Stanford bioengineers close to brewing opioid painkillers

A decade-long effort in genetic engineering is close to re-programming yeast cells to make palliative medicines
August 27, 2014

Stanford Bioengineer Christina Smolke has been on a decade-long quest to genetically alter yeast so they can “brew” opioid medicines in stainless steel vats, eliminating the need to raise poppies and then industrially refine derivatives of opium into pain pills (credit: Rachel Sakai)

Stanford bioengineers have hacked the DNA of yeast, reprograming these simple cells to make opioid-based medicines* via a sophisticated extension of the basic brewing process that makes beer.

Led by Associate Professor of Bioengineering Christina Smolke, the Stanford team has already spent a decade genetically engineering yeast cells to reproduce the biochemistry of poppies, with the ultimate goal of producing opium-based medicines, from start to finish, in fermentation vats.

“We are now very close to replicating the entire opioid production process in a way that eliminates the need to grow poppies, allowing us to reliably manufacture essential medicines while mitigating the potential for diversion to illegal use,” said Smolke, who outlines her work in the August 24 edition of Nature Chemical Biology.

Smolke added five genes from two different organisms to yeast cells. Three of these genes came from the poppy itself, and the others from a bacterium that lives on poppy plant stalks.

The final missing link

This replicates two, now-separate processes: how nature produces opium in poppies, and how pharmacologists use chemical processes to further refine opium derivatives into modern opioid drugs such as hydrocodone**.

In her new paper, Smolke started with thebaine obtained from poppies, put this into her bioengineered yeast and got refined opioids at the end of the process.

Bioengineer Christina Smolke: reprogramming nature to create essential drugs

Now her team must extend the 2008 process from sugar to thebaine. Once she forges this missing link in the chain of biochemical synthesis, she will have produced a bioengineered yeast that can perform all 17 steps from sugar to specific opioid drugs in a single vat.

“We are already working on this,” she said.

Smolke said it could take several more years to perfect the last steps in the lab and scale up the process to produce large-sized batches of bioengineered opioids that are pharmacologically identical to today’s drugs that start in a field and are refined in factories.

“This will allow us to create a reliable supply of these essential medicines in a way that doesn’t depend on years leading up to good or bad crop yields,” Smolke said. “We’ll have more sustainable, cost-effective, and secure production methods for these important drugs.”

* Morphine is one of three principal pain killers derived from opium. As a class they are called opiates. The other two important opiates are codeine, which has been used as a cough remedy, and thebaine, which is further refined by chemical processes to create higher-value therapeutics such as oxycodone and hydrocodone, better known by brand names such as OxyContin and Vicodin, respectively.

Today, legal poppy farming is restricted to a few countries–including Australia, France, Hungary, India, Spain and Turkey — supervised by the International Narcotics Control Board, which seeks to prevent opiates like morphine, for instance, from being refined into illegal heroin.

** The thrust of Smolke’s work for a decade has been to pack the entire production chain, from the fields of poppies, through all the subsequent steps of chemical refining, into yeast cells using the tools of bioengineering.

What Smolke’s team has now done is to carefully reprogram the yeast genome—the master instruction set that tells every organism how to live—to behave like a poppy when it comes to making opiates.

The process involved more than simply adding new genes into yeast. Opioid molecules are complex three-dimensional objects. In nature they are made in specific regions inside the poppy. Since yeast cells do not have these complex structures and tissues, the Stanford team had to recreate the equivalent of poppy-like “chemical neighborhoods” inside their bioengineered yeast cells.

It takes about 17 separate chemical steps to make the opioid compounds used in pills. Some of these steps occur naturally in poppies and the remaining via synthetic chemical processes in factories. Smolke’s team wanted all the steps to happen inside yeast cells within a single vat, including using yeast to carry out chemical processes that poppies never evolved to perform—such as refining opiates like thebaine into more valuable semi-synthetic opioids like oxycodone.

So Smolke programmed her bioengineered yeast to perform these final industrial steps as well. To do this she endowed the yeast with genes from a bacterium that feeds on dead poppy stalks. Since they wanted to produce several different opioids, the team hacked the yeast genome in slightly different ways to produce each of the slightly different opioid formulations, such as oxycodone or hydrocodone.

Abstract of Nature Chemical Biology paper

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.