The tetrahydroisoquinoline (THIQ) moiety is a privileged substructure of many bioactive natural products and semi-synthetic analogs. Plants manufacture more than 3,000 THIQ alkaloids, including the opioids morphine and codeine. While microbial species have been engineered to synthesize a few compounds from the benzylisoquinoline alkaloid (BIA) family of THIQs, low product titers impede industrial viability and limit access to the full chemical space. Here we report a yeast THIQ platform by increasing production of the central BIA intermediate (S)-reticuline to 4.6 g L −1 , a 57,000-fold improvement over our first-generation strain. We show that gains in BIA output coincide with the formation of several substituted THIQs derived from amino acid catabolism. We use these insights to repurpose the Ehrlich pathway and synthesize an array of THIQ structures. This work provides a blueprint for building diverse alkaloid scaffolds and enables the targeted overproduction of thousands of THIQ products, including natural and semi-synthetic opioids.

A fundamental undertaking of metabolic engineering involves identifying and troubleshooting metabolic bottlenecks that arise from imbalances in pathway flux. To expedite the systematic screening of enzyme orthologs in conjunction with DNA copy number tuning, here we develop a simple and highly characterized CRISPR-Cas9 integration system in Saccharomyces cerevisiae. Our engineering strategy introduces a series of synthetic DNA landing pads (LP) into the S. cerevisiae genome to act as sites for high-level gene integration. LPs facilitate multicopy gene integration of one, two, three, or four DNA copies in a single transformation, thus providing precise control of DNA copy number. We applied our LP system to norcoclaurine synthase (NCS), an enzyme with poor kinetic properties involved in the first committed step of the production of high-value benzylisoquinoline alkaloids. The platform enabled rapid construction of a 40-strain NCS library by integrating ten NCS orthologs in four gene copies each. Six active NCS variants were identified, whereby production of (S)-norcoclaurine could be further enhanced by increasing NCS copy number. We anticipate the LP system will aid in metabolic engineering efforts by providing strict control of gene copy number and expediting strain and pathway engineering campaigns.

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The ever-increasing quantity of data deposited to GenBank is a valuable resource for mining new enzyme activities. Falling costs of DNA synthesis enables metabolic engineers to take advantage of this resource for identifying superior or novel enzymes for pathway optimization. Previously, we reported synthesis of the benzylisoquinoline alkaloid dihydrosanguinarine in yeast from norlaudanosoline at a molar conversion of 1.5%. Molar conversion could be improved by reduction of the side-product N-methylcheilanthifoline, a key bottleneck in dihydrosanguinarine biosynthesis. Two pathway enzymes, an N-methyltransferase and a cytochrome P450 of the CYP719A subfamily, were implicated in the synthesis of the side-product. Here, we conducted an extensive screen to identify enzyme homologues whose coexpression reduces side-product synthesis. Phylogenetic trees were generated from multiple sources of sequence data to identify a library of candidate enzymes that were purchased codon-optimized and precloned into expression vectors designed to facilitate high-throughput analysis of gene expression as well as activity assay. Simple in vivo assays were sufficient to guide the selection of superior enzyme homologues that ablated the synthesis of the side-product, and improved molar conversion of norlaudanosoline to dihydrosanguinarine to 10%.

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