posted on 2021-11-08, 22:15authored byMeghan
E. Davies, Daniel Tsyplenkov, Vincent J. J. Martin
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.