An enzyme-coupled assay enables rapid protein engineering for geraniol production in yeast

Geraniol is an important monoterpene alcohol with various industrial applications. The biological synthesis of geraniol requires the activity of geraniol synthase (GES). Despite several engineering efforts to improve catalytic rates of GES, overall efforts have been limited by the lack of a high-throughput screen. Here, we developed a coupled enzyme-based fluorogenic assay that can detect geraniol as well as other medium to long chain alcohols (C4-C9). Aided by this rapid screening capability, we performed saturation mutagenesis of GES of Catharanthus roseus and identified a mutation of F418 to Q that improved production of geraniol. This robust screening assay enables more high-throughput analysis and engineering of geraniol and other alcohols in S. cerevisiae and E. coli.