A microbial biomanufacturing platform for natural and semisynthetic opioids

Abstract

Opiates and related molecules are medically essential, but their production via field cultivation of opium poppy Papaver somniferum leads to supply inefficiencies and insecurity. As an alternative production strategy, we developed baker's yeast Saccharomyces cerevisiae as a microbial host for the transformation of opiates. Yeast strains engineered to express heterologous genes from P. somniferum and bacterium Pseudomonas putida M10 convert thebaine to codeine, morphine, hydromorphone, hydrocodone and oxycodone. We discovered a new biosynthetic branch to neopine and neomorphine, which diverted pathway flux from morphine and other target products. We optimized strain titer and specificity by titrating gene copy number, enhancing cosubstrate supply, applying a spatial engineering strategy and performing high-density fermentation, which resulted in total opioid titers up to 131 mg/l. This work is an important step toward total biosynthesis of valuable benzylisoquinoline alkaloid drug molecules and demonstrates the potential for developing a sustainable and secure yeast biomanufacturing platform for opioids.

Figure 1. Engineering a heterologous morphine biosynthesis pathway in yeast

(a) The schematic depicts observed transformations of thebaine by the morphine biosynthesis enzymes - thebaine 6-O-demethylase (T6ODM), codeine O-demethylase (CODM), and codeinone reductase (COR) from opium poppy P. somniferum. Two routes to morphine which pass through intermediates codeinone and codeine (route i) and oripavine and morphinone (route ii) occur in opium poppy. Our work demonstrates that route (i) and a newly-identified route to neomorphine (iii) occur in the heterologous context of a yeast cell, revealing a broader substrate range for COR and CODM than previously reported. (b) LC-MS analysis of opiates produced from an engineered yeast strain (CSY907) expressing T6ODM, COR, and CODM from a pYES1L vector. Cells were cultured with 1 mM thebaine for 96 h in deep-well plates prior to analysis of the culture medium. Labeled peaks corresponding to opiate molecules are shown on the extracted ion chromatograms as: 8, codeinone (m/z 298, RT=4.5 min); 2, codeine (m/z 300, RT=4.0 min); 11, neopine (m/z 300, RT=3.2 min); 1, morphine (m/z 286, RT=1.4 min); 12, neomorphine (m/z 286, RT=0.8 min). The identities of opiates detected in the culture medium were confirmed by comparison to the MS2 spectra and retention times of purchased standards where available, or by NMR analysis (Supplementary Figs. 1-3, Supplementary Table 2). Empty vector control strains did not perform transformations of thebaine to other opiates (Supplementary Fig. 1).

Figure 2. Altering gene copy number and localizing COR1.3 to the endoplasmic reticulum increases pathway specificity for morphine

(a) Titers of the target product morphine (black bars) and non-target neomorphine (grey bars) were analyzed from strains harboring different numbers of copies of T6ODM, COR1.3, and CODM. The culture medium was analyzed by LC-MS for opiate production after 96 h growth in deep-well plates with 1 mM thebaine. Each strain expressed one copy of T6ODM, COR1.3, and CODM (Supplementary Table 1), on a pYES1L vector (Supplementary Fig. 8). Additional gene copies were integrated into the host cell genome at the ura3, his3 and leu2 loci (Supplementary Table 3). (b) A spatial engineering approach was applied to delocalize COR1.3 from T6ODM activity. A COR1.3-GFP fusion protein was observed to localize to the yeast cytoplasm, similar to T6ODM and CODM (Supplementary Fig. 6). An endoplasmic reticulum localization tag (ER1) was fused to the C-termini of GFP (GFP-ER1) and a GFP-COR1.3 fusion protein (GFP-COR1.3-ER1). Confocal microscopy confirmed ER-localization of these ER1-tagged proteins. Cytoplasmic, untagged COR1.3 and ER-localized COR1.3-ER1 were each expressed together with T6ODM and CODM from a pYES1L vector. Strains were cultured in optimized media with 1 mM thebaine, grown for 96 h, and the culture medium analyzed for morphine (black bars) and neomorphine (grey bars) by LC-MS. Bars represent mean values ±1 s.d. of three biological replicates. The percentage of morphine (out of the sum total of morphine and neomorphine) is displayed above each bar. Image scale bars, 4 μm.

Figure 3. Incorporating bacterial enzymes allows for the biological synthesis of semi-synthetic opioids

Schematic depicting the extended transformations of thebaine in yeast by incorporating morA, morphine dehydrogenase, and morB, morphine reductase, from Pseudomonas putida M10 into the heterologous pathway.

Figure 4. Optimized yeast strains for the production of diverse opioids

(a) Total opioid molecule concentration in the culture medium after closed-batch fermentation. Yeast strains CSY950, CSY951, and CSY952 (Supplementary Table 3) were optimized for the production of morphine, hydromorphone, and hydrocodone/oxycodone, respectively. The indicated strains were cultured in closed batch fermentations in media supplemented with 1 mM thebaine (equivalent to 311 mg/L). Culture medium was analyzed at the end of the fermentation for a panel of opioids through LC-MS. (b) Cell density and concentrations of key opioids (hydrocodone, dihydrocodeine, and oxycodone) as a function of time for the fermentation of yeast strain CSY952. At indicated time points, samples were taken, diluted, and analyzed for cell density through spectrometry and opioid production through LC-MS.