New & Noteworthy

Yeast Reveals a Vicious Circle Between Sugar and Cancer

November 10, 2017


The housing crisis that started in 2006 is a classic example of a vicious circle. As prices went down, some people couldn’t afford their mortgages and so were foreclosed upon. These foreclosed houses entered the market and caused prices to drop further which caused more foreclosures and so on. Only a Herculean effort by the government and the Federal Reserve was able to break this cycle.

ForeclosedHouse

A vicious circle between foreclosed homes and home prices popped the housing bubble starting in 2006. A similar vicious circle between glucose fermentation and cancer cell aggressiveness makes for particularly nasty cancers. (Wikimedia Commons)

Vicious circles can happen in biology too.

In a new study in Nature Communications, Peeters and coworkers have used our friend Saccharomyces cerevisiae to uncover one of these in cancer cells. And it turns out that this yeast is the perfect model organism for this study. (Funny how often that is true…)

Both S. cerevisiae and cancer tend to utilize their sugar through fermentation instead of respiration. This process in yeast is obviously great news for beer and wine drinkers. However, it is not such great news for people with cancer.

In the Warburg effect (named after the scientist who discovered it), the more aggressive a cancer is, the more sugar it ferments. This new study suggests that the consumption of sugar via fermentation and the aggressiveness of the cancer are related in a vicious circle.

Fermentation creates the byproduct fructose-1,6-bisphosphate (Fru1,6bisP) which, according to this study, activates the oncogene Ras which causes the cell to grow faster. As it grows faster, it ferments sugar faster creating more Fru1,6bisP which activates more Ras and so on. The cancer cells grow out of control until doctors apply some Herculean treatment to put a stop to it.

While yeast and cancer cells have a lot in common, wild type yeast isn’t quite up to cancer’s standards when it comes to cancer’s unbridled intake of glucose into the glycolysis pathway. These authors turned yeast a bit more cancer-like in this regard by deleting the TPS1 gene. Tps1p is a yeast hexokinase that tightly regulates yeast’s glucose intake into glycolysis.

Yeast cells without TPS1 deal poorly with glucose and need to be grown in galactose. When Peeters and coworkers added glucose to tps1Δ cells that had been previously grown in galactose, the cells activated Ras, a potent oncogene. This activation caused the cell to undergo apoptosis as assayed by cytochrome c release from the mitochondria, exposure of phosphadityl serine on the plasma membrane and generation of reactive oxygen species.

Deletion of hexokinase 2 eliminated this activation of Ras and suppressed the apoptosis. This suggests that perhaps some buildup of an intermediate metabolite in the glycolytic pathway might be to blame for the Ras activation.

The authors tested this hypothesis by removing the cell wall of wild type yeast cells and adding glycolysis metabolites to the resulting spheroplasts. They found that one metabolite, Fru1,6bisP, activated Ras. Supporting this finding, they found that deletion of both PFK1 and PFK2, two genes that encode phosphofructokinase 1, the enzyme responsible for making Fru1,6bisP, eliminated Ras activation in tps1Δ cells. Together, these data support the idea that Fru1,6bisP is the culprit behind Ras activation in yeast.

coffee_cycle

Vicious circles like the one behind the Warburg effect are everywhere! (Twisted Doodles)

All well and good, but what about human cancer cells? Is something similar going on there? You have probably guessed that the answer is yes.

The authors first depleted the level of Fru1,6bisP in two different cell lines by starving them of glucose for 48 hours. They then added back glucose, which has been shown to increase the levels of Fru1,6bisP in cells, and measured the activation level of Ras and two of its downstream targets, MEK and ERK. All three were transiently activated in both HEK293T and Hela Kyoto cell lines. Looks like Fru1,6bisP activates Ras in cancer cells as well.

So this might explain the Warburg effect that I mentioned earlier. The more glucose a cancer cell uses during fermentation, the more Fru1,6bisP it generates. And the more Fru1,6bisP that is made, the more Ras gets activated prompting the cell to grow faster. Which of course results in more glucose getting fermented and so on.

I don’t have time to go into the work these authors did to try to uncover the mechanism by which Fru1,6bisP activates Ras, but suffice it to say that it does not appear to be a direct interaction with Ras. Instead, it appears to work at least partially by disrupting the interaction between Ras and one of its guanine nucleotide exchange factors, Cdc25p (or Sos1 in humans).

And the story is not yet complete. There is still a lot of work to do in pinning down the specifics of this vicious circle. Yeast will undoubtedly be instrumental in helping us work through the rest of the details.

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: cancer , Warburg Effect , PFK1 , PFK2 , TPS1 , fermentation

Creating an Ethanol-Making ‘Super Bowl’ Championship Team

May 24, 2017


New England v. Miami NFL

Tom Brady makes a team better by making the players around him play better. The same thing can be said for a mutant RPB7 gene that makes other genes work together better as an ethanol-making team. By Paul Keleher [CC BY 2.0 (http://creativecommons.org/licenses/by/2.0)%5D, via Wikimedia Commons.

There are a few ways to turn a failing sports team around. One is to tailor individual training to make each player better. Now, the team is better overall because of the changes each player makes.

Another way to improve a team is to change a player in a key position who makes everyone better. A classic example of this is the American football team, the New England Patriots.  

On September 23, 2001, Drew Bledsoe, then the starting star quarterback of the New England Patriots, took a savage hit from New York Jets linebacker Mo Lewis. The Patriots replaced Bledsoe with his backup, Tom Brady, and some might argue, the team (whom Brady led to their first Super Bowl win that year) and the NFL, has not been the same since.

Quarterback Tom Brady, along with head coach Bill Belichick, makes whomever the New England Patriots bring in better. Wide receivers, tight ends, and running backs can be replaced in the lineup without the team missing a beat. He just makes the players around him better than they might be on another team.

In a new study, Qiu and Jiang take a “Patriots” approach to ethanol production in the yeast Saccharomyces cerevisiae. Rather than improving individual genes on their own, these authors instead decided to “bring in” a new version of RPB7, a gene that encodes a key subunit of RNA polymerase II, the molecular machine responsible for making messenger RNA (mRNA).

They hoped that changing this pivotal transcriptional player would cause lots of other genes to do “better” so that “team” yeast would make a lot more ethanol.  Their hopes were realized in their Tom Brady equivalent—a mutant they called M1. Yeast bearing this mutant RPB7 gene became the Super Bowl champs of ethanol production.

One of the keys to increasing ethanol production in yeast is to find strains that are more tolerant of high levels of ethanol. The more ethanol they can withstand, the more they can make.

These authors used error prone PCR mutagenesis of the RPB7 gene to find their game-changing mutant. They then took their library of ~108 clones and cultured them in increasing amounts of ethanol, selecting for more ethanol-resistant strains.

After 3-5 rounds of subculture, they plated the cells onto media containing ethanol. Around 30 colonies were picked and sequenced with the best mutant being the one with two mutations—Y25N and A76T. They named this mutant M1.

This mutant grew a bit better than the parental strain background, S288C, in the absence of ethanol, but where M1 really shined was when ethanol was around. It grew around twice as fast in 8% ethanol and could grow at 10%, a concentration that completely inhibited the parental strain from growing.

Being able to withstand high levels of ethanol is important, but it isn’t all that yeast have to deal with. There are multiple other stressors around when you are swimming in 20 proof media.

For example, yeast can suffer from high levels of reactive oxygen species (ROS). M1 not only tolerated 3.5 mM hydrogen peroxide, a proxy for ROS, better than the parental strain, but it also had around 37% of ROS levels inside cells than that of the parental strain. M1 can deal with high levels of ethanol and ROS.

The authors then tested how this mutant dealt with other potential fermentation problems. For example, acetate, a fermentation byproduct, and high levels of NaCl both inhibit yeast growth. M1 tolerated 80 mM acetic acid and 1.5 M NaCl better than the parental strain did.

drunk Gingy

A couple of mutations in the RPB7 gene makes yeast able to tolerate alcohol way better than this guy. By jerome Chua [CC BY 2.0 (http://creativecommons.org/licenses/by/2.0)%5D, via flickr.

M1 appeared to be a champion mutant for making ethanol, and the fermentation studies bore this out.

Under a wide variety of conditions, M1 outperformed the parental strain in terms of growth rate, cell mass, and amount of ethanol made. For example, after 54 hours, yeast containing the M1 mutation of RPB7 managed to make 122.85 g/L of ethanol, 96.58% of the theoretical yield. This is a 40% increase over the control strain. Quite the ethanol producer!

Finally, Qiu and Jiang used microarray analysis of the parental and M1 strains at high levels of ethanol to discover the genes that M1 affected. They found 369 out of a total of 6256 genes behaved differently between the two strains. Of the 369, 144 were up-regulated and 225 were down-regulated.  

I don’t have time to go over all the genes they found but a great many of them make sense. As the authors write, “…a significant set of genes are associated with energy metabolism, including glycolysis, alcoholic fermentation, hexose transport, and NAD+ synthesis.”  M1 seems fine-tuned for making ethanol.

A mutant subunit in RNA polymerase II has made yeast better at making high levels of ethanol, most likely by affecting many key genes at once. It is a fascinating way to quickly affect a whole suite of genes involved in a process. In the ethanol-making Super Bowl, we have a new champion yeast strain, M1.

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: RPB7 , Super Bowl , NFL , RNA polymerase II , Patriots , Tom Brady , ethanol , fermentation

Yeast Tackles Climate Change

November 29, 2016


With its new superpower of turning xylose into ethanol, yeast is just about ready to provide the low carbon fuel we need to help us stave off climate change. Image from pixabay.

One of the best parts about doing outreach with a museum is creating a successful hands on activity for the visitors. This is not an easy thing to do.

You first create something that you think will appeal to and educate visitors. When that falls flat on its face, you then do a series of tweaks until it is working smoothly. In the end you have smiling kids who understand DNA better (or at all)!

Genetic engineering can be similar. You can import the genes for a complex pathway into your beast of choice but it may not work first try. A bit of tinkering, evolution style, is often needed to get the engineering working well enough to be useful.

This is exactly what happened when a group of researchers tried to get our favorite beast, the yeast Saccharomyces cerevisiae, to turn the sugar xylose into ethanol. They added the right genes from either fungi or bacteria, but S. cerevisiae couldn’t convert enough xylose into ethanol to be useful.

And it is important for all of us that some beast be able to do this well. A yeast that can turn xylose into ethanol means a yeast that can turn a higher percent of agricultural waste into a biofuel. Which, of course, means lots of low carbon fuel to run our cars so that we have a better shot of limiting the Earth’s heating up by 1.5-2 degrees Celsius.

To get their engineered yeast to better utilize xylose, Sato and coworkers forced it to grow with xylose as its only carbon source. Ten months and hundreds of generations later, this yeast had evolved into two new strains that were much better at turning xylose into ethanol. One strain did its magic with oxygen, the other without it.

In a new study in PLOS Genetics, Sato and coworkers set out to figure out which of the mutations that came up in their evolution experiments mattered and why.

Two genes were common in both the aerobic and anaerobic strains – HOG1 and ISU1. Both needed to be nonfunctional in order to maximize ethanol yields from xylose. They confirmed this by deleting each individually and together from the parental strain.

HOG1 encodes a MAP kinase, and ISU1 encodes a mitochondrial iron-sulfur cluster chaperone. These probably would not have been the first genes to go after with a more biased approach. The benefits of evolution and natural selection!

Further experiments showed deleting each gene individually was not as good as deleting both at once when oxygen was around. In fact, while deleting only ISU1 had a small effect on the ability of this yeast to convert xylose into ethanol, deleting HOG1 alone had no effect at all. Its deletion can only help a strain already deleted for ISU1.

In the absence of oxygen, yeast needs a couple of additional genes mutated – GRE3 and IRA2. GRE3 is an aldose reductase and IRA2 is an inhibitor of RAS. Again, not very obvious genes!

Still, once you find the genes you can come up with reasonable hypotheses for why they are important.

Some are easier than others. Hog1p, for example, is known to enhance a cell’s ability to turn xylose into xylitol, which shunts the xylose away from the ethanol conversion pathway. GRE3 is involved in this as well. Deleting either should make more xylose available to the yeast.

This doesn’t mean this is Hog1p’s only role in boosting this yeast’s ability to turn xylose into ethanol of course. It also probably “…relieves growth inhibition and restores glycolytic activity in response to non-glucose carbon sources.” Consistent with this, the authors found that the xylose-utilizing strain deleted for HOG1 was also better at using glycerol and acetate as carbon sources.

activity

The tweaks needed to make a successful museum activity are often nonintuitive. Much like the tweaks needed to maximize the performance of genetically engineered yeast. Image courtesy of The Tech Museum.

Other genes were less obvious. For example, perhaps mutating ISU1 frees up some iron so extra heme can be made. Or alternatively, it may have increased the mass of mitochondria available. Again, probably would not have been the first gene to go after to improve yeast’s ability to convert xylose to ethanol.

Which again underlines the importance of letting natural selection improve an engineered organism as opposed to only trying to pick and tweak the genes you think are important. Biology is simply too complicated and our understanding too limited to be able to know which are the best genes to go after. This is reminiscent of prototyping museum activities.

Some tweaks are obvious but others you would never have guessed would be needed. For example, we had visitors spreading bacteria on a plate and found that if they labeled their plate first, they almost always put their transformation mixture on the lid instead of on the LB agar. This problem was solved by having them add their mixture first and then labeling their plate.

It would be very hard to predict something like this from the get-go. The activity needed to evolve on the museum floor to work optimally. Much like the yeast engineered to utilize xylose needed to evolve in the presence of xylose to work optimally. And to perhaps take a big step towards saving the Earth from warming up too much.

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: evolution , biofuel , xylose , ethanol , glucose metabolism , fermentation

A Radical Discovery About a Well-Known Enzyme

May 22, 2013

Living your life puts a lot of wear and tear on you. A big reason is that as your cells go about their business, they churn out lots of damaging chemicals. 

This radical wanted us to rethink our role in Vietnam. The radical superoxide is making us rethink what the enzyme Sod1p does in a cell.

One of the worst offenders is the free radical superoxide, O2. Cells can’t help producing this powerful oxidant during normal metabolism, but it’s so toxic that it can destroy proteins and damage DNA.

Cells have come up with a two-step process to deal with this toxic waste. In the first step, they use the enzyme superoxide dismutase (Sod1p is the cytosolic form in yeast) to convert superoxide into the less harmful hydrogen peroxide (H2O2) and water. The cells then use catalases to take care of the H2O2, converting it to water and molecular oxygen. 

We’ve known about the first enzyme, superoxide dismutase, for decades. It has always been thought to have a simple role, sitting in the cytoplasm and detoxifying O2. But new research shows that its job is considerably more interesting than that: it also has a role in a regulatory process known as the Crabtree effect.

The Crabtree effect is named after the scientist who first described it way back in 1929. Some types of cells are able to produce energy by either fermentation or respiration in the presence of oxygen. Since these two processes have different metabolic costs and consequences, which one to use is a critically important choice.

If lots of glucose is around, yeast cells choose fermentation. They prevent respiration by repressing production of the necessary enzymes, and this glucose-dependent repression is the Crabtree effect. It happens not only in yeast, but also in some types of proliferating cancer cells.

A new study by Reddi and Culotta shows that Sod1p is actually a key player in the Crabtree effect. In response to oxygen, glucose, and superoxide levels, it stabilizes two key kinases that are involved in glucose repression.

It was recently found that the sod1 null mutant can’t repress respiration when glucose is around.  This is different from the wild type, which is subject to the Crabtree effect. 

Reddi and Culotta started by investigating this observation and found that SOD1 is part of the glucose repression pathway that also involves the two homologous protein kinases Yck1p and Yck2p. They found that Sod1p binds to Yck1p, which wasn’t totally unexpected since this interaction had been seen before in a large-scale screen. The unexpected part was that Sod1p binding actually stabilizes Yck1p and Yck2p.  These stabilized kinases can now phosphorylate targets that propagate the glucose signal down the pathway and ultimately repress respiration.

Now the question is why does Sod1p binding stabilize the kinases? It turns out that its enzymatic activity is crucial for stabilization. One idea is that the hydrogen peroxide that Sod1p makes in the neighborhood of the kinases could inactivate ubiquitin ligases that would target them for degradation. Ubiquitin ligases are rich in cysteine residues, and so could be especially sensitive to oxidation by H2O2.

This regulation might also feed into other pathways: these kinases are also involved in response to amino acid levels, and the sod1 null mutant was seen to affect the amino acid sensing pathway in this study.

Most excitingly, this mechanism is not just a peculiarity of yeast Sod1p. The authors mixed and matched yeast, worm, and mammalian superoxide dismutases and casein kinase gamma (the mammalian equivalent of Yck1p/Yck2p), and found that binding and stabilization works in the same way across all these species.

Superoxide dismutases may have been drafted into this regulatory role during evolution because they are the only molecules that sense superoxide, whose levels reflect both glucose and oxygen conditions. A radical idea indeed!

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight

Tags: regulation , Saccharomyces cerevisiae , respiration , fermentation

Better Beer Through Beards

October 16, 2012

Small time craft brewers are always looking for ways to push the envelope of beer taste.  They are trying to find variations in beer’s fundamental ingredients — hops, barley, and yeast — that will make their beer distinctive.  Of these three, the most important is probably yeast (of course, we’re biased here at SGD!).

Think of the tasty beers that could come out of those beards.

Something like 40-70% of beer taste comes from the yeast used to make it alcoholic.  This is why brewers search high and low for new strains of yeast that will give their beer that special something which will make it stand out.  They have looked on Delaware peaches, ancient twigs trapped in amber, Egyptian date palms, and in lots and lots of other places. 

But brewers don’t always have to go far away because sometimes the best yeast is right under their noses.  Literally.

A brewery in Oregon found the yeast they were looking for in one of their master brewers’ beards.  They are now using this yeast to brew a new beer!  This seems uniquely revolting but the beer supposedly is quite tasty.  Perhaps if they don’t advertise the source of their yeast, this beer could become popular.

They aren’t sure where the yeast in his beard came from, but they think it may have come from some dessert he ate in the last 25 years or so (he hasn’t shaved his beard since 1978).  What would be fun is if his beard wasn’t just an incubator, but a breeding ground for new yeast.  Maybe yeast from a dessert from 1982 hooked up with a beer yeast blown into his beard while he was working at the brewery.  The end result is a new improved hybrid yeast! 

Of course we won’t have any real idea about this yeast until we get some sequence data from it.  And all kidding aside, the more yeast that are found that are good for making beer, the better the chances that scientists can home in on what attributes make them beer worthy.  So this beard borne yeast may help many beers in the years to come despite its troubling beginning.

Perhaps brewers also need to start searching through more beards to look for likely beer yeast candidates.  Beard microbiome project anyone?

More information

About beard yeast

What’s in a beer, anyway?

Original lager yeast found in Patagonia

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: beer , Saccharomyces cerevisiae , fermentation

Ethanol from Waste

June 16, 2012

Scientists are coming up with ways for yeast to use waste like this to generate ethanol.

Biofuels hold the promise to significantly slow down global warming.  But this will only be the case if they come from something besides corn.

We don’t want them to come from the parts of other plants we eat, either.  Shunting food towards fuel will only jack up food prices and put the lives of the poorest at risk.  Policy makers should not have to decide between feeding the poor and running their cars.

No, to make biofuels worth our time, we need to be able to turn agricultural waste, grass, saplings, etc. into ethanol.  Unfortunately this stuff is mostly cellulose and lignin and we don’t have anything that can efficiently ferment this “lignocellulosic biomass.” 

Many groups are working towards creating strains of Saccharomyces yeast, the predominant fungal organism used for large-scale industrial processes, to do this job.  None have yet been created that can do the job well enough to be industrially viable. They are either poor fermentors or are genetically modified so that they include non-yeast genes.  Ideally any strain would include only Saccharomyces genes, to avoid the public’s fear and loathing of genetically modified organisms.  

This is where a new study in GENETICS by Schwartz and coworkers comes in.  This group is working towards engineering a yeast that can ferment the pentoses like xylose that make up a good chunk of this otherwise inedible biomass, using genes that are naturally occurring in Saccharomyces.  They haven’t yet created such a yeast, but they have at least identified a couple of key genes involved in utilizing xylose.

The researchers took what seemed to be a straightforward approach.  Collect and screen various yeast strains for their ability to grow on xylose and isolate the relevant gene(s) from the best of them.  Sounds easy enough except that most of the strains they’ve found are terrible sporulators.  This means that they couldn’t use conventional methods to isolate the genes they were interested in and so had to come up with new methods.

First they needed to find some way to get the strain to sporulate.  They were able to force sporulation by creating a tetraploid intermediate between the xylose fermenting strain, CBS1502, and the reference strain, CBS7001, by adding an inducible HO gene.  During this process, they noticed that the ability to utilize xylose segregated in a 3:1 pattern.  This usually means that two genes are involved.

They next needed a way to identify these two genes.  What they did was to pool 21 spores that could ferment xylose and 21 that could not.  They then purified the DNA from each pool and compared them using high throughput sequencing.  They eventually found two genes that were key to getting this yeast to use xylose as its carbon source.  (They also found at least two other “bonus” genes that seemed to boost its ability to use xylose).

One of the genes, GRE3, was a known member of a xylose utilization pathway.  But the other gene, the molecular chaperone APJ1, was not known to be involved in metabolizing xylose.  The authors hypothesize that APJ1 might stabilize the GRE3 mRNA.

These two genes may not be enough to create an industrially viable, xylose fermenting Saccharomyces just yet.  But the novel methods of gene isolation presented in this study may allow researchers to find additional genes that might one day get them there.  Then we will have a way to get ethanol without the large carbon footprint and without the human cost.

 

A genetic engineering approach to getting yeast to ferment agricultural waste

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: ethanol , biofuel , yeast , lignocellulosic biomass , biomass , Saccharomyces , Saccharomyces cerevisiae , fermentation

Finally Great Tasting, Low Alcohol Beer

February 17, 2012

A lab engineered strain of yeast may make low alcohol, great tasting beer a reality.

Let’s face it: low alcohol beer just doesn’t taste that great.  This is because the alcohol is either diluted or removed chemically after fermentation.  Both methods wreak havoc with a beer’s flavor.

Dr. John Morrissey of University College Cork is trying to change this.  His lab is working to generate a strain of yeast that turns some but not all of its sugar into alcohol.  That way the beer process is the same, just with less alcohol at the end.

This is different from stopping fermentation early.  In that case there are still sugars in the final product which ruin a beer’s taste even more than removing the alcohol!  Here the same amount of sugars are used up, it is just that only part of that energy has gone into making the alcohol.  Same sugar content, less alcohol.

Although we don’t have all the details because of intellectual property issues, what we do know is that he compared the genomes of yeast species that make a lot of alcohol and those that don’t.  In an email he stated that he focused on genes that would affect carbon metabolism without perturbing redox balance in a significant way.  Presumably he then swapped the appropriate genes between strains and created his low alcohol strain.

This is not only a godsend for low alcohol beer, but it may be useful for other fermentation processes as well.  For example, maybe something similar can be done for low or no alcohol wines which, apparently, are even less tasty than low alcohol beer.  Designated drivers everywhere will be thanking Dr. Morrissey profusely if he can make decent tasting, low alcohol drinks a reality.

And apparently it isn’t just designated drivers that want this stuff.  Judging by recent upticks in sales of the relatively low quality low alcohol beers currently on the market, there is definitely a market out there for such beverages.  A cool science project, decent low alcohol beer and nice profits to boot!  Who could ask for more? 

How beer is made, from Modern Marvels, http://www.history.com

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: low alcohol beer , carbon metabolism , redox , fermentation , yeast

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