So, you want to join the Real Vegan Cheese project? - Orientation for new folks

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. Real Vegan Cheese is not a cheese substitute -- the cheesemaking process is just like using cow or goat milk! 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.

What is Cheese?

Cheese consists of four main milk proteins, called caseins: Alpha-S1, Alpha-S2, Beta and Kappa casein. Milk contains several other proteins, but those wind up in the whey during the cheesemaking process. All four casein protein are somewhat hydrophobic so in milk they cluster together in small spherical blobs called casein micelles. Kappa casein has a hydrophilic (water loving) tail end, so these casein micelles wind up covered with kappa casein, with the hydrophilic tails sticking out, which makes the micelles stable in milk. The casein micelles in milk scatter the light and are responsible for the white color of milk. During cheesemaking, we use acids to collapse those hydrophilic tails, or enzymes such as rennet to cut them off. This causes the micelles to come out of suspension and stick together, forming cheese curds.

The coagulating casein micelles also trap a lot of the fat globules floating around in whole milk, as well as some of the lactose. And the casein proteins themselves also bind a lot of Calcium and Phosphate.

Making cheese without cows

The four main ingredients of cheese are water, milkfat, cheese protein (casein) and sugar (lactose). There are vegan sources for water, (vegan) milkfat, and sugar. That just leaves casein! (At least, that’s the idea…). If we can produce casein proteins in another organism like yeast, we should be able to construct a cheese which is virtually identical to cow’s cheese, but without any animal suffering and a lot fewer greenhouse gas emissions!

So that is the main goal for our project: Make casein proteins without cows; put those caseins together into casein micelles; make real cheese from those micelles.

Where are we currently?

Making casein proteins

We have been able to express all four casein proteins in E. coli - see here for a more detailed technical update. However we lost some critical mass for the wetlab component of the project, and we are looking for someone who can help us restart this work, either at BioCurious in Santa Clara, or at Counter Culture Labs in Oakland.

Our immediate next steps would be to retrieve the plasmids from the -80 freezer, transform them into E. coli, and store glycerol stock of the E. coli strains in the -80. Then we’ll do some expression and purification runs, and measure yield for each. We are also working on designing a new set of plasmids to express the casein proteins in yeast.

Subprojects

Casein Protein Production

We take the genes for cheese proteins (caseins) and use synthetic biology to add them to yeast and other microflora, which then produce the casein proteins. We then brew up a large batch of these engineered microflora in a bioreactor, and purify the casein proteins. 

Making Animal-Free Cheese from Recombinant Casein

After producing the casein proteins, now it is time to make cheese! First we need to combine the purified caseins into micelles - the tiny protein spheres that make milk white. We are also researching plant-based fats and sugars to replace the butterfat and lactose in traditional cheese. From there, we should be able to make any type of cheese we want using traditional cheesemaking techniques -- with some tweaks. 

Cheese Ripening and Flavor

In the aging process, diverse bacterial strains break down sugars, proteins and fat in fresh cheese into a complex molecular profile. This helps intensify the flavor and develop unique textures. We’re researching the molecular mechanisms behind this process and the strains needed to better understand how to create your favorite aged cheeses.

All the differences in flavor between a bland cottage cheese and a sharp cheddar are due to microbial action, so we have a natural interest in cheese ripening bacteria as well. In particular, if we use plant-based fats and sugars instead of butter fat and lactose, we will make sure that those ripening bacteria will produce the same flavor and aroma profiles that we expect in cheese.

Community Engagement

We’ve helped pave the way for international community-based research efforts, hosting and presenting at events around the globe. We also want to engage the public to discuss new food technologies in a transparent and open way. We aim to build trust in these new methods of food production, which show promise for ethical and environmental reasons.

Advancing Open Science

Most new food science is developed under patents and brought to market using a traditional intellectual property and investment business model. RVC is investigating and developing ways to use an Open Science business model. We don’t want to see promising technology locked up with a single company - we want it to be accessible to the wide spectrum of innovators, entrepreneurs, and cheese producers to have the largest impact. 

Narwhal Cheese and Evolutionary Genomics

We can make cheese from any genome sequenced mammal - how about… Narwhal whale cheese! Part whimsy, part hard-core science, this sub-project is studying topics from the evolution of the casein gene cluster and tooth development genes, to unicorn mytho-phylogenetics. This project is a great introduction to evolution, genome science, and bioinformatics. Click for more about this project

How can you contribute?

It can feel overwhelming to find a way to contribute to such a large and long running project but it is intended for anyone of any skill level to be a part of.  We do not assume that everyone will join and be at the same skill/knowledge level.  

Some skills that are most useful include: 

• Note taking

• Literature search

• Molecular biology

• Bioinformatics

• Evolutionary genomics

• Social Media (Surveys, Blog Posts, Social media posts)

How do meetings run?

Our online zoom meetings are every Monday, alternating between Science and Admin sessions.  During the science meetings we cover a specific science topic related to the project or go over recent science of the project. Admin meetings are where we determine the science topics and do some basic discussion of non science topics from blogs to the backend running of the project.  If you have non science questions then Admin weeks are the perfect time to ask questions.

In-person lab sessions at Counter Culture Labs or BioCurious are scheduled whenever we can get some folks willing to lead a session - stay tuned!

Join us next Monday…

You can join us every Monday at 7pm PDT. Sign up though Meetup.com at either of the following:

• https://www.meetup.com/counter-culture-labs/

• https://www.meetup.com/biocurious/ 

How can you learn more?

We have a wiki at https://wiki.realvegancheese.org/wiki/Real_Vegan_Cheese. It includes an introductory textbook that should give you a solid introduction to what’s going on.

You can also browse our FAQ.

The Fermentation-Enabled Dairy industry: how to make milk in a lab, and who are the major startups in the field?

By Maria Andrea Gonzales

Dairy has been a part of our daily routine throughout our entire lives — milk is the main thing we drink as infants, and after outgrowing plain milk consumption we still find it in the cheese, butter and desserts we all love. However, with the recent surge in the popularity of plant-based milk and awareness of the dairy industry’s environmental impact, it’s clear that consumers are looking for more eco-friendly ways to enjoy the creaminess of traditional dairy. This is where alternative dairy, also known as lab-made dairy, comes in. In this article, we will dive into the science of making lab-made dairy and take a look at the main companies leading the change into a cow-free dairy world. 

Making milk in a lab?

At first, it might seem nearly impossible to make milk in the laboratory exactly as it is produced by cows. However, startups have already started creating dairy alternative products acclaimed for being “indistinguishable” from the real thing. The secret behind the making of these alternative dairy products lies in what gives milk its characteristic creaminess and flavor: proteins. Casein and whey proteins are the main solids found in milk after sugars, and their structure is what gives milk its flavor and texture. When talking about lab-made dairy, startups usually only focus on producing the proteins found in traditional dairy. The other components of milk — fats, sugars and minerals — are usually obtained from plant-based sources which are then to be combined with the milk proteins. That’s right — milk made in the lab isn’t 100% identical to “‘real milk” just yet, but it can replicate its properties a lot better than plant-based alternatives can. 

The proteins themselves are produced via synthetic biology, that is, by genetically modifying microorganisms to make the proteins for us. First, we find the genetic sequences for bovine casein and whey proteins in online databases such as NCBI. Then, we synthesize those genes in DNA and insert them into suitable microorganisms, turning them into little milk protein factories. Once these are produced, they are harvested and purified to be mixed with fats, sugars, minerals and water, the other main components of milk. With that, a vegan yet creamy lab-made milk is obtained. This process is sometimes referred to as “precision fermentation” in media and articles. After that, if you wish to make milk-based products such as cheese or yogurt, the standard fermentation process that is used with cow milk can be used.

What are some of the startups in the field?

The idea to make a vegan cheese using recombinant protein had been circulating in the San Francisco Bay Area biohacker community since at least 2011. Members of the current Real Vegan Cheese team actually tried to start a Highschool team for the International Genetically Engineered Machine (iGEM) competition on the topic in 2012, followed by our 2014 entry as a team competing in iGEM (look for an upcoming blog post on the early days of th e Real Vegan Cheese project!). Our iGEM project aimed to make vegan cheese from lab-produced casein proteins, and we continued to pursue this goal even after the competition ended. There are several startups that have begun their journey into lab-made dairy production in the last few years. 

Perfect Day, a Bay Area-based startup, also started out in 2014, initially named “Muufri'' before rebranding to its current title in 2016. With $350 million in seed funding and valued at $1.5B, they are the only company currently working in the lab-made dairy market, producing a whey protein (b-lactoglobulin) and two casein proteins (kappa and beta casein). Since the company operates under a B2B model,  proteins are sold to alternative dairy companies such as Brave Robot and Modern Kitchen, ice cream and cream cheese producers respectively, who use Perfect Day’s proteins to incorporate into their groceries. Perfect Day is also looking to expand its frontiers to produce animal-free fats in the lab for their use in dairy alternative products along with their milk proteins.

After 2014, the next big years for animal-free dairy were 2017 and 2018, as four major cheese startups: Nobell Foods, Formo, Biomilk and New Culture were founded. Having raised $75M in a Series B funding round and $100M so far, San Francisco-based startup Nobell Foods - founded by former Real Vegan Cheese member Magi Richani - aims to produce milk proteins, specifically caseins. However, they achieve this in a different way from the previous organizations mentioned so far: by genetically engineering soybean plants to express caseins. These are later harvested from the plant and can be processed into cheese. According to the company, they chose the soybean plant as it makes protein more efficiently than any other crop while potentially being cheaper than cow milk. They plan on releasing mozzarella and cheddar cheese in the market by 2023. Formo, formerly known as Legendairy Foods, is a Berlin-based startup focusing on lab-made cheese. With a seed funding of $50 million and having gained a total of $101M in 4 funding rounds since, they are also aiming to have their lab made mozzarella and ricotta cheese in the market by 2023. Similarly, the San Francisco-based startup New Culture is also working in the vegan cheese department. They work with engineered yeast to produce alpha, beta and kappa caseins to mix with coconut, sunflower and canola oil along with water, sugar and minerals to ferment into mozzarella. The startup, founded in late 2018 received a $3.5M seed funding and has obtained $5.1M up to date. The last startup to have started in 2018 is Biomilk, an Israel-based company a bit different from the rest. Biomilk, unlike most companies in the list, doesn’t work with microbes or plants. Instead, they work with mammalian cells. They isolate bovine mammary cells and culture them in bioreactors that mimic the conditions found inside of the animal, which enables them to grow and develop until they secrete milk, like they would inside the cow. The company received $2 million as funding to make their innovative idea possible.

Remilk and Fantastic Farms, founded in 2019, are two other Israeli dairy alternative startups to join the race. The former is working with microbes to express caseins for the making of milk. The latter is instead working with plants, engineering them to produce dairy proteins to later extract them and make dairy products. In that same year, another San Francisco-based company, Change Foods, was born with an ambitious goal — producing milk fats through synthetic biology for their use in cheese. The startup, with $3M in seed funding, aims to make not only caseins, but also milk fats to replace the plant-based oils used in the making of animal-free dairy cheese. Like fellow startups Formo and Nobell Foods, they are also planning on releasing their first consumer-available product in 2023. 

Even through the COVID-19 pandemic, two other companies have managed to start out in 2020, with the most recent one having announced its first appearance in August this year. We are talking about Those Vegan Cowboys, Imagindairy and Renegade Creamery; Dutch, Israeli, and Minneapolis-based startups respectively. Those Vegan Cowboys, like a lot of startups in this list, specialize in casein production for cheesemaking as well as milk. Imagindairy follows a similar path to Perfect Day, as they also use a B2B model and are starting out with selling whey proteins to various companies interested in making vegan dairy products. Finally, the latest startup to join the crew is Renegade Creamery, an initiative developed by General Mills in 2021 aiming to commercialize cheddar and cream cheese obtained from animal-free milk proteins.

Starting with Real Vegan Cheese before 2014, the lab-made dairy industry has grown to have more than a dozen startup companies currently in business (check out the excellent 2020 State of the Industry Report by the Good Food Institute). Some of these companies have even announced their first commercial cheeses to come to the market as soon as 2023. Here in RVC, we have great pride in being the first organization to have started working on this idea and being able to say we have mentored people in our weekly meetings that later went off to fund some of the startups mentioned in the list above. We hope to continue working towards this common goal and inspiring people to join us in the journey. To learn more about the early days of RVC and how we began, tune in to our next blog post: The History of Real Vegan Cheese.

Maria Andrea Gonzales is a 4th year Bioengineering undergraduate in UTEC (Lima, Peru). She is currently an intern in Real Vegan Cheese, helping out in the search for hypoallergenic caseins and media article writing. Maria Andrea is passionate about synthetic biology, working on various research projects in the food and agrotech sector.

Launching Our New Website!

Today we are excited to launch our new, completely revamped realvegancheese.org website! Sharing our work and resources with the world is a key part of the Real Vegan Cheese Project. Our previous website served us well since 2014, but was overdue for updates and new material. This new website is also easier for us to manage.

One of the added features of the new website is this blog, which makes it easy for us to share content and substantive updates about the project. We have already included several blog/news posts with significant updates from the past several years. However, note that this blog doesn’t cover all of our activity going back to the start of the project.

Narwhal Cheese at Biosummit 4.0

On Narwhal Cheese, the Evolution of Whales, and Imaginary Phylogeny of Unicorns

An introduction to the research being done by the narwhal phylogenetics team for the Real Vegan Cheese project.

We will start with a high level introduction into the research being done by the narwhal phylogenetics team for the Real Vegan Cheese project. After that we'll explore some of the specific tools and approaches that are being used to conduct including BLAST and Biopython. To close things out, there will be time for some questions.

What to bring:

Might be useful to install the latest version of Python and Biopython if you want to follow along with some of the steps.

Check out the video recording of this session here!