IRCAEB Dept. of Basic Research, Tokio Tani

Research PR Showcase Vol.4 will feature the work of RNA researcher Prof. Tokio Tani, on the creation of novel alcoholic beverages using fission yeast.

Did you know that almost all of the alcoholic beverages in the world are fermented using an organism called budding yeast?

“Fermentation” is a food-processing technique utilized by mankind since antiquity, with many types of fermented foods still consumed today. Nattō, vinegar, miso, wine, yogurt, cheese and kimchi are all examples of fermented food products.

Fermentation is basically a specialized form of respiration. When oxygen is available, it is used to break down sugars and generate energy, while ejecting carbon dioxide as a by-product. Fermentation refers to the break-down of sugars in the absence of oxygen, which generates less energy in return. Pending on the by-products made, fermentation can be divided into alcoholicand lactic acid fermentation. Even though humans require oxygen to live, humans can still perform lactic acid fermentation. Muscle pain after strenuous exercises is the consequence of lactic acid fermentation when oxygen level is low.

Of course, fermentation is also of interest because the by-products can be useful to humans. Many types of break-down processes similar to fermentation occur in nature, though processes that produce by-products harmful to humans are termed “decomposition”. In another word, humans are known to purposefully grant cultural significance to natural break-down processes that occur by chance, by modifying them in incorporate them into our everyday lives.

The raw ingredients for making alcoholic beverages are very diverse. Japanese sakeis made from rice, shōchū from barley, sweet potato or rice, beer from malt and hop, and wine from grapes. All of these require fermentation by budding yeast, the very same microorganism known for making breads. Yeasts are related to molds, and are defined assingle-cell organisms that possess cell walls and undergo alcoholic fermentation. Based on recent research on natural yeast-made breads, yeasts can be found essentially everywhere. In particular, environments rich in sugar such as the surfaces of fruits, flowers and tree saps are often found to be colonized by yeasts. As such, the discoveries of yeast-mediated alcohol fermentation of rice or grapes are most likely coincidental. Even though yeasts are single-cell organisms they are not bacteria, but are eukaryoticorganisms like us humans. Over the long history of human-yeast interaction, humans have become skilled with yeast-handling, and now use yeasts as a model organism in life science.

Two types of yeasts, budding yeast and fission yeast often used in laboratories. Unlike budding yeasts that produce small daughter cells through budding, fission yeast cells split into two daughter cells of equal size. Fission yeast (Schizosaccharomyces pombe) has been used to brew traditional African millet beer, but aside from this exception essentially all alcoholic beverages in the world are made with budding yeast. Specific yeast strains determine the distinct tastes of alcohols from different breweries, with changes in the strains (i.e. changes in the yeast’s genome sequences) profoundly affecting the flavor and aroma. Since no one has attempted to brew alcohols using fission yeasts, the Tani laboratory have decided to take up the serendipitous challenge.

Gene activity starts with the synthesis of RNA from DNA (transcription), then the conversion into amino acids to form proteins (translation). During this process, mRNA molecules are translocated from the nucleus to the cytoplasm. The Tani laboratory studies this process using the fission yeast Schizosaccharomyces japonicus, which was discovered from strawberries in 1928, and is one of the four currently known fission yeast species. Unlike budding yeast, the fission yeast genome is complex similar to the human genome. This gives fission yeasts the advantage when addressing questions that cannot be solved using simple genetic systems. Furthermore, fission yeast cells are larger than budding yeast cells, making the visualization of fluorescent proteins easier.

For research, large fission yeast cultures are often grown in flasks in 26°C overnight, shaken constantly to oxygenate the culture. One morning when Prof. Tani came into the lab, he found the incubator was broken and the flask-shaking machineries were stopped last night. When the culture flasks were opened, a wonderful scent filled the room. Apparently ithese yeasts underwent alcoholic fermentation in the absence of oxygen. Prof. Tani wondered how would alcohols taste if brewed with fission yeasts instead. This marks the beginning of the Kumamoto University artisanal brewery project.

First, the fermentation capacity of fission yeast was tested by using fission yeast to rise bread dough. Even though the fermentation activity of fission yeast wasn’t remarkable, it was enough to produce gas and make normal-looking bread.

Next comes the fun part of alcohol-brewing! But first, do you know what makes alcoholic beverages delicious? Even though all Japanese sakeare made from rice, they still come in many varieties each with distinct flavors. These differences are due to variations in the starting ingredients (variety of rice and degree of rice grain polish) and the brewing process (temperature, time, etc.). However, even sakebrewed from the same rice using the same process can be vastly different, depending on the yeast strains used. If such diversity in alcohol can already be created with different budding yeast strains alone, what sort of exotic drinks can fission yeast, which is not even closely related to budding yeast, possibly brew?

The aroma of Japanese shōchū and sake are often described with the term “ginjōka” (吟醸香). Currently 3 types of aromas are used to characterize Japanese liquors: appleginjōka (scent of Daiginjo sake), bananaginjōka, and roseginjōka. With the compounds responsible for each aroma identified, the aroma of liquors can be quantifiedby measuring the concentrations and balance of these compounds.

First fission yeasts were mutagenized to isolate strains that produce the desired scent in excess. For example, the compound responsible for apple ginjōka is ethyl caproate, therefore we aim to find mutants that make more ethyl caproate than usual. During ethyl caproate synthesis, intermediates are converted into the final product in step-wise fashion by a series of enzymes (i.e. metabolism). After fission yeasts are randomly mutagenized, some mutants have these ethyl caproate synthesis enzymes disrupted. Among these strains, some can synthesize ethyl caproate using a different pathway, others have mutations that mask the defects in ethyl caproate synthetic enzymes. Out of the 100 million yeast cells mutagenized, 36 mutant strainswere selected. In order to identify mutants that produce ethyl caproate in excess, the metabolite profiles of these mutants were analyzed.

Two mutant strains with sufficiently high alcohol fermentation and growth rates, CR11 and CR28, were re-named Kumadai-T11 and Kumadai-U28, respectively. Preliminary shōchū production test-runs were done with these two strains in collaboration with the Kumamoto Industrial Institute. This was to test whether the mutants can ferment actual brewing ingredients instead of culture media used in laboratories, and produce the desired aroma. The results showed that these two mutant strains indeed produce more aromatic compounds than budding yeasts traditionally used for brewing. Sensory test results suggest Kumadai-T11 produced shōchū superior than those made with budding yeast in terms of aroma and taste, even though identical starting ingredients and brewing procedures were used.

Once the preliminary tests were finished, Prof. Tani considered scaling up the production for commercial purposes. Due to the lack of appropriate equipment it’s not practical to brew alcohol at the industrial scale in the university, and so we sought out collaboration opportunities with commercial breweries instead. However since yeasts are microscopic organisms, introducing a lab-grown yeast strain to a brewery remains challenging. It is difficult to know how the introduced fission yeast will interact with the brewery’s original budding yeast, and whether the introduced fission yeasts can be completely removed from the brewery equipment afterward. These are exceptionally important, as contaminations may permanently change the flavor of the brewery’s other products. Kumadai-T11 does not propagate efficiently in breweries (the cells divide but do not form spores). Nevertheless, many brewers were still concerned about the Kumadai strains contaminating their breweries.

Fortunately we were able to establish a collaboration agreement with Amakusa Shuzo, a Kumamoto-based shōchū brewery. After a year of hard work, the world’s first sweet potato shōchū brewed using the fission yeastS. japonicus strain Kumadai-T11 was created. The shōchū was christened “Ikenotsuyu Yushima The Highest Yeast” and hit the shelf in the spring of 2022. With a unique aroma not found in budding yeast-brewed shōchū, it was sold out almost immediately. Amakusa Shuzo plans to restock this spring, please don’t miss this opportunity to pick one up and try it for yourself!

After the success of Kumadai-T11, next we plan to collaborate with the Kumamoto-based craft beer maker Diamond Brewing to develop other yeast products, and the craft beer preliminary tests are currently underway. The prototype was imbued with a lovely ginjōka fruity aroma, and we foresee the end product to be commercially available by the spring of 2023.

As mentioned earlier, currently the vast majority of alcoholic beverages are brewed with budding yeast, yet we have discovered that brewing alcohol with fission yeast vastly improved the taste and aroma. In pursuit of the unknown culinary experiences, the Tani laboratory will continue to strive and seek out more novel yeast strains with fascinating aromatic products.