Evolution‐Inspired Engineering of Diterpene Biosynthesis via Chloroplast Genome Modification

Terpenes constitute the largest and most structurally diverse class of plant secondary metabolites, with critical roles in plant-environment interactions and broad industrial applications. Although nuclear genome engineering of terpene pathways has been extensively explored, chloroplast genome engineering remains largely undeveloped, with all reported studies restricted to the model plant Nicotiana. Here we report successful chloroplast genome engineering for diterpene production in the crop plant potato (Solanum tuberosum) guided by evolutionary principles. First, we identified the trnT/trnL plastomic locus as a new transgene integration site with minimal integration-associated growth penalties. Insertion of a bifunctional diterpene synthase gene from a fern that is absent in flowering plants into this plastomic site yielded transplastomic potato plants with successful production of new diterpenes, but with reduced growth. The co-expression of an algal geranylgeranyl diphosphate synthase gene of chloroplast genome origin to enhance precursor supply restored normal growth while elevating diterpene production. Transplastomic plants were otherwise agronomically comparable to wild-type. This work expands chloroplast engineering as a viable strategy for evolution-inspired terpene pathway engineering in crop improvement and high-value terpene production.