The process of beer-making is complicated. The brewing process has an array of steps that need to be followed fairly precisely or you risk ruining the flavor of your concoction. A general description of the process is that brewer’s yeast, usually specially cultivated strains of the model organism Saccharomyces cerevisiae, is used along with sugars from chosen grains in order to ferment them into ethanol and the other flavors that make up your typical beer.

The Problems With Hops

Hops are a critical component for most beer makers, as the compounds in the flowers of the Humulus lupulus plant are what give beer its characteristic bitter flavor and the majority of its fragrance. This is why beer with hops are commonly referred to as having a “hoppy” flavor profile. And these types of beers have only become more popular over time, leading to more farmers planting hops in order to sell to breweries.

This isn’t actually a good thing, however, even with the increase in domestic sales prices. Hop plants require a significant amount of water and irrigation to grow and they have to be grown rather particularly if you want to even approximate a consistent flavor production. So, in terms of environmental impact, they are firmly in the negative. Add to this that said flavor is largely not able to be consistent over time, due to differences in how much of the essential oils in the flowers are produced year by year, meaning that beer brewed with hops tastes at least a little altered with each batch. When trying to sell beer to a general market, this isn’t a good thing.

Due to this, a fair amount of research has gone into hops in order to identify what the specific compounds are in the oils that impart that characteristic flavor. It hasn’t been an easy taste, due to the cornucopia of compounds found in the oils. But, over time and much effort, the determinants of flavor were narrowed down to two terpenes, linalool and geraniol. They appear to be the basis of the aroma and flavor that hops present in beer and it was also found that they are the main mixture used in the most popular brand of hops in the US known as Cascade hops.

With that figured out, the next step was to find out how to produce those compounds separate from the hop plant itself. Luckily, there was plenty of insight on how to do that already. Experiments with wine yeast in the past had succeeded in making monoterpene compounds such as those by overexpressing a transgene for them in yeast. However, that was only with one compound. Producing both and in the right quantities to make a precise balance of flavor would be more complicated.

Synthesizing Some Synthases

Scientists at the University of California and the Lawrence Berkeley National Laboratory felt up to the task though. They first sought out the synthases responsible for making the two aforementioned compounds, as the genes and enzymes for those two in particular had yet to be identified in the hop plant. Though they had been found in other unrelated plants, but the question is whether they would produce enough of it or even any of it at all when expressed in yeast.

Six separate linalool synthase genes from a variety of plants were taken and expressed in a lab strain of yeast. But the experimental groups for each one were not able to have enough gene and protein activity in order to meet the needed concentrations. The main reason for this is that these monoterpenes are usually produced in the chloroplasts of plants, where the N-terminal end is cleaved to make the mature protein. Without this cleavage, the proteins are only able to work at a minimal level of activity.

The method to know where to cleave on the protein hasn’t been perfected yet, as the answer varies between plant species. So the researchers continued looking through linalool synthase genes and used a bioinformatics algorithm to try and predict the proper truncation site, along with finding a synthase that had a high amount of activity. They finally found what they were looking for in a tomato cultivar named Lycopersicon esculentum. A similar search for geraniol synthases found another suitable gene for that purpose.

Now that the synthases with a high enough activity in yeast had been found, next was to move beyond the lab yeast strain that was being used and try to do the same monoterpene production in a specially chosen brewer’s yeast. They had to take care to ensure production also continued during the process of beer fermentation.

Finalizing the Hops-Less Beer

A number of pathways were investigated to find limiting steps in the process of terpene production and several genes were overexpressed in order to provide plenty of the pre-curser molecules needed to make the terpenes. To get around the difficulty of making such a multi-gene transgene construct, they used an existing toolkit in the Golden Gate genetic building blocks assembly method.

The last problem they came across was delivering all of these desired transgene into the brewer’s yeast. The size at over 10 kilobases was too large for older methods to accomplish, so they used a Cas9-mediated method in order to integrate the transgenes, along with using a colorimetric visual assay so selective markers weren’t needed in order to confirm successful integration.

Some refining of the brewer’s yeast and several generations later and the researchers had accomplished their goal. A comparison of consistency between the yeast and hops sent in from different farms around the US, all of which were meant to be made for consistent Cascade hops type beer, showed that only the yeast managed to have consistent combination levels of the terpenes between multiple samples.

A Taste of Success

The final test was to see if beer brewed using the yeast and no added hops tasted as the beer should. A blinded panel of taste testers was used, along with different developed strains of the yeast with variations in terpene output. The testers confirmed the beer has the “hoppy” favor they wanted and that each of the beers had a unique flavor profile.

Overall, the scientists noted that this only forms a foundation for future research and terpene producing brewer’s yeast production. The two terpenes used make up just a simple form of the flavor from hops, with there being many other aroma and flavor compounds left to incorporate to make alternative kinds of hops-less beer. They hope that future yeast biosynthesis research will be able to improve on their work and make an even broader amount of hops molecules for use in beer brewing.

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Photo CCs: Hops-1 from Wikimedia Commons

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