Engineering Yeast for De Novo Synthesis of the Insect Repellent Nepetalactone

While nepetalactone, the active ingredient in catnip, is a potent insect repellent, its low in planta accumulation limits its commercial viability as an alternative repellent. Here we describe a platform for de novo nepetalactone production in Saccharomyces cerevisiae, enabling sustainable and scalable production. Nepetalactone production required introduction of eight exogenous genes including the cytochrome P450 geraniol-8-hydroxylase, which represented the bottleneck of the heterologous pathway. Combinatorial assessment of geraniol-8-hydroxylase and cytochrome P450 reductase variants, as well as copy-number variations were used to overcome this bottleneck. We found that several reductases improved hydroxylation activity, with a higher geraniol-8-hydroxylase ratio further increasing 8-hydroxygeraniol titers. Another roadblock was the accumulation of an unwanted metabolite that implied inefficient channeling of carbon through the pathway. With the native yeast old yellow enzymes previously shown to use monoterpene intermediates as substrates, both homologs were deleted. These deletions increased 8-hydroxygeraniol yield, resulting in a final de novo accumulation of 3.10 mg/L/OD600 of nepetalactone from simple sugar in microtiter plates. Our pathway optimization will aid in the development of high yielding monoterpene S. cerevisiae strains.