Natural products are the principal source of and inspiration for pharmaceuticals. Because of their complex regio- and stereo- chemistry, many natural products are most economically made by cultivating the producer. Plant natural products from opium poppy, which include morphine and codeine, are produced in this way. Agricultural supply of drugs and drug precursors from opium poppy is an annual process with a large demand on land, subject to high variability due to weather, pests, soil conditions, and natural variation, and requiring intensive mechanical and chemical processing for product isolation. Therefore, there is a need for alternative processes to provide opiates with greater supply stability and less resource use. Heterologous microbial biosynthesis has been used successfully to make plant natural products, most notably the sesquiterpene artemisinic acid, and could provide a more stable supply of opiates due to greater process control and shorter batch times. The objective of this research was to design and build yeast strains to biosynthesize plant natural products found in opium poppy, including morphinan alkaloids, using a synthetic biology framework. One challenge to implementing these pathways is the decrease in titers observed with increasing pathway length, which must be tackled with strain engineering efforts. Another challenge was that a key enzyme in morphine biosynthesis was not yet discovered.<br/>We developed yeast strains to convert either sugar or a commercially available substrate to morphinan alkaloids, which required the discovery of a novel epimerase from opium poppy. We co-discovered this missing enzyme and demonstrated its activity in the context of our engineered yeast strain. Rational protein engineering of a plant cytochrome P450 enzyme increased product titers. The engineered yeast strains are able to biosynthesize the morphinan alkaloid thebaine and the opiate- derived semi-synthetic drug hydrocodone <i>de novo </i>at proof-of-principle levels. Further protein and strain engineering will lead to improved strains for microbial biosynthesis of opium poppy alkaloids and their congeners.

Complete biosynthesis of opioids in yeast

251st National Meeting (2016)

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