March 18, 2015
Microbiologists in the lab spend a lot of time and effort keeping each microbial strain and species separate. The conventional wisdom is that if you want to really understand an organism, you need to study it in isolation, as a pure culture. If contaminating colonies of some other bug appear on your Petri dishes, you’d better melt down those plates in the autoclave and trash them!
On the other hand, amateur microbiologists have known for centuries that mixtures of microbes can do great things. Sourdough bread, for example, is made using a culture of Lactobacilli and yeasts. They complement each other during the fermentation: the bacteria metabolize sugars that the yeast can’t use, and make new compounds that can be fermented by the yeast. The result is extremely tasty.
A new study from Zhou and colleagues brings a microbial community into the lab to make a medicine called paclitaxel that is used to treat cancer. Yes, bread bowls for your clam chowder are cool, but this is obviously way more important for human health.
Paclitaxel is an incredibly successful drug for treating breast and ovarian cancer, but unfortunately there isn’t an easy way to make it. It can be purified from the bark of the Pacific yew (killing the trees in the process), synthesized by plant cells cultured in vitro, or synthesized chemically. But all of these processes are expensive and complicated, which means this life saving drug is always in short supply.
The researchers wanted to produce it more cheaply and easily, and an obvious solution was to let microbes do most of the work. But neither of the most commonly used microbial workhorses, S. cerevisiae and E. coli, was exactly right for the job.
E. coli had already been engineered to overproduce the compound taxadiene. The taxadiene then needs to be oxidized to create oxygenated taxanes, which are paclitaxel precursors. This oxidation can be done by membrane-bound oxidoreductase enzymes called cytochrome P450s. But these enzymes are not found naturally in bacteria, and getting them expressed and functional in E. coli is challenging.
The researchers decided to see whether they could coax these two microbes into cooperating to produce oxygenated taxanes. After creating an E. coli strain that produced taxadiene and an S. cerevisiae strain that produced a P450 oxidoreductase, they grew them together in the same culture, with glucose as the carbon source.
As planned, the E. coli pumped out taxadiene and it was able to diffuse into the yeast cells, where it became oxygenated. However, the two species weren’t as happy together as the researchers had hoped.
One of the things that humans love about yeast is that when it grows on glucose, it produces ethanol. However, the E. coli cells didn’t love being bathed in ethanol: their yield of taxadiene went way down as the ethanol levels in the culture went up.
So Zhou and coworkers switched the carbon source to xylose. S. cerevisiae cannot consume xylose, but E. coli can. When growing on xylose, E. coli produces acetate, which the yeast can use—and they don’t produce ethanol under these conditions.
Growing the microbes in xylose doubled the yield of oxygenated taxanes over that of the glucose-grown culture. But still, only 8% of the taxadiene that was produced was getting oxygenated.
To be sure that the yeast cells were producing the P450 enzyme as efficiently as possible, the researchers tried driving transcription of the gene using several different promoters. Using the promoter that was strongest in the co-culture conditions made a significant difference in the proportion of taxadiene that was oxygenated.
The researchers guessed that another factor limiting the final yield was that the yeast cells were not growing as well as they could. Tweaking the ratio of the two species in culture and the contents of the media resulted in a three-fold increase in oxygenated taxanes. But Zhou and colleagues hoped to improve things even more.
Thinking that the limiting step in yeast growth might be the supply of acetate, the researchers tried to beef up the acetate synthesis pathway in E. coli. They engineered the bacteria to overproduce several of the enzymes in the acetate biosynthesis pathway, but this didn’t make a large difference.
They reasoned that if they forced the E. coli to rely on the acetate biosynthesis pathway for energy, the cells might ramp up their acetate production. To do this they blocked oxidative phosphorylation by deleting the the atpFH gene that encodes a subunit of ATP synthase. Now more acetate was produced, the yeast cells grew better, and 75% of the taxadiene that was produced got oxygenated. They were in business!
Zhou and coworkers went on to show that the co-culture environment could be modified to generate several other isoprenoids. This class of naturally-occurring molecules includes some that are in use, or in development, as pharmaceuticals (paclitaxel, and others currently in clinical trials) and compounds that have other applications, from fragrances to fuels.
There’s much more work to be done, and the potential of microbial communities is just beginning to be realized. Harnessing the power of multiple organisms means that different steps of pathways can be optimized separately and then mixed and matched for a desired result. This approach could turn out to be the best thing since sliced bread! But then again, sourdough bakers already knew that.
by Maria Costanzo, Ph.D., Senior Biocurator, SGD
Categories: Research Spotlight