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Team:SF Bay Area DIYbio/Project
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Overall project summary There are currently over 260 million dairy cows worldwide, each producing 115 kg CO2 equivalent per day, or about 2.7% of global greenhouse gas emissions1,2,3. In South America, Africa and Asia, deforestation to make way for pastureland is an urgent concern. About 83% of the deforestation in the Amazon rainforest is used for pasture4. The Real Vegan Cheese Project intends to reduce dependence on large-scale commercial dairy in the production of cheese and casein-derived products by replacing the dairy cow with baker’s yeast, Saccharomyces cerevisiae. We are engineering the synthesis of bovine and human Alpha-S1, Alpha-S2, Beta and Kappa casein proteins as well as a kinase enzyme that may help the process of making a solid cheese. It is our hope that we can synthetically produce cheese that is more ethical, healthier and produces lower emissions than traditional dairy. By replacing dairy cows with carbon-sequestering bioreactors, we could save thousands of doe-eyed Holsteins and Jerseys from the misfortunes of the industrial farm, reduce the incentive for deforestation and drastically lower the GHG emissions of casein and cheese production. We estimate that we could reduce emissions per gram of casein by 40-90%. Furthermore, Real Vegan Cheese is committed to open source and open patent biology. It is our hope that this process can be employed and improved upon by enterprising biologists, hackers and epicures the world over. Project Details 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. Materials and Methods The Experiments Results Data Analysis Conclusions We have gone through numerous hurdles; we have strived and put our utmost effort into keeping this project open source and non-profit. We represent something different - something revolutionary. Powered by citizen science and representing a community spirit, we are attempting to do something that will provide vegans, lactose intolerant people, and people with cow’s milk allergies around the world with an alternative- real vegan cheese. We are attempting to make a real vegan cheese by engineering normal baker's yeast (S. cerevisiae) to express milk proteins (caseins), purifying the proteins, creating a milk-substitute by blending in vegan replacements for lactose and milk fat, and finally turning the resulting milk-substitute into semi-hard cheese like gouda using the regular cheese-making process. In doing so we are not only creating something that will revolutionize the food industry, but we are also representing the groundbreaking concept of citizen science and community labs. We are giving people of all sorts of ages and experience the opportunity to learn and take part in something revolutionary. Citations
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