1 Department of Biology, Concordia University, Montréal, Québec H4B 1R6, Canada.
2 Centre for Applied Synthetic Biology, Concordia University, Montréal, Québec H4B 1R6, Canada.

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 for the first time de novo nepetalactone synthesis in Saccharomyces cerevisiae, enabling sustainable and scalable production. Nepetalactone production required introducing eight exogenous genes including the cytochrome P450 geraniol-8-hydroxylase, the bottleneck of the heterologous pathway. Combinatorial assessment of geraniol-8-hydroxylase and cytochrome P450 reductase variants, and copy-number variations were used to overcome this bottleneck. We found that several reductases improved hydroxylation activity and increasing geraniol-8-hydroxylase gene copy number improved 8-hydroxygeraniol titers. The accumulation of an unwanted metabolite implied inefficient channeling of carbon through the pathway. With the native yeast old yellow enzymes previously shown to use monoterpene intermediates as substrates, both homologues were deleted. These deletions increased 8-hydroxygeraniol yield, resulting in 3.10 mg/L/OD₆₀₀ of nepetalactone from simple sugar in microtiter plates. This optimized pathway will benefit the development of high yielding strains for the scale up production of nepetalactone.