Questions and Answers

What is Real Vegan Cheese?

Real Vegan Cheese (RVC) is a grassroots, non-profit research project working to produce real cheese using cellular agriculture. We add the genes for cheese proteins to yeast and other microflora, and turn them into little protein factories, then make real cheese by adding plant-based fats and sugars. We are dedicated to Open Science and making sure the results of our research are available to the global community to enable a sustainable animal-free dairy industry.

Why do we need Real Vegan Cheese?

We believe that using animals as large-scale food production machines is ethically and environmentally irresponsible. The dairy industry takes a heavy toll on the environment, including an enormous carbon footprint. It also causes a lot of suffering for the animals involved. However, people crave cheese! That’s why we think it is so important to develop animal-free cheese with a lower carbon footprint that is just as delicious as traditional cheese.

How is this different from other vegan cheeses?

There are plenty of companies making nut, soy, or even mushroom based “cheeses”, and we have tried several of these ourselves. There are even some plant based cheeses that melt nicely. But in our opinion, none of these have the combination of texture and flavor that really satisfy our cheese cravings. That is why we want to make a Real cheese, not a plant-based spread that is vaguely reminiscent of cheese.

How is Real Vegan Cheese different from companies developing similar products?

Since our project started in 2014, a whole industry of exciting cellular agriculture start-ups has emerged. This includes some companies developing dairy and egg products from proteins produced in cell culture, similar to Real Vegan Cheese. However unlike these companies, all of our work is “Open Science” (see question below), and we have a broad mission to help build a global community to enable a sustainable animal-free dairy industry. We are also one of the only groups focused on cheese.

What does it mean to do “Open Science”?

We believe that this technology should not be locked up in proprietary patents monopolized by a single company. Rather, we want to make sure that anyone with the right resources and expertise can start up a cheesemaking company. All information is published under free-culture licenses (e.g. Creative Commons). Any and all patentable material is put in the public domain; and all research is published via our wiki and mailing list as it is generated.

Where does the cheese protein DNA come from?

None of the genes we use have been inside an animal. To make our proteins, we study animal genomes (such as the cow genome, which has already been sequenced), select the gene sequence that produces milk proteins, and then synthesize those genes.

Does this cheese contain GMOs?

While our cheese proteins are produced by genetically engineered yeast or bacteria, the resulting cheese should not contain any engineered organisms or DNA. Note that the microbially produced rennet used in 80% of the world’s cheese is produced by genetically engineered bacteria or fungi, so you are likely already eating cheese produced with the help of genetic engineering. We encourage people to have an open mind about genetic engineering: it can help us address some of the world’s most pressing problems with safe, environmentally friendly, nutritious food.

Is this safe for the environment?

The resulting cheese will be made without animal suffering, and with far fewer greenhouse gas emissions than regular dairy cheese. The yeast will be contained in bioreactors, not grown freely in the environment. This also means that we can control the waste output from our process with more precision. Additionally, the strains of yeast will be engineered to prevent them from growing outside of the intended bioreactors. This will prevent environmental contamination and contamination of the products of nearby yeast-farmers (brewers and bakers).

Is this actually vegan?

Yes! Our cheese and cheesemaking process are completely free from animal products, meaning no animals are harmed or exploited when making our cheese. We use yeast and other microflora growing on vegan media to express our milk proteins.

What about people who are lactose intolerant?

Good news! Since we will be producing pure milk protein, there will be NO lactose in Real Vegan Cheese whatsoever. We will likely be adding some other type of sugar (not lactose), both for flavor, and to feed the bacterial cultures that are used in cheese ripening.

What if I have a dairy allergy?

Cow’s milk protein allergy is an entirely different issue from lactose intolerance. Because the cheese protein we’re initially producing is identical to the animal-derived version, these proteins may trigger a reaction for those who are allergic to milk proteins. However, because we have the luxury of engineering everything from scratch, we can choose the least allergenic naturally occurring casein variants from cows or other dairy animals.

How much will it cost?

In the long-run, this animal-free cheese needs to be affordable to everyone. Eventually, the price should be competitive or cheaper than traditional cheese. Achieving a low cost will require extensive scale-up and process optimization. We expect the first products will be priced like a high-end organic cheese, and then the price will fall from there. Cheese also offers a more favorable path to commercialization than liquid milk: it has a higher retail cost per gram of protein, and is a more differentiated product (less of a bulk commodity).

Can I try some cheese? When will it be available?

We have made very small quantities of protein and cheese so far -- not yet enough for a tasting event. Our current focus is on developing the science and production process.

What are Real Vegan Cheese’s plans for commercialization?

As a non-profit organization, Real Vegan Cheese is dedicated to developing this technology, including some initial scale-up, and making sure it remains available to everyone. We do have plans to spin off a commercial venture to commercialize the cheese while keeping the IP open - help us develop a business model!

Can I get involved?

Absolutely! No specific background expertise required - there are plenty of volunteer spots we can fit you into. If you are in the SF Bay Area, the easiest way to jump on board is to join us at one of our weekly organizational meetings Monday evenings at Counter Culture Labs or BioCurious. If you cannot join us in person, you can connect to those meetups online over Zoom.

I want to get started! What do I need to know?

We’ve spent some time working on making the process of joining as easy as possible. We’ve got a blog post going in-depth on background information on cheese and how the project works, with resources for anyone from a middle school student fascinated with science to a PhD in microbiology looking to contribute their expertise. We suggest you start with the blog post here, or just show up at a meeting and jump right in!

What particular tasks do you need help with?

Anyone is welcome to help out, but definitely reach out to us if you can help us with these specific skills:

• Milk/cheese/food science
• Website and social media
• Legal issues, especially around protecting Open IP
• Molecular biology, especially protein production and purification
• Developing a for-profit, but Open Source business model for Cellular Agriculture (there are good models for Open Source Software, and Open Source Hardware, but hardly any for “Wetware”)

Where does the funding come from?

The Real Vegan Cheese project is currently a completely volunteer-run non-profit organization that runs on a shoestring budget. We initially raised $37K USD of funding on Indiegogo back in 2014. We also received a $50K grant from the Tarshis Family Foundation in 2018.

How can I donate to the project?

You can contribute to the project via our donate page.

Biology

Why we need to optimize the codon usage for yeast

The short answer is: Because each codon has multiple possible tRNAs that recognize that codon, and the abundance of tRNAs differs between species.

As an example, there are several different codons that code for Serine (UCU, UCC, UCA, UCG) but in one species it may be that there are lots of tRNAs that recognize UCU, but very few for UCU, UCA and UCG. Thus, if you want high expression, you'll want to use UCU as your codon for serine.

Here's the mapping of codons to amino acids:

 http://en.wikipedia.org/wiki/Genetic_code#RNA_codon_table

and here's the codon usage table for S. cerevisiae:

 http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=4932

Now, getting high expression isn't quite as simple as just choosing the most-used codon for each amino acid. There are other factors. For example, it may actually give higher expression to use some less-used codons at the beginning of a gene, giving the ribosome a slow start, and some proteins require that the ribosome slows down in certain regions in order for the protein to fold correctly (presumably one part needs time to fold before the next part is added).

Another important factor is mRNA secondary structure. If the mRNA sequence is such that there are sequences complimentary bases on the same mRNA strand, then it can fold in on itself and prevent or limit ribosome binding. So, codon optimization tools try to take a set of factors into account at the same time and create the optimal compromise codon usage that is expected to get the highest expression.

Even though codon usage optimization can be important it's often not necessary at all. For many proteins you will get some level of expression if you simply copy the gene from its host organism with PCR and transform it into your cell with an appropriate plasmid vector. It's likely however that we'll get much lower expression levels, but that's not always a bad thing (an expression that's too high may put a high burden on the cell and reduce survivability or for secreted proteins it may saturate the secretory pathway).

We're going to optimize codon usage for high expression and use an inducible promoter to limit how much is expressed. That allows us to experiment with expression levels without changing the DNA again. It also allows us to wait until a culture is in its exponential growth phase before inducing expression, which is one way of getting a higher over-all protein yield from a batch culture.