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Enhance thiol biotransformation with Tropics

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Our collection of Tropics strains have been engineered for dramatically enhanced thiol biotransformation. This means that during fermentation, these strains release large quantities of two thiol molecules: 3MH and 3MHA. These two molecules impart strong tropical fruit flavors, and beer made with Tropics strains has strong sensory notes of guava, passionfruit, and grapefruit. On this page, we’ll describe the science behind the creation of our Tropics technology, and we’ll also highlight brewing strategies to maximize the flavors that these strains produce

A primer on volatile thiols and biotransformation

In order to understand how Tropics strains work, let’s first talk about how 3MH and 3MHA molecules are formed, and the role of biotransformation in this process. The formation of 3MH and 3MHA begins in plants. Many plants –including the barley and hops used for brewing– naturally produce a flavorless molecule called Glut-3MH as a byproduct of glutathione metabolism. Some of this Glut-3MH is converted into a second flavorless molecule, Cys-3MH, through the activity of naturally occurring plant enzymes. Finally, a different plant enzyme converts some of this Cys-3MH into the flavor-active thiol, 3MH. This biosynthetic pathway is depicted below.

Figure 2. Biosynthesis of 3MH in plants. Red arrows indicate enzymatic reactions. In barley and hops, the final two reactions are very inefficient.

A critical point here is that the efficiency of each step in this biosynthetic pathway varies greatly across different plant species. For example, in tropical fruits like guava and passionfruit, the conversion of Glut-3MH to Cys-3MH and 3MH is very efficient, and these plants contain a high concentration of 3MH as a result. In barley and hops, the situation is very different. In barley and hops, the enzymes that catalyze each conversion are inefficient, and few 3MH molecules are produced (Figure 2, red arrows). Even in hops that are renowned for tropical fruit flavors, –like Citra and Mosaic – conversion rates are low, resulting in less than 1% of the Glut-3MH converted to 3MH1. A consequence of this inefficient conversion is that barley and hops contain a low concentration of 3MH, but high concentrations of Glut-3MH and Cys-3MH2. In fact, for every molecule of 3MH in hops, there are on average 68 molecules of Cys-3MH, and 865 molecules of Glut-3MH3!
cys-3mh 3mh glut-3mh barley hops biotransformation

Figure 3. Average percentages of Glut-3MH, Cys-3MH, and 3MH, in barley and hops.

The large quantities of Glut-3MH and Cys-3MH contained within barley and hops has recently become a subject of great interest for brewers. The reason for this is that while these “thiol precursor” molecules are flavorless, it only takes one or two enzymatic reactions to convert them into flavor-active 3MH. And because these molecules are so abundant in barley and hops, their conversion to 3MH would release a tremendous amount of tropical flavor. 

This is where yeast, and biotransformation come in. It turns out that some yeast strains express enzymes that are able to convert Glut-3MH and Cys-3MH into 3MH. During beer fermentation, these enzymes can bind to thiol precursors in the wort, then convert these precursors into 3MH! This process – the conversion of plant-derived thiol precursor molecules into 3MH by yeast enzymes – is referred to as “thiol biotransformation”.

thiol biotransformation brewing schematic

Figure 4. Overview of thiol biotransformation during brewing.

Unfortunately, normal brewing strains are not very good at performing thiol biotransformation. The reason for this is that the yeast enzyme that catalyzes the conversion of Cys-3MH to 3MH is a weak enzyme that is not very active. Past studies have estimated that this enzyme only converts about ~1% of the available Cys-3MH into 3MH4. This means that in beers made with standard brewing strains, over 99% of the thiol precursors are never converted to 3MH, and instead remain in their flavor-inactive forms in the finished beer.

Enter CSL, and Tropics 

In 2019, Berkeley Yeast identified an enzyme that is able to convert Cys-3MH to 3MH over 100 times more efficiently than any existing yeast enzyme. We discovered this enzyme, named CSL, in the genome of Citrobacter freundii, a commensal bacterium found in the healthy human microbiome. Using precise genetic modification techniques, we isolated the DNA encoding the CSL gene, then inserted this gene into the chromosomes of a London Ale brewing yeast. In R&D brewing trials with the CSL-expressing yeast, it was immediately clear that CSL dramatically enhanced the efficiency of thiol biotransformation: Beer made with this strain contained 113-fold more 3MH than beer made with a control strain, and its aroma was dominated by guava, passionfruit, and grapefruit flavor notes. After confirming that this new strain was genetically stable and produced no off-flavors, we released it for commercial use under the name London Tropics. In commercial scale fermentations, London Tropics produces between 50- and 500-fold more 3MH than beer made with an unmodified London Ale strain. The exact amount of 3MH London Tropics produces depends on the specific hops and barley used in the brewing recipe (more on this below).

London Tropics 3MH biotransformation 3MHA

Figure 5. London Tropics expresses the CSL enzyme, which converts Glut-3MH and Cys-3MH into 3MH (left panel). 3MH production by London Tropics is generally >100X greater than that of the parental London Ale strain (right panel).

Production of a second volatile thiol, 3MHA

In addition to dramatically increasing 3MH production, London Tropics also produces a second volatile thiol molecule, 3MHA. It does this through the natural expression of an alcohol acyltransferase (AAT) enzyme that forms 3MHA by acetylating 3MH (see Figure 6 below). How does production of 3MHA affect beer flavor? Well, like 3MH, 3MHA also imparts tropical flavors, especially passionfruit and grapefruit. But unlike 3MH, you can smell and taste 3MHA even when it is present at extremely low concentrations. In more specific terms, the olfactory detection threshold of 3MHA is 14-fold lower than it is for 3MH. This means that a single molecule of 3MHA imparts as much tropical aroma and flavor as 14 molecules of 3MH!

 
3mha acyltransferase biotransformation tropics

Figure 6. Diagram of 3MHA production by London Tropics.

Since that 3MHA is such a potent flavor molecule, beers containing both 3MH and 3MHA are brighter, and have more tropical fruit character than beers containing 3MH alone. This improved sensory profile results from the high flavor potency of 3MHA, as well as synergistic effects between these two flavor molecules. So, how much 3MHA does London Tropics make? As is the case with 3MH, it depends on the specific barley and hops used, but anywhere from 50 to 100 times as much as an unmodified London Ale strain.

London Tropics 3MH 3MHA berkeley yeast

Figure 7. 3MHA and 3MH production by London Tropics.

Other Tropics strains

London Tropics is great for making hazy IPAs, but we didn’t want our enhanced thiol biotransformation technology to only be available in a London Ale strain. Recently we added three additional strains to our Tropics line. These are: Chill Tropics (an Augustiner lager strain), Hornindal Tropics (a Hornindal Kviek strain), and Vermont Tropics (a juicy hazy IPA strain). Each of these strains expresses the same CSL enzyme used in our original London Tropics strain, and they each produce similar levels of 3MH. Importantly, each of these strains also expresses an AAT enzyme, and so they also produce 3MHA. 

How to get the most out of Tropics strains

As discussed above, Topics strains produce 3MH and 3MHA through the process of thiol biotransformation. Since thiol biotransformation begins with the precursor molecules Glut-3MH and Cys-3MH, the amount of 3MH and 3MHA produced by Tropics strains depends on the concentrations of these precursors in the hops and barley used for brewing. Importantly, recent research has discovered that the concentrations of these precursors vary widely among different barley and hop cultivars. 
 

In order to help brewers maximize the tropical flavor potential of their beer, we and others are actively conducting research to quantify the thiol precursors present in a variety of different hops and barleys. This work is ongoing, but preliminary results suggest the following: 

  • Even without hop addition, Tropics strains produce over 100-fold more 3MH than control strains, indicating that a majority of thiol precursors are derived from barley.
  • Lightly kilned barleys appear to contain the highest amount of thiol precursors2
  • Hops can also contribute thiol precursors, but to a lesser extent than barley2.
  • Hops like Cascade and Simcoe consistently have high concentrations of thiol precursors1,3. To facilitate biotransformation, add these hops during the whirlpool. 
  • It has been suggested that mash hopping might help convert Glut-3MH to Cys-3MH prior to fermentation, but we strongly recommend against this practice. Tropics strains already express enzymes that convert Glut-3MH to Cys-3MH during fermentation, and mash hopping is prone to creating astringent, bitter, and vegetal off-flavors.

To try out one of our Tropics strains, visit our strains page. For any unanswered questions about these strains, or to learn more about how to brew with them, get in touch!

References

1. Christina Schönberger and Laurent Dagan. Development of Thiols and Thiol Precursors in Different Hop Varieties during Hop Harvest and Their Impact on Beer Flavor. Craft Brewer’s Conference 2020. Link. 
 
2. Roland, Aurélie. et al. First Evidence of Cysteinylated and Glutathionylated Precursors of 3-Mercaptohexan-1-Ol in Malts. World Brewing Congress 2016. Link. 
 
3. Roland, Aurélie. et al. First Identification and Quantification of Glutathionylated and Cysteinylated Precursors of 3-Mercaptohexan-1-Ol and 4-Methyl-4-Mercaptopentan-2-One in Hops (Humulus Lupulus). Flavour and Fragrance Journal 31 (6): 455–63 (2016). Link. 
 
4. Santiago, Margarita, and Richard C. Gardner. Yeast Genes Required for Conversion of Grape Precursors to Varietal Thiols in Wine. FEMS Yeast Research 15 (5) (2015). Link.