Bottom fermentation and top fermentation

Lagers are by far the most common type of beer. More than 90% of the beer brewed in the world and over 99% in Japan is lager. Lager beers are produced by bottom-fermenting yeast*. Because of its ubiquitous popularity, Suntory chose to sequence the genome of the bottom-fermenting Saccharomyces pastorianus Weihenstephan Nr.34.

Hybrid yeast

Beer yeast (scientific name: Saccharomyces pastorianus) is actually a hybrid of two different kinds of yeast: Saccharomyces cerevisiae (most commonly used as baker’s yeast) and the related species Saccharomyces eubayanus. Beer yeast has an extremely complicated genome structure.

The beer yeast genome

What is the difference between the beer yeast genome and that of the already-sequenced Saccharomyces cerevisiae?

Our genetic research yielded two beer yeast sequences: the S. cerevisiae (Sc) sequence, which is very similar to the S. cerevisiae genome (homology: ≥ 98%) and the non-S. cerevisiae (non-Sc) sequence (also referred to as the S. eubayanus (Sb) sequence), which shows about 80% homology to S. cerevisiae (Sc) sequence. We also identified a large number of Sc/non-Sc chromosomes produced by translocation between Sc and non-Sc sequences (shown by the combination of blue and orange in the figure titled “Beer Yeast Chromosomes”). The findings indicate that translocation between many heterologous chromosomes occurred after the natural hybridization between S. cerevisiae and related species, which led to the production of our modern beer yeasts with their complex chromosomal structures.

Suntory develops the world’s first DNA microarray for beer yeast

A DNA microarray is a chip that has a collection of target gene fragments attached to a surface several centimeters in size (see photo at left). It allows scientists to simultaneously analyze the expression of extremely large numbers of genes. Using information from the sequenced genome, Suntory developed a DNA microarray that can monitor the expression of all 12,000 or so beer yeast genes.

The use of this microarray allows us to uncover the relationships between beer yeast genes and the resulting beer flavors. Analyzing with the chip has also allowed us to classify beer yeasts and examine changes in their genome structure through genetic diagnostic techniques.

Benefit #1 Select the best yeasts to make delicious beer

Genetic diagnosis allows us to select the best yeast for producing delicious beers. We can also determine the ideal fermentation conditions for these yeasts by identifying the genes involved in brewing and their functions. As an example, a beer yeast–specific gene (non-Sc-type SSU1) has been found to contribute substantially to the production of sulfite, which is a natural flavor stabilizers.

Advanced genome mapping: Analyzing the functions of beer yeast genes

Because we have now mapped the entire genome for beer yeast, it is now easy to obtain information on beer yeast genes. This information allows us to analyze the functions of characteristic genes to come up with a group of them that contribute to the fermentation traits. We can also get clues on how to ensure the reliable production of delicious beer by identifying the control mechanisms for these genes. Most importantly, bottom-fermenting yeast produced by the natural hybridization between S. cerevisiae and related species has a set of genes derived from an ancestor that it does not share with top-fermenting yeast. We can assume that it is this distinctive set of genes that give bottom-fermenting yeast its unique characteristics.

Unique sulfite production mechanisms in beer yeast

1. Oxidation is the enemy of good beer

Oxidation is one of the key factors in deteriorating beer quality. When various substances in beer react with oxygen, the beer loses its freshness and develops unpleasant tastes and flavors. Decreasing the oxygen concentration in the brewing process reduces the risk of oxidation, but it also inhibits yeast activity, making it extremely difficult to drop oxygen concentration while maintaining good fermentation. The challenge is developing ways to prevent oxidation while maintaining robust yeast activity.

2. Sulfite produced by beer yeast are natural flavor stabilizers

Sulfites are used as antioxidants in everything from foods and beverages to pharmaceuticals. Our research revealed that the concentration of sulfites in beer correlates to consistent flavor and taste. Sulfite, which are produced by beer yeast, therefore act as natural stabilizers that ensure the most delicious brews.

3. How do sulfite work, and how can we produce more of them?

Sulfite are produced as an intermediate metabolite in the synthesis of substances containing sulfur, such as methionine and cysteine. It has been reported that sulfite is increased by overexpression of the genes upstream of sulfite synthesis and suppression downstream. However, sulfite themselves actually damage yeast because they are powerful deoxidizers, meaning that excessive synthesis of sulfite interferes with good fermentation. Our challenge was to find a mechanism by which the sulfite produced in yeast cells could be efficiently excreted from them.

In 1994, Xu et al. discovered the SSU1 gene. Subsequent analysis clarified that SSU1 gene products were localized on cell membranes, and they functioned as sulfite pump (Park and Bakalinsky, 2002). This led us to suspect that the SSU1 gene might affect the production of sulfite in beer as well.

4. Powerful sulfite pump unique to beer yeast

The top-fermenting yeast used to produce ales produces relatively few sulfite, while the bottom-fermenting yeast used for lagers can produce 10–20 ppm of sulfite. As indicated earlier, bottom-fermenting yeast has a set of genes derived from an ancestor strain that hybridized with S.cerevisiae. We compared the effects of cerevisiae-derived (Sc-type) SSU1 genes with those of SSU1 genes unique to bottom-fermenting yeast in order to analyze sulfite production.

Experimental method

The most effective way to conduct a functional genetic analysis is generally to estimate the functions of target genes by examining changes that occur after disruption or overexpression. We therefore prepared strains in which Sc-type and non-Sc-type SSU1 genes were disrupted or overexpressed, and then conducted beer fermentation tests using these strains.

Results and discussion

Disruption in each SSU1 gene revealed that the examined beer yeast had two Sc-type and three non-Sc-type SSU1 genes. We prepared strains in which we reduced the number of a single gene by one, two, or three, and also prepared strains in which each of the SSU1 genes were overexpressed, using strong promoters that trigger constitutive expression.

With these strains, we conducted fermentation tests using wort, and then compared the amount of sulfite production.

The strain missing the two Sc-type SSU1 genes (strain B) produced almost the same amount of sulfite as the parent strain, but the strains missing two or all non-Sc-type SSU1 genes (strains D and E) produced little sulfite. Because yeast proliferation was slow in strains D and E and fermentation did not progress well in these strains, we reconfirmed that efficient excretion of sulfite out of yeast cells was important for yeast proliferation and good fermentation.

Further, the strain in which Sc-type SSU1 genes were overexpressed (strain F) produced 1.5 times more sulfite than the parent, and the strain in which non-Sc-type SSU1 genes were overexpressed (strain G) produced about 4 times more, suggesting that the non-Sc-type SSU1 genes and their gene products substantially affected sulfite production in beer yeast.

Benefit #2 Using genetic diagnostics to analyze yeast health

Genome sequencing tells us that beer yeast is an unstable organism at the genetic level. Changes in genome structure at breweries can delay fermentation and lead to undesirable flavors and tastes.

To address this concern, Suntory created a way to monitor structural changes in the yeast genome using a DNA microarray. We also developed a method of detecting these structural changes even faster and more easily than with the DNA chip, and used it to set up a system to monitor these changes at our breweries.

How beer yeast developed

Beer yeast acquired its complex chromosomal structure as the result of natural hybridization between S. cerevisiae and related species and subsequent translocations between many heterologous chromosomes.

Using a DNA microarray to compare chromosomal structures

We use enzymes to cleave genomic DNA and hybridize the resulting fragments with a DNA microarray in order to approximate the structure and number of the respective chromosomes.

For example, this method identifies how some kind of stress to the Y chromosome structure passing into the X or Z chromosome structure triggers structural changes in the strain.

We also developed a method called the MD plate method, which is able to monitor structural changes in the yeast genome at breweries even faster than the DNA microarray.

We can distinguish healthy yeast (white colony) from variant yeast (red colony) on an MD plate.

Conducting these “health checkups” for yeast allow us to create better conditions for our yeast, which in turn creates a more delicious product for our customers.