초록
<P><B>Abstract</B></P> <P>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 <I>Catharanthus roseus</I> 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 <I>S. cerevisiae</I> and <I>E. coli</I>.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A coupled enzyme assay using geraniol dehydrogenase and diaphorase can detect alcohols. </LI> <LI> Detection of geraniol and medium- to long-chain alcohols (C4-C9) is possible. </LI> <LI> Assay is robust for high-throughput analysis and can function in yeast and bacterial media. </LI> <LI> An F418Q mutation in geraniol dehydrogenase enhanced production by 1.2-fold in <I>S. cerevisiae</I>. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>The coupled enzyme-based fluorogenic assay detects geraniol and aliphatic alcohols (C4-C9). Its robustness for high throughput screening enables rapid screening of enzyme variants for high geraniol production. In this work, we discovered the mutation (F418Q) improves the activity of the truncated geraniol synthase from <I>C. roseus.</I> This newly developed assay offers opportunities for protein and pathway engineering for microbial production of geraniol and medium- and long-chain alcohols.</P> <P>[DISPLAY OMISSION]</P>