Making Cheese Without Cows Part 1

When I was 13, I went vegetarian. Mostly for the standard, well worn reason of the fact that our current farming system is environmentally destructive and unethical.

And as a vegetarian, I never once felt the temptation to eat meat. It lost most of the appeal I had once craved. I didn’t have a desire to try to eat soy products that highly resemble burger patties or tofurky or anything of the kind. I was quite content to just sit around snacking on chickpeas and tofu for the rest of my days.

So content, that one day I contemplated making the full plunge into veganism.

That would mean that I needed to give up eggs, milk, butter, gelatin (doable, doable, doable and doable)…and cheese.

Completely, definitely, absolutely undoable. I am never giving up cheese.

Which basically means that I can’t be a true vegan or I have to find an alternative.

Most cheese alternatives are based on nuts (cashews being the most popular). And while they are quite delicious in their own right, they lack that distinctly cheese quality.

But, what if we were to still make cheese — the exact same thing we’ve been making for centuries — in a way that isn’t as environmentally destructive?

Well, what’s so bad about cheese — what part about it makes it so unsustainable or “bad” for the environment?

It’s not so much the cheese that’s bad for the environment but the way it’s made. Cheese comes from cows, and cows take a lot of land, water, food and energy to raise. 33 times more calories are invested into a dairy cow than she will return in her lifetime!

And if we were to look at cows like machines (which unfortunately we have come to do), they would be a pretty inefficient kind of machine.

Sure we can cut down on the inputs by squishing cows closer together, but this is beyond unethical. And the fact of the matter is that we already do this, but we are still wasting so many resources.

So, what if we just eliminate the cow?

Such began my quest to produce cheese without the cow. This is the beginning of a series in which I document how I try to use something called acellular agriculture to create cheese that is biologically identical to the creamy cheddar, cambert, swiss or havarti we have all come to love just made without the dairy cow.

Naturally, this might seem impossible. But, we can actually do it using something called acellular agriculture.

Acellular agriculture has to do with using microbes as little machines to fabricate the proteins found in animal derived products (like milk, cheese, eggs and fats).

On average, using acellular agriculture takes 91% less land, 98% less water, has 65% less global warming potential, and emits 84% less greenhouse gasses than producing the same product conventionally.

And given that it outright bypasses the animal, there is not even a slight chance of abusing one’s welfare.

Sustainability, check (or at least in comparison to the way we do things now).

So, how does it work?

Acellular agriculture works in four main steps.

1. The genes encoding for the protein we want to produce are taken from a natural source. It is called the recombinant DNA.
2. Through genetic transformation, the recombinant DNA is added to the bacteria or yeast as a plasmid — a piece of DNA which can replicate on its own.
3. Through a process called fermentation, the yeast or bacteria is fed sugar and begins to multiply. As it multiplies, it expresses its DNA including the protein that of the protein we inserted.
4. The proteins are then separated from the residual yeast and bacteria using purification and are used to produce final products.

So, let’s just use cellular agriculture to make cheese. But to do that, we’ll need to understand what proteins cheese is made of, so that we can use this process on them.

What is Cheese?

When cheese is made traditionally, we start out with milk. Now milk is made up of a mixture of water, milk sugar (lactose), fats, enzymes and proteins.

About 80% of the proteins found in milk are called casein. This along with a bit of fat and sugar is what eventually becomes a whole block of cheese.

Now casein itself is not a protein but a term applied to a family of four proteins: alpha_1 casein, alpha_2 casein, beta casein and kappa casein. Together, these four proteins are suspended in the milk as tiny structures called micelles.

These micelles are like little bubbles with an outer wall of kappa-casein. This kappa-casein has hair-like structures called hydrophilic glycomacropeptides which attract water and repel other micelles. This is what keeps each of the micelles from binding to one another in one solid mass and instead remain happily floating all by itself.

But to make cheese, we need all of these micelles to join together. So, we somehow have to get rid of these hydrophilic glycomacropeptides.

The first way we can do this is by acidifying the milk. Casein proteins are made up of a bunch of different amino acids, some of which have positive charges and some of which have negative charges. The isoelectric point of a substance is the pH at which it’s net charge becomes neutral. Casein has an isoelectric point of 4.6, but milk naturally has a pH of 6.6. When the right amount of acid is added to the milk, it’s pH will drop to 4.6.

This causes the casein to denature — or the micelles begin to unwind — and instead of bonding exclusively with itself, it begins to form loose bonds with other ex micelles. Hence, the big chunk of protein.

Option number two is to add rennet. Rennet is derived from the chymosin enzyme which when added to milk effectively snips off the hydrophilic glycomacropeptides from the kappa casein.

Now remember that these little tails are what prevented the micelles from binding to one another so once they are gone, the micelles will attract each other and boom, big chunks of protein.

This alone won’t get you a finished block of cheese because, well, the casein is still sitting in all the other proteins that make up milk — i.e. the whey.

Whey is made up of two proteins called alpha-lactoglobulin and beta-lactoglobulin. Both proteins do not react as the casein does when exposed to rennet or acidified to that extent, so they simply remain a liquid substance.

As the casein micelles begin to precipitate — i.e. come together — they coagulate into solid curds on top of the whey. Hence the phrase ‘curds and whey’. Once they clump together, they are cut into smaller blocks, strained out of the solution, salted, flavoured, aged or whatever it is that makes cheese so darned delicious.

Now, rennet is actually already produced using acellular agriculture, and it has been since the 1990s. So, we could approach culturing cheese in two ways — we could either produce milk and then add rennet or we could just go straight for the cheese.

The downside of the first one is that we would also have to figure out how to culture all the other proteins and fats and sugars that go into the whey. And since we don’t really care about these components at the moment, we might as well just go for the cheese.

Cheese meats Acellular Agriculture

As we saw above, cheese is primarily made up of the four different kinds of casein (with a bit of sugar and water), so producing them using acellular agriculture seems like a pretty good place to start.

Let’s focus on beta-casein for a minute. In order to culture it we’ll need to

1. Get the gene — called CSN2 — which codes for it.
2. Genetically transform bacteria so that it now contains the CSN2 gene.
3. Ferment the bacteria by feeding it sugar so that it expresses the CSN2 gene as beta casein and can replicate itself to produce more copies of the CSN2 gene which also express themselves.
4. Purify this mixture to separate the beta-casein from the bacteria.

So now of course, this begs the question (or three) how do we get the CSN2 gene and for that matter, the genes that code for the other proteins, the fat and the sugar? How do we genetically transform the bacteria to contain each of these genes? How do we purify this mixture to get rid of the bacteria?

Alright, one thing at a time.

Getting the CSN2 Gene

Nowadays with the wonders of modern technology, rather than trying to get a gene by zooming into a cow cell and magically pulling out a perfectly intact strand of DNA, we can sequence it in a lab and fabricate it.

This means figuring out what the “molecular code” of the gene is — i.e. the order of A, C, G and Ts that make it up and then stringing these bases together in real life.

The entirety of the Bos Taurus. (cow) genome has actually been sequenced, and from this, scientists have figured out what specific part of it are the casein genes.

So, we can take this code, send it to one of the many organizations that fabricate genes commercially (pretty cool), and for about $10, they’ll send you back the DNA as a plasmid within a couple weeks.

Genetically Transforming Bacteria

Now, we have to get the plasmid into our bacteria and to do that, we need our bacteria to be competent — able to intake foreign, extra cellular DNA. It also needs to be able to horizontally transfer genes — i.e. integrate the foreign genes into it’s own DNA.

Horizontal gene transfer is much more difficult in eukaryotic organisms than prokaryotic organisms. This is because eukaryotic organisms, have both a cell membrane and a nuclear membrane which the plasmid needs to get through. In prokaryotic organisms, the plasmid just has to penetrate the cell membrane.

So, let’s consider a non naturally competent prokaryotic cell like E. coli. In order to make it competent, we must

1. Expose it to a salt such as CaCl_2 which neutralizes the negative charges on the cell membrane’s phosphate heads as well as the negative charges on the plasmid. This prevents the two from repelling.
2. Heat shock it by incubating it in warm water for a few seconds. This opens up large pores on the surface of the cell which the plasmid can get into.

So in order to stick our casein genes into the bacteria, we will do just this.

Seems like something easier said than done.

But, infact, it is something that you can do at home. A wonderful and controversial self identifying biohacker named Josiah Zayner started a company called “The Odin” which aims to democratize gene editing technology. As part of this whole mission, he created the DIY Bacterial Gene Editing kit which for the price of about $150, allows you to engineer the DNA of non pathogenic e-coli strains.

This kit does come with a built in experiment (and an option of a course) to help you get a feel for how gene editing works in the first place, so I’ll be doing that before I attempt to jam another gene into a living organism on my own.

Expressing Casein

Now that the casein genes are in the e.coli, we can culture the bacteria — feed it nutrients — so that it multiplies and expresses it’s genes. The easiest way to do this is by growing the bacteria on LB agar plates — mounds of LB agar, a substance which contains a whole bunch of essential nutrients.

But, how exactly does the bacteria express it’s genes? Through a process known as transcription & transformation.

The first part of this process, transcription, starts off with something called RNA or ribonucleic acid which is made up of

• a sugar-phosphate backbone including a sugar called ribose
• one of four nitrogenous bases: adenine, uracil, cytosine and guanine.

Now, let’s consider a part of DNA that we want to make a protein out of (in this case our plasmid). This is called the “promoter DNA”.

1. RNA polymerase binds to the promoter DNA.
2. RNA polymerase forms a replication bubble (a section of the DNA where the hydrogen bonds are split).
3. RNA polymerase goes down a strand of the DNA strand and attaches the complementary RNA bases.
4. The hydrogen bonds between the DNA and RNA are broken.
5. A special kind of guanine 5’ cap is added to the 5’ end of the RNA.
6. An enzyme adds a bunch of adenines to the 3’ end of the RNA known as the poly-A tail.
7. Snrps (ribonucleoproteins) come along and figure out where the RNA have irrelevant sequences of bases called introns. They bind to a bunch of proteins called the spliceosome which cuts the RNA at these locations and joins the remaining parts back together.

The resulting strand of RNA is called messenger RNA — mRNA.

Now, for translation.

1. Ribosomes are organelles found within the cell which are made up of a combination of ribosomal RNA and different proteins. They have a couple binding sites into which the mRNA is fed.
2. tRNA — or transfer RNA — goes into these slits and sort of acts like a key ring. On one end, it has three nitrogenous bases and on the other end, it has an amino acid.
3. The mRNA is divided up into codons which are sequences of three consecutive nitrogenous bases. tRNA skims along these codons and tries to match up it’s three bases with that of the mRNA.
4. When it finds its match, it adds the amino acid attached to its other end to a queue. This queue of amino acids eventually becomes our protein.

Purifying the Bacteria

Now once, this is done, it doesn’t necessarily mean that all of the bacteria we have actually contains the beta casein gene. In order to get rid of the e.coli that doesn’t, we can expose it to an antibody raised against beta-casein. In this way, everything that doesn’t contain beta-casein will die off.

Then we have to purify the bacteria to separate out the e.coli from the casein.

The most common way is using something called a centrifuge. This basically spins a mixture around an axis with a lot of force in order to separate out solids from liquids.

Then, soak your solution in a well buffered ionic solution (like sodium chloride). This employs a principle called osmosis to suck all the water out of the bacteria, eventually killing them.

Now, of course this is far from the ultimate technique to produce entire blocks of cheese as we think of them, mostly because cheese is not just casein. It is also a careful mixture of fat cells, sugars and salts, although in smaller quantities.

This is just the first of many steps for me to try and figure out what the heck is going on with the principles involved before I try to get everything in the correct ratios.

More to come with Part 2 where I’ll be modifying e-coli using The Odin.