Structure-Guided Engineering of a Scoulerine 9-O-Methyltransferase Enables the Biosynthesis of Tetrahydropalmatrubine and Tetrahydropalmatine in Yeast

Benzylisoquinoline alkaloids (BIAs) are an important class of plant natural products with diverse pharmacological properties. Microbial platforms can produce BIAs through heterologous biosynthesis more efficiently than native plant hosts and allow for the generation of currently inaccessible BIA biosynthetic intermediates and unnatural derivatives. However, much remains unknown regarding the structures, substrate scopes, and mechanisms of many of the enzymes involved in BIA biosynthesis, which hampers efforts toward engineering these enzymes to produce alternative products or act in non-native biosynthetic contexts. Here, we present multiple crystal structures of two scoulerine 9-O-methyltransferase (S9OMT) variants from Thalictrum flavum which catalyze an essential step in the biosynthesis of berberine. The crystal structures revealed the structural basis for substrate and cofactor recognition by TfS9OMT and provide further insight into the structure and function of S9OMTs. A structural comparison of the TfS9OMT and T. flavum norcoclaurine 6OMT (Tf6OMT) crystal structures identified important residues for enzyme regiospecificity, which were confirmed via mutagenesis and in vitro assays. Several TfS9OMT mutants with expanded substrate scopes toward various 1-benzylisoquinoline substrates were generated. The rationally engineered TfS90MT mutants with altered regiospecificity were tested in a yeast-based scoulerine production platform and enabled de novo production of tetrahydropalmatrubine and tetrahydropalmatine.