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Abstract  Monoterpene indole alkaloids (MIAs) are a class of natural products comprised of thousands of structurally unique bioactive compounds with significant therapeutic values. Due to difficulties associated with isolation from native plant species and organic synthesis of these structurally complex molecules, microbial production of MIAs using engineered hosts are highly desired. In this work, we report the engineering of fully integrated Saccharomyces cerevisiae strains that allow de novo access to strictosidine, the universal precursor to thousands of MIAs at 30–40 mg/L. The optimization efforts were based on a previously reported yeast strain that is engineered to produce high titers of the monoterpene precursor geraniol through compartmentalization of mevalonate pathway in the mitochondria. Our approaches here included the use of CRISPR-dCas9 interference to identify mitochondria diphosphate transporters that negatively impact the titer of the monoterpene, followed by genetic inactivation; the overexpression of transcriptional regulators that increase cellular respiration and mitochondria biogenesis. Strain construction included the strategic integration of genes encoding both MIA biosynthetic and accessory enzymes into the genome under a variety of constitutive and inducible promoters. Following successful de novo production of strictosidine, complex alkaloids belonging to heteroyohimbine and corynantheine families were reconstituted in the host with introduction of additional downstream enzymes. We demonstrate that the serpentine/alstonine pair can be produced at ∼5 mg/L titer, while corynantheidine, the precursor to mitragynine can be produced at ∼1 mg/L titer. Feeding of halogenated tryptamine led to the biosynthesis of analogs of alkaloids in both families. Collectively, our yeast strain represents an excellent starting point to further engineer biosynthetic bottlenecks in this pathway and to access additional MIAs and analogs through microbial fermentation. One Sentence SummaryAn Saccharomyces cerevisiae-based microbial platform was developed for the biosynthesis of monoterpene indole alkaloids, including the universal precursor strictosidine and further modified heteroyohimbine and corynantheidine alkaloids. 

Summary In plants, the biosynthetic pathways of some specialized metabolites are partitioned into specialized or rare cell types, as exemplified by the monoterpenoid indole alkaloid (MIA) pathway ofCatharanthus roseus(Madagascar Periwinkle), the source of the anticancer compounds vinblastine and vincristine. In the leaf, theC. roseusMIA biosynthetic pathway is partitioned into three cell types with the final known steps of the pathway expressed in the rare cell type termed idioblast. How cell‐type specificity of MIA biosynthesis is achieved is poorly understood.We generated single‐cell multi‐omics data fromC. roseusleaves. Integrating gene expression and chromatin accessibility profiles across single cells, as well as transcription factor (TF)‐binding site profiles, we constructed a cell‐type‐aware gene regulatory network for MIA biosynthesis.We showcased cell‐type‐specific TFs as well as cell‐type‐specificcis‐regulatory elements. Using motif enrichment analysis, co‐expression across cell types, and functional validation approaches, we discovered a novel idioblast‐specific TF (Idioblast MYB1,CrIDM1) that activates expression of late‐stage MIA biosynthetic genes in the idioblast.These analyses not only led to the discovery of the first documented cell‐type‐specific TF that regulates the expression of two idioblast‐specific biosynthetic genes within an idioblast metabolic regulon but also provides insights into cell‐type‐specific metabolic regulation.