Real Vegan Cheese

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Intro

Kappa casein is the protein responsible for milk coagulation into cheese when it is cleaved by rennet (chymosin). If we could express this protein in yeast, we could essentially make vegan cheese.

A 2005 paper showed that the C-terminal part of the human kappa casein protein can be overexpressed in S. cerevisiae (baker’s yeast) and Pichia pastoris, and they were able to get it secreted in sufficient quantity that they could run the culture medium on a gel, and see a clear band for the protein. (“This macropeptide has various biological activities and is used as a functional food ingredient as well as a pharmaceutical compound.“ - valuable stuff, not just for vegan cheese!)

Our first task would be to replicate this result - ideally in the BioBricks format. Next would be to express and secrete the full-length protein. If we can get it to express and secrete in sufficient quantity, we should be able to show that you get a single band on the gel for the intact protein, but two bands after cleaving the protein with chymosin.

We can have some people working simultaneously on trying to express the other casein proteins (alpha-s1, alpha-s2, and beta). But since these are hydrophobic, we may not be able to express and secrete them without kappa-casein to hold them in suspension in a casein micelle. We can also attempt to modify the yeast lipid biosynthesis pathways to produce milk fats.

Essential reading

• Rennet coagulation of milk
• Mechanisms of Coagulation: The principles, the science and what they mean to cheesemakers
• Production of human caseinomacropeptide in recombinant Saccharomyces cerevisiae and Pichia pastoris (on seafile)
• VitaYeast project, by Johns Hopkins’ 2011 iGEM team
• Yeast parts in the BioBricks parts registry

Kim YJ, Oh YK, Kang W, Lee EY, Park S. Production of human caseinomacropeptide in recombinant Saccharomyces cerevisiae and Pichia pastoris. J Ind Microbiol Biotechnol. 2005 Sep;32(9):402-8.

“Caseinomacropeptide is a polypeptide of 64 amino acid residues (106-169) derived from the C-terminal part of the mammalian milk k-casein. This macropeptide has various biological activities and is used as a functional food ingredient as well as a pharmaceutical compound. The gene encoding the human caseinomacropeptide (hCMP) was synthesized and expressed with an alpha-factor secretion signal in the two yeast strains, Saccharomyces cerevisiae and Pichia pastoris. The complete polypeptide of the recombinant hCMP was produced and secreted in a culture medium by both the strains, but the highest production was observed in S. cerevisiae with a galactose-inducible promoter. In a fed-batch bioreactor culture, 2.5 g/l of the recombinant hCMP was obtained from the S. cerevisiae at 97 h.” “Escherichia coli XL1-Blue (Stratagene, USA) was used for cloning and propagating genes. Host strains for recombinant hCMP were S. cerevisiae 2805 (his-, ura-) [...] For S. cerevisiae, pYIGP (containing a constitutive GAP promoter) or pYEGa (containing a galactose-inducible GAL promoter) was used.”

Yeast expression notes:

Galactose induction is the most common form of controlling heterologous expression. Common vectors are the pYES2 and pESC series vectors.

pESC contains a bidirectional promoter which may allow expression of both parts of the protein without cleavage being required.

Secretion of proteins will require a secretion signal ( triggering processing through the ER, golgi and then exocytosis).

Casein

Patrik D says: In a bout of procrastination from grant writing, I actually stumbled upon some more really good references on casein proteins and their recombinant expression. Turns out they're not quite as insoluble as I had feared, especially if you keep the Ca concentration low enough. They've even been explored as chaperones to get other problematic proteins to fold better. There's also some papers on alpha- and beta-casein expression in E. coli and yeast, and on reconstitution of casein micelles from beta and kappa casein (beta casein is the major component in human milk).

Reasons why the vegan cheese in yeast project is awesome

1. It will get a lot of attention to Counter Culture Labs (great PR angle)
2. It could be used to show the local environmentalist organizations that GMO itself is not pure evil and that we (CCL) are on their side (which I feel that we are). E.g. Sierra Club, Earth First, Greenpeace, etc.
3. It can involve people who don’t have much lab experience, since we’ll have to actually make cheeses both with milk (to ensure that we can make cheese at all), with dried casein from milk and butter (to ensure that we can reconstitute the casein into something that can be used to make cheese) and with dried casein and a milkfat replacement.
4. It is something that will be potentially useful over a relatively short timespan.
5. It is ethically awesome
6. It could turn into a commercial product
7. Selling the worlds first real vegan cheese at an auction could fetch CCL quite a bit of money.
8. Everyone loves cheese (ok maybe not everyone).
9. Nutritional yeast is already used as a cheese substitute by many vegans, so we could make a cheaper less purified product and still have it be a viable cheese substitute.
10. It is fairly realistic to at least express the protein in yeast and the project can easily be divided into separate phases with their own independent success criteria, many of which can be worked on in parallel (express protein, export protein from cell, express a lot of protein, purify protein, make cheese from protein, find viable replacement for milkfat, scale up production).
11. We could label the cheese “Ethical GMO” or “Non-evil GMO” or invent our own term. Maybe a backronym Genetically Magnificent Organism. Genetically Modified and Open.
12. We will have to use another sugar than lactose (probably not a problem, lactobacteria can live on other sugars), which will make our cheese lactose free.
13. Marc has already built a wifi-enabled cheese cave out of a wine cooler that we can use to age our cheese prototypes.
14. There is _so_ much info available on cheese chemistry because cheese industry.
15. Chymosin, the rennet enzyme, is already commercially produced by genetically modified organisms. They label it “microbially produced” and most major cheeses use it so also producing the casein protein shouldn’t make any difference with regards to law/regulation.