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Our whole warehouse smelled like a guava.It was crazy, it was shocking, and it was really inspiring and thrilling.
We've made a pretty big, what we think at least, is a pretty big advance in biotransforming those thyles, and so that's why we're talking about it in this poster.
Just last week, Jennifer Doudna from UC Berkeley became a recipient of the Nobel Prize in Chemistry for the development of a method for genome editing known as CRISPR-Cas9.
This week on the show, we catch up with Charles Denby from Berkeley Yeast, who is using a similar set of miraculous genetic scissors on brewer's yeast.
Pat Brown may be saving the planet with Impossible Burgers, but Charles Denby is helping brewers make Impossible Beer.
Hi, my name is Charles Denby and I'm with Berkeley Yeast.
First of all, I can't believe it's been two and a half years since we last talked.That just blows my mind.
Yeah, I know.I know.It seems like just yesterday.I don't know if it feels weird to you or not, but... I remember very vividly our last conversation.
I remember very specifically, we went around in a circle thinking about different terms to describe, like where we ended up talking about DNA sisters with Brian.That's right.
You know, I knew you'd be back though. You're solving too many interesting problems to not be a regular on the show.So, I guess anyone who's been with us that long will certainly remember you from episode 84.
And if anybody listening hasn't already heard Charles on episode 84, I definitely recommend hitting stop now and going back and listening to that one first. So Charles, you're at it again.
I'm looking at your WBC poster, which covers two new engineered yeast strains that do very different things.What was the goal with the first strain?
One of the big differences between last time we were at WBC, which is obviously in 2016, and this time is that we've really like right now we are focused on
Solving the problems or creating solutions to challenges that are the most important to the brewing community, right?
So we come from kind of an academic background and in academia, a lot of times you read the literature to find the problems that you want to work on.But now we're very much like
going out and chatting, especially before COVID, like we're going out and chatting with brewers and trying to understand the things that they're the most excited about.
And the overwhelming consensus from all the breweries that we talked to was that they love these flavors and aromas that you guys know from like tropical fruits and from various different hop cultivars.
That's really the set of flavors that's most prized in the industry from our survey, informal survey.One of the things that we knew about those flavors is that a lot of them come from these thiol compounds.
And we're also aware that hops and malt have these thiol compounds in a different form, like a precursor form.We refer to them as bound, either glutathione or cysteine bound thiols.And so we figured that, we knew that the yeast is
is able to break that chemical bond that differentiates a bound thiol from an aromatic thiol.Typically, yeasts range in their ability to cleave those chemical bonds.
Some of them don't do it at all, and some of them do it at 1% efficiency, others up to 5% efficiency. We were interested to see whether we could get a yeast that does it even more efficiently.That's why we really set about working on this project.
It's been a really interesting project and we worked really actively on it over the last two and a half years since we last talked.
There are several thiols that have been identified as important when the goal is to achieve those strong tropical flavors and aromas.Tim Wallin's poster, which we talked about on the show just a few weeks ago, focused on 4-MMP, for example.
Which thiol or thiols did you set out to amplify here?
Yeah, so the enzyme that is liberating those thiols will actually act on 4-MMP, 3-MH, and what we've observed is there is a lot of 3-MH precursor in malt and there's lots of precursor for both 4-MMP and 3-MH in hops.
this enzyme will basically do what your ingredients... it will liberate whatever bound thiols your ingredients have.
Wow.So this is not just working on 3MH.I didn't realize that.Yeah.
Yeah.The data and the poster is on 3MH.And the reason for that is just because, um, we have like, uh, built our, built our, uh, GCMS methods around 3MH and, um, it's a pretty, it's a pretty good one for us.
Like it's, it's a pretty strong indicator for us.
Sure, that's awesome.Okay, well, talk about why it's difficult to get a lot of 3MH and some of these other files in beer the old-fashioned way.
Yeah, so... The biggest challenge really is that these guys are bound up on peptides, either on a glutathione peptide or on cysteine.When they're bound to those amino acids, basically they're not volatile and they don't have those flavors.
It's only when you break that chemical bond and now you have just the free 3MH where they start to work their magic.
Okay.Now there's some Australians in the wine industry that figured out how to increase 3MH.Talk about that.
Yeah, okay, cool.So, there's a really cool study from over 10 years ago now where folks at AWRI developed a yeast strain that has an enzyme that basically does the same activity that our enzyme is doing, where it basically cleaves the free thiol.
But the thing that's interesting about that is that It also generates a very potent off flavor.That flavor smells like poop, actually.We were doing some early experiments just to
kind of validate what they had observed previously, and we found, oh man, like we're getting these beautiful, bright, guava, tropical flavor and aromas from these strains, but we're also getting these fecal notes, and obviously we were pretty concerned about that.
So, the first thing we want to do is figure out what that's coming from, and it turns out that that's coming from the fact that that enzyme can also convert tryptophan into indol, which is one of the things that drives the poop aroma.
And so, the next challenge we set out to tackle, and that's kind of figure two of this poster, is trying to engineer that enzyme so it would make the guava flavor but not the poop aroma.
And we were able to find, we were able to engineer the enzyme such that it was specific and it only makes the guava flavor and aroma.You can see that in the result in figure three.
Now you just said tryptophan, isn't that the stuff in turkey that's supposed to make people sleepy?
Heck yeah, yeah, that's one of the amino acids, and um, yeah, it's, it's uh, there are, the reason that tryptophan, well the reason that indol, uh,
is, you know, humans have evolved to like recognize indol is that a lot of your gut microbiome will convert tryptophan into indol.And like, that's literally why poop smells like poop.
All right, fair enough.Okay, so after you sort of solve the tryptophan problem, the abstract says that you ended up seeing thiol increases over tenfold with this strain.That's a pretty big increase.Talk about that.
Yeah, honestly, even we were surprised when we got these results, like some of our very first batches.Our work stream is basically, we'll work with brewers to try and figure out problems that they want to solve.
Then we'll figure out a strategy for how we can tackle that.Then we'll generate strains that we think will solve that problem.Then we'll basically ferment pilot batches at 20 liter scale in our pilot brewhouse.
a couple dozen fermenters so that we can test lots and lots of different strains.
Sometimes you do have to test a bunch of strains and sometimes it's an iterative process where the first batch you produce doesn't do the thing that you want and you have to go back and try and figure out, this is a great example of that.
Anyway, when we finally did get to a point where It was just producing that 3MH.Our whole warehouse smelled like a guava.It was crazy, it was shocking, and it was really inspiring and thrilling.
That's awesome.Okay, what can you tell us about how the wort was hopped for these trials?
Yeah, so pretty sparingly, like we used a bit of bittering hop addition.
But otherwise, like in this particular in figure three, that's just the 3MH from the malt that we use, which is pretty, pretty, you know, standard, like craft grain bill for like a pale ale or an IPA.
Wow, that's amazing.So I've not heard a lot about this, about the possibility of malt being leveraged for production of these dials.Talk more about that.I mean, no one's talking about that as far as I know.Totally.It's all about the hops, right?
Yes, yes.You're absolutely right.I remember It was either 2017, something like that, when I went to the HOPS conference at OSU, which is a really cool conference.
A lot of folks were talking about the bound files that are present in HOPS, but I remember distinctly seeing one talk that said, well, actually a lot of this precursor compound can come from Malt as well.
And we didn't necessarily, like, I wasn't really sure whether to believe that or not.
And it really wasn't until we were getting these huge guava bombs out of just, like, a pretty bare malt-based recipe that we were so confident that there is a lot of that 3MH precursor in malt.There is, of course, even more if you add particular
And I think that's one of the things that I find most exciting about what's to come is that right now we have a pretty good sense for how this strain plays with
typical malt bill, but I think it's going to get even more interesting when brewers use different hops and see how this strain interacts with those hops, and also other different substrates, like if you have other grains that you want to ferment, seeing how it will play with those.
Wow.That's crazy.So, um, I guess just real quick, do you mind saying, I don't think you said what varieties you use, um, hop varieties you use.
Um, so, I mean, there were no, like you weren't, there were no, you know, Southern hemisphere hops in this or anything, right.
No, no, but they had those like, you know, crazy Guava aromas that you would get from those, you know, Australian, New Zealand, uh, hop cultivars for sure.
So what variety did you use in these trials?
You know, we bittered with something like northern, like, hmm. It's something very boring, honestly.It was like, I can't remember the exact Goldor.
You can't even remember it.That's great.Okay.So, I guess I want to ask you a question about malt and sort of like understanding the level of precursor in malt, right?
Because obviously there's so many different malts out there coming from so many different places.Like, how does a brewer,
figure like is all malts going to have this precursor enough of this precursor in it or is this something that we need to start looking at and figuring out how do we does that need to be on my next malt coa yeah i mean i think probably both like on the one hand i can say that we've tried this with a variety of different grain bales and we see it across the board but i think what we are going to start finding out is that
Yeah, they're certainly going to range, like there's going to be a range in the amount of bound 3MH and other thiols.So, I can't say that we know a ton about that yet.
I know that I can say that a handful of different grain bills have produced really impressive flavors and aromas, but I do think we still have a lot to learn on that.
Okay.Wow.All right.Well then I don't have to ask my next question, which was going to be how important is hop selection, a hop variety selection with this strain.
And, and I was going to say, presumably you'd want to avoid varieties that are low in, in 3MH precursors, but sounds like that doesn't matter.
Yeah, no, I mean, but, but both like the other thing is that, well, I can say, you know, very confidently that there's a lot of 3MH precursor in a lot of different malts.
You know that there are tons of bound files in HOP cultivars, like BEYOND 3MH, whether it be 4MMP.The most famous ones that have been reviewed in literature are 3MH, 3MHA, which is just an acetylated version of 3MH, 4MMP.
But just like with anything, if you try and break down this super complex flavor and aroma that is, you know, beer ingredients.There's a lot going on.There's a lot going on, exactly.
So, like, you know, those are the heavy hitters, but guaranteed there's another dozen thiol compounds that are impacting the flavor and quality of beer. Coming up.And he was like, you know what, dude, diastole sucks.
Like, can you do something about that?And I was like, yeah, actually, we can totally do something about that.
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The original strain that you engineered that we already talked about back on episode 84, that was designed to produce linalool and geraniol.
And while that was a remarkable feat, one of the criticisms, correct me if I'm wrong, was that the resulting hop flavor and aroma was somewhat one-dimensional.That while those are important
compounds, hop flavor and aroma, just as we just said, is more complex than just linalool and geraniol.
So, I'm assuming that with what's going on with this enzyme now, that any concerns about this being sort of one-dimensional are very much mitigated.
Yeah, totally.Um, so I mean, I think that that is a really good, uh, really good, like a very legitimate criticism of that first set of strains that we produced.
Like, um, you know, obviously that was just when we had gotten started and that was kind of like our first proof of concept that it was even feasible to make excuses.That was pretty amazing.
Yeah, but like to look back on that, I mean those, and this kind of gets at exactly what we were just talking about with like, you know, we talk a lot about 3MH, 3MHA, and 4MMP as the thiols that drive a lot of flavor, whatever.
And the same with like, if you go back to the 80s and 90s and look at the flavor compounds that people were attributing hot flavor and aroma to, a lot of it was, yeah, linalool, geraniol, citronellol, those terpenes that were like, you know, the heavy hitters.
But in reality, there are Beer is a complex matrix.Hops are a complex matrix.There's tons of stuff going on there.
A big part of what we're interested in is expanding the portfolio of flavor and aroma compounds that we can get yeast either to make from sugar, like in the case of linoleum geraniol, or to make from
the substrates, the malts, the hops that you're adding or whatever other ingredients you're adding to your beer.
By the way, I'm sure you've got plenty on your plate, but someday you should do a study and present at one of these conferences that shows like, you should do some trials where you take one of the sexy hop varieties and do it as a control at like a very high
pounds per barrel rate and then figure out just how low you can get that rate with this enzyme.
So if you could say like, I took a recipe that was three pounds per barrel galaxy and I basically was able to achieve the same results at a tenth of a pound per barrel with this enzyme, that would be a pretty awesome presentation, paper, poster, whatever.
Yeah, I fully agree.Yeah, that might be ongoing in the lab right now.I don't know.
Biotransformation is a topic that's come up on the show often.Just a few months ago, we heard about the enzyme trials that Maddy Kavana and Leandro Miners did down in Argentina. We also talked with Eric Abbott last year on episode 119.
The focus that time was on yeast strains that are relatively high producers of beta-glucosidase.
Without getting too far into the weeds, could you maybe paint the big picture for us in regards to the different strategies for biotransformation and describe why you wanted to focus your efforts on releasing bound thiols rather than hydrolyzing glycosides?
Yeah, I mean, I think that the reason we focused on thials is because when we go talk to brewers, they're always talking about the flavors that we know come from those thials.
And the reason that they're always talking about it is because their customers are always giving them feedback on those beers that end up producing those thial bombs.
So, you can go talk to a brewer that has a GCMS and is quantifying all these different flavor and aroma compounds that are coming from their hops, and they have this sense that the beers that they make that chart particularly high on the thiol spectrum are the ones that their customers are coming back for more and more of.
And to get back at one of your other questions, which is like, well, why is it difficult to get those flavors into your beer?
The hopping rate that is required to achieve the levels that are very distinctive, like, okay, obviously it depends on the hop cultivar, it depends where it was grown, it depends on the year that it was grown, but generally speaking, like, getting those intense, like, guava, passion fruit, those flavors and aromas requires extraordinary hopping rates.
And then once you get to those really high rates, first of all, it's expensive.But second of all, it starts to present all these other challenges.
One of them that is also kind of what I'm going to talk about or I talk about in this poster is the hop creep issue.But others are like, you know, when you start really pushing the limits, you get like, hop burn.
I know that's something that a lot of brewers are talking about.
So, I think the thing, the key thing about what we're working on here is we're trying to be able to get those intense flavor and aromas that brewers and customers really love, but without having to overdo it on some of the other parts that kind of piggyback along with those heavy hopping loads.
You used cow-ale yeast as a control for this study, I guess because it's ubiquitous, but that strain is a pretty low bar for biotransformation.
How does your engineered strain stack up to other strains that are more widely used for producing hazy IPAs?
Yeah, so in terms of like thiol content, they are blowing like all available strains out of the water.I can say that with a lot of confidence, like a lot of certainty.Having said that,
those hazy strains do a lot of other things, like a lot of things that might be related to various other compounds.So, we have also introduced this enzyme into hazy strains as well, and that's something that's
kind of ongoing and we're continuing to experiment with.But basically, we're trying to make this technology or this enzyme available in all different strains that a brewer might find useful.
Okay, that's awesome.Yeah, I have another kind of question about that, too.
I was kind of curious about how easy or difficult it is, like, once you've figured out how to do this for one strain, like, you know, how much of a challenge it is to, you know, engineer the same effect in a different strain.
Sounds like it's maybe not that hard.
You know, it's kind of like a lot of what we do, like in concept, it's not that hard.You know, the same thing for like, you know, this research, like, oh, you know,
In concept, it's like figuring out a gene that you can put into a yeast strain that makes an enzyme that converts bound thiols into free thiols.
Seems pretty straightforward, but as soon as you start peeling the onion, you realize that there are layers.The first layer, if you will, is like, oh, this enzyme doesn't just produce that
like awesome guava aroma, it also produces these not desirable flavors and aromas.So, the same thing is true when you switch from like a California bait, like a cow ale strain to another strain.It might not
work the exact same if you just port the engineering from one strain to another, there is oftentimes some additional tinkering that you have to do with the genetics.That's why we do what we do.To go back to it, we'll create
you know, dozens of strains if we're trying to achieve some goal.
And then we'll take those dozens of strains, we'll test them at small scale, see how they're doing, scale them up to 20 liter scale, see how they're doing, make sure they're making like really delicious beer, and then that's when we pass them on to some of the brewers that we work most closely with.
And they'll, you know, scale it up anywhere from like 5 to 20 to more barrels of beer.
And then we just make sure with those first kind of like pilot brews that those are going well before we kind of like open it up and release those strains to the broader brewing community.
And the last thing that I'll say that I at some point in this podcast I'd love to get into is just like we make it like I also make it sound as though we're We're creating this diversity of strains and then we're just giving them to brewers.
We also have a very rigorous safety evaluation analysis process that we go through before it gets from our brew house out into the world.
Okay, so moving on, we've talked a lot about one of the two strains on your WBC poster.The other strain on your poster eliminates a process aid that I regularly buy and dump into my fermentations.Talk about that.
Yeah, so we have a strain of yeast that we're calling Hop Creep Killa, and what it does is it reduces the production of diacetyl, especially as it pertains to when you are charging your beers up with big dry hop loads.
So, you know, we call it hop, creep, kill it.
We understand that it doesn't completely eliminate hop, creep, but it does eliminate the worst part of it, which is, you know, that diacetyl formation that you can either get, that you get, like, especially after packaging can be particularly pervasive.
And that was a strain where Again, that's just us listening to our friends who are brewers and deal with these challenges on a day-to-day basis.I was having a conversation with one of my buddies who's a brewer at Bale Breaker, and he was like,
You know what, dude, diastole sucks.Like, can you do something about that?And I was like, yeah, actually, we can totally do something about that.
And like, a couple months later, I hit him up and I was like, hey, we got something if you want to try it, you should you should check it out.And they've got a little 10 barrel system over there.
And he brewed, you know, five somewhere between five and 10 batches of it.And we're just like, This thing is a freaking powerhouse.They do forced diacetyl tests on all their beer ad nauseum and this basically eliminates that issue for them.
That's how we like to work with breweries.We like to hear about the challenges and we like to figure out how we can help them solve those issues.
With regard to your comment of you use an enzyme to reduce that diacetyl after your dry hops, I'm assuming that's when you throw it in.
No, so you can buy, I mean, commercial ALDC, you know, exists.A lot of people use it.And typically, typically it's added at fermenter full.
And so, you know, it's going to function, it's going to have, you know, like any enzyme is going to have a, you know, its own temperature and pH optimums and whatnot.
But a lot of the folks who have, who have attempted to add the enzyme when dry hopping often, We may have even talked about this in another episode, I'm not sure, but often those folks don't get the intended results.
A lot of times there's too much alpha-acetylactate already formed at that point.And so, I think the professional advice is to always add the enzyme at the start of fermentation if you want to get the maximum benefit.
Yeah, so I mean, this is a I feel like I feel like reasonable people could argue a lot about how this works.A lot of folks get it when you dry hop though.That's where
That's where the diacetyl that they can't clear up really produces a serious problem for them.This particular strain eliminates that problem for the breweries that we've been working with.
The cool thing about having ALDC inside the cell instead of outside the cell, like when you add the enzyme exogenously, is that the cell never makes acetyl lactate in the first place.You can add your big hop charge
And whereas a normal yeast would make a bunch of acetyl lactate and then you have to wait for the yeast to clean it back up, you know, with our strain, you never make the acetyl lactate in the first place.
So, you know, the analogy I like to use is with normal strain, you're basically making a mess and then you can clean it up with enzyme additions.Whereas with our strain, you never make the mess in the first place.
You don't have to worry about cleaning it up. The reason I think that this is a more elegant solution than adding an enzyme to your fermenter is because enzymes, as soon as you add an enzyme into your wort, it's going to start degrading.
it's hard to predict how it's going to behave in the particular substrate that you're putting it into.
The thing that's great about having the ALDC enzyme inside of the yeast cell is that yeast is really good at controlling the environment inside of its cell wall. it's always going to be roughly the same pH inside of a yeast cell.
The protein is going to be much more stable inside of a yeast cell than it's going to be outside of a yeast cell.The way I think about it is the second you add your
if you if you're using an like a an exogenous enzyme like the second you add it to the fermenter it's going to start stopping working like it's going to start degrading it's going to start and it might not even work that well in the first place in that particular environment the thing that's nice about having inside the cell is the cell knows how to control it it knows how to make sure that it's active um so it's much more effective that way and i mean that's that's just what we've seen um with the you know
dozen or so breweries that we've been working with on this.
Well, beyond that, I would say that your solution is more elegant because it's a whole set of other things that the brewer doesn't have to worry about.You don't have to go buy commercial enzyme elsewhere.
You don't have to make sure it's shipped and stored properly and that it has the right shelf life and all that.You don't have to make sure that you're adding the correct dose.You don't have to, if you aren't adding it right at the
the fermenter full, you don't have to worry about oxygen ingress as you're adding it, all those things, right?Because just as you said, it's there, it's in the cell.So, that's what's elegant about it in my mind.
I love it.Thank you for saying those things.I mean, that's exactly how we feel about it.
Cool.And now this strain, you did some trials with this both at Drake's and Russian River that I understand had pretty stellar results, right?
Yeah, and I should also say like the good folks at Bailbreaker were the real pioneers.I mean, these guys are all pioneers, but those guys really took the plunge as well with these two guys.
Awesome.Okay, so... I kind of got at this earlier, but maybe this is a very different question.I'm not sure.
So earlier I kind of asked a little bit about how easy or difficult it was once you have something that you've engineered to just plop it into different yeast strains.
The other question that is maybe kind of obvious and maybe is a stupid question, maybe the answer is it's just not that easy. Why not engineer a strain that has both of these modifications instead of separate ones?
And along those lines, how difficult is it for you to take several of the different modifications that you've made and put it all together in a single strain?Or do I just need to go do co-fermentations to get all these effects?
It is not hard to mix and match.Part of our business is just selling strains off the shelf and another part of our business is working with breweries on custom strains.If you're a brewery and you want
you want more intense biotransformation or you want to dial it to a certain level, and you also want to eliminate diacetyl formation, then we'll build that strain for you and make sure that it works just right for your process.
That was Charles Denby here on the Master Brewers podcast.If you're wondering how you can get either of the yeast we discussed today into your brewery, the strain that produces intracellular ALDC is available now on the Berkeley yeast website.
And Charles tells me the file releasing strain will be commercially available in early 2021.
If you want to hear more about some of Berkeley yeast strains, you should check out what was a really great WBC workshop with Charles, Chaz and Molly from Lalamont, who you might remember from episode 149, as well as Sean Sasser from Cigar City, Cole Hackbarth from Rheingeis Brewing, and Philip Emerson from Almanac Brewing.
During that workshop, the panelists discussed a variety of topics including how the various brewers are using GM yeast, how Charles and Chaz ensure GM yeast are safe to use, and so much more.
It's really great content and it's available for on-demand viewing along with a whole lot of other great content for folks who snagged premium or boundless WBC registrations. Are you enjoying the Master Brewers podcast?
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Charles, since we recorded this episode, you got back in touch with me to let me know that it's awesome as what the Hopkilla yeast strain does.There's something that brewers have told you is not so awesome about it.What is that?
That's the name.It's a little bit of a misnomer.A bunch of brewers have pointed out to us, look, you're not getting rid of hop creep, you're getting rid of the worst part of hop creep, which is that diacetyl formation in the packaged product.
One of the breweries that we work with, they just got rid of the name and started calling it diastole free.So we we love that name.So we're going to start using diastole free.And so you can look for strains that are called DF 2.0.
That's that's that's kind of how we've evolved there based on the feedback that we've been getting from our partners and our customers.
