Hacking yeast to produce high CO2 / low ethanol

[Bryan Hugill]

Hi there everyone - has anyone over here playing or be willing to play with the idea of hacking the biological pathway in yeast (Saccharomyces cerevisiae) to produce high CO2 and low ethanol? If yes, I'd very much like to talk to you. Cheers! Bryan

[Nathan McCorkle]

hmm, I thought that was affected by respiration vs fermentation, so
presumably you could look into breaking fermentation or mutants
other's have made like this.

On Thu, Aug 2, 2018 at 11:09 PM Bryan Hugill <bryan....@gmail.com> wrote:
>
> Hi there everyone - has anyone over here playing or be willing to play with the idea of hacking the biological pathway in yeast (Saccharomyces cerevisiae) to produce high CO2 and low ethanol? If yes, I'd very much like to talk to you. Cheers! Bryan
>

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-Nathan

[Skyler Gordon]

If you’d be willing to give the end-goal of your project I would be willing to share my ideas on how to use fungal enzymes to convert products to CO2. Typically, the conversion of CO2 to biomass is much more desirable than producing CO2 from
biomass.

-SG

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[Bryan Hugill]

Hi Skyler - We're be using the yeast to produce a fermented (for flavour and CO2), but ultra-low alcoholic (or zero ethanol, if possible) gingerbeer (max. 0.1 ABV). I really want the flavours and gas of the fermentation process (vs. CO2 injection). Any suggestions?

[Bryan Hugill]

Can you explain a bit more about what you mean?

[Skyler Gordon]

Have you considered just providing the yeast oxygen? The yeast only begin creating alcohol at the end of their life cycle in an attempt to survive the oxygen free environment they’ve created through oxidative respiration. If you provide them oxygen, they should continue to break down your food (sugar, wheat, rice, etc.) in their usual way.

This is going to change the acidity levels at the end, and probably your taste profile along with it - not to mention you’re skipping ‘fermenting’ as a whole.

Does this answer your question?

-SG

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[Bryan Hugill]

At the moment, the CO2 that's produced is being used to force carbonate the drink at the same time, saving the extra step/expense of capturing the CO2 and either trying to reinject it (expensive) or discarding the CO2 from the fermentation and injecting CO2 from a tank (time consuming and expensive...it would need to be a very slow injection process of 5-7 days with the liquid at around 1-3 deg. C to ensure tiny bubble size).

And yes, by off-gassing the CO2, the drink's acidity (and by extension, shelf-life / risk of spoilage) would be significantly reduced. So, not ideal.

Also, wouldn't fermenting the drink in an oxygen-rich environment encourage the growth of other critters, which may then outcompete the yeast and increase the chances of spoiled batches?

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[Nathan McCorkle]

On Fri, Aug 3, 2018 at 6:07 PM Bryan Hugill <bryan....@gmail.com> wrote:
> And yes, by off-gassing the CO2, the drink's acidity (and by extension, shelf-life / risk of spoilage) would be significantly reduced. So, not ideal.

I think he was saying that by avoiding the alcohol production pathway,
you're going to affect the pH with other metabolites, aside from CO2
modulating this.

>
> Also, wouldn't fermenting the drink in an oxygen-rich environment encourage the growth of other critters, which may then outcompete the yeast and increase the chances of spoiled batches?

my guess is the lack of alcohol as an antimicrobial would contribute
more than excess oxygen for competitors

[Bryan Hugill]

So, looking at this basic diagram, it appears that Pyruvate is able to produce CO2 in two separate processes, and it seems to be determined by the enzymes that are responsible for breaking it down, triggered by the environmental factors in which the yeast cell is located. Am I reading this correctly?

So then the question is: Is it possible to denature/prevent the enzyme responsible for converting Pyruvate into Acetadehyde + CO2, and rather have the activity redirect itself towards the mitochondria? Or is Acetyl-COA critical for mitochondrial functioning?

[Nathan McCorkle]

On Fri, Aug 3, 2018 at 6:54 PM Bryan Hugill <bryan....@gmail.com> wrote:

So, looking at this basic diagram

you can check here too:

https://metacyc.org/ 

[Skyler Gordon]

Well, if oxygen is present the yeast don’t actually go into fermentation. Fermentation is what happens when the critters have sugars (pyruvate is really just half a glucose, so it can be thought of as a 3 carbon sugar) but don’t have a good enough electron acceptor (like oxygen) to break that 3 carbon sugar all the way down to CO2.

So, when all the oxygen is gone (and only once all the oxygen is gone) they can only break down the pyruvate part way to get some energy, and they end up with things like ethanol and acetate - that’s fermentation! This only starts happening at the end of the process. It’s called “fermentation” the whole time, but the yeast only perform alcoholic fermentation once they run out of oxygen. Your specific gravity is going to change the whole time, but will be more significantly effected at the end. At the beginning your “bricks” are going to change a lot more than your specific gravity. That means the yeast are eating the sugar, but not yet producing that much alcohol.

When they have oxygen, the pyruvate gets broken down into CO2 in the mitochondria - I would look more closely at the citric acid cycle, it should give you a better idea of how to make the yeast eat sugar better. That being said, you are right that if there is more oxygen then more things can survive. That can be avoided by proper sterilization.

If you’re looking to just mess with the existing pathways in yeast, you might be out of luck. They absolutely need Acetyl-CoA to survive and have evolved over millions of years to eat sugar pretty efficiently.

If you want to introduce a new pathway, you could look at cutting off the pathway to ethanol and driving it to acetate and then introducing a methanogen bacterial pathway. There’s no telling if that will work in fungi, and you’ll also end up converting your acetate to methanol or methane - which will make you go blind if you drink it.

That’s the trick with trying to get down to a single carbon without oxygen present - you get methanol. My suggestion would be to bubble in oxygen, which is going to increase the pressure so I don’t know how that will effect the forced carbonation, because that’s the only way you’re going to get more CO2 without producing things that might kill you.

Sorry for the rant.

-SG

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[Skyler Gordon]

Just a thought - if you time it right, you could have the yeast use up all the oxygen and then heat shock them before they start making alcohol.

Since you’re in bottles already you’d have to not heat them too much (pop!), but hopefully killing the yeast before they start fermenting will get the effect you want. It would take some work to get the timing right.

You could also remove the enzymes that produce alcohol in the yeast and just not get as much carbonation! Knockouts (KO) aren’t always simple or stable though!

-SG

[Ravasz]

Hi,

Metabolic modelling is one of my favourite hobbies (yes, I'm a weirdo) but even I would suggest you bite the bullet and carbonate the drink with external CO2. There are numerous industrial solutions for doing this and all the big breweries use them. Producing large amounts of CO2 and only that with yeast would be a very complex task as you need to mess with some of the most fundamental pathways of yeast metabolism.

"So then the question is: Is it possible to denature/prevent the enzyme responsible for converting Pyruvate into Acetadehyde + CO2, and rather have the activity redirect itself towards the mitochondria? Or is Acetyl-COA critical for mitochondrial functioning?"

As Skyler says, this is the switch between anaerobic and aerobic energy production. If there is oxygen present, then yeast will prefer using the mitochondria as it is more efficient. Without oxygen, anaerobic fermentation will take place and yeast will produce ethanol via acetaldehyde. So the only way you could operate the mitochondria constantly is to provide a steady supply of oxygen to your culture. This will lead to numerous problems.

To answer your second question, Acetyl-CoA is absolutely critical for metabolic function. Luckily, because its so important there are numerous ways of generating it. Glucose, fatty acids, amino acids, other things can all be channeled into Acetyl-CoA production. I don't know by heart what yeast likes to do, but I guess in a high oxygen, high glucose environment it will get it from the krebs cycle via citrate mostly, whereas in low oxygen via the pyruvate dehydrogenase complex. So you can probably knock out pyruvate dehydrogenase and still live with high oxygen, but your glucose uptake (and therefore growth) will be somewhat lower.

TL;DR: just carbonate your brew with external CO2, engineering yeast for it is a faff.