New & Noteworthy

Creating an Ethanol-Making ‘Super Bowl’ Championship Team

May 24, 2017

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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.

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

Yeast Tackles Climate Change

November 29, 2016

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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.

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

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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