Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine. As MIAs are difficult to chemically synthesize, the world’s supply chain for vinblastine relies on low-yielding extractions of precursors vindoline and catharanthine from the plant Catharanthus roseus, followed by chemical coupling and reduction to form vinblastine. Here, we demonstrate de novo microbial biosynthesis of vindoline and catharanthine from renewable feedstocks such as simple sugar and amino acids using highly engineered yeast. The study showcases the longest biosynthetic pathway refactored into a microbial cell factory to date, including 29 enzymatic steps from the yeast native metabolite geranyl pyrophosphate to catharanthine and vindoline. We made 44 genetic edits to yeast that include expression of 35 heterologous genes from plants as well as deletions, knock-downs, and overexpression of 10 yeast genes or variants thereof to improve the precursor supply. Finally, we demonstrate one-step in vitro vinblastine production using chemical coupling and reduction of vindoline and catharanthine. Not only is the yeast a scalable platform for production of vinblastine, it is also a platform for production of more than 2,000 different natural and new-to-nature MIAs.

Cells (about 15 OD x mL units) were pelletized by centrifugation at 5,000 g for 3 min and kept at -80 ℃ until sample preparation. Cells were lysed and protein extracted as described previously. Briefly, it consists of a bead-beating step, cell lysis, protein precipitation, protein resuspension, protein quantification, and normalization of protein concentration followed by standard bottom-up proteomic procedures of reducing and blocking cysteine residues and tryptic digestion. Peptide samples were analyzed on an Agilent 1290 UHPLC system coupled to an Agilent 6460QQQ MS (Agilent Technologies) as described previously. Briefly, peptide samples were loaded onto an Ascentis® ES-C18 Column (Sigma–Aldrich) and introduced to the MS using a Jet Stream source (Agilent Technologies) operating in positive-ion mode. The data were acquired with Agilent MassHunter Workstation Software, LC/MS Data Acquisition operating in dynamicMRM mode. MRM transitions for the targeted proteins were generated by Skyline software (MacCoss Lab Software) and selection criteria excluded peptides with Met/Cys residues, tryptic peptides followed by additional cut sites (KK/RR), and peptides with proline adjacent to K/R cut sites.

All yeast strains were grown in 2 mL SC or YPD media in three biological replicates at 30 °C 300 rpm for 2 days and then harvested for proteomic analysis.