Biosynthesis of Medicinal Tropane Alkaloids in Yeast
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Article Biosynthesis of medicinal tropane alkaloids in yeast https://doi.org/10.1038/s41586-020-2650-9 Prashanth Srinivasan1 & Christina D. Smolke1,2 ✉ Received: 20 April 2020 Accepted: 23 July 2020 Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are Published online: 2 September 2020 used for treating neuromuscular disorders and are classifed as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in Check for updates frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfres5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker’s yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafcking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines. Tropane alkaloids (TAs) such as cocaine and atropine are present in chloroplast, peroxisome, ER membrane, vacuole), cell types (root plants from the nightshade (Solanaceae), coca (Erythroxylaceae) and pericycle, endodermis, cortex) and tissues (secondary roots)11. bindweed (Convolvulaceae) families. Some TAs, including hyoscyamine Reconstitution of such pathways in yeast is thus made challenging by and scopolamine, are used to treat neuromuscular disorders ranging incompatibilities of enzymes adapted for specific spatial or regulatory from nerve agent poisoning to Parkinson’s disease1,2. Direct chemical contexts, and metabolite transport strategies that are not readily real- syntheses of TAs are not economically viable owing to challenging ste- ized in microbial hosts. reochemistries9. Thus, intensive cultivation of Duboisia shrubs from the Hyoscyamine and scopolamine comprise an arginine-derived nightshade family in Australia, India, Brazil and Saudi Arabia undergirds 8-azabicyclo[3.2.1]octane (‘tropine’) acyl acceptor esterified with a the global supply for medicinal TAs2,10,11. This agriculture-based supply phenylalanine-derived phenyllactic acid (PLA) acyl donor (Fig. 1a). The chain poses three risks to public health. First, overall increasing demand identification of a type III polyketide synthase (PYKS) and cytochrome for TA-based medicines already results in recurring supply shortages3,4. P450 (CYP82M3) catalysing the cyclization of N-methylpyrrolinium to Second, regional events, such as the 2019–2020 Australian wildfires, can tropinone in Atropa belladonna13 enabled us and others to engineer threaten global supply5. Third, global crises, such as the ongoing COVID- yeast strains for de novo production of tropine14,15. The recent report of a 19 pandemic, can threaten local availability owing to demand spikes and UDP-glucosyltransferase (UGT84A27) and serine carboxypeptidase-like disruption to supply chains6,12. The urgency of having options for quickly (SCPL) acyltransferase (littorine synthase) catalysing the condensation scaling production of essential medicines to match regional and local of tropine and phenyllactate to littorine16 resolved a debate about the demand, free of geopolitical dependencies and robust to environmental nature of the acyl transfer reaction9. However, functional expression and socioeconomic upheaval, is widely recognized7,8. of plant SCPL acyltransferases (SCPL-ATs) in non-plant hosts has not Phytochemical production using engineered yeast can address many been reported. Also, although the cytochrome P450 (CYP80F1) that of the vulnerabilities associated with crop cultivation. The rapid genera- catalyses rearrangement of littorine to hyoscyamine aldehyde17,18 and the tion times and high cell densities achieved in microbial fermentations 2-oxoglutarate-dependent hydroxylase/dioxygenase (H6H) that cataly- enable production of target compounds with reduced time, space ses epoxidation of hyoscyamine to scopolamine are established19,20, and resource requirements relative to plant extraction. Cultivation no enzymatic activity for reduction of hyoscyamine aldehyde to hyos- in closed bioreactors can also reduce supply chain susceptibility to cyamine is known, necessitating discovery of such an enzyme (Fig. 1a). environmental and geopolitical disruption, while providing improved batch-to-batch consistency and active ingredient purity. However, biosynthesis of TAs in Solanaceae exhibits extensive TA acyl acceptor and donor biosynthesis intra- and intercellular compartmentalization, with enzymes active We designed a biosynthetic pathway comprising five functional across specific sub-cellular compartments (cytosol, mitochondrion, modules for hyoscyamine and scopolamine production from simple 1Department of Bioengineering, Stanford University, Stanford, CA, USA. 2Chan Zuckerberg Biohub, San Francisco, CA, USA. ✉e-mail: [email protected] 614 | Nature | Vol 585 | 24 September 2020 a Arg2 NH O O O Mitochondrion H2N N OH HO OH H NH NH 2 Module I: 2 Arginine Car1 Putrescine Glutamic acid ADC biosynthesis As O H N NH2 NH2 H N N 2 H NH2 H2N OH H2N N Spermine NH2 Agmatine Ornithine speB Fms1 O pe1 Meu1 S Oaz1 H OH NH2 Fms1 N NH2 NH2 H2N H2N Putrescine Spermidine Phenylalanine Module II: Tropine biosynthesis AbPMT1 Module III: Aro8 DsPMT1 H H PLA glucoside Aro9 N N biosynthesis H2N H2N O AbPYKS N Spontaneou N-methylputrescine ΔC-PTS1 OH 2× malonyl s DmMPO1 -CoA N-methyl- O pyrrolinium H H N N Phenylpyruvic acid O O Hfd1 O O Ald2 Ald3 4-methylaminobutanal HO N Ald4 Ald5 Egh1 WfPPR Peroxisome O O 4-(1-Methyl-2-pyrrodinyl)- N DsTR1 N 3-oxobutanoic acid AbUGT AbCYP82M3 O OGlc OH AtATR1 OH Tropinone Tropine OH OH Phenyllactic acid Phenyllactic acid glucoside NtJAT1 O N OGlc Nucleus ER OH OH Module V: DsRed TA scaffold biosynthesis -AbLS Vacuole N N AbCYP80F1 OH OH AtATR1 O O O O N Littorine O N OH O Module IV: Medicinal TA biosynthesis O O O Ds O Hyoscyamine HDH H6H aldehyde Prev N OH Ds Scopolamine unknowio n usly O O Hyoscyamine b 300 ** c CSY#Ļ Tropine PLAPLA d e 2.5 glucoside 2.0 * 250 1251 *** ) –1 2.0 l 200 1.5 1287 150 1.5 15 glucoside titre titre (mg 1.0 *** glucoside titre A A 1.0 * PL 10 1288 * * * 0.5 5 0.5 Stds Relative PL NA Relative PLA 0 0.0 0.0 H H R B R 0 123 4 5 6 7 345 6 LD PP hcx PP UGP1 PGM2 EXG1SPR1EGH1 ControBcl LDHLcLD Lp Lp Ab WfPPR Time (min) Time (min) Time (min) Control ControlΔ Δ Δ Fig. 1 | Engineered biosynthetic pathway for de novo production of scopolamine biosynthesis via module III. Chromatogram traces are representative of three in yeast and optimization of PLA-glucoside biosynthesis. a, Modular pathway biological replicates. d, Relative titres of PLA glucoside in yeast engineered for construction for scopolamine biosynthesis in yeast. Enzyme/protein colour scheme: overexpression of UDP-glucose biosynthetic enzymes. Enzymes or negative control orange, yeast (overexpressed); green, plant; purple, bacteria; red, other eukaryote; (BFP) were expressed from low-copy plasmids in strain CSY1288. e, Relative PLA grey, spontaneous/non-enzymatic. Red boxes indicate disrupted yeast proteins; glucoside titres in CSY1288 with disruptions to endogenous glucosidases. In d and e, dotted or solid lines of vacuole membrane delineate functional biosynthetic PLA glucoside accumulation was compared using relative titres owing to lack of an modules. DsRed–AbLS, Discosoma sp. red fluorescent protein fused to the N authentic chemical standard. Strains were cultured for 72 h before liquid terminus of A. belladonna littorine synthase. b, PLA production in yeast engineered chromatography–tandem mass spectrometry (LC–MS/MS) analysis of metabolites for expression of PPRs or LDHs. Heterologous enzymes or negative control (BFP) in culture supernatant. Data in b, d and e represent the mean of n = 3 biologically were expressed from low-copy plasmids in strain CSY1251. c, Multiple reaction independent samples (open circles), error bars denote s.d. *P < 0.05, **P < 0.01, monitoring (MRM) and extracted ion chromatogram (EIC) traces from culture ***P < 0.001, Student’s two-tailed t-test. Statistical significance is shown relative to medium of yeast engineered for step-wise reconstitution of PLA glucoside controls. Exact P values are in Supplementary Table 5. precursors in yeast (Fig. 1a). Modules I/II and III enable de novo biosyn- tropine via modules I and II14 (Extended Data Fig. 1). A putrescine bio- thesis of the acyl acceptor and donor moieties; module IV enables TA synthesis module (I) designed to increase putrescine accumulation scaffold modifications to produce hyoscyamine and scopolamine; mod- incorporated (i) overexpression of glutamate N-acetyltransferase ule V comprises the central acyltransferase reaction linking upstream (Arg2), arginase (Car1), ornithine decarboxylase (Spe1) and polyamine acyl acceptor/donor biosynthesis to downstream scaffold modifica- oxidase (Fms1);