Maxence Holtz, Simon D'Oelsnitz, Cecília Castellví Domingo, Niklas G Madsen, Mars Yu Hong, Jonathan Asmund Arnesen, Aafke C A van Aalst, Samantha de Haan, Christopher K Weingarten, Ditte H Welner, Pamela A Silver, Yan Jessie Zhang, Michael K Jensen, Carlos G Acevedo-Rocha
Monoterpene indole alkaloids (MIAs) are medicinally valuable plant natural products, but their scarcity and structural complexity hinder conventional production. Microbial cell factories offer a sustainable alternative, although optimization of their long biosynthetic pathways is constrained by costly, low-throughput analytics. In this article, we evolved the promiscuous bacterial transcription factor RamR to detect five MIAs, obtaining sensitive and selective biosensor variants with EC50 values below 10 μM. We functionalized RamR in yeast and used it to screen a 188-gene overexpression library for improved strictosidine (STR) production, the common precursor for all MIAs. Biosensor fluorescence strongly correlated with HPLC measurements (R2 = 0.932), enabling identification of engineering targets that, when combined, increased STR titers more than threefold to over 220 mg/l. This RamR-based biosensing platform enables cost-effective screening and makes large biological design spaces experimentally accessible, accelerating strain and enzyme engineering for MIAs and other complex natural products.