Using structurally fungible biosensors to evolve improved alkaloid biosyntheses

A key bottleneck in the microbial production of therapeutic plant metabolites is identifying enzymes that can greatly improve yield. The facile identification of genetically encoded biosensors can overcome this limitation and become part of a general method for engineering scaled production. We have developed a unique combined screening and selection approach that quickly refines the affinities and specificities of generalist transcription factors, and using RamR as a starting point we evolve highly specific (>100-fold preference) and sensitive (EC50 <30 μM) biosensors for the alkaloids tetrahydropapaverine, papaverine, glaucine, rotundine, and noscapine. High resolution structures reveal multiple evolutionary avenues for the fungible effector binding site, and the creation of new pockets for different chemical moieties. These sensors further enabled the evolution of a streamlined pathway for tetrahydropapaverine, an immediate precursor to four modern pharmaceuticals, collapsing multiple methylation steps into a single evolved enzyme. Our methods for evolving biosensors now enable the rapid engineering of pathways for therapeutic alkaloids. ### Competing Interest Statement S.D., K.J., R.T. and A.D.E. have filed two patent applications on materials described in this manuscript. R.T. and A.D.E have equity in GRO Biosciences, a company developing protein therapeutics. The other authors declare no conflict of interest.