New & Noteworthy

Too Big for the Oven

March 23, 2017


I love lucy, Pioneer Women episode picture

Just like baking bread, “oven” size matters when trying to increase yields of fatty acids in yeast.

In a classic skit from I Love Lucy, Lucy bakes a loaf of bread that is so big it ends up coming out of the oven, pushing her against the far wall. Definitely one of the funniest moments from early TV.

In a new study in Nature Communications, Gajewski and coworkers show that the yeast enzyme fatty acid synthase (FAS) complex, is a bit less comical when it comes to the fatty acid chains it makes. When these authors engineer the enzyme complex to “shrink” the oven, the “bread” it makes becomes smaller. A more constrained active site that holds onto its growing fatty acids less tightly makes shorter-chained fatty acids.

This is an important finding because these shorter-chained fatty acids are so useful as precursors for products like biofuels. Engineered yeast like these may, among other things, one day help us get to a carbon neutral future.

Gajewski and coworkers were able to pull this off because the 3-D structure of this enzyme complex is so well understood. They engineered changes that would be predicted to restrict the growth of the fatty acid chain.

For example, a key player in the elongation step happens in the condensation domain (KD) of the complex. They focused initially on the amino acid methionine at position 1251 (M1251).

This amino acid sticks out into the active site and needs to rotate out of the way when the fatty acid chain elongates. The authors reasoned that if they made it bigger and harder to rotate, the enzyme complex  wouldn’t be as good at elongating the fatty acid chains it makes. The loaf would stop growing when it ran out of room.

They were right. When they mutated a nearby glycine to serine (G1250S), the yeast now made 15.3 mg/liter of fatty acids with 6 carbons (C6 fatty acids), a 9-fold increase over wild type. FAS usually makes C12-C18 fatty acids.

two chemostats with yeast cultures

Tomorrow’s oil wells? By (Image: Maitreya Dunham) [CC BY 2.5], via Wikimedia Commons

They got even better results when they bulked up position 1251 by changing the methionine to a tryptophan (M1251W). Now the yeast made 19.1 mg/liter of C6 fatty acids, a 12-fold increase, and 32.7 mg/liter of C8 fatty acids, a 56-fold increase. They made a third mutation, F1279Y, that ended up affecting growth adversely when combined with G1250S.

This was good, but not good enough. To be commercially viable, they needed higher yields. They next wanted to make mutations that would cause the enzyme complex to hold onto the fatty acids less tightly, like having the baker remove the bread from the oven before it got too big.

For this, they focused on the malonyl/palmitoyl transferase (MPT) domain. They reasoned that by making the complex less able to bind malonyl, it would also bind the growing fatty acid chain less well too. Again, they were right.

When they replaced an arginine with a lysine at position 1834 (R1834K), the yeast made 100 mg/liter of mostly C8 fatty acids, a 23-fold increase. They made an additional mutation, I306A, that had little effect on its own.

Things got really interesting when they looked at different combinations of these mutations. When they mixed and matched them, they pushed yields up even more. And different combinations made different proportions of various sized fatty acids. Scientists can pick the mutant that gives them their desired product.

The best yield was with yeast carrying the triple mutant, I306A-R1834K-G1250S. These yeast managed to make 118 mg/liter of shorter-chained fatty acids.

Gajewski and coworkers boosted this mutant’s yield even more by changing out the promoter. Doing so resulted in the yeast making 464.4 mg/liter of the enzyme.

We now have a strain of yeast that can make a lot of the precursors needed for making biofuels and other chemicals. And scientists can pick the mutant that gives them more of their desired precursor. If they want more C6, they should use one mutant and if they want more C8 they should use a different one.  

#APOYG is helping to create designer molecules that could, among other things, help steer us toward a more carbon neutral future.  

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

Apply Now for the 2017 Yeast Genetics & Genomics Course

March 17, 2017


For almost 50 years, the legendary Yeast Genetics & Genomics course has been taught each summer at Cold Spring Harbor Laboratory.

For almost 50 years, the legendary Yeast Genetics & Genomics course has been taught each summer at Cold Spring Harbor Laboratory. (OK, the name didn’t include “Genomics” in the beginning…). The list of people who have taken the course reads like a Who’s Who of yeast research, including Nobel laureates and many of today’s leading scientists.

The application deadline is April 15th, so don’t miss your chance! Find all the details and application form here.

This year’s instructors – Grant Brown, Maitreya Dunham, and Elçin Ünal – have designed a course (July 25 – August 14) that provides a comprehensive education in all things yeast, from classical genetics through up-to-the-minute genomics. Students will perform and interpret experiments, learning about things like:

  • How to Find and Analyze Yeast Information Using SGD
  • Isolation and Characterization of Mutants
  • Transformation of Plasmids & Integrating DNAs
  • Meiosis & Tetrad Dissection as well as mitotic recombination
  • Synthetic Genetic Array Analysis
  • Next-Gen. whole-genome and multiplexed DNA barcode sequencing
  • Genome-based methods of analysis
  • Visualization of yeast using light and fluorescence microscopy
  • Exploring synthetic biology with CRISPR/CAS9-directed engineering of biosynthetic pathways

Techniques have been summarized in a completely updated course manual, which was recently published by CSHL Press.

legendary plate race

There’s fierce competition between students at CSHL courses in the Plate Race, a relay in which teams carry stacks of 40 Petri dishes (used, of course).

Scientists who aren’t part of large, well-known yeast labs are especially encouraged to apply – for example, professors and instructors who want to incorporate yeast into their undergraduate genetics classrooms; scientists who want to transition from mathematical, computational, or engineering disciplines into bench science; and researchers from small labs or institutions where it would otherwise be difficult to learn the fundamentals of yeast genetics and genomics. Significant stipends (in the 30-50% range of total fees) are available to individuals expressing a need for financial support and who are selected into the course.

Besides its scientific content, the fun and camaraderie at the course is also legendary. In between all the hard work there are late-night chats at the bar and swimming at the beach. There’s a fierce competition between students at the various CSHL courses in the Plate Race, which is a relay in which teams have to carry stacks of 40 Petri dishes (used, of course). There’s also a sailboat trip, a microscopy contest, and a mysterious “Dr. Evil” lab!

Last year’s Yeast Genetics & Genomics Course was loads of fun – don’t miss out!

In Yeast, Being Old Can Be a Good Thing

March 13, 2017


Like this marathon runner, some older yeast are able to win out over their younger counterparts. In the right environment, that is! Image from Wikimedia Commons.

A square slab can make an excellent door stop. Over time, though, the corners can get chipped, making the slab a bit rounded. This new bit of rock makes a less useful doorstop, but a much better wheel! The chipping and aging of the original stone has made it worse in some situations, but better in others.

A new study by Frenk and coworkers in Aging Cell shows that something similar can happen in yeast. Young yeast are much better at utilizing glucose, but older yeast have them beat with galactose (as well as with raffinose and acetate).

One way to think about this is that age turns yeast from a glucose specialist into a sugar generalist. Aging chips away at a yeast cell’s ability to use glucose, but this loss results in a gain in its ability to use galactose.

So at least in the right environment (i.e., when there’s lots of galactose around), with our old friend Saccharomyces cerevisiae, there can be advantages to getting older.

What makes this particularly fascinating is that at least in yeast, this suggests that there may be a positive selection for aging because of the advantage it can give in certain environments. Those yeast who are ageless would compete less well compared to their aging counterparts when their glucose was taken away. The aging process wins out over immortality!

Frenk and coworkers used a relatively simple experimental set up. Take young cells and old cells, mix them together, and see which outcompetes the other using various sugars.

They used yeast that had been aged for 6, 24, and 48 hours in glucose. This is a nice range as 6-hour “old” yeast are fully viable, 24-hour “old” yeast are starting to suffer a bit in the reproductive viability department, and the 48-hour “old” yeast have passed the median lifetime of a yeast cell. Young adult, middle aged, and elderly yeast.

In the first experiment, they compared these yeast to log-phase yeast which the authors refer to as young (vs. the other three which are referred to as aged).

While the 6 hour yeast could hold its own against the young yeast in glucose, the 24-hour and 48-hour yeast grew much more slowly. This is what you would expect, the younger yeast growing faster than the older yeast. The young guns outdoing the older generations.

The situation was different in galactose. Here, the elderly, 48-hour yeast, ran circles around the young yeast. They blew them out of the water.

And it appears to be an age thing. When they compared 6- and 48-hour yeast that were aged in galactose instead of glucose, the more aged yeast still won. So, it wasn’t the shift in environment that caused the difference, it was in the older yeast cells all along.

The change is also not permanent. The offspring of the older yeast weren’t any better at growing in galactose than the younger yeast were. Only the cells that had lived a longer life could use galactose so well.

flintstonemobile

The cylindrical stone may not be as good a doorstop, but it makes a much better wheel! Image from flickr.

A concern here is that yeast as old as 48 hours are pretty rare in the wild. But when they changed assays and looked at colony size as opposed to competition, they saw that even 18-hour yeast had an advantage over the young whippersnappers.

This was such a surprising result that they also looked at cell cycle times of individual aged cells and their daughters. The older mother cells cycled faster in galactose than their daughters. And the opposite was true in glucose.

So it really looks like there are advantages to growing older. Things break down a bit, but that breakdown uncovers new talents that had previously lain dormant.

If you’re a yeast, growing old is not a one-way decline into dotage. You gain new abilities that, under the right conditions, let you outcompete your children! The older cells are selected for in the right environments. #APOYG shows us something good about growing older.

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

Feed a Cold, Starve a Cancer?

March 2, 2017


Unlike a fever, starving some cancers is actually a good treatment. And our friend yeast may be able to help. Image from http://maxpixel.freegreatpicture.com.

You may have heard the old wives’ tale of feed a cold, starve a fever. Turns out that this isn’t particularly good advice (although some studies do suggest that with a fever, you shouldn’t force-feed yourself). It also turns out to have probably originated in the 19th century and not from Chaucer in the 14th as many websites claim.

But while starving a fever is probably never a good idea, starving a cancer can be. Not by following the medical myth that since cancers use a lot of sugar, you can starve them by cutting down on sugar in your diet. Instead you can starve some cancers by denying them the amino acid asparagine (Asn).

On their way to becoming cancerous, acute lymphoblastic leukemia (ALL) cells lose their ability to make Asn. This means that unlike the cells around it, they need to pull Asn from the blood to make their proteins and to survive.

Doctors exploit this weakness by injecting L-asparaginase amidohydralase (L-ASNase) into patients which starves the cancer cell by depleting Asn levels in the blood. The cells around the cancer cells are fine because they can still make Asn.

Right now doctors use L-ASNase from two different bacterial sources: Escherichia coli and Erwinia chrysanthemi. But if a recent study by Costa and coworkers in Scientific Reports holds up, they might want to think about switching to using the Saccharomyces cerevisiae L-ASNase encoded by the ASP1 gene.

An older study had suggested that the yeast enzyme might be too weak to be useful. This new study finds that this is not the case.

The difference between the older study and this one was the purification protocol. The older study purified the native enzyme through multiple chromatography steps while this study used a single affinity chromatography step. The purified yeast and E. coli versions have comparable activity in this study.

They are also comparable in terms of being able to work with very low concentrations of Asn. This is important as Asn levels are very low in the blood.

What makes the yeast enzyme potentially better is that it is much worse at hydrolyzing a second amino acid, glutamine, than are the bacterial versions. This higher specificity for Asn is important because one of the major side effects of the current treatment is neurotoxicity caused by decreased levels of glutamine in the blood. Since the yeast version hydrolyzes glutamine at a lower rate, they predict patients may not suffer as badly from this side effect with the yeast version.

Of course this is all for naught if the yeast enzyme can’t kill cancer cells! Or if it kills cells indiscriminately.

The S. cerevisiae version was nearly as good as the E.coli version in tissue culture. After 72 hours of incubation, both versions had little effect on normal cells (HUVEC), and both were cytotoxic to the L-ASNase-sensitive cell line MOLT-4 with the E. coli version killing 95% of MOLT-4 cells and the yeast version killing 85% of them.

puppy

Move over dog, yeast is humanity’s best friend now. Image from pixabay.

Taken together these results suggest that the S. cerevisiae version may be an alternative to the bacterial versions. It may be able to kill cancer cells with fewer side effects.

But the yeast version is not the only alternative in town. Another group is engineering the E. coli version to lessen its propensity for hydrolyzing glutamine. Either way it looks like certain leukemia patients may be getting an effective cancer treatment with fewer side effects.

Beer, wine, bread, chocolate, and now maybe a treatment for a nasty form of leukemia. Yeast may be humanity’s best friend. #APOYG!

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

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