Just created the blueprint for yeast-made milk. Any takers?
Genetically engineering a yeast plasmid to produce milk — without the cow.
I really love dairy.
However, in the last few years, I’ve come to understand the climate impacts and ethical violations left in the dairy industry’s wake. Greenhouse gas emissions, surface runoff, biodiversity loss, animal cruelty…now, in retrospect, it seems to be a difficult thing to love.
People’s first instinct when I discuss these topics is to turn to alternatives. “If dairy and meat are so bad — what, do you expect me to turn vegan?”
This, now-common reaction to information on food sustainability displays the immense progress of the plant-based industry, but in my opinion, also leaves little space for new conversations.
Plant-based alternatives are only part of the answer because the world has too large of an appetite for real dairy, myself included. In fact, world dairy production is projected to grow at 1.7% per year by 2030, faster than most other main agricultural commodities. So despite the prevalence of emissions, waste, and subsequent climate effects from the dairy industry, there continues to be a significant demand for dairy products.
Additionally, dairy has an incredible amount of cultural and social significance, being at the core of traditions, knowledge and practices performed by people around the globe.
This industry is deeply entrenched in our world due to these socio-economic factors. In other words, the products themselves aren’t the problem, but rather the process of production.
If there’s anything I’ve realized from writing and learning in the food climate sector, is that for every good or service we create, a plethora of climate impacts are generated throughout the entire life cycle of the product, from cradle to grave.
In the case of milk, the major climate impacts are emitted by the production of feedstock (corn, wheat) for cows, livestock manure, and the natural production of various harmful gases throughout the process, like ammonia and nitrous oxide. But as detailed in the high level overview in Figure 1, there are a variety of other inputs required to complete the various steps required throughout the entire traditional milk life cycle.
It’s clear that it isn’t dairy that’s got to go — but instead, dairy practices.
This is where this project comes in. And no, this is not some half-baked technology of the future, but rather a real, patented present idea. Many companies have already made this process happen, for a variety of food products, not just dairy. Products that can be bought in stores.
Welcome to the exciting world of precision fermentation, the usage of natural fermenting microbes to produce fully fledged proteins and end products. In this case, giving yeast the ability to produce real milk.
In this article, I aim to fully explain the scientific process of producing real milk proteins using this process by doing it alongside you. Procedural knowledge (information gained from doing the thing) is in my opinion one of the most tangible ways to understand complex ideas. Accordingly, I can’t wait to share the small amount I have gained, alongside my immense excitement for the future of this tech!
Summary
I created the genetic blueprint for a yeast cell to produce milk on Benchling, a genetic engineering platform, harnessing the power of biology and precision fermentation.
My plasmid can give a yeast cell the ability to create equal parts of the four main proteins found in milk. For the sake of staying within legal, un-patented ground, I used the genome of a reindeer instead of a cow.
My methodology was based upon some of the incredible work by Justin from The Thought Emporium and Katherine Hubbard. Both of these sources are a must-view as they facilitated my entry into plasmid design with relevant high quality content.
✩ Additionally, this article assumes a baseline knowledge of biology. I define terms that are pertinent to understanding and specificity of the project, but my target audience are people that are as nerdy as I am about biology and food.
Finally, this whole project was heavily inspired by an awesome company called Perfect Day, who were one of the first to make this process real, now to the point of being able to create a beverage alongside Nestle with their brewed milk proteins. Their work catalyzed my current trajectory in food, and continues to fill me up with insane optimism for the future it will unlock.
The Overarching Process — Fermentation
Fermentation — the process where yeast metabolize sugar into alcohol — is the baseline of this project. But before animal-free milk, it’s been in practice for 10,000 years, in industries like beer, kombucha and a variety of other products we’ve consumed for years.
Using the example of beer, the process of fermentation (Figure 2) is carried out using wort, a mashed liquid containing sugar from barley or wheat. The wort is then metabolized by yeast to produce the final, foamy alcohol product. Depending on the type of wheat or barley, yeasts used, and wort produced, many different varieties of beer can be produced.
Fermentation can more specifically be broken up into two main steps: glycolysis and NAD+ regeneration.
Glycolysis is the breakdown of the single chain molecule of sugar (like glucose) into two “half” chains called pyruvates, and NAD+ regeneration is the process of transforming those pyruvates into ethanol using enzymes.
There is more nuance and chemistry behind this process shown briefly in Figure 3.
In this application of fermentation, called precision fermentation, the exact same process is followed, but instead harnesses the power of genetic engineering to produce another end product instead of alcohol.
Microorganisms, like yeast, are like incredibly useful cell factories. By programming the right DNA, we can optimize their machinery to produce a wide array of amazing products. In our case, these products would be the wonderful proteins that make up milk.
Technical Overview:
In order to program proteins, or the little machines that complete all cellular work, I had to create the code for their production, the DNA. I executed this by using a yeast plasmid. Then, through the process outlined by steps 2, 3, 4 in Figure 4, I genetically engineered my plasmid by inserting the code for reindeer milk proteins inside of it (in red). After the created plasmid was ready, it would be inserted within the yeast cell using a process called transformation. Finally, using the biological processes of transcription and translation, my yeast cell could theoretically now manufacture the desired milk proteins.
Although I created the code on my computer, I didn’t complete this project in practice by actually synthesizing the DNA in yeast cells and producing the milk. Regardless of this limitation, I will still explain how each of these steps could be completed tangibly in the lab, as it makes the significance of this project more real.
Context: Plasmids 101
This project centred around one plasmid, but it really made me appreciate the beauty and power in these circles of code.
Plasmids are circular pieces of DNA that exist within many different organisms, primarily bacteria, but occasionally in eukaryotes like yeast! They are incredibly useful cloning vectors. A cloning vector is just a fancy name for a carrier of inserted foreign DNA, like the turquoise piece in Figure 5.
Plasmids have been my focus in the last few months because they are very valuable tools in genetic engineering. This is because they replicate independently of chromosomal DNA (DNA found in the nucleus with all the vital survival components) and are generally less tightly packed than chromosomal DNA, so scientists and wannabe genetic engineers like me are able to manipulate them more effectively.
Plasmid Capability Breakdown
1) Independent replication
Plasmids can replicate independently from the host cell chromosome because they have their own origin of replication (ORI). The ORI is the place where DNA replication begins, and because it is unique for each plasmid, the enzyme of replication, DNA polymerase, completes DNA replication separately.
2) Easy Manipulation
Plasmids are easy to manipulate for bioengineers as a result of their nature — because they aren’t tightly coiled around histone proteins, like the packed linear DNA found in the nucleus, their circular unpacked DNA is easy to edit. Plasmids are also generally much smaller in length, around 5–500 kilobase pairs vs 200 to 2000 kb in the yeast chromosome.
Additionally, plasmids contain areas called restriction sites where new genetic material can be inserted. This can occur with the help of restriction enzymes that recognize these sites and are able to create a “cut” in the plasmid gene.
Now that the plasmid context has been established, let’s dive deep into each step outlined in Figure 4.
Technical Deep Dive: Steps 1–5 (Restriction Cloning)
Restriction cloning, or the insert of new foreign DNA for replication in a plasmid, consists of four main steps: digestion, amplification, ligation and transformation.
Digestion is the process where a restriction enzyme “cuts” the plasmid at a restriction site; amplification is the preparation of the desired gene for insertion; ligation is the “glueing” of the plasmid and desired gene; and transformation is the insertion into a real cell. The full process is outlined in Figure 6.
Digestion + Amplification
Digestion occurs at the restriction sites detailed earlier. In the lab, you would select which restriction site to use depending on what enzymes were available, the nature of your inserted gene, and the type of cut present at each site.
At blunt restriction sites, like that of EcoRV, a straight cut is completed by the EcoRV enzyme, allowing for any gene to be inserted, no matter the base pair.
At “sticky” restriction sites, like that of SalI, a staggered cut is completed by the Sal1 enzyme, requiring the inserted gene to be amplified with cloning primers. Cloning primers would contain the corresponding base pairs of the sticky ends to align properly.
If we were creating a cloning primer for the SalI method above for example, this would mean the corresponding base pairs of TCGAC for the first sticky end, and the corresponding base pairs of CAGCT for the second. This creation of primers (amplification) can be completed using a process called PCR, or polymerase chain reaction.
For my plasmid I decided to use the SalI restriction site because sticky ends allow for greater accuracy, as only the prepared gene can be inserted. My restriction site selection didn’t really matter because I wasn’t actually making the plasmid in the lab.
Ligation
Once the inserted gene is prepared with the correct matching cloning primers, all the cracks are sealed by a “glueing” enzyme called ligase. The plasmid now contains the genetic information to code for milk proteins!
Transformation
Transformation is the process of actually inserting the created plasmid into the yeast cell. There are a variety of methods to do so (see Figure 8) with varying degrees of efficacy. For example, heat shock is a common method that reduces the repulsion between the plasma membrane and the plasmid allowing it to enter and become a transformant (a cell with the plasmid).
A key takeaway about this process is that not all yeast colonies will contain the plasmid by the end. This is a result of the inefficiencies present in each method, and other host cell factors.
Understanding the process of actually inserting this gene was a necessary prerequisite to creating it. But now, let’s design our plasmid, using the awesome methodology provided by The Thought Emporium!
DESIGN STEP 1: Finding a plasmid
Some criteria for a high-quality plasmid for this project were an effective selectable marker, a strong promoter in yeast, and a multiple cloning site. I was also looking for a yeast expression vector that was high copy, because this would ensure the stability of the plasmid in each of the daughter cells, and also create more viable copies of the plasmid if I were to execute this in the lab.
My explanations for each component are as follows:
Selectable Marker
A selectable marker acts like an indicator in the petri dish for which yeast contain the desired plasmid. After cellular replication, only a certain amount of yeast cells become transformants because of the nature of transformation, so by using a selectable marker, I would be able to determine which colonies contained the milk protein gene.
In practice, a selectable marker is usually in the form of an antibiotic resistance gene. Scientists will utilize a gel substance called agar to generate cell growth, and if an agar with the antibiotic is applied in the lab, only the plasmids with the selectable marker will survive.
For example, if my plasmid contained the Kanamycin resistance selectable marker, I would be able to isolate the colonies that contained it using a Kanamycin agar.
Multiple Cloning Site
A multiple cloning site is a spot on the plasmid with multiple insertion “slots” called restriction sites, mentioned in the digestion stage of restriction cloning.
Promoter
A promoter is the initiator of transcription of the gene inserted in a plasmid. It is the site where enzymes like RNA polymerase are given the green light to start transcribing the DNA into mRNA that will then be translated into a protein. Depending on the different promoters used, transcription strength can vary.
After learning about these components, all that was left to do was find the plasmid on Addgene, one of the best databases with a variety of cloning vectors.
I selected the Saccharomyces cerivisiae plasmid used in the methodology by The Thought Emporium because it contained a strong TEF1 yeast promoter, multiple cloning site with a variety of different enzymes, and the kanamycin selectable marker. An added bonus of this plasmid was that it also contained a bacterial promoter and selectable marker, so it had the dual capability of also being able to be expressed or transformed in bacteria, like E. coli!
NOTE: In full transparency, the specifics of the plasmid used weren’t of great importance to me as long as it had the main components outlined above. My intention was to learn the true science behind precision fermentation and solely perform the genetic engineering.
In reality, when completing a true synthesis of this DNA to be used in the lab, many considerations would have to be taken into account, including strain of yeast used, cost, efficiency, enzymes on hand, preferred promoter…the list goes on. It is worth noting that these were not factors I had to consider, but are just as important in the realization of this product outside of the digital world.
It was now time to create the gene that would be inserted through restriction cloning.
DESIGN Step 2: Creating the Inserted Gene
Now that I’d found my plasmid backbone, all that was left to do was create the inserted gene. The gene for this project produced equal parts of the four main proteins in milk: beta-casein, kappa-casein, alpha lactalbumin, and beta-lactoglobulin.
To create these four proteins, the inserted gene contained 11 components: 4 secretion signals, the code for 4 proteins, and 3 self-cleaving peptides.
1. 𝝰 Mating Factor Secretion Signal
Before I could import the DNA sequences of each protein, I had to account for a simple fact: where and how this protein would be manufactured. If I just inserted the protein sequences without this crucial component — the alpha mating factor — my yeast cells would have manufactured this protein until it filled itself up and burst, because most proteins are manufactured for use inside the cell.
The alpha mating factor is an important hormone in the mating of yeast. The most important fact to know about it in this context, is when it is produced by a cell, it is generally secreted (discharged) to mate and bind to other yeast cells. By inserting its DNA sequence into my plasmid, my yeast cell was granted the ability to produce this fantastic hormone that opens the cells secretory passages. Because the milk proteins follow it in the line of transcription, they will thus also be packaged by vesicles and discharged from the cell, preventing cell burst and promoting increased protein production.
I sourced the DNA sequence for the 𝝰 mating factor from another plasmid found on Addgene, and copied it in after the promoter to ensure it would be transcribed accordingly.
2. Protein Code
Before diving into this new world of genes, something I hadn’t previously realized was the abundance of genetic data available online. The internet is truly a goldmine of sequence information with incredible data sources like Uniprot, NCBI, and Addgene.
I sourced all the amino acid sequences for each of the four proteins from Uniprot, and then used NCBI’s BLAST (essentially a protein/nucleotide finder) feature to source their according sequences in the reindeer (Rangifer tarandus) genome.
I then back-translated these amino acid sequences back into nucleotide sequences using Benchling’s back-translate feature, and copied them in after each alpha mating factor signal to ensure secretion.
3. p2a Self-Cleaving Peptide
Another component I had to learn about was the p2a self-cleaving peptide. If I placed the genetic sequence for each protein consecutively, they would all be continuously translated, creating a monster “ultraprotein” with all four combined. This could create errors in aggregation and folding, and was not what I wanted for the components of my milk because within the beverage each protein is separate.
For this reason, I needed a tool that could stop translation between the proteins to ensure that the ribosomes wouldn’t continuously fuse the proteins all together. To generate this desired separated result, a self cleaving peptide was used.
A self cleaving peptide is a sequence that induces ribosomal skipping during translation. It completes this by preventing the formation of the peptide bond (amino acid ↔ amino acid bond) between itself and the subsequent protein, modelled by Figure 10.
All together:
Thanks to the wonderful work of those mentioned, I was able to recreate the full inserted gene sequence to produce the four main proteins that make up hypothetical reindeer milk! The final inserted gene is illustrated in Figure 11, with the magenta pieces being the alpha mating factor signals, the cool toned pieces being each of the four proteins, and the yellow pieces being the p2a peptides. Here is the link to my final Benchling file.
Now that I’ve created the blueprint, all the yeast cell has to do is manufacture the protein goods. It accomplishes this through two core processes — transcription and translation, that can be read about in more detail here, if needed.
Future Considerations
Although these four proteins are the main part of the protein component of our favourite creamy white beverage, milk also has many other components, outlined with percentages in Figure 12.
To make this animal-free dairy truly milk, these other components would need to be added and factored in to the final mixture after the secreted proteins were separated from the yeast cells and processed.
This project focused on the proteins instead of the other components because they were the most straightforward to engineer, and also are the easiest to transform into a variety of products, not just milk.
Additionally, this plasmid produces equal parts of all four of these proteins, when in reality, this isn’t the case. If this design were to be refined, I could utilize different promoters with varying strengths to produce the correct ratio of proteins in true milk composition.
Conclusion
All in all, I learned a lot through this process of producing a design that’s could make an innovative food product a reality. My brain had to venture down a lot of new neural pathways during this project.
I’ve now been exposed briefly to the extent of detailed information on these components we call genes. I can’t wait to keep swimming towards the bottom of our knowledge on this biological code. With new breakthroughs every year, who knows how far we’ll keep extending it?
My main takeaway from this whole process is that biology is an insanely powerful agent in realizing new and interesting ideas. Whether it’s in an application that aims to solve an actual problem like this one, or a truly magical experiment, the positively endless possibilities with this technology fill me up with so much energy and enthusiasm.
It’s been incredibly exciting seeing all the new food companies popping up in precision fermentation! I think that with enough time, transparency, and development at scale, this field has the potential to narrow the delta between the world’s growing appetite and the climate impacts of food.
Thank you so much for reading my article! My list of references can be found here. I hope you extracted some value, and if you’d like to connect with me in other places, here’s my twitter, linkedin and substack where I occasionally post mini reflections. ✩
I approached the writing of this piece with the core value that there is much that I don’t know I don’t know that I want to learn, so if there are any misconceptions you feel are present in this article, please reach out by email here, always happy to iterate and expand my brain. Appreciate your time — your most valuable resource — and look forward to many more future moments!
