Taxol is a widely used chemotherapy drug – it has been used to treat millions of ovarian, breast, and lung cancer patients. Today, it's mainly produced by extracting its chemical precursor, baccatin III, from yew trees. But, yew trees grow slowly, so the amount of medicine produced per tree pales in comparison to its demand.
Taxol is large and complex, making it expensive to manufacture synthetically. That's why scientists since the 1990s have sought to identify the enzymes trees employ to make Taxol, which can then be inserted into organisms such as industrious yeasts that can churn out the drug.
"We really need enzymes to build this molecule," said Conor McClune , a postdoctoral scholar in chemical engineering. "Enzymes are often the most efficient and cleanest way of doing a chemical reaction."
Now, McClune and colleagues have unlocked a new means of peering at plant genes. The effort revealed several key enzymes for creating Taxol, also known as paclitaxel. The findings bring researchers much closer to the goal of producing the drug efficiently using industrial microbes, the team reported in the journal Nature on June 11. "Taxol has been the holy grail of biosynthesis in the plant natural products world," said the study's senior author, Elizabeth Sattely , an associate professor of chemical engineering. "Being able to use a bioproduction strategy to manufacture a molecule like Taxol is a really exciting prospect."
Mysterious tree chemistry
Scientists have strained to peek into the yew's laboratory. Compared to a bacterium like E. coli – whose chromosome carries about 4,000 genes – the yew tree genome is massive, about 50,000 genes. Narrowing down which is responsible for making Taxol has proven difficult. Prior to the study, 12 genes had been identified, but the goal of producing Taxol or baccatin III was still out of reach.
To speed up the search, the Stanford team developed a method of filtering the thousands of enzymes for just those needed to make the medicine, inspired by the work of co-author Polly Fordyce , an associate professor of bioengineering and of genetics. They snipped needles off of yew trees and plopped them into plates with wells of water and fertilizer. Then, they intentionally stressed out their samples, adding hormones and microbes that induced the needles to produce defensive compounds – including Taxol.
The researchers ground up the needles and pulled out about 10,000 nuclei from their cells. They sequenced the nuclei and counted their messenger RNA. This allowed the scientists to see which genes were switched on from the stressors – the more RNA, the more of a particular gene is being transcribed and made into proteins.
In this way, the team could see which genes flickered on together, indicating they might be partnering to produce proteins. Starting with the 12 genes already identified in Taxol production, the scientists searched for genes that this initial bunch might work with. They made lists of promising genes, and then inserted those candidates into tobacco plants to see if they furthered the chemical reaction that outputs Taxol.
Inserting enzyme recipes into industrial microbes
The experiment yielded eight new genes critical for making the drug. One, called FoTO1, plays an especially important role in streamlining and channeling the reaction. The newly identified enzymes were the missing puzzle pieces needed to produce baccatin III. In fact, the tobacco plants produced baccatin III at a concentration higher than found in yew trees. "Theoretically, with a little more tinkering, we could really make a lot of this and no longer need the yew at all to get baccatin," said McClune, who is a co-lead author of the paper.
The team also identified an enzyme catalyzing one of the chemical steps between baccatin and Taxol, which helped push the pathway even further beyond baccatin – leaving only two final steps missing to Taxol. Coincidentally, in April, scientists at the University of Copenhagen identified those two final enzyme puzzle pieces that move the reaction from baccatin III to Taxol. Put together, the 22 genes now uncovered may represent the yew's chemical recipe. "We now have the full set of genes that would allow us to synthesize Taxol from scratch," said McClune.
In the near future, the researchers plan to verify in tobacco plants whether these final two enzymes work with the other 20 genes to complete Taxol synthesis. If the recipe is indeed complete, the genes encoding these enzymes can be inserted into a microbe. Strains of yeast could be engineered into "extremely efficient chemical factories" producing the drug at commercial scale, said McClune.
More broadly, this new method for testing thousands of cell nuclei may enable further discoveries in plant chemistry. Yew trees are not the only enigmatic plant chemists. McClune and colleagues are now studying the genomes of common crops. These vegetables are "full of enzymes that are doing interesting chemistry," said McClune, "but we just don't know what they're up to."
Sattely is also a Howard Hughes Medical Institute investigator, a member of Stanford Bio-X , and a faculty fellow at Sarafan ChEM-H . Fordyce is also a member of Stanford Bio-X, a member of SPARK at Stanford , and an institute scholar at Sarafan ChEM-H. PhD student Jack Chun-Ting Liu is a co-lead author of the article; other Stanford co-authors include PhD student Chloe Wick and former PhD student Ricardo De La Peña (now at biotech startup Amyris). Bernd Markus Lange, associate professor at Washington State University, is also a co-author.
The research received funding from the Howard Hughes Medical Institute, the National Institutes of Health and the Damon Runyon Cancer Research Foundation.
