COVER STORY TAPPING YEAST S GENOME

Transcription

1 CVER STRY TANKS FR THE DRINKS Fermentation tanks are where beers get their alcohol, along with hundreds of others aromatic and flavorful compounds. SHUTTERSTCK TAPPING YEAST S GENME ACS MEETING NEWS: Advanced technology is bringing new science to the ancient art of beer brewing MATT DAVENPRT, C&EN WASHINGTN N THE SURFACE, Colorado brewmasters Keith Villa and Max Filter are very different people. The clean-shaven Villa, founder of Blue Moon Brewing Co., sometimes rides to work in his purple limited-edition Dodge Challenger. Filter, the dude of brews at Renegade Brewing Co., sports a gnarly brown beard and drives a dusty black Toyota 4Runner. Villa s SandLot brewery sits just past right field in the 50,000-seat Coors Field, home of Major League Baseball s Colorado Rockies. Filter s is located in a gravel lot across from what used to be a lumberyard. Cabdrivers become incredulous when passengers ask to be left at the address. Blue Moon opened in 1995 and produces about 2 million barrels of beer annually, or roughly 4 million kegs, operating within the MillerCoors beer conglomerate. The independently owned Renegade, founded in 2011, filled about 1,250 barrels in C&EN visited Villa and Filter to learn about modern beer brewing last month during the American Chemical Society national meeting in Denver. Despite their obvious differences, the two men have the same passion and wonderment for beer and the brewing process. It s tempting to think that they share some sort of brewers genes. They do, in a way. Those genes just belong to yeast. Yeast are perhaps the best understood organisms on the planet. Scientists have been studying the microscopic eukaryotes CEN.ACS.RG 8 APRIL 20, 2015

2 for decades and have correlated nearly 80% of their genes with specific functions. Now, as DNA-sequencing technologies become faster and more affordable, scientists are exploring and exploiting the genetic codes of yeast like never before. Brewers such as Villa and Filter are capitalizing on this growing genetic knowledge base to exercise better control over their product. And scientists may soon deliver designer yeast strains to brewers, who could in turn serve up engineered beers along with the sticky questions that accompany genetic tinkering. BEER ME Much of a brewer s job boils down to making yeast happy, Filter told C&EN as he sipped a velvety imperial stout inside his brewery, surrounded by palettes of empty cans. Yeast make the beer, he said. Yeast are our partners. Brewers hold up their end of the bargain by making wort essentially sugar water that feeds the yeast and serves as fuel for fermentation. Fermentation s fundamental equation is simple: Yeast plus sugar yields alcohol. Yeast metabolize sugar molecules glucose, maltose, and maltotriose to produce ethanol and carbon dioxide, Filter said as C 2 bubbles percolated noisily from nearby fermentation tanks. The organisms also contribute hundreds of flavorful and aromatic compounds to beer. As millions of yeast cells at Renegade performed the reactions their ancestors honed over millions of years, chemists and craft brewers congregated a few miles north of the brewery at the ACS meeting to discuss how new tools and techniques could advance the practice of beer making. ne session began with a look back at the ancient symbiosis between yeast and humanity, a relationship that likely started MRE NLINE How do yeast and brewers crank up the alcohol content in beer? Visit brewing to find out. MATT DAVENPRT/C&EN by accident, said Robert A. Sclafani, a biochemist at the University of Colorado (CU), Denver. He and Carrie Eckert of the National Renewable Energy Laboratory organized a symposium on Colorado s craft brewing industry for the Division of Biochemical Technology. Sclafani opened the session by hypothesizing the origin story behind humanity s alcoholic endeavors, which began thousands of years ago, probably with a piece of broken fruit. Yeast would have covered the skin of the fruit Sclafani imagined Yeast turn out to be the best model organisms out there. it as a peach because wild yeast will cover anything filled with sugar. When Sclafani s peach fell from its tree and smooshed on the ground, yeast would have swarmed to the liberated juice and fermented like crazy, he suggested. As the yeast produced ethanol and other volatile chemicals, Sclafani posited, someone must have walked by and thought, Hey, that smells pretty good. From this happy accident, wine was born. Humans then rounded out their liquor cabinets, making beer and spirits by replacing wine s fruit with malt grains whose starches had been broken down into sugars by their own enzymes. When humans began intentionally fermenting fruits and grains, they introduced an element of artificial selection to yeast s evolution. Certain wild strains were better suited for different libations, and alcohol makers played favorites. Distillers want yeast that make lots and lots of alcohol, Sclafani said. Beer and wine makers want something different. As humans unwittingly but intentionally recruited specific yeast strains for specific beverages, the strains mutated differently, influenced by their fermentation conditions. Brewer s yeast thus adapted to brewing. Armed with a modern understanding of genetics and fermentation, today s scientists are more direct in their manipulation of yeast. Researchers have engineered yeast to produce biofuels, vaccine candidates, and biological pharmaceuticals. Remarkably, the same species of yeast, Saccharomyces cerevisiae, a name derived from a Greek phrase meaning sugar fungus, can make ales, fuels, wines, spirits, medicines, even bread. ne notable exception is lager beers, which include pilsners and bocks. These rely on a different yeast species: a hybrid in the Saccharomyces genus that ferments better at lower temperatures than its ale-making counterparts. Although industrial yeast is largely uniform at the species level across fermented beverages, there is great diversity at the strain level. There s even significant genetic diversity within the branch of S. cerevisiae that s come to be known as brewer s yeast. As of early April, the yeast distributor White Labs offered more than 30 ale yeast strains to professional brewers. Different strains bring different characteristics to the fermentation tank different aromas, flavors, and alcohol tolerances. And all of these readily observable differences are rooted in genomic variation between strains. Some of Colorado s craft brewers have already taken advantage of this genetic variation to keep undesirable microbes out of their beer. DN T YU MEAN REAL-TIME PBR? In brewing, it s common knowledge that there are a lot of contaminants that can ruin a beer, said Dan Driscoll, a microbiologist with Avery Brewing Co. during a presentation at the Denver meeting. Bacteria and wild yeast can infiltrate fermentation tanks and fill beer with foul flavors. Avery keeps its tanks as isolated from the outside world as possible, minimizing the risk posed by wild yeast and bacteria. During his presentation, Driscoll revealed that Avery s biggest contamination risk is its own yeast. Avery puts out more than 30 beer varieties every year using six different yeast strains. For comparison, Blue Moon s Villa said he sticks to two strains. At Avery, Driscoll said, we pride ourselves on using a lot of different yeast and turning our fermentation tanks around quickly. nce Avery completes one beer, it can start another beer of a different style in the same tank. The brewers sterilize the CEN.ACS.RG 9 APRIL 20, 2015

3 CVER STRY 5a Yeast cells consume oxygen, nitrogen, and sugars. Glucose is their main food source, but yeast can also digest maltose and maltotriose. Additionally, yeast gobble up and metabolize the fatty acids from hops as well. 5b Di erent strains handle the nutrients di erently, but all brewer s yeast have genes that encode enzymes to convert sugar into ethanol and C 2. There are as many ways to make beer as there are breweries, but every brewer follows the same basic process. Here s how beer makers turn amber waves of grain into amber ales, IPAs, and other beer styles. H CH 2 H H H H Glucose Yeast CH 2 H H H H Maltose CH 2 H H H H CH 2 H CH 2 H H H H H H Maltotriose CH 2 H H H H Metabolism H H C H C H H H Yeast C 2 Barley/malt Water Hops Yeast Cooler Mash tun Mash kettle Lauter tun Brew kettle C&EN/J. BISCI. BIENG./SHUTTERSTCK 1 Mashing Brewers need sugar to feed the yeast. This sugar comes from malted grains grains that have started germinating and producing starch-processing enzymes. Brewers mill the grains and toss them into warm water, where enzymes break the grains starches down into sugars. This process is called mashing. 2 Lautering The lauter tun separates grain husks from sugary liquid. This liquid is next fed to a brew kettle. 3 Boiling Brewers boil the liquid, called wort, inside the brew kettle, along with hops and other flavorful ingredients. Hops bestow a bitterness to beer, along with fatty acids that yeast can metabolize to create more flavors and aromas during fermentation. 4 Cooling After the wort is boiled, brewers remove the solids from the liquid. The liquid is cooled to the optimal fermenting temperature for yeast. Ale strains ferment best around room temperature (20 C); lager strains function better at lower temperatures (10 C). 5 Fermenting The fermentation tank is where the magic happens: Yeast meet the wort and turn the sugary stock into alcoholic beer. nce the fermentation is complete, yeast aggregate and settle out of the liquid. Brewers can pull beer o the tank s cylindrical top and reclaim yeast from the tank s conical bottom to use in the next fermentation. 6 Finishing/Bottling Brewers might filter their beer and/or store it in resting tanks to age and fully develop its flavors, but the liquid that leaves the fermentation tank is drinkable beer. tanks between beers, but it s not always 100% effective. For instance, a yeast strain used to brew Avery s Belgian witbier could linger in a tank that s slated to brew India pale ale (IPA). The brewery s witbier strain produces a compound called 4-vinylguaiacol, which tastes and smells of cloves. Cloves, along with other spices, can add delightful dimensions to wheaty witbiers, but they detract from hoppy IPAs. Although it s a rare occurrence, a witbier strain could hide out in an IPA tank until beer tasters detect the vinylguaiacol with their mouths or noses, which could be weeks into the brewing process. Avery has to dump contaminated batches, but it could save time and money if it caught the problem sooner. ne method capable of detecting rogue strains before they change a beer s profile is real-time polymerase chain reaction. Real-time PCR makes copies of low-level contaminant DNA in a sample and uses fluorescent probes to signal its presence. The technique can thus look for low-level biological contaminants. Researchers have already identified the CEN.ACS.RG 10 APRIL 20, 2015 gene that codes for the enzyme responsible for producing 4-vinylguaiacol. That enzyme is called phenylacrylic acid decarboxylase, and its gene bears the name PAD1. PCR techniques could thus ferret out a foreign strain, but Driscoll still needed the specific biochemical tool kit to detect an invader s PAD1 sequence. Developing PCR assays is costly, especially for a craft brewery, so Driscoll turned to a time-tested tenet of academia. I know from personal experience that grad students really like beer and getting a new project every once in a while, said

4 5c Yeast can also produce flavor and aroma compounds more than 500 of them. Here are some of the more common esters: Ethyl acetate fruity, solventlike Isoamyl acetate banana, pear Phenyl ethyl acetate roses, honey Fermenting tank 5 6 Used yeast SINCE % ABV Proudly Brewed in Washington, D.C. Ethyl caproate apple, aniseed Ethyl caprylate apple Driscoll, who graduated with a master s in microbiology from regon State University and spent several years at biotech startups before finding a home at Avery. ne night, Driscoll grabbed a beer with members from the Next-Generation Sequencing Facility at CU Boulder s BioFrontiers Institute. The conversation turned to Avery s cross-contamination problem. That led to CU Boulder genetics and computational biology researcher Robin D. Dowell looping in professional research assistant Phillip A. Richmond, who was eager to tackle the problem, pro bono. YEAST 101 With an average diameter around 5 µm, yeast are larger than most bacteria. Yeast, like humans, are eukaryotes, meaning they store their genetic material inside a cell nucleus. Humans have 23 chromosomes; yeast have 16. For example, brewer s yeast often display what s known as aneuploidy. They may have one or two copies of one chromosome, but three of another. In humans, aneuploidy can cause birth defects. It s also a common trait in cancer cells, but it s not a problem for simple brewer s yeast. I like beer, Richmond told C&EN. Richmond, who had the capability to sequence genomes rapidly, developed a test that can differentiate between yeast strains based on their PAD1 sequences. He s preparing to submit the protocol and analysis for publication in the Journal of the American Society of Brewing Chemists. Sequencing each yeast strain s 12 million base pair genome is the biggest resource sink, he said. But it is much cheaper and much quicker than it would have been a decade ago. Richmond is confident he will soon extend the assay to uniquely identify contamination from any of Avery s yeast. With his expertise and the technology at his disposal, he added, six strains is kind of a cakewalk. Driscoll doesn t believe this sort of PCR-backed quality control is the right fit for every brewery. It s expensive, and most breweries don t capitalize on yeast diversity as aggressively as Avery does. But the idea of brewers working with academics is one he hopes catches on. Yeast still hold many mysteries, and many of those are bound to have an impact on beer. Brewers should contact academia, Driscoll said. That s the best idea ever. TASTE THE STRAIN-BW Academia has a pretty solid understanding of yeast. The organisms genes are some of the best-studied DNA sequences on the planet. That s in part because many of those genes, and the biochemical functions they orchestrate, are also found in higher eukaryotes, humans included. Yeast turn out to be the best model organisms out there, CU Denver s Sclafani said. We can take them apart and put them back together. Researchers have used biochemical tricks to disable, or knock out, just about every gene inside yeast to get at what their functions are, he went on. We can t do that with humans. Yeast studies have helped researchers understand human health and disease and have garnered multiple Nobel Prizes. The flip side of this historical, humancentric interest in yeast is that there is a lot of unexplored territory in yeast s genome as it relates to brewing. An international collaboration that spans academia and industry is now trying to map that ground. These researchers from the University of Leuven; Flanders Institute for Biotechnology (VIB); the yeast distributor White Labs; and Illumina, a pioneering company in next-generation sequencing are sequencing the majority of brewing strains and annotating these sequences. That is, they are identifying what different chunks of the genome do. Then they will try to decipher how those data correlate with how different strains brew in different conditions, providing a fuller understanding of when and how yeast impart different characteristics to beers. It s the microbiology and chemistry of yeast that makes beer beer, said Troels Prahl, head of White Labs research and development. All this work is driven by the love of beer and the love of science. The international team is working toward publishing some initial results for about 200 strains. It ll be very rewarding to offer those data to the community of brewers and brewing scientists, Prahl told C&EN. With this knowledge, he and his colleagues can recommend the yeast that will best accommodate a brewer s tastes. It s like being a librarian and having read all the books, he said. DAVID SCHARF/SCIENCE SURCE CEN.ACS.RG 11 APRIL 20, 2015

5 CVER STRY Illumina s HiSeq high-throughput sequencing systems have helped get this massive undertaking off the ground. The sequencing is very fast and inexpensive, said Clotilde Teiling, a marketing programs manager at Illumina. The first sequencing run she and a colleague performed with yeast took about three days and cost roughly $3,500, she said. That run provided data on 96 yeast strains. All told, the project has now accounted for about 500 different beer flavor profiles influenced by yeast, said Kevin J. Verstrepen, who studies yeast genetics and genomics at the University of Leuven and VIB. We think we ve pretty much covered the major part of diversity in brewing yeast, Verstrepen said. In the family tree of yeast, brothers and sisters often company Sapporo has brewed with yeast collected from honeybees, which pick up yeast as they pollinate plants. Brewer s yeast also mutate as they feed on wort and reproduce asexually. As these mutations accumulate, yeast can start to produce different flavor and aroma compounds. Yeast are still evolving naturally, in the wild and during the accidental autohusbandry that is brewing. But, of course, humans are wont to tinker. RENEGADES F FUNGUS We re entering the age of designer yeast, Verstrepen said. With a wealth of genetic knowledge and high-tech sequencing tools, scientists don t have to wait for nature to give them the yeast they want. SEAN PARSNS/ACS PRDUCTINS MATT DAVENPRT/C&EN T THE MAX Filter, the head brewer at Renegade, plays a big role in helping the company live up to its name. share the same flavor or aroma profile, Verstrepen went on, adding that he was taking some subtle liberties with the definition of family tree. But as soon as you go a little bit further to the aunts and uncles they can already have very different characters. Verstrepen and his colleagues didn t organize this project to be the be-all, endall study in brewing yeast diversity. It can t be, he said, pointing out that wild yeast, the myriad yeast strains living in nature that are largely unused in industry, will always be a wild card in adventurous brewers decks. For instance, the Japanese brewing Verstrepen s team has created a genetically modified yeast strain that s programmed to make about 100 times as much of a banana-flavored compound, isoamyl acetate, as natural yeast. We actually brewed with this one, he said. It was like a banana milk shake. It was quite cool. But he stressed that there are also natural ways of tweaking brewer s yeast. The genes of asexually reproducing yeast can be groomed by controlling their growth environment, Verstrepen explained. His group and other groups are working on breeding strains with desirable properties. Researchers led by Kristoffer Krogerus and Brian Gibson of VTT Technical Research Centre of Finland recently created new lager strains by mating S. cerevisiae with another species, S. eubayanus. In addition to tolerating the lower lager CEN.ACS.RG 12 APRIL 20, 2015 ARTS AND CRAFT BREWS Villa relies on science and his senses to brew his Blue Moon flagship and limited-release beers. fermentation temperatures, the hybrid yeast fermented faster and produced more alcohol than either of its parents (J. Ind. Microbiol. Biotechnol. 2015, DI: / s ). We weren t even sure what to expect, said Krogerus, who s also a researcher at Aalto University, in Finland. It was a pleasant surprise when the hybrids outperformed the parents, he said. This is really the tip of the iceberg, Gibson added. Lager brewers have been using one strain for hundreds of years, but we re now in a position where we can generate hundreds of strains, essentially made-to-order. Such natural modifications guided by human hands can take brewers to untapped levels of yeast performance, but these levels are still restricted by nature. Crossbred yeast probably won t make banana milk-shake beers. Exotic brews such as that will likely require more invasive engineering, the kind that s likely to be labeled GM genetically modified. Many scientists are skeptical that anyone will brew on a commercial scale with GM yeast anytime soon, but that s because of the social stigma of the GM tag rather than the availability of GM yeast. Jef D. Boeke of New York University s Langone Medical Center is one of those skeptics. Last year, Boeke and Srinivasan Chandrasegaran of Johns Hopkins Bloomberg School of Public Health led a team of researchers who created the first synthetic

6 eukaryotic chromosome. It belonged to yeast. Boeke said the inspiration for this project was basic scientific curiosity. The team wanted to see whether it could reveal any gaps in the fundamental doctrines of biology by creating a chromosome from scratch. At some level, you hope to fail, Boeke mused. But the team didn t fail, and researchers are getting closer to creating a completely synthetic yeast genome. Emerging third-generation sequencing technologies, such as Pacific Biosciences RS II machine, are letting researchers read longer and longer strings of DNA sequences. This enables scientists to verify synthetic DNA codes more quickly and more routinely, Boeke said. Although researchers are primarily using these tools to answer basic biological questions, there s no doubt brewers could use the knowledge, Boeke said. He just doesn t think they will. Not yet. If it s GM, it s very unlikely to gain acceptance, Boeke explained. Pervading public opinion is that engineered organisms are inherently dangerous. I think that s a ridiculous argument, though, he said, adding that humans have been engineering yeast since they first made wine. Blue Moon s Villa also believes that the public opinion toward genetically modified products will remain frosty for the foreseeable future and that brewers will cater to that. The brewing industry is very, very conservative, he said, adding that Blue Moon does not use GM products in its beers. Villa said that he modified yeast strains during his doctoral studies in Belgium. ne produced three times the isoamyl acetate that banana-flavored compound as natural yeast. But those strains sit on liquid nitrogen, cryogenically frozen and waiting for a future willing to accept beers with GM on their labels. It makes sense to use it, Villa told the audience at the ACS meeting. But I don t think anybody wants to be the first and be singled out for making a GM product. So Villa, who holds a doctorate in brewing, challenges himself to create unique flavors by brewing with natural ingredients such as vanilla, chai tea, lemon grass, orange peel, and even marijuana. He s developing that marijuana beer in his own personal home-brewing setup; MillerCoors will not work with something that s on the 2015 Thermo Fisher Scienti c Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scienti c and its subsidiaries. Drug Enforcement Administration s list of Schedule I drugs. But marijuana is legal in Colorado. Villa wants to have a recipe ready if and when cannabis gains more universal acceptance. It might seem strange that Villa described his marijuana home brew moments after calling the brewing industry conservative for its stance on GM yeast. But sitting in Denver, it was easy to feel like the locals would have a hand in lugging a Luddite industry toward a more liberal future. Maybe brewers are a little different in Colorado. Maybe it s something about their water, cold as the Rocky Mountains. Maybe it s in their genes. Whatever it is, there seems to be an awful lot of renegades around. What can you achieve Today s analytical labs face a host of new challenges. Demands to analyze more trace compounds in less time, while keeping pace with changing regulations. Meeting lower levels of detection while facing constrained budgets and increased competition. Now there s a GC-MS/MS solution to address the changing analytical landscape. The Thermo Scienti c TSQ 8000 Evo uses innovative hardware and software to deliver outstanding productivity, analyzing thousands of trace level compounds with high sensitivity in less time. Evolve with us. with all this speed? Discover more at thermoscienti c.com/tsq8000evo CEN.ACS.RG 13 APRIL 20, 2015