Directed Evolution of a Generalist Biosensor Accelerates Alkaloid Pathway Engineering

In the past decade microbial engineering for production of complex therapeutic plant metabolites has significantly advanced. However, a key bottleneck in the engineering process is screening to identify variants with improved activity, which is typically performed using low-throughput chromatography-based methods. Genetic biosensors can overcome this limitation and increase throughput by several orders of magnitude, but few biosensors exist in Nature for plant metabolites with therapeutic potential. We address this gap by synergizing the extreme promiscuity of a multidrug resistance regulator with a custom directed evolution scheme to create a series of highly specific biosensors for the plant alkaloids tetrahydropapaverine, papaverine, glaucine, rotundine, and noscapine. High resolution structures of these biosensor variants elucidate key adaptations acquired during evolutionary specialization. We subsequently apply one biosensor to evolve a plant methyltransferase, enabling the first microbial production of tetrahydropapaverine, an immediate precursor to four modern pharmaceuticals. Previously studied as virulence factors, multidrug resistance regulators can be repurposed as high throughput analytical tools to accelerate pathway engineering for therapeutic plant metabolites.