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. A new biosynthetic branch to neopine and neomorphine was discovered, which diverted pathway flux from morphine and other target products. Strain titer and specificity was optimized by titrating gene copy number, enhancing cosubstrate supply, applying a spatial engineering strategy, and performing high-density fermentation, resulting 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.

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…Therefore, feeding monosodium glutamate (MSG) and 2-oxoglutarate was sought. [273] By cosubstrate titration, the morphine titer was increased up to tenfold. [272] For both MIA and BIA biosynthesis, BH 4 redox cofactor is required for the activity of TyrH-a mammalian tyrosine hydroxylase with higher substrate and product specificity.…”

“…This tag allowed COR1.3 to be localized in the endoplasmic reticulum, allowing the specificity to rocket as high as 86% while sacrificing the morphine titer. [273] Other than the spatial organization, simply changing sources of an enzyme can significantly reduce the promiscuity of the enzyme. For the production of S-reticuline, activity of tyrosinase is crucial since it converts L-tyrosine to L-DOPA.…”

“…In order to increase the morphine production, Thodey et al manipulated the ratio of T6ODM, COR, and CODM, the three key enzymes that lead the conversion from thebaine to morphine. [273] The gene copy numbers of these promiscuous enzymes were varied, resulting in the optimal gene copy numbers of 2:1:3; from this optimal gene expression ratio, 5.2 mg L −1 of morphine and 4.8 mg L −1 of neomorphine were produced. [273] It is interesting to note that the high CODM level biases the pathway toward morphine since this enzyme has a higher specificity to codeine.…”

“…In this piece of work, the authors also produced the semi-synthetic morphinan alkaloids including hydromorphone, hydrocodone, and oxycodone by expressing morA and morB isolated from P. putida M10. [273] Another group also attempted morphinan alkaloids production via S. cerevisiae. For the conversion of R-reticuline to salutaridine, heterologous salutaridine synthase (SalSyn) and CPR were expressed.…”

“…[266,270a] As for the production of morphinan alkaloids, since their whole biosynthetic pathway from simple carbon sources had not been identified until recently, [272] conversion from BIA intermediates has been sought. Thodey et al managed to produce both natural and semi-synthetic morphinan alkaloids by feeding thebaine, [273] which is the feedstock for chemical synthesis of opioids. [274] Three enzymes responsible for this conversion-2-oxoglutarate/Fe +2 -dependent dioxygenases (thebaine 6-O-demethylase, T6ODM, and codeine O-demethylase, CODM) and NADPH-dependent aldo-keto reductase (codeinone reductase, COR)-were cloned from Papaver somniferum for morphine production.…”