Techniques To Develop Innovative New Yeasts

At Escarpment Labs, we're not just propagating any old yeast—we're developing new ones. We design and create new yeast strains in response to gaps in the landscape of available strains. Say, for example, a brewer loves Vermont Ale in their IPA, but wishes it was hazier and peachier.

To make new yeast strains, we first listen to our customers. Then we head to the lab and use techniques such as yeast breeding, adaptive laboratory evolution (ALE), and precision gene editing. All of these techniques are used in our R&D laboratory, based in Guelph, Ontario, Canada.

Let’s explore these advanced methods in detail, highlighting how each contributes to developing unique and efficient yeast strains that enhance the beer fermentation process.

Oh, and I’m going to cram in as many Jurassic Park references as possible, so watch out for that too.

Before We Do Anything: Yeast Strain Characterization!

It’s important to characterize yeasts before deciding to develop any new ones. At Escarpment Labs, we believe that the vast majority of conventional brewing yeasts have critical weaknesses and flaws that can be overcome using scientific tools. Examples include STA1 (diastatic), POF+ (phenolic off-flavour), and nutritional shortcomings (also known as auxotrophies).

We collect a ton of data on all of our yeasts. We collect whole genome sequencing data as well as detailed aroma compound production and growth rate data.

We can use all this data to better understand our yeasts, and leverage the data to dream up new combinations or improve traits. For example, a phenolic yeast could be turned into a non-phenolic yeast with one simple gene edit.

Beer yeasts produce varying levels of phenolic aroma (POF/phenolic off flavour) based on the presence or absence of genetic mutations.

 

Yeast Breeding: Harnessing Natural Genetic Diversity

Yeast breeding, also known as yeast hybridization, is a cornerstone of our strain development program. Breeding leverages the natural genetic diversity of yeasts to produce hybrids with desirable traits. This method involves mating two different parental yeast strains to combine their attributes, such as flavour production or stress tolerance, in their offspring hybrids. The resulting hybrids are stable, and can be repitched like any other brewing yeast. This gets around the major drawback of yeast blends, which are unstable when repitched, making blends a poor option for most commercial brewers.

Technical Aspects of Yeast Breeding:

Spore-to-spore mating and rare mating are the two techniques we use to breed new yeasts at Escarpment Labs.

 

• Selection of Parent Strains: Choosing strains with complementary traits that can enhance the hybrid. We have found that not every yeast strain is capable of sexual reproduction, so yeast strain selection can be limited. Escarpment Labs has contributed to research that makes it easier to mate certain brewing yeasts (like Foggy London Ale) that otherwise present challenges. We did this by making it easier to change and stabilize the mating type of yeast (mating types are similar to how sex chromosomes work). In most mating schemes, we will pick one strain with the efficiency characteristics we want, and one strain with the flavour characteristics we want. We also use whole genome sequencing data and analytics to improve our predictions and strain pairings.
• Mating Process: Facilitating the natural sexual cycle of yeast under controlled conditions to encourage the fusion of genetic material. There are two main approaches to yeast breeding: Rare mating, and spore-to-spore mating.
  • Spore-to-spore mating involves creating spores of the chosen parental yeasts, then using a specialized tool called a yeast tetrad dissector to combine spores to create new hybrids directly (hence, spore-to-spore). This method is more efficient but requires expensive equipment.
  • Rare mating involves mixing populations of thousands of yeast cells from two parental strains together, and selecting/screening for the “rare” occurrence of hybrid formation. First, you have to modify the parental strains to be deficient in making key amino acids (say, for example, lysine - they used the same idea in Jurassic Park), so you can select for “complemented” progeny without deficiencies later. This method is more labour intensive, but can be done using relatively simple lab tools (agar plates, centrifuge, basic microbiology techniques).
• Screening Hybrids: Evaluating the progeny for desired traits through phenotypic and genetic assays. For example, we may select strains based on traits like attenuation, flocculation, ester production, or haze stability. In most breeding projects, we will screen 8 or more progeny for our desired traits.
• Stabilizing Hybrids: Hybrids are not genetically stable when they are first created. Over time, the yeast strain loses redundant genetic material which can impact key characteristics in beer fermentation such as flocculation or attenuation. We take extra steps to stabilize hybrids through sequential wort fermentations (repitching) for at least 8 generations and up to 50 generations. This ensures consistent results in the brewery.
• Hybrid Vigor: Hybrids can show better performance than the parental strains, which is an example of hybrid vigor. The same effect has resulted in all of the modern malting barley and hop varieties on the market, which often have traits more extreme than their parents.

Examples of strains made using Yeast Breeding:

Hydra & JÖTUNN:

Our first hybrids were developed using the rare mating technique. With Hydra, we combined Cerberus and Vermont, both great IPA strains with key limitations. The goal was to produce a haze-positive and non-diastatic strain with limited maltotriose metabolism and strong aroma production, to help brewers produce juicy IPAs when they have limited control over brewhouse variables. With JÖTUNN, the goal was to produce a saison-type strain with maximum aroma capability and temperature range and with good flocculation, achieved by breeding a Saison yeast with a Kveik yeast.

Thiol Libre:

Our popular Thiol Libre strain was developed using spore-to-spore mating. We combined a kveik strain with great fermentation characteristics and good thiol release with a wild strain (non-diastatic) with strong thiol release potential. The hybridization resulted in a new strain that balances thiol production - key for achieving tropical fruit and white wine flavours in beer - while maintaining vigorous fermentation characteristics.

Elysium:

For Elysium, we targeted one flavour in particular: the pineapple ester, ethyl hexanoate (and related esters). This strain was also developed using spore-to-spore mating. We combined a kveik yeast with strong pineapple ester with another beer yeast that had strong ester production, leveraging hybrid vigor to boost this aroma. The new hybrid Elysium also shows good fermentation efficiency and superior acid tolerance, lending itself well to IPAs, fruit beers, and sour beers alike.

 

Adaptive Laboratory Evolution (ALE): Tailoring Yeasts Through Directed Evolution

Adaptive Laboratory Evolution is a dynamic method where yeast strains are evolved in the lab to adapt to specific environmental conditions or to enhance certain metabolic capabilities. This technique involves growing yeast continuously under controlled stress (e.g., high alcohol, temperature extremes) to promote mutations that confer advantageous traits.

Or in the eternal words of Jeff Goldblum in Jurassic Park, “Life, uh… finds a way.”

 

Steps in Adaptive Laboratory Evolution:

• Initial Culture and Stress Induction: Starting with a genetically diverse population or a specific parent strain, yeast is cultured under selective pressure. This could be sugar content (osmotic stress), ethanol (ethanol stress), hops (terpene/hop oil stress), nutrients (nutrient limitation stress),
• Continuous Cultivation: Yeast is propagated or fermented repeatedly over the course of the experiment, sometimes for more than 100 transfers or “repitches”.
• Isolation and Selection: Regular sampling and screening to isolate colonies that thrive under stress, followed by genomic sequencing to identify beneficial mutations. For example, strains adapted for higher wort attenuation often show mutations in the MAL (maltose utilization) genes. Another side effect of lab evolution is that yeast loves to evolve stronger flocculation. If we are lucky, we end up with an improved strain that can be used to make beer better or faster!

Examples of strains developed with Adaptive Laboratory Evolution:

KRISPY: Our KRISPY yeast improvement project in 2021 is a prime example of ALE in action. Originally, KRISPY was appreciated for its clean fermentation profile suitable for pseudo-lagers. However, we needed to blend 2 strains in the original product (one attenuative and one flocculent), which led to instability. Through ALE, we evolved a single-strain KRISPY under selective conditions that favoured higher attenuation and flocculation. The result was a strain that not only showed improved attenuation but also maintained its clean flavor profile, enhancing its suitability for crafting lager-like clean beers more efficiently, which is especially important in markets like Canada where beer demand is extremely seasonal and brewers need to turn around beer quickly in the busy season.

TERPS: Another winner from our lab evolution efforts, TERPS was developed to maximize hop aroma impact through enhanced terpene biotransformation. This strain was evolved from an American ale baseline to increase its capacity to biotransform terpenes like geraniol into more aromatic compounds such as beta-citronellol. When paired with appropriate hops, this strain imparts a stronger citrus and mango character to beers. This strain also developed faster and more complete attenuation, lending it perfectly to modern West Coast IPAs where dryness is desired and hop aromatics must be punchy.

Pomona: A strain developed with a combination of techniques

Gene Editing: Precision at the Molecular Level

Gene editing, an example being CRISPR/Cas9 technology, offers unprecedented precision in yeast strain development. This method allows for targeted edits to the yeast genome, enabling the addition, removal, or alteration of specific genetic sequences.

By employing gene editing techniques, we can precisely eliminate genes responsible for undesirable off-flavours or enhance genes that contribute to flavour complexity and fermentation efficiency. For example, one could use gene editing to inactivate the genes that cause phenolic off-flavours in Belgian strains, thus enhancing their flavour profiles without affecting other desirable characteristics.

Back in my day (2016 - biology moves fast) we had to do yeast genetic engineering “uphill both ways”. Now with gene editing tools and cheap sequencing, it’s much easier to test targeted improvements. We’re in the hands of engineers.

Process of CRISPR/Cas9 in Yeast Editing:

• Design of Guide RNA (gRNA): Creating a small piece of RNA with a short "guide" sequence that binds to a specific target sequence in the yeast genome.
• Cas9 Enzyme Delivery: Introducing the Cas9 enzyme into yeast cells, where it uses the gRNA as a guide to find and cut the DNA at the target location.
• DNA Repair and Modification: After the DNA is cut, the yeast naturally repairs the break. By providing a template DNA sequence during repair, specific edits (insertions, deletions, or changes) are made to the genome.

 

Conclusion: Crafting the Future of Fermentation

We are dedicated to using scientific tools and knowledge to improve yeast strains, with the goal of helping brewers worldwide harness efficiency and embrace new flavours. By using diverse strain development methods and merging traditional brewing knowledge with cutting-edge science and our own perspective on the future of brewing, we aim to set new standards in fermentation and offer brewers new yeasts they can use to craft their best beers.

Which strain will you try next? Let us know in the comments!