New & Noteworthy

Using Yeast to Discover Treatments for ALS

November 01, 2012

What do Lou Gehrig, Stephen Hawking, David Niven and Mao Zedong have in common?  They all suffered (or in Hawking’s case, continue to suffer) terribly from a disease called amyotrophic lateral sclerosis or ALS.  And now the humble yeast S. cerevisiae may help scientists find new treatments so that others do not need to suffer similarly.

Patients with ALS gradually lose use of their motor neurons and generally die within 3-5 years of diagnosis.  While there are some rare forms that run in families, most are sporadic.  There is no history of the disease in the family and then suddenly, it just appears.

The causes of ALS have remained a mystery for many years but recent work has suggested that RNA binding proteins and RNA processing pathways are somehow involved.  In particular, an RNA-binding protein called TDP-43 appears to be a key player.  Mutations in its gene are associated with ALS, and aggregates of the protein are found in damaged neurons of ALS patients. Unfortunately, since this protein is needed for cell survival it is not an easy target for therapies.  This is where yeast can help.

Scientists have managed to mimic the effects of TDP-43 in yeast.  When this protein is overexpressed, the yeast cells die just like the motor cell neurons do. In a recent Nature Genetics paper, Armakola and coworkers use this model system for finding better therapeutic targets.  And it looks like they may have succeeded.

These authors used two different screens to systematically look for proteins that when deleted or expressed at lower levels rescued yeast overexpressing TDP-43.  They found plenty.  One screen yielded eight suppressors while the other yielded 2,056 potential suppressors.  They decided to focus on one of the stronger suppressors, DBR1.

The first thing they wanted to do was to make sure this wasn’t a yeast specific effect.  If lowering the amount of DBR1 has no effect in mammalian models, it is obviously not worth pursuing!

To answer this question, they created a mammalian neuroblastoma cell line with an inducible system for making a mutant version of TDP-43, TDP-43 Gln331Lys, found commonly in ALS patients.  As expected, these cells quickly died in the presence of inducer.  They could be rescued, though, when DBR1 activity was inhibited with siRNA.  The authors confirmed that decreasing the activity of DBR1 in primary neurons decreased TDP-43 toxicity as well.

So decreasing the amount of DBR1 appears to rescue cells that die from the effects of mutant TDP-43.  This suggests that targeting DBR1 may be useful as a therapy for ALS.  But this study doesn’t stop there.  It also tells us a bit about how lowering DBR1 levels might be rescuing the cells.

DBR1 is an RNA processing enzyme involved in cleaning up the mess left behind by splicing.  It cleaves the 2’-5’ phosphodiester bond of the spliced-out intron (called a lariat).  Previous studies in yeast have shown that when Dbr1p levels are reduced or its catalytic activity is disrupted by a mutation, there is a build up of these lariats. This study showed directly that the accumulated lariats interact with TDP-43 in the cytoplasm to suppress its toxicity. So in ALS, the accumulated lariats may serve as a decoy for the mutant TDP-43 protein, preventing it from binding to and interfering with more essential RNAs.

This last result may also suggest another potential therapy.  If scientists can find other ways to increase the amount of decoy RNA, then they may not need to depend on reducing levels of DBR1.  There may be many possible approaches to soaking up rogue TDP-43.

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: ALS, DBR1, Lou Gehrig's Disease, RNA binding, Saccharomyces cerevisiae, yeast model for human disease

When Polymerases Collide

October 25, 2012

Lots of recent studies are showing that transcription happens over way more DNA than anyone previously thought. For example, the ENCODE project has shown that most of a genome gets transcribed into RNA in humans, fruit flies and nematodes. This transcriptional exuberance was recently confirmed in the yeast S. cerevisiae as well.

There is also a whole lot of antisense transcription going on. Taken together, these two observations suggest that there are lots of opportunities for two polymerases to run headlong into each other. And this could be a big problem if polymerases can’t easily get past one another.

Imagine that the two polymerases clash in the middle of some essential gene. If they can’t somehow resolve this situation, the gene would effectively be shut off. Bye bye cell!

Of course this is all theoretical at this point. After all, smaller polymerases like those from T3 and T4 bacteriophages manage to sneak past one another. It looks like this isn’t the case for RNA polymerase II (RNAPII), though.

As a new study by Hobson and coworkers in Molecular Cell shows, when two yeast RNAPII molecules meet in a head on collision on the same piece of DNA, they have real trouble getting past each other. This is true both in vitro and in vivo.

For the in vivo experiments, the authors created a situation where they could easily monitor the amount of transcription close in and far away from a promoter in yeast. Basically they pointed two inducible promoters, from the GAL10 and GAL7 genes, at one another and eliminated any transcription terminators between them. They also included G-less cassettes (regions encoding guanine-free RNA) at different positions relative to the GAL10 promoter, so that they could use RNAse T1 (which cleaves RNA at G residues) to look at how much transcription starts out and how much makes it to the end.

When they just turned on the GAL10 promoter, they saw equal amounts of transcription from both the beginning and the end of the GAL10 transcript. But when they turned on both GAL10 and GAL7, they saw only 21% of the more distant G-less cassette compared to the one closer to the GAL10 promoter.

They interpret this result as meaning the two polymerases have run into each other and stalled between the two promoters. And their in vitro data backs this up.

Using purified elongation complexes, they showed that when two polymerases charge at each other on the same template, transcripts of intermediate length are generated. They again interpret this as the polymerases stopping dead in their tracks once they run into one another. Consistent with this, they showed that these stalled polymerases are rock stable using agarose gel electrophoresis.

Left unchecked, polymerases that can’t figure out how to get past one another would obviously be bad for a cell. Even if it were a relatively rare occurrence, eventually two polymerases would clash somewhere important, with the end result being a dead cell. So how do cells get around this thorny problem?

One way is to get rid of the polymerases. The lab previously showed that if a polymerase is permanently stalled because of some irreparable DNA lesion, the cell ubiquitinates the polymerase and targets it for destruction. In this study they used ubiquitin mutants to show that the same system can work at these paused polymerases too. Ubiquitylation-compromised yeast took longer to clear the polymerases than did their wild type brethren.

The authors think that this isn’t the only mechanism by which polymerases break free though. They are actively seeking factors that can help resolve these crashed polymerases. It will be interesting to see what cool way the cell has devised to resolve this dilemma.

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

Categories: Research Spotlight

Tags: RNA polymerase II, Saccharomyces cerevisiae, transcription, ubiquitin-mediated degradation

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!).

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, fermentation, Saccharomyces cerevisiae

How To Remember Without a Brain

October 02, 2012

Single celled beasts like the yeast S. cerevisiae can “remember” previous insults and so respond better to environmental changes in the future. For example, a yeast cell treated with 0.7M NaCl will respond better in the future to hydrogen peroxide. Not only that but so will its daughters, granddaughters and even its great, great granddaughters.

In a new study out in GENETICS, Guan and coworkers show at least a couple of ways that this can happen. One is what anyone in biology might expect these days (although with an interesting twist). The NaCl treatment causes a rewiring of the regulatory network at an epigenetic level and this affects future responses to environmental insults.

But fancy epigenetic changes aren’t the only way that yeast remembers things.  No, it also uses a simple, elegant solution—protein stability.
 
Long Live The Protein!
 

The researchers did a set of experiments that showed that a yeast’s memory of a salt treatment did not rely on new protein synthesis and that it slowly faded with each generation.  One possible explanation was that the salt induced a stable factor that was divvied up and diluted with each passing generation. Guan and coworkers found that this was the case and that at least one of these factors was the cytosolic catalase 1 protein, Ctt1p.

The cytosolic catalase 1 or CTT1 gene is induced by salt but quickly returns to normal levels when the salt is removed.  However, Ctt1p is so long lived that it hangs around for at least six hours.  In that time the yeast has budded off multiple daughters, all of which are still better at dealing with hydrogen peroxide than their untreated sisters.

What a marvelously simple way to adapt!  Just make something that hangs around a long time and you and your kids will do better when the next insult comes.  The elegance of evolution.

This explains in part how yeast cells can remember the salt treatment of their ancestors, but a single long-lived protein isn’t the whole story.  No, there is something a bit more complicated going on at the nuclear pore too.

 
Attached for Quick Access
 

Guan and coworkers looked at the gene expression pattern of salt stressed and naïve yeast when exposed to hydrogen peroxide.  They found that 449 genes responded more quickly to hydrogen peroxide treatment if the cell had been pretreated with salt.  Importantly, 51 of these hadn’t reacted previously to the salt treatment, meaning that previous activation wasn’t required.

One idea is that these genes are more accessible to transcription because they are associated with the nuclear pore.  The idea is that faster response happens because the gene is closer to the nuclear envelope and/or because it has been looped near some sort of activator.

This is what has been proposed with inositol starvation and it looks like it may be true here too.  In both cases, eliminating Nup42p, a nuclear pore protein, eliminates the more rapid response to hydrogen peroxide.

So in this case it looks like cells can remember a previous insult with just a long-lived protein and a bit of genetic rewiring.  It will be interesting to see how universal these sorts of mechanisms are for cell memory.

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

Categories: Research Spotlight

Tags: catalase, cellular memory, Saccharomyces cerevisiae, stress response

Wintering in a Wasp’s Gut

September 24, 2012

Anyone reading this blog probably knows how important the yeast S. cerevisiae is.  It makes our bread better, our beer and wine more spirited, and our genetics more understandable. 

Because it is such an important beast, this yeast is also incredibly well characterized.  It was the first non-bacterial organism whose genome was sequenced and is a key model organism for teasing apart how eukaryotes like us work. We may know more about the molecular biology and genetics of S. cerevisiae than about any other organism on the planet.

And yet we know surprisingly little about S. cerevisiae in the wild.  We know that it isn’t on unripe fruits but suddenly appears once they ripen. We also know it doesn’t tolerate winter particularly well.  So where does yeast hang out when there isn’t ripe fruit around and/or it gets chilly?  A group of researchers in Italy thinks a key place is inside a hibernating wasp.

When Stefanini and coworkers looked, they found lots of yeast (including S. cerevisiae) in wasp intestines. They were also able to show that the S. cerevisiae remained viable in a hibernating queen over the winter and that that the queen transferred the yeast to new wasps in the spring by regurgitation.  With this one study, these scientists managed to find at least one way that yeast can survive the winter and get to ripe fruit.

To figure this out, Stefanini and coworkers did experiments both in the field and in the lab.  They first collected wasps and bees from around the Italian countryside and showed that wasps, but not bees, harbored yeast in their gut.  In all they found 393 yeast strains in the 61 wasps they dissected, 17 of which turned out to be S. cerevisiae.   By sequencing and comparing the genes URN1, EXO5, and IRC8, they were able to conclude that these yeast were related to wine, beer, bread, and laboratory strains of S. cerevisiae.

The researchers figured out that the yeast could survive for three months and be passed on to the next generation of wasps with a couple of controlled experiments they did in the lab.  They fed queens GFP labeled yeast and then let them hibernate.  After three months they dissected some of them and found lots of viable yeast in their intestines.

The rest of the queens were allowed to wake up and find new nests.  Larvae were removed from the nests and were found to contain GFP yeast as well.  The yeast not only lived through the winter but passed on to the next generations!

Of course this doesn’t mean that this is the only way that it can happen.  But it is the first time anyone has managed to get such a detailed look at feral yeast.  And this kind of work is important if we want to use S. cerevisiae as a way to study evolution. 

To understand its evolution, we have to understand the natural forces that shaped S. cerevisiae into the organism it now is.  Only then can we piece together why S. cerevisiae has evolved the way that it has and so learn fundamental lessons about the mechanisms of evolution. 

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

Categories: Research Spotlight

Tags: evolution, Saccharomyces cerevisiae, wild yeast

Few Genetic Paths From Here to There

September 12, 2012

Everyone knows that when the environment changes, those individuals with certain DNA differences useful in this new environment thrive while others wither.  But there hasn’t been a lot of work done to investigate how many DNA differences are available to a population for adapting to a particular environmental change.

This may sound esoteric but the answer has real implications for speciation.  If there are few mutations possible and these mutations are very similar in terms of phenotype, then different populations will travel similar routes in their adaptations to the same environmental change.  This will definitely slow down speciation.  If on the other hand there are many genetic ways to adapt to the same change, then isolated populations will head down different paths leading to faster speciation.

In a new study out in GENETICS, Gerstein and coworkers found that at least for the environmental insult they used (low levels of the fungicide nystatin), there were very few paths to resistance. In fact, just four genes in the ergosterol biosynthesis pathway turned up in the 35 resistant lines they surveyed using whole genome sequencing.

Now that isn’t to say that there were just a few mutations.  There weren’t.  They found eleven unique mutations in the ERG3 gene, seven in ERG6, and one each in ERG5 and ERG7.  There were duplications, deletions, premature stop codons and missense mutations.  So there are lots of ways to mutate these few genes.

The small range of genes affected might suggest that adaptation favors populations evolving along similar paths since the same environmental effects result in the same adaptative mutations.  And yet, not all of these mutations in these few genes are created equally.  Different lines responded differently to other stressors.

For example, lines with mutations in the ERG3 gene responded poorly to ethanol while the other lines did very well.  And the lines with mutations in ERG5 and ERG7 responded less well to salt than the other lines.  So if one population was subjected to salt and nystatin and the other to ethanol and nystatin, the strains would almost certainly adapt with mutations in different genes.  Even within this narrow set of genes, there is room for adaptation by different routes.

While a useful first step, we don’t want to infer too much from this single study.  The researchers used a very specific environmental insult known to work through a specific pathway and found only mutations in that pathway.  The next study might want to focus on something like salt tolerance, a trait predicted to be achieved through multiple pathways.  Then we can get an even better feel for how many options a population has for adaptation.

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

Categories: Research Spotlight

Tags: ergosterol biosynthesis, evolution, nystatin, Saccharomyces cerevisiae

What Happens in Genes, Stays in Genes

September 06, 2012

Chromatin proteins, primarily histones, are a great way to control what parts of a cell’s DNA are accessible to its machinery.  These proteins coat the DNA and are marked up in certain ways to indicate how available a piece of DNA should be.  A methyl group here, an acetyl group there and a cell “knows” where the genes are that it is supposed to read!

Of course this structure needs to be maintained or a cell might start to misread parts of its DNA as starting points of genes.  Then RNA polymerase II (RNAPII), the enzyme responsible for reading most protein-coding genes, would start making RNA from the wrong parts of the DNA, wreaking havoc in a cell.

One place where maintaining chromatin structure might be especially tricky is within the coding parts of genes.  It is easy to imagine RNAPII barreling down the DNA, knocking the proteins aside like pins in a bowling alley.  But it doesn’t.  For the most part the chromatin structure stays the same and survives the onslaught of an elongating RNAPII.

Two key marks for keeping histones in place are the trimethylation of lysine 36 of histone H3 (H3K36me3) that is mediated by Set2p, and a general deacetylation of histone H4 that is mediated by the Rpd3S histone deacetylase complex.  We know this because loss of either complex causes an increase in H4 acetylation and transcription starts from within genes.

In a recent study in Nature Structural & Molecular Biology, Smolle and coworkers identified two key components that help chromatin resist an elongating RNAPII in the yeast S. cerevisiae.  The first, called the Isw1b complex, binds H3K36me3 and the second, the Chd1 protein, binds RNAPII itself.  That these two were involved wasn’t surprising since previous work had suggested they helped prevent histone exchange at certain genes.

What makes this work unique is that the researchers showed the global importance of these proteins in the process and were able to tease out some of the fine details of what is going on at the molecular level. They used electrophoretic mobility shift assays to show that Isw1b bound the trimethylated form of H3 via its Ioc4p subunit and used chromosome immunoprecipitation coupled to microarrays (ChIP-chip) to show that Isw1b localized to the middle of genes in vivo. They also showed that when Set2p was removed, the localization disappeared (presumably because of the loss of the trimethylation of lysine 36).  They clearly demonstrated that Isw1b is found primarily in the middle of genes.

While these results indicate that the Ioc4p-containing Isw1b complex is moored to the middle of genes via its interaction with H3K36me3, it does not establish what it is doing there.  For this the researchers knocked out Isw1b and Chd1 and showed via genome tiling arrays a global increase in cryptic transcription starts.  The DNA in the middle of genes was now being used inappropriately by RNAPII as starting points for transcription.  Further investigation with Isw1b and Chd1 knockouts showed an increase in chromosome exchange and an increase in acetylated H4 in the middle of genes.

Whew.  So it appears that Isw1b and Chd1 inhibit inappropriate starts of transcription by keeping hypoacetylated histones in place over the parts of a gene that are read.   They are two of the key players in maintaining the right chromatin structure over genes.  They help keep RNAPII from railroading histones aside as it elongates, thus protecting the cell from inappropriate transcription starts.

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

Categories: Research Spotlight

Tags: Chd1, chromatin, Isw1b, RNA polymerase II, Saccharomyces cerevisiae

Yeast with Sticking Power

July 16, 2012

Most strains of Saccharomyces cerevisiae don’t stick together very well. And hardly any of them form biofilms. But it would be very useful to have a better understanding of why some strains like to stick together and others do not. 

Stickiness helps in any process where you want the yeast to do something and then get rid of it. An obvious example is ethanol production either for energy or to make our beer and wine. After sticky yeast are done with their job of making the alcohol, they simply fall to the bottom of the fermentor or float on the surface in a biofilm (the “flor”). This makes the step of separating the yeast from the finished product that much easier.

Understanding more details of yeast stickiness would also be useful for studying harmful yeast. Adhesion to other cells and to substrates is an important factor in pathogenesis. It would be nice to investigate this phenomenon in the more tractable brewer’s yeast.

The Ibeas lab has decided to figure out why most strains of S. cerevisiae can’t flocculate by comparing one of the few that can (the “flor” strain used to make sherry) to a reference strain that can’t.  They previously showed that a key gene in the process, FLO11 (also known as MUC1), is expressed at much higher levels in flor.  They were also able to show that a large part of this increased expression comes from a 111 base pair deletion in the FLO11 promoter in this particular strain.

In a recent paper in GENETICS, Barrales and coworkers set out to investigate why the loss of these 111 base pairs leads to increased gene expression.  They were able to conclude that the deletion does not significantly affect histone occupancy at the promoter.  What they could see was that histone placement was affected and that PHO23 may play a significant role in this.

The researchers had previously shown that the histone deacetylase complex (HDAC) Rpd3L was important for maximal FLO11 activity.  They next wanted to determine if this complex was the major player in explaining the increased activity of the 111 bp deletion FLO11 promoter (Δ111) over the wild type (WT) one.  They did this by comparing the level of mRNA made by each promoter in strains lacking either the Pho23p or the Rpd3p subunits of the Rpd3L complex.  They found that the Δ111 construct was much more severely compromised by the loss of PHO23 than was the WT one.  (A bit confusingly, neither was much affected by the loss of RPD3.) 

Given that PHO23 is part of a complex that affects chromatin, the next thing the researchers did was look at the histones in and around both FLO11 promoters.  They found that PHO23 was involved in maintaining an open chromatin structure at the FLO11 promoter but that deleting the 111 base pairs didn’t affect this process significantly.

Where they started to see subtle differences was when they looked at histone placement as opposed to occupancy.  Using micrococcal nuclease protection to map chromatin structure, they found a number of differences between the two promoters, centered on the deletion and the TATA box, and deleting PHO23 affected the two promoters in different ways.

It appears that FLO11 is upregulated in the flor strain because the deletion of 111 base pairs leads to an altered chromatin structure.  The next steps will be to figure out what this means and then to use that knowledge to create stickier yeast. We’ll end up with a better understanding of transcriptional regulation and adhesion, and beer and wine makers may end up with even better self separating yeast.

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

Categories: Research Spotlight

Tags: biofilm, flocculation, Saccharomyces cerevisiae, transcription

Yeast on the Brink

July 02, 2012

How scientists are using baker’s yeast to discover the warning signs of impending financial, climate, and species collapse.

Tipping points are all the rage these days. They are discussed with regard to global warming, financial collapses, ecosystems and lots of other situations too.

A tipping point is a point from which something can’t return to what was before. In other words, it is the point at which a new equilibrium is reached.

One of the more interesting tipping points occurs when a population of organisms becomes so low that it may collapse and not be able to recover. This can happen because the beasts are all so interrelated that a disease can wipe them all out.  Or they become so few in number that potential mates have trouble finding one another. Many other reasons can bring a population to this point.

Theory makes a number of predictions about how populations at the tipping point will behave.  Dai and coworkers decided to create a model system using S. cerevisiae to study what populations at the tipping point actually look like experimentally. And to perhaps find easy to study signs that a population is veering close to one of these tipping points.

Their experiments ended up faithfully reproducing a population in the lab that was at a tipping point. This is a big deal in and of itself.  But while they were able to identify signs that a population was at a tipping point, none would be very easy to spot in a wild population. 

Their model system involved using dilutions of yeast grown in sucrose. Since sucrose is hydrolyzed by yeast outside of the cell, a sucrose molecule hydrolyzed by one yeast cell can be used by another. This cooperative effect means that yeast grow better in sucrose at higher cell densities than they do at lower ones. This mimics the effects of low population density in other systems.

The researchers then did a set of simple dilution experiments with this system. They diluted a starting population of yeast by varying amounts into replicate samples and determined how each sample did with subsequent dilutions over time. They found that they reached their tipping point in their system at dilutions of between 500 and 1600. At these dilutions, some replicates survived while others went extinct. 

They confirmed they were at a tipping point by shocking their cultures with high salt. If a population is near a tipping point, it is less able to survive environmental shocks compared to a more robust system. The researchers found that those samples near the tipping point were indeed more vulnerable to salt shock. 

Taken together, these two findings suggested that the researchers had successfully engineered a model system for tipping points. They were now ready to study their population at or near its tipping point to look for any tell tale warning signs.

They found that their model system agreed with a lot of the theory. As a population neared the tipping point it tended to fluctuate more, and to take longer to reach a new stable population. Unfortunately, neither of these is an obvious sign of an impending tipping point. Both effects require lots of observations over a long time period to see.

Given the consequences of going past a tipping point (sea level rise, coral bleaching, the Great Recession, species extinction), recognizing when we are getting close to one is of paramount importance. Perhaps research like this will help us see the warning signs before it is too late to pull back from the brink.

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

Categories: Research Spotlight

Tags: Allee effect, collapse, cooperativity, Saccharomyces cerevisiae, sucrose, tipping point

Ethanol from Waste

June 16, 2012

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

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