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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. Plants synthesize more than 3000 tetrahydroisoquinoline (THIQ) alkaloids, but only a few of them have been produced by engineered microbes and titers are very low. Here, the authors increase ( S )-reticuline titer to 4.6 g/L and repurpose the yeast Ehrlich pathway to synthesize a diverse array of THIQ scaffolds.

Eukaryotic cells compartmentalize metabolic pathways in organelles to achieve optimal reaction conditions and avoid crosstalk with cytosolic factors. We found that cytosolic expression of norcoclaurine synthase (NCS), the enzyme that catalyzes the first committed reaction in benzylisoquinoline alkaloid biosynthesis, is toxic in Saccharomyces cerevisiae and, consequently, restricts ( S )-reticuline production. We developed a compartmentalization strategy that alleviates NCS toxicity while promoting increased ( S )-reticuline titer. This strategy is achieved through efficient targeting of toxic NCS to the peroxisome while, crucially, taking advantage of the free flow of metabolite substrates and products across the peroxisome membrane. We demonstrate that expression of engineered transcription factors can mimic the oleate response for larger peroxisomes, further increasing benzylisoquinoline alkaloid titer without the requirement for peroxisome induction with fatty acids. This work specifically addresses the challenges associated with toxic NCS expression and, more broadly, highlights the potential for engineering organelles with desired characteristics for metabolic engineering. Increased production of ( S )-reticuline and other alkaloids is achieved through alleviating norcoclaurine synthase toxicity by targeting the enzyme to the peroxisome plus enlarging peroxisomes by expression of engineered transcription factors.

The tetrahydroisoquinoline (THIQ) moiety is a privileged substructure of many bioactive natural products and semi-synthetic analogues. The plant kingdom manufactures 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 THIQ platform by increasing yeast production of the central BIA intermediate (S)-reticuline to more than 3 g L-1, a 38,000-fold improvement over our first-generation strain. Gains in BIA output coincided with the formation of several substituted THIQs derived from host amino acid catabolism. Enabled by this activity, we repurposed the yeast Ehrlich pathway and demonstrate the synthesis of an array of unnatural THIQ scaffolds. This work provides a blueprint for synthesizing new privileged structures and will enable the targeted overproduction of thousands of THIQ products, including natural and semi-synthetic opioids.

Researchers in the field of metabolic engineering aim to develop processes to produce useful chemicals, sustainably and responsibly, using biotechnology. These processes areoften designed to replace products derived from fossil fuels, which are unsustainable and contribute to climate change, or plant-based products, which compete with food production for scarce land and are subject to supply uncertainty due to weather, crop disease, and climate change. Here, we present two research projects in metabolic engineering. First, we demonstrate microbial production of the red food dye betanin by engineering the betalain biosynthesis pathway into yeast. Betanin is currently manufactured through extraction from red beets specifically grown for dye production. We achieved betanin production levels of 17 mg/L, which is equivalent to the amount of betanin found in 10 g/L of beet extract. With further production improvements, this bioprocess may become cost-competitive with agricultural production and is likely to lead to a purer product. We also demonstrate the synthesis of a suite of non-natural betalain dyes achieved through feeding of diverse amines to a yeast production host, including several which have never been reported. In the second research project, we discover that an enzyme that limits production levels of a drug family is toxic to the yeast production host. This enzyme, norcoclaurine synthase, is critical to the production of benzylisoquinoline alkaloids, an important family of medicines that are extracted from plants like the opium poppy. We devised a novel subcellular compartmentalization strategy, sequestering norcoclaurine synthase in the peroxisome to alleviate cytotoxicity while maintaining access to the enzyme’s substrates. By targeting norcoclaurine synthase for organellar compartmentalization, we achieved improved cell growth, final titer, and culture productivity. These projects highlight the potential of engineering complex plant pathways into microbial hosts for economical and sustainable chemical production.

Eukaryotic cells compartmentalize metabolic pathways in organelles to achieve optimal reaction conditions and avoid crosstalk with other factors in the cytosol. Increasingly, engineers are researching ways in which synthetic compartmentalization could be used to address challenges in metabolic engineering. Here, we identified that norcoclaurine synthase (NCS), the enzyme which catalyzes the first committed reaction in benzylisoquinoline alkaloid (BIA) biosynthesis, is toxic when expressed cytosolically in Saccharomyces cerevisiae and, consequently, restricts (S)-reticuline production. We developed a compartmentalization strategy that alleviates NCS toxicity while promoting increased (S)-reticuline titer, achieved through efficient targeting of toxic NCS to the peroxisome while, crucially, taking advantage of the free flow of metabolite substrates and product across the peroxisome membrane. We identified that peroxisome protein capacity in S. cerevisiae becomes a limiting factor for further improvement of BIA production and demonstrate that expression of engineered transcription factors can mimic the oleate response for larger peroxisomes, further increasing BIA titer without the requirement for peroxisome induction with fatty acids. This work specifically addresses the challenges associated with toxic NCS expression and, more broadly, highlights the potential for engineering organelles with desired characteristics for metabolic engineering.