Friday, August 26, 2011

The missing lager yeast strain has been found!

You probably know that beer is alcoholic because of yeast. Yeast grow and divide in sugary starting solutions, and the byproducts of their metabolism (or fermentation) include ethanol.

If you make beer yourself, you also know that the two major categories, ale and lager, are fermented at very different temperatures. Ales are fermented at room temperature (68-72 degrees Fahrenheit), while lagers are fermented at much cooler temperatures (45-55 degrees). This difference is because the yeast "like" to grow and ferment at different temperatures, but why? A research group thinks they figured it out, and they're publishing their findings next week in the journal PNAS (Proceedings of the National Academy of Sciences).

Ale yeast is the species known as Saccharomyces cerevisiae (the same yeast that people use to make bread). Lager yeast is slightly different. It's a hybrid species that came about because S. cerevisiae mated and hybridized with another species. This hybridization allowed the lager yeast (known as Saccharomyces pastorianus) to ferment at much cooler temperatures than would be possible with S. cerevisiae alone.

The problem was, the other contributing yeast species was unknown. People tried looking at the genome of S. pastorianus to figure out the identity of the other species, but because many yeast species are closely related and have similar gene sequences, these efforts were not conclusive. It was thought that the other strain was Saccharomyces bayanus because this yeast grows much better than S. cerevisiae at lower temperatures. However, S. bayanus is also a hybrid species, which complicates the analysis. Based on gene sequence similarities, it was also proposed that the other strain is Saccharomyces monacensis, but this identification is also controversial because S. monacensis might also be a hybrid species.

Researchers from Argentina, the US and Portugal seem to have finally identified the mystery lager yeast. This species, named Saccharomyces eubayanus, was found on large nodules (galls) that grow on beech trees in Patagonia (southern South America). Tree galls are full of sugary compounds that the yeast like to feast on, so the researchers collected galls from different tree species in different locations. When they isolated the yeast from these Patagonian beech trees, they discovered that this yeast's genome was 99.5% identical to the non-cerevisiae portion of the S. pastorianus genome. The most interesting piece of evidence supporting the true identification of S. eubayanus as the "lager" portion of the hybrid yeast species is that these beech forests exist in an Alpine environment with consistently low temperatures, and they seem to be the yeasts' preferred habitat.

A yeast species that is 99.5% identical to a large portion of the S. pastorianus genome and naturally grows at lager temperatures seems like pretty convincing evidence. There's one giant question remaining, though: how did a yeast species from Patagonia make it to Germany, where lagers were born, in the 1400s? The prevailing thought is that lagers have been made in Germany since the early 1400s, which is long before Patagonia was explored by Europeans. There are several possible explanations...

First, maybe the Germans got their dates wrong. It's possible that lagers were not produced in Germany until after South America was explored by Europeans.

Second, Patagonia might have been explored by other groups before the Columbian era. There is some evidence that Vikings and other seafaring groups found parts of North and South America a long time before the Western Europeans. That alone doesn't explain why lagers were first made in certain regions of Germany. You'd expect Viking lagers if this were true, right?

Third, and the most likely possibility in my mind, S. eubayanus might not be specific to Patagonian forests. There are plenty of Alpine environments in Europe, particularly regions of Europe that are easily accessible to Germans. Now that people know to look at tree galls in cold climates, we may discover that S. eubayanus is common to all forests in cool climates.

Cited paper:

Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast

Monday, August 8, 2011

The buckyball traps a single water molecule!

I thought this was sort of a cute story.

Researchers at Kyoto University in Japan published an article in Science in which they determined that a fullerene C60 (buckminsterfullerene, buckyball, etc.) carbon cage molecule can trap a single water molecule. So what? Well, water molecules are notoriously difficult to separate (maybe impossible using other isolation methods?). This capture in a fullerene cage may be a way to study water molecules without the influence of the ever-present effects of hydrogen bonding. I'm not sure if this is going to be a successful way to study the properties of individual molecules of water, but it's a great start.

Literature cited:

Kurotobi K and Murata Y. A single molecule of water encapsulated in fullerene C60. Science. 2011 Jul 29;333(6042):613-6.

Monday, August 1, 2011

Zinc Finger Nucleases, TALENs and Genesis: the future of human gene manipulation

A major problem in treating genetic disorders is the inability to precisely target and remove or change the offending genes, known as gene therapy. If you can take a person's problem cells out of his or her body, modify the dysfunctional genes, and put the modified cells back into the person's body, then you can most likely treat/cure the disorder. All of the steps in this process are complicated, though, and it's been extremely difficult to "cure" a genetic disorder in this way. First, due to the lack of homologous recombination, it's not easy to target genes in somatic cells, so tricks need to be used to modify the genes of interest. Second, most of the targeted cells would not be "fixed" due to the low efficiency of successful modification. Third, it's extremely challenging, maybe impossible, to modify cells in organs or other solid tissues because they can't be manipulated in a lab or efficiently exposed to the therapy, so gene therapy has generally been restricted to blood disorders, where a person's blood cells can be removed and modified in a lab setting, while all residual dysfunctional blood cells in the body are killed through radiation.

Early ideas of gene therapy involved the use of viruses that could modify or introduce DNA into human cells, theoretically removing the dysfunctional genes or introducing helpful genes to cells that could be re-introduced into the patient's body. However, there were problems with these early trials, mainly that they sometimes caused these cells to grow out of control, like a cancer. It was too high a risk to take with human lives, so this research has not moved forward as much as people would have liked.

A new group of tools has been developed in the last few years that might now allow researchers to use gene therapy to treat or cure genetic disorders. These tools help with the first (and to a lesser extent, the second) problem: they can specifically target genes of interest inside cells, allowing specific mutations or deletions to be made in dysfunctional genes, possibly at a higher efficiency than was possible with the older virus-based tools.

The first tool is known as the ZFN, or zinc finger nuclease, developed by Sangamo BioSciences in Richmond, CA (disclosure: members of my lab are collaborating with Sangamo to test ZFNs in human cell lines and recently published an article on their research). This is a combination of two different proteins, the zinc finger transcription factor and a DNA-modifying endonuclease. The zinc finger is what targets a specific three-letter DNA sequence (codon) in the genome, and the endonuclease makes a cut at that specific region. This can be used to both disrupt a gene and add to a gene. Sangamo has licensed its technology to Sigma-Aldrich, which sells ZFN constructs for $25,000 apiece.

The second tool, called the Genesis system, is based on adeno-associated viruses (AAVs) and has been developed by Horizon Discovery in the UK. Their technology, named Genesis, seems to be more flexible than the ZFNs in that they can add genes as easily as they can delete them. The technology uses AAVs that contain single-stranded DNA sequence that is homologous to the sequence of the target gene. This company has not licensed its technology to a large biotech, but it has collaborated with Novartis on a pilot study and is working with a number of academic institutions (called Centers of Excellence) to use this technology. Horizon Discovery seems to collaborate with these academic institutions free of charge, but it charges industry clients between $20,000 and $50,000 per cell line, depending on the level of customization.

The third tool has been developed by the French company Cellectis. This technology, similar to ZFNs, is called transcription activator-like effector nucleases, or TALENs. They make use of enzymes called meganucleases, which cut at specific DNA regions. The major difference is that TALENs can target individual DNA nucleotides rather than the three-nucleotide codons that are targeted by ZFNs. Cellectis offers TALEN constructs for $5,000 and up.

Together, these new technologies represent the next leap in human genetic disease (and animal model of human disease) research. These technologies could solve some of the problems plaguing gene therapy research, and they could set us forward many years in our ability to precisely modify human and mouse genes for the purposes of treating and curing diseases.

Interesting reading:

Sangamo BioSciences
Horizon Discovery