Microbial production of 5- <i>epi</i> -jinkoheremol, a plant-derived antifungal sesquiterpene

Synthetic biology using microbial chassis is emerging as a powerful tool for the production of natural chemicals. In the present study, we constructed a microbial platform for the high-level production of a sesquiterpene from <i>Catharanthus roseus</i>, 5-<i>epi</i>-jinkoheremol, which exhibits strong fungicidal activity. First, the mevalonate and sterol biosynthesis pathways were optimized in engineered yeast to increase the metabolic flux toward the biosynthesis of the precursor farnesyl pyrophosphate. Then, the transcription factor Hac1- and m⁶A writer Ime4-based metabolic engineering strategies were implemented in yeast to increase 5-<i>epi</i>-jinkoheremol production further. Next, protein engineering was performed to improve the catalytic activity and enhance the stability of the 5-<i>epi</i>-jinkoheremol synthase TPS18, resulting in the variant TPS18^I21P/T414S, with the most improved properties. Finally, the titer of 5-<i>epi</i>-jinkoheremol was elevated to 875.25 mg/L in a carbon source-optimized medium in shake flask cultivation. To the best of our knowledge, this is the first study to construct an efficient microbial cell factory for the sustainable production of this antifungal sesquiterpene.IMPORTANCEBiofungicides represent a new and sustainable tool for the control of crop fungal diseases. However, hindered by the high cost of biofungicide production, their use is not as popular as expected. Synthetic biology using microbial chassis is emerging as a powerful tool for the production of natural chemicals. We previously identified a promising sesquiterpenoid biofungicide, 5-<i>epi</i>-jinkoheremol. Here, we constructed a microbial platform for the high-level production of this chemical. The metabolic engineering of the terpene biosynthetic pathway was firstly employed to increase the metabolic flux toward 5-<i>epi</i>-jinkoheremol production. However, the limited catalytic activity of the key enzyme, TPS18, restricted the further yield of 5-<i>epi</i>-jinkoheremol. By using protein engineering, we improved its catalytic efficiency, and combined with the optimization of regulation factors, the highest production of 5-<i>epi</i>-jinkoheremol was achieved. Our work was useful for the larger-scale efficient production of this antifungal sesquiterpene.