetase (LeuRS), acting as a competitive inhibitor of leucine binding. Collectively, these findings reveal a mechanistic basis by which isobutanol inhibits yeast growth, offering new insights into enhancing isobutanol tolerance and production in yeast.