LAUSR

Significance We report the de novo microbial biosynthesis of (S)-tetrahydropapaverine (THP) and the semisynthesis of papaverine using microbially biosynthesized THP. We used protein engineering to develop variants of two enzymes with improved nonnative activity on pathway intermediates. The biosynthesis of THP demonstrates the ability to use protein homologs and protein engineering to replace the activity of unknown enzymes in heterologous biosynthetic pathways. We improved pathway flux by knocking out two yeast multidrug resistance (MDR) transporters, which reduces the export of pathway intermediates. MDR knockouts may be applied to increase flux through other heterologous pathways. The strain engineering in this work provides a demonstration of fermentation-based production of the clinically significant molecules THP and papaverine, which have experienced recent supply chain shortages.