The opium poppy plant Papaver somniferum is currently used as the sole commercial source of the most powerful therapeutic, prescribed opiate pain killers. There has been a long time interest in using synthetic biology to produce opiates as bioproducts, with the goal of controlling the drug, and hopefully producing cheaper, less addictive and more secure medicines. The family of molecules known as benzylisoquinolone alkaloids (BIA) include many plant products derived from tyrosine, such as pain killers, antimicrobials, anti-cancer drugs muscle relaxants and cough suppressants.

Within the first six months of 2015, three reports have described a complete biosynthetic pathway using yeast to convert glucose into morphine1,2,3. Although there have been numerous previous advances in the last several years, it is now possible to engineer the entire morphine biosynthesis pathway in yeast in order to brew morphine instead of beer. While most enzymes in the pathway were derived from the poppy plant, there was also the use of other plant and bacterial enzymes. The ability to combine these enzymes in a single engineered strain still requires significantly more work, but it does bring to the forefront numerous ethical issues. The most intriguing issue revolves around how to prevent these engineered strains from being obtained or possibly reproduced by the drug cartels. Although this area of research has generated gossip of another season of the television series Breaking Bad, featuring a chemistry teacher turned methamphetamine cook and dealer, we will focus here on the synthetic biology behind these discoveries.

Glucose to (S)-Reticuline

During normal metabolism and growth of yeast on glucose, the amino acid tyrosine is naturally produced during the process of amino acid synthesis. The researchers first tackled a long standing problem in yeast, converting tyrosine to L-DOPA1. A plant enzyme was introduced that converts L-DOPA to a fluorescent pigment, whose production can be easily monitored in a biosensor assay looking for pigmented colonies, or using fluorescence to detect yellow yeast cells. It was initially expected that a high throughput screen of cDNA libraries would be necessary, but a bioinformatic analysis revealed two candidate enzymes, one from the button mushroom and another from the sugar beet, that might convert tyrosine to L-DOPA. Codon optimized versions of these enzymes were introduced and yeast strains expressing the sugar beet enzyme were shown to produce L-DOPA, which was converted to the yellow pigment. Further enzyme engineering was carried out using error-prone PCR to generate a library of mutant enzymes, which were successfully screened for variants that had improved yields of L-DOPA1.

The next step was to produce (S)-Reticuline, a central precursor in the biosynthesis of opiates. Using a combination of one bacterial and several poppy enzymes, most of which were previously identified and many shown to function in yeast, (S)-Reticuline was produced. This required the introduction of many non-yeast genes, expressed from strong promoters either from the chromosome or low-copy plasmids, and the use of unique yeast background strains that produced higher than normal amounts of key metabolites required for (S)-reticuline synthesis.

(R)-Reticuline to Morphine

Using knowledge of the morphine biosynthesis pathways in the poppy, a yeast strain was engineered to express 7 poppy plant enzymes, which led to the production of the critical intermediate thebaine, and ultimately the final products, codeine and morphine2. Cultures were supplemented with (R)-reticuline, because at this time the enzyme that converted (S) to (R)-reticuline had not been described. This kind of pathway engineering can also be tweaked to produce other important final products derived from thebaine, including opiod antagonists, which are used to treat opiate addiction. Similarly, thebaine is also a starting point for the chemical synthesis of oxycodone, which has a better side-effects profile, compared to morphine.

The most recent paper identified the poppy enzyme responsible for the S-to-R epimerization of reticuline3. With this finding, all enzymes required for morphine biosynthesis were identified. The pathway contains fifteen enzymatic steps, compared to a previous synthetic biology accomplishment of expressing five enzymes needed for the synthetic production of the antimalarial compound, artemisin.

Final thoughts on brewing morphine.

While the current morphine production from the poppy plant does meet demand, synthetic biologists have made great progress in synthesizing this compound in both bacteria in yeast. In comparison, bacteria still make higher yields of important intermediates like L-DOPA, when compared to yeasts, but the overall yield of opiates are still very low. However, the stage is set to produce a single yeast strain with all necessary enzymes and to optimize the production of high morphine yields from yeast fermentation reactions.

Another major advance in the field of synthetic biology is the ability to synthesize custom stretches of DNA. The relative low cost to order “DNA to go” may lead to abuses from individuals with sinister motives in attempting to create bioweapons, and possibly to attempt to construct their own morphine brewing microbial strains. DNA synthesis companies do regular screening of the sequences they produce, and it has been suggested to now search these orders for enzymes in the morphine synthesis pathway.

References

1. An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose. DeLoache WC, Russ ZN, Narcross L, Gonzales AM, Martin VJ, Dueber JE. Nat Chem Biol. 2015 Jul;11(7):465-71. doi: 10.1038/nchembio.1816. Epub 2015 May 18.

2. Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae. Fossati E, Narcross L, Ekins A, Falgueyret JP, Martin VJ. PLoS One. 2015 Apr 23;10(4):e0124459. doi: 10.1371/journal.pone.0124459.

3. Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy. Farrow SC, Hagel JM, Beaudoin GA, Burns DC, Facchini PJ. Nat Chem Biol. 2015 Jul 1. doi: 10.1038/nchembio.1879

Canadian Synthetic Biology Researchers

Vincent Martin, Professor, Concordia University

Peter Facchini, Professor, University of Calgary