Evolutionary hijacking of fatty acid biosynthesis drives insecticidal acylsugar diversity in tomato.

The evolution of plant defensive specialized metabolites often involves repurposing existing primary metabolic pathways for novel roles. However, the relative contribution of changes in protein function versus gene regulation and localization in driving such divergency remains less well understood. Here, we investigate the evolutionary origin of long-branched-chain acylsugars-a class of insecticidal metabolites found in wild tomatoes but absent from their domesticated counterpart. We show that this chemical divergency is enabled by a single enzyme, an acyl-CoA synthetase (SpBACS1), that was repurposed to bridge two distinct primary metabolic pathways. We demonstrate that SpBACS1 activates products of amino acid catabolism and primes them as non-canonical starters for elongation by the plastidial fatty acid synthase machinery for acylsugar assembly. Strikingly, the loss of this trait during domestication was not due to impaired enzyme function. Instead, we describe two distinct regulatory mechanisms. First, the cultivated ortholog, SlBACS1, lacks expression in acylsugar-producing trichomes due to promoter sequence divergence, despite retaining enzyme catalytic activity. Concurrently, their paralogs, BACS2, were neolocalized to the mitochondria, functionally isolating them from the chloroplast-based primary fatty acid metabolic elongation pathway. These findings demonstrate how divergence in cis-regulatory elements and subcellular targeting, in the absence of protein function modification, were potent drivers of metabolic evolution, providing a strategy for re-engineering valuable chemical diversity into cultivated crops.