New & Noteworthy
Yeast as a Painkiller Factory
September 04, 2014
Imagine you were designing a factory to make a very special product. You’d study the process carefully, buy the right equipment, and bring in the right people.
To make a tricky product you need to have the right factory, workers and machinery. And if you’re making opiate drugs, then yeast makes a great factory! Image from Wikimedia Commons
So if one step made a lot of dust, while another step had to be dust-free – you’d be sure to separate them into different rooms of your factory. And you’d make sure that the instructions were written in a language that your experts could understand!
In a new paper in Nature Chemical Biology, Thodey and coworkers designed a factory in just this way to make some very important molecules: the opiate drugs that millions of people rely on every day to control pain. Because of this new factory, opium poppies won’t be needed for making these drugs (although they’ll still be very pretty!). The factory’s location: inside cells of our favorite yeast, S. cerevisiae.
The researchers first tried to coax the yeast to produce the natural opiates morphine and codeine. They recruited experts in the field (or, from the field), taking three opium poppy genes for enzymes in the opiate synthesis pathway: thebaine 6-O-demethylase (T6ODM), codeine O-demethylase (CODM), and codeinone reductase (COR).
Of course, simply transforming yeast with a plant gene doesn’t do much good. Yeast and poppies don’t speak the same language at the transcription level (and even their translation dialects are hard to understand). So the researchers put the poppy genes under the control of efficient yeast transcriptional regulatory sequences such as promoters and terminators, and optimized their codons for yeast.
Thodey and colleagues tweaked the system to try to steer it in the direction of the products they wanted. They fed the yeast additional monosodium glutamate and glutamine to increase intracellular levels of 2-oxoglutarate, which is required during catalysis by the T6ODM and CODM enzymes. They also varied the relative expression levels of the three poppy enzymes by varying the copy numbers of their genes in yeast.
Although these tweaks improved things, almost half the product was still the undesirable neomorphine. To address this, the researchers looked even more closely at the details of the pathway.
When morphine synthesis is going right, the neopinone made by T6ODM spontaneously rearranges to the codeinone that COR uses to continue along the pathway. But if COR grabs the neopinone before there is time for the rearrangement, the end result of the pathway is neomorphine, which does no one any good.
When you design a factory, it’s important that your assembly line doesn’t move too fast! In the yeast factory, when neopinone gets to the COR enzyme too quickly, the end result is not what you want – although maybe not this messy.
Going back to their blueprint, Thodey and colleagues decided to separate T6ODM and COR into different parts of the factory, to allow more time for this rearrangement. They added a tag to COR that would direct it to the endoplasmic reticulum membrane, while T6ODM stayed in the cytoplasm. Now it would take longer for neopinone to reach COR, giving it plenty of time to rearrange into codeinone. Sure enough, morphine production went way up.
This was great, but the researchers decided to take it a step further. Semisynthetic opioids such as hydrocodone, oxycodone, and hydromorphone are medically useful because they work better in some cases than the natural opiates. Currently, these are produced by chemical modification of the opiates produced by poppies. Could yeast do this job too? Of course!
Turning to different expert workers, Thodey and colleagues tried expressing the enzymes NADP+-dependent morphine dehydrogenase (morA) and NADH-dependent morphinone reductase (morB) from the bacterium Pseudomonas putida* along with the poppy enzymes. Again, the process needed a lot of tweaking, more than we can describe here. But the end result was a strain that produced both hydrocodone and oxycodone.
Putting together all their results, the researchers were able to construct three yeast strains, each like an assembly line tailored for different products. One assembly line is optimized for codeine and morphine, another for hydromorphone, and one for hydrocodone and oxycodone.
The next steps will be to scale up this process to industrial levels, and also to construct yeast strains that carry out the entire process starting from simple sugars, rather than needing to be fed the precursor thebaine. Substituting yeast cultures for opium poppy fields will have a huge global impact that goes far beyond pharmaceutical production.
It’s important to note that this factory could never have been constructed without knowing how to make its fundamental building blocks. Basic research in yeast molecular biology and genetics, which may seem arcane to some, was essential to provide the knowledge necessary to express and manipulate these foreign genes in yeast. Just another reason that we’re “high” on yeast research!
* Read more about Pseudomonas putida, a bacterial workhorse with an appetite for all kinds of weird substances.
by Maria Costanzo, Ph.D., Senior Biocurator, SGD
Categories: Research Spotlight
Tags: opiate biosynthesis , pathway engineering , Saccharomyces cerevisiae