The recent award of the Nobel Prize for the anti-malaria drug artemisinin prompted us to interview Dr. Jay Keasling, whose work led to the industrial production of this important drug in yeast using synthetic biology.
Discovery of Artemisinin
The 2015 Nobel Prize in Physiology or Medicine has been awarded in part to a Chinese scientist, Dr. Youyou Tu, for development of the anti-malaria drug artemisinin. The award was notable in that it is the first Nobel awarded to a China-based scientist, and the recipient was a woman. Dr. Tu was tasked in the 1960s by the Chinese government to find new treatments that would overcome resistance to the quinine-based malaria drugs.
Her approach was to start with 2000 traditional Chinese remedies with potential anti-malarial activity. She eventually chose 380 for testing in mice. One extract from the plant Artemisia annua (commonly known as sweet wormwood) showed promise, but initial results were inconsistent. She took the unusual next step of reviewing a 1600-year-old Chinese text that provided clues that led to the isolation of the active anti-malaria ingredient artemisinin in 1972. The Nobel Prize was awarded not for the discovery of the anti-malarial properties of this plant, but for the use of sophisticated research methods to develop a new therapy from traditional Chinese medicine.
The significance of the development of this drug cannot be underestimated. Nearly half the world’s population is at risk of contracting malaria. In 2013, there were approximately 200 million malaria cases and more than 500,000 global malaria deaths. Pregnant women and children under the age of five are the most severely affected. Artemisinin-based treatments have played a major role in reducing the average infection prevalence in sub-Saharan African for children aged two to ten from 26% in 2000 to only 14% in 2013.
Stabilizing the Supply of Artemisinin Drug
While artemisinin has saved millions of lives that would have been taken by malaria, the fact that it is derived from a plant source can limit its availability. The World Health Organization (WHO) recommended artemisinin-based combination therapy as the therapeutic regimen for the treatment of malaria in 2001. When artemisinin went into large scale production as a result of this recommendation, demand for the plant material source outpaced supply, driving up the price. As farmers greatly increased production in response to higher prices, supplies of raw material exceeded drug production capacity and its price dropped more than 80%.
The lower value led to decreased cultivation, which then caused a shortage of plant material and a resulting drop in production of the drug. The consequence has been a two- to three-year cycle in which the availability of the main ingredient has fluctuated as much as 400%. As long as the drug is produced from plant-based material, availability will be subject to this fluctuation. A more stable supply of the raw material to produce artemisinin was needed.
Using Synthetic Biology to Make Artemisinin
Synthetic biology provided an alternative means to obtain artemisinin, using a yeast-based synthesis approach developed in the laboratory of Dr. Jay Keasling, Professor of Chemical Engineering and Bioengineering at the University of California, Berkeley. Twist recently interviewed Dr. Keasling to get his perspective on the biosynthetic journey to artemisinin, in light of Dr. Tu’s recent Nobel Prize.
Dr. Keasling’s team is focused on engineering production of chemicals in microbes by utilizing and modifying their metabolic pathways. In particular, his interest centers around isoprenoids, the largest family of natural chemicals found in virtually all organisms. They provide scents, flavors, and even colors in many plants, including the yellow in sunflowers and the red in tomatoes. Around 2001, one of Dr. Keasling’s graduate students found a paper on artemisinin, and they felt that they might be able to coax yeast into making it.
They received a $42 million grant from the Bill and Melinda Gates Foundation in 2004 to define the metabolic pathway in the sweet wormwood plant for synthesis of artemisinin, and to and engineer it into yeast. The resulting biosynthetic process for making the drug would then be licensed at no cost to a pharmaceutical company to produce it on a non-profit basis.
In order to synthesize artemisinin, a pathway of multiple enzymatic reactions needed to be constructed. The gene for the first enzyme had been identified, but it was not yet commercially available. The gene had to be made in the laboratory, starting from nucleotides and oligonucleotides, the building blocks of DNA. After a laborious process, the gene was synthesized and cloned into yeast where it produced the required enzyme.
Some surprises during the Keasling team’s efforts made the subsequent steps in the pathway to artemisinin easier to attain. Three enzymes were required in the sweet wormwood plant for the production of artemisinin. But when the first one was cloned into yeast, it replaced the functions of all three plant enzymes, greatly reducing the effort required.
In yeast, the final product of the pathway is artemisinic acid, which could be toxic if it accumulated in the microbe. Luckily, the yeast secreted the acid into the fermentation tank and continued to grow. In fact, so much artemisinic acid was produced that it crystallized in the tank, aiding the first step in purification of the material. The crystals could simply be collected for further processing.
Microbial Production of Artemisinin is a Reality
Once a functioning synthetic pathway for artemisinic acid was engineered into yeast, the next step was to optimize it for industrial production. The chemical reactions for conversion of the acid to artemisinin were optimized as well. These steps were accomplished by Amyris, a company founded by Dr. Keasling for this purpose. The complete process was then licensed to Sanofi for production on a non-profit basis.
In August of 2014, Sanofi distributed the first shipments of artemisinin made from yeast. As of May 2015, 15 million treatments have been made available to African nations severely challenged by malaria outbreaks. Sanofi has the capacity to produce 150 to 200 million treatments per year. According to Dr. Keasling, “within 10 years about one billion people should have received the microbially-produced drug.”
Advantages of Microbially-Sourced Artemisinin
The amount of artemisinin made by microbial means is currently low. Production is being ramped up carefully so as not to discourage farmers from planting, which could lead to rapid fluctuations in supply of the drug made from the natural product. However, the lower cost of artemisinin made by microbial means provides a very meaningful advantage.
As proved to be the case with HIV, the most effective treatments for malaria are combinations, or “cocktails” of drugs, in order to reduce the incidence of drug resistance that sometimes occur with single-drug approaches. As Dr. Keasling stresses, “the world cannot afford development of resistance to artemisinin by the malaria parasite”. In fact, WHO has recommended that only combinations of other drugs with artemisinin be used to treat malaria.
However, there are many manufacturers producing malaria drugs that contain only artemisinin, and some countries still allow this monotherapy approach. The fact that artemisinin can be produced by microbes at the lowest cost provides control over how drug manufacturers use it by allowing access only to those manufacturers who agree to use artemisinin strictly in multi-drug therapies. Market pressures will then encourage producers of monotherapy drugs to adopt a multi-drug approach.
Since microbial production of artemisinin can be ramped up in weeks rather than years, its biggest advantage is improved access. This is achieved by eliminating long lead times for artemisinin produced from plants, as well as avoiding production shortages caused by wild swings in price.
“If drug of very high quality and efficacy was accessible by everyone who needed it, it would not matter if it was made by yeast or from a natural product”
Commitment was the Key to Success
Dr. Keasling credits the dedication of many highly-qualified members of his team and the companies involved for the success of the effort to produce artemisinin in yeast. This was the case even though no one involved with the project stood to profit from it, including the university, Amyris or Sanofi. Speaking for his team, Dr. Keasling feels that the knowledge that their efforts could save lives provided extra incentive.
When asked if he felt that artemisinin will eventually be made entirely by microbial means, he responded that that what was important was universal access to high quality drug. “If drug of very high quality and efficacy was accessible by everyone who needed it, it would not matter if it was made by yeast or from a natural product”. As for Dr. Keasling himself, he feels that the greatest reward from this project is “children’s lives saved, and adult lives made better because of it”.
What does the Future Hold?
Synthetic biology holds great promise for the production of everything from life-saving drugs to fuels, flavors, fragrances and chemicals currently produced from oil or scarce natural products—as well as a myriad of other applications. Dr. Keasling says, “This is just the start of what will be possible through engineered biology, and the kind of work that Twist Bioscience is doing to make DNA (genes) inexpensive and widely available is key to this future.” Dr. Keasling sees his mission as “making sure that we have the all the science we need to do all that we need to do to make the planet better” with synthetic biology.
