December 16, 2016
Some people get the jitters from a single espresso while others need a triple shot just to get started in the morning. Some of this is due to caffeine tolerance—a buildup of resistance to the marvelous effects of that wonderfully addictive substance, caffeine. But the rest has to do with genetic differences that affect how well each of us processes caffeine—our caffeine sensitivity.
Our best buddy Saccharomyces cerevisiae is a real wimp when it comes to caffeine. In fact, like a lot of other microorganisms, caffeine actually kills this yeast. S. cerevisiae is indeed a sensitive soul when it comes to caffeine.
In a new study in the Journal of Agricultural and Food Chemistry, Wang and coworkers were able to toughen up budding yeast against caffeine by adding bfr1, a gene from Schizosaccharomyces pombe that encodes the ABC transporter that shunts caffeine out of the cell. And then, using random mutagenesis, they were able to make bfr1 even better at its caffeine-exporting job. Although the yeast don’t get any of the pleasurable effects of caffeine, at least they can now happily grow in cultures that have more caffeine than a strong cup of coffee.
This new attribute could prove to be incredibly useful if caffeine producers ever want to start making caffeine biologically instead of synthetically. You can imagine adding the caffeine pathway from coffee to yeast and having the yeast merrily exporting caffeine to the culture medium where it can be harvested. And who knows, maybe they can have the yeast make caffeine and alcohol at the same time creating the equivalent of a vodka and Red Bull in a single step!
Previous research had shown that bfr1 was an important player in helping S. pombe deal with caffeine. When Wang and coworkers added the gene to S. cerevisiae, this newly engineered yeast could now better tolerate caffeine. For example, whereas wild type yeast barely grew with 8 mg/ml caffeine, the engineered yeast did OK.
These authors next turned to random mutagenesis of the bfr1 gene to screen for mutants that could tolerate even more caffeine. And boy did they win the lottery on this one! A mutant that they named bfr1-B did great even at concentrations of 25 mg/ml caffeine. Now they were getting somewhere.
Bfr1 doesn’t just export caffeine; it actually exports many different compounds. The authors found that bfr1-B was fairly specific for increased resistance to caffeine. For example, when they tested the bfr1-B mutant with theophylline, a structurally similar compound, and atropine, a structurally distinct compound, they found that S. cerevisiae expressing the mutant were, if anything, more sensitive to these compounds. They found what looked like a caffeine-specific mutant.
When they looked at the mutant, Wang and coworkers found that there were 11 amino acid substitutions scattered across the protein. The next step was to figure out which ones mattered and which ones didn’t.
Using a bit of modeling with the 3-D structure of other ABC transporters, they settled on testing three mutations individually. Two of the mutations, S36 and D340 were in the nucleotide binding domain (NBD) and the third, Y497, was in the transmembrane domain (TMD). The NBD is where ATP binds to the transporter to supply the energy to move caffeine across the membrane.
Of the three, only D340 in the nucleotide binding domain conferred caffeine resistance. While not as robust as bfr1-B, this mutant allowed yeast expressing it to tolerate caffeine concentrations up to 15 mg/ml, conditions under which cells with wild type bfr1 failed to grow.
So while this mutation explains a lot of why bfr1-B is so good at dealing with caffeine, it is not the whole story. At least some of those other 10 mutations contribute to how well bfr1-B does with caffeine.
In the end we have a bullet-proof yeast when it comes to caffeine that should prove useful for anyone who wants yeast to synthesize caffeine for them. Of course unlike even the most grizzled 30 year coffee drinker with ideal genetics, the yeast almost certainly gets no joy from its morning Joe. But at least that cup of coffee won’t kill it!
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight