Chapter 9. Real Vegan Cheese

Patrik D’haeseleer, Marc Juul, and Craig Rouskey

Team photo
Figure 9-1. Team photo

Cheese. The food that all people love. Unless, of course, you’re lactose intolerant, have an autoimmune reaction to cow, goat, or sheep milk proteins, or for a variety of reasons have chosen to be vegan. While a number of vegan cheese products exist, they all rely on a combination of alternate protein sources and thickeners to approximate the coagulation phenomenon that occurs during cheesemaking [1]. Thus, while vegan cheese manufacturers have created many delicious products, and even passable substitutes for some types of cheese, none has so far been able to deliver anything resembling a sharp chedder or aged Gouda. In response to the needs of people with dietary restrictions around animal-derived cheese products, the Real Vegan Cheese team has emerged from the San Francisco Bay Area to deliver broadly consumable real vegan cheese. The team is a collabortive effort between Oakland-based Counter Culture Labs and South Bay–centered BioCurious. These biohacker organizations have joined forces to enter their Vegan Cheese project into the International Genetically Engineered Machine (iGEM) competition taking place in October 2014.

On paper, making Real Vegan Cheese should be simple. First, genetically engineer brewer’s yeast to produce cheese protein. Then, grow the yeast in a bioreactor and purify the protein. Combine the cheese proteins with a vegan milkfat replacement, a (nonlactose) sugar to feed the ripening bacteria, and water to produce a sort of vegan milk. From there, proceed with the age-old traditional cheesemaking process for the desired type of cheese.

In practice, it gets a bit more complicated. Milk, it turns out, is a fairly complex substance. It is nature’s solution for packing large quantities of protein, calcium, and fat into liquid form that can turn into a solid for prolonged nutrient release in a suckling mammal’s stomach—a pretty impressive bit of biochemistry.

The Structure of Cheese

Time for some dairy science: given the right pH and calcium concentration, four of the more hydrophobic milk proteins assemble into micellar aggregates that include calcium ions and milkfat molecules. One of these caseins, kappa-casein, acts as a sort of built-in surfactant by making up the surface of the micelle and extending its hydrophilic tail into the watery solution. Making hard and semi-hard cheese usually involves the use of the rennet enzyme Chymosin, which cuts kappa-casein’s hydrophilic tail, making the now hydrophobic micelles link together into a network, forming cheese curd. This process is so efficient that it takes as little as 40 minutes to convert milk into something akin to a block of soft tofu floating in a watery solution.

It’s not surprising that vegan cheese is hard to make. The cheesemaking process relies on specific protein interactions that simply do not occur with proteins from any nonmammalian source.

The obvious solution is to invent a nonmammalian source of the required proteins.

Genetic Engineering

To make the cheese-protein-producing yeast, first, the genetic sequences that code for milk proteins in mammals have to be analyzed and the DNA sequence optimized for expression in yeast. The important four proteins for cheese are the four caseins: kappa, beta, alpha-s1, and alpha-s2. The genetic sequences for the yeast versions of these proteins are combined with a secretion signal (alpha-factor) that will cause the proteins to be secreted from the yeast cells. The sequences are synthesized and inserted into a plasmid with an inducible promoter so the expression can be controlled during growth, and the plasmid DNA is transformed into baker’s yeast (Saccharomyces cerevisiae). The yeast is then grown in vegan broth media, where it expresses and secretes cheese protein, which can be separated and purified.

So far this is all standard genetic engineering, and while getting the yeast to express and secrete enough protein may prove challenging, this part of the project should not present any extraordinary problems—in fact, we’ve already discovered published papers demonstrating that most of the casein proteins can be expressed in yeast or E. coli. The difficult part, it seems, is getting the purified protein to form correctly into micelles in imitation of the structure in milk. Getting transgenic proteins to fold correctly is often a problematic endeavor, usually involving considerations of protein secondary structure, tertiary structure, and post-translational modifications such as phosphorylation and glycosylation. So far, our research indicates that folding of individual proteins will not be an issue. With regard to glycosylation, its influence on micelle formation remains an open question. But even a glancing review of dairy science literature will show that correct phosphorylation is likely to make or break the project. Interestingly, though protein phosphorylation was originally discovered in casein and discovered way back in 1883 [2], the kinase enzyme responsible for phosphorylation wasn’t identified until 2012 [3]. In mammals, this unusual kinase—no, not "casein kinase," but one named Fam20C—is actually secreted along with the cheese proteins, and our team is designing for a similar effect in yeast. By altering the kinase secretion sequence, optimizing the kinase sequence for yeast, and co-expressing it with cheese protein, the secreted proteins should emerge correctly phosphorylated.

Molecular Gastronomy

Even with a perfect set of mammalian cheese-proteins, our experiments have shown that the process of turning purified proteins into micelles in solution is not as simple as mixing and stirring. Our research suggests that precisely controlling pH and calcium concentration while using a combination of techniques such as ultra-sonication and colloid mill homogenization are likely to achieve the desired results.

Narwhal Milk?

Each of the four casein proteins found in dairy milk has dozens of different genetic variants that occur in different cow breeds, not to mention goats, sheep, yaks, camels, water buffalos, and more. Many of these genetic variants are associated with different coagulation properties of the milk, various health effects (check out the hype around "A2 milk," for example), or even allergic reactions. Since we have full control over the DNA sequences, we have the option to pick and choose exactly which of these genetic variants to incorporate. Since one concern is minimizing the chance of an allergic response, an obvious choice is to use human genes, although we have noticed a sociologically interesting "ick factor" around the idea of drinking human-derived milk. Finally, for packing the maximum amount of protein per volume into milk, nothing beats a whale. Thus, our team has opted to engineer three varieties based on genes from three different species: human, cow, and narwhal.

Social and Ethical Concerns

Factory farming often entails treating animals less well than most of us would like, and it is likely that providing better alternatives will decrease demand for traditional products and thus decrease the number of poorly treated animals. Using genetic engineering to achieve such a goal makes this an interesting ethical quandry for many of those who oppose GMOs and champion the ethical treatment of animals. There are three important points that should be considered when addressing this issue:

  1. Real Vegan Cheese will not contain any GMOs.

    The genetically engineered yeast is only used to produce milk-protein.

    The yeast itself stays behind while only the milk-protein becomes part of the cheese.

  2. The yeast will be contained in bioreactors, not grown freely in the environment.

    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).

  3. This method of production has been used for more than three decades, safely, successfully, and at large scale to produce anything from vanillin (vanilla flavor) [4] to life-saving drugs such as insulin [5] and affordable malaria medicine [6].

These issues should be taken into account when evaluating whether the dangers of genetic engineering outweigh the potential for reducing animal mistreatment.

As a relevant comparison, most of the cheese produced today is made with a rennet enzyme manufactured using genetically engineered organisms grown in bioreactors, which has limited the need for harvesting rennet from the stomach linings of young cows [7].

Environmental Impact

The cost of yeast-based production of cheese protein makes it unlikely that it will pose a threat to traditional methods in the near future. It is likely, however, that this method will provide an alternative for those with dietary restrictions, whether ethical, religious, or health related.

That being said, production of milk and cheese using milk from factory-farmed animals has a host of environmental and ethical problems, and it is important to understand if this method of production will be preferable from an environmental standpoint.

The conclusion of the 400-page UN report "Livestock’s long shadow" has this to say:

…the livestock sector is a major stressor on many ecosystems and on the planet as whole. Globally it is one of the largest sources of greenhouse gases and one of the leading causal factors in the loss of biodiversity…

While our team is still working on a comparison of the expected impact on climate change per gram of cheese produced using traditional methods versus yeast-based production, it will likely be relatively simple to contain the carbon dioxide released from large bioreactors, while doing the same for the methane produced by grazing cattle poses a unique and difficult challenge. At first glance, the required food source for yeast is potentially less favorable than that preferred by cows, since yeast’s preferred diet of sugars makes it compete with humans for arable land capable of supporting sugar-producing plants, whereas ungulates are able to digest foods that grow on land less suited for traditional crops. In reality, both bioreactors and livestock are often fed with various industrial byproducts not fit for human consumption, which complicates the comparison and makes it difficult to ascertain how bioreactors compare to cows in their effect on biodiversity and on the global food supply. A more thorough analysis is part of our effort, and we welcome anyone who wishes to collaborate or critique.

How to Support

Contributors are invaluable to us and throughout this project have taken many forms. Our collaborators and supports provide financial resources, time, or even scientific resources. This is an open community project, and meetings are held every week in Oakland or Sunnyvale, and remote participation is possible via video conferencing. Discussions take place on our mailing list: http://groups.google.com/group/ccl-igem. All of our research notes and meeting minutes are available on our wiki: http://wiki.realvegancheese.org. If you would like to support us by turning your dollars into cheese, visit http://support.realvegancheese.org/.

References

  1. Daiya lactose-free cheddar cheese alternative slices. Retrieved from http://bit.ly/1xcifBS.
  2. Hammarsten, O. (1882–83). "Zur Frage, ob das Casein ein einheitlicher Stoff sei." Zeitschrift für Physiologische Chemie 7 (3): 227–273.
  3. Tagliabracci VS, Engel JL, Wen J, et al. (2012). "Secreted kinase phosphorylates extracellular proteins that regulate biomineralization." Science 336 (6085): 1150–3.
  4. Rouhi, A. Maureen (2003). "Fine chemicals firms enable flavor and fragrance industry." Chemical and Engineering News 81 (28): 54.
  5. Humulin - Life Sciences Foundation. Retrieved from http://www.lifesciencesfoundation.org/events-Humulin.html.
  6. Ro DK, Paradise EM, Ouellet M, et al. (April 2006). "Production of the antimalarial drug precursor artemisinic acid in engineered yeast." Nature 440 (7086): 940–3. doi:10.1038/nature04640.
  7. Chymosin - GMO Database. Retrieved from http://www.gmo-compass.org/eng/database/enzymes/83.chymosin.html.