Efficient nepetalactone production in Saccharomyces cerevisiae via metabolic engineering and bioprocess optimization

Nepetalactone, a natural insect repellent comparable to N,N-diethyl-meta-toluamide (DEET), is challenging to produce through plant extraction or chemical synthesis. This study achieved the de novo synthesis of nepetalactone in Saccharomyces cerevisiae without expensive precursors or inducers. Initially, the metabolic pathway for nepetalactone synthesis was successfully established in Saccharomyces cerevisiae. A metabolic pathway was established using strategies such as iridoid synthase (ISY) source screening, enzyme fusion, and cofactor regeneration to optimize nepetalactone production. Bioprocess optimization through chromosomal integration and two-phase fermentation prevented its conversion to dihydronepetalactone, resulting in a high-yield strain, NTE21, with a titer of 2.5 g/L. A record titer of 4.5 g/L was achieved in 5.0 L fed-batch fermentation via continuous batch feeding. This study documents the potential of microbial platforms for the sustainable, cost-effective, and scalable production of nepetalactone, paving the way for its commercial application as a natural insect repellent.