Synthetic microbial sensing and biosynthesis of amaryllidaceae alkaloids

A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimer's medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from low-yielding plants, including the wild daffodil Narcissus pseudonarcissus. Engineered biocatalytic methods have the potential to stabilize the supply chain of amaryllidaceae alkaloids. Here, we propose a highly efficient biosensor-AI technology stack for biocatalyst development, which we apply to engineer amaryllidaceae alkaloid production in Escherichia coli . Directed evolution is used to develop a highly sensitive (EC50= 20 uM) and specific biosensor for the key amaryllidaceae alkaloid branchpoint 4-O'Methylnorbelladine. A machine learning model (MutComputeX) was subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which were rapidly screened with the biosensor. Functional enzyme variants were identified that yielded a 60% improvement in product titer, 17-fold reduced remnant substrate, and 3-fold lower off-product regioisomer formation. ### Competing Interest Statement The authors declare the following competing financial interest(s): S.d.O., D.D. and A.D.E. have filed a patent application on materials described in this text. S.D. is the founder of Retna Bio, a company that commercializes genetic biosensors. D.D. is the founder of Intelligent Proteins, a company that engineers proteins with machine learning algorithms.