Complete biosynthesis of opioids in yeast

Abstract

Opioids are the primary drugs used in Western medicine for pain management and palliative care. Farming of opium poppies remains the sole source of these essential medicines, despite diverse market demands and uncertainty in crop yields due to weather, climate change, and pests. We engineered yeast to produce the selected opioid compounds thebaine and hydrocodone starting from sugar. All work was conducted in a laboratory that is permitted and secured for work with controlled substances. We combined enzyme discovery, enzyme engineering, and pathway and strain optimization to realize full opiate biosynthesis in yeast. The resulting opioid biosynthesis strains required the expression of 21 (thebaine) and 23 (hydrocodone) enzyme activities from plants, mammals, bacteria, and yeast itself. This is a proof of principle, and major hurdles remain before optimization and scale-up could be achieved. Open discussions of options for governing this technology are also needed in order to responsibly realize alternative supplies for these medically relevant compounds.

Fig 1. Engineered biosynthetic pathway for de novo production of thebaine and hydrocodone and optimization of reticuline-producing platform strains

(A) Biosynthetic scheme for production of thebaine and hydrocone from sugar. Thebaine is a starting material for many opioid drugs through biosynthetic and semi-synthetic routes. Block arrows indicate enzyme-catalyzed steps. Light grey arrows, unmodified yeast enzymes; dark grey arrows, overexpressed and modified yeast enzymes; purple arrows, mammalian (Rattus norvegicus) enzymes; orange arrows, bacterial (Pseudomonas putida) enzymes; green arrows, plant (Papaver somniferum, Papaver bracteatum, Coptis japonica, Eschscholzia californica) enzymes. Yellow outline indicates DRS-DRR; red outline indicates engineered SalSyn. E4P, erythrose 4-phosphate; PEP, phosphoenolpyruvate; DAHP, 3-deoxy-D-arabino-2-heptulosonic acid 7-phosphate; 4-HPP, 4-hydroxyphenylpyruvate; 4-HPAA, 4-hydroxyphenylacetaldehyde; BH₄, 5,6,7,8-tetrahydrobiopterin; Tkl1p, transketolase; CPR, cytochrome P450 reductase; Aro4p^Q166K, DAHP synthase; Aro1p, pentafunctional arom enzyme; Aro2p, bifunctional chorismate synthase and flavin reductase; Aro7p^T226I, chorismate mutase; Tyr1p, prephenate dehydrogenase; Aro8p, aromatic aminotransferase I; Aro9p, aromatic aminotransferase II; Aro10p, phenylpyruvate decarboxylase; TyrH^WR, feedback inhibition-resistant tyrosine hydroxylase (R37E, R38E, W166Y); DoDC, L-dopa decarboxylase; NCS, (S)-norcoclaurine synthase; 6OMT, norcoclaurine 6-O-methyltransferase; CNMT, coclaurine N-methyltransferase; NMCH, N-methylcoclaurine hydroxylase; 4’OMT, 3’-hydroxy-N-methylcoclaurine 4’-O-methyltransferase; DRS-DRR, 1,2-dehydroreticuline synthase-1,2-dehydroreticuline reductase; SalSyn, salutaridine synthase; SalR, salutaridine reductase; SalAT, salutaridinol 7-O-acetyltransferase; T6ODM, thebaine 6-O-demethylase; morB, morphinone reductase. Details on the BH₄ biosynthesis, recycling, and salvage pathway, conversion of (S)-norcoclaurine to (S)-reticuline, and genetic pathway modules are provided in figs. S1–2. (B) Optimization of the reticuline-producing platform strain through pathway and strain engineering. Reticuline in the growth media was analyzed by LC-MS/MS MRM and quantified with an external standard curve. Error bars represent standard deviation of three biological replicates (C) Chiral analysis of reticuline produced by the platform strain. Reticuline was isolated from the growth media of strain CSY1061 and separated on a chiral column. This chromatogram is one of two similar traces from replicate yeast cultures. The chromatogram was smoothed using a 7-point boxcar moving average.

Fig. 2. DRS-DRR converts (S)-reticuline to (R)-reticuline

(A) Biosynthetic scheme for the reaction catalyzed by DRS-DRR. (B) Identification of DRS-DRR via bioinformatic analysis of COR-like sequences. Bioinformatic query was COR VIGS sequence and subject sequences were the P. bracteatum PhytoMetaSyn transcriptome; P. bracteatum P. setigerum, P. somniferum, P. rhoeas 1000 Plants Project transcriptomes; and all deposited sequences in Genbank belonging to Papaveraceae. The scale bar indicates amount of genetic change in amino acid substitutions per site. Branches highlighted in red indicate sequences containing both CYP and COR-like domains. Phylogenetic tree was generated using ClustalX bootstrap NJ tree with 1000 trials. (C) Chiral analysis of reticuline produced by yeast strains expressing and not expressing DRS-DRR. Chiral analysis of reticuline accumulated in the growth media of strain CSY1071 with empty vector or DRS-DRR (pCS3301) was performed as described in Fig. 1C. This chromatogram is one of two similar traces from replicate yeast cultures. The chromatogram was smoothed using a 7-point boxcar moving average.

Fig. 3. Engineered SalSyn chimeras improve conversion of (R)-reticuline to salutaridine

(A) Schematic of the chimeric SalSyn engineering strategy to address incorrect processing and glycosylation of the wild-type SalSyn in yeast. Orange diamonds represent glycosylation. (B) Comparison of salutaridine produced from SalSyn variants, site-directed glycosylation mutants, and engineered fusions in yeast. Yeast strains expressing the indicated SalSyn variant were fed 10 µM (R)-reticuline, and the growth media was analyzed by LC-MS/MS MRM. Peak areas were normalized to wild-type SalSyn (black). (C) Comparison of thebaine produced from SalSyn variants in yeast. Yeast strains were fed 1 mM rac-norlaudanosoline, and thebaine in the growth media was quantified by LC-MS/MS MRM with an external standard curve. Bars outlined in black denote wild-type and best engineered variant. Error bars are standard deviation of at least three biological replicates.

Fig. 4. Complete biosynthesis of the opiate thebaine and semi-synthetic opioid drug hydrocodone in yeast

(A) Chromatograms of thebaine detected in CSY1064 media and 7.8 µg/L (25 nM) thebaine standard. (B) Spectra of eight multiple reaction monitoring (MRM) transitions of thebaine produced by engineered yeast and standard. (C) Chromatograms of hydrocodone detected in CSY1064+pCS2765 media and 0.3 µg/L (1 nM) hydrocodone standard. (D) Spectra of four MRM transitions of hydrocodone produced by engineered yeast and standard. Growth media was analyzed for opioids by LC-MS/MS MRM. Traces are representative of four biological replicates.