Precision Fermentation Perfected: Fermentation 101

Precision Fermentation

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Precision fermentation is a promising innovation that enables us to create animal products using microorganisms and a computer database.

Rapidly gaining prominence for its ability to produce food ingredients at scale, precision fermentation is fast becoming a key pillar in the alternative protein space.

Still, public awareness of this relatively new technology has yet to catch up.

Keen on getting up to speed? Here’s everything you need to know about precision fermentation—and more!

What IS fermentation anyway?

Traditional fermentation has been around for centuries as a way to preserve and increase the nutritional value of foods. Fermentation uses microorganisms (also called microbes) to chemically transform the composition of food through enzymes, which are a type of protein that start a series of chemical reactions. Common foods made via fermentation include cheese, yogurt, beer, miso, kombucha, and kimchi.

One of the earliest known instances of fermentation dates back to 1700-1100 BCE with the production of honey wine in Asia. People at the time understood the concept of allowing foods to transform over time, such as the transformation of grapes and barley into alcoholic beverages, but it wasn’t until much later that the science behind fermentation was fully understood.

In 1876 CE, French microbiologist Louis Pasteur discovered the role that microbes play in fermentation. Through his experiments, Pasteur found that microorganisms ferment to produce the nutrients they need in oxygen deficient environments.

This is called anaerobic respiration. In the absence of oxygen, microbes convert sugars into alcohol or lactic acid to create a fermented final product. In this way, fermentation has been a key method of food production for centuries.

What’s the difference between traditional fermentation and precision fermentation?

Let’s start with an analogy: Traditional fermentation is like a basic calculator, while precision fermentation is like a sophisticated computer. While a calculator can only perform basic calculations, a computer offers the ability to customize and manipulate data.

Similarly, precision fermentation leverages biotechnology to fine-tune the molecular output of microorganisms, giving scientists greater control over the fermentation process. Just as a computer can produce an array of outputs, precision fermentation can produce a variety of nutritious and delicious foods.

One major difference between traditional and precision fermentation is that in traditional fermentation, the inputs become the outputs. Think of how grapes aged in a wooden barrel turn into wine.

On the contrary, in precision fermentation, the inputs can be customized to create an entirely new output. As is the case with a computer, the number of modifications made to the inputs—and therefore the number of possible outputs—is nearly endless.  Thus, precision fermentation offers the opportunity to produce a plethora of new ingredients never before made through fermentation.

What was the first precision-fermentation-derived product?

The introduction of precision fermentation revolutionized the world of biotechnology in 1982 when Genentech debuted the first precision-fermentation-derived protein: a recombinant human insulin produced by Escherichia coli (E. coli).

This breakthrough was made possible by the recent advent of recombinant DNA technology, which allowed scientists to dictate what proteins the microbes produced. The FDA rapidly approved this novel insulin as the first commercially available product from recombinant DNA technology for human use, marking a major milestone in the evolution of precision fermentation.

The first food application of precision fermentation came in 1990, when Pfizer created precision fermentation derived rennet. Rennet is an enzyme found naturally in the lining of nursing calves’ stomachs, one that is commonly used to clot and curdle milk proteins during cheese making. Precision-fermentation-derived rennet was ground-breaking because it could be made without the use of animals. Since the FDA approved this animal-free rennet as a food substance in 1990, it has become the norm in cheese production globally, replacing rennet derived from cows.

What is a recombinant protein? How does recombinant DNA technology work?

Recombinant proteins are a result of genetic engineering through recombinant DNA technology. This technology enables scientists to instruct microbes to produce a new end product by inserting a customized genetic sequence into their existing DNA. The term “recombinant” refers to the combination of the microbe’s original genetic sequence with the added, synthetic DNA to mimic another organism.

To produce dairy proteins, for example, one would create a strand of DNA modeled after cow DNA and insert this into the microbe’s DNA to create a recombinant production host. Once this set of “instructions” has been given, the microbes will produce this new product automatically.

How does precision fermentation work?

Fundamentally, precision fermentation consists of just a few steps. Steps 1 and 2 are considered part of “upstream processing”, or the steps that create the product of interest. Step 3 is considered “downstream processing”, which pertains to how the product is purified and finalized for commercialization.

Precision fermentation in three steps:

  1. We create a string of DNA that contains the instructions on how to produce the desired product and insert this DNA strand into the DNA of the microorganism, also referred to as the production host.
  2. We place the modified production host under ideal growing conditions in a bioreactor, giving it all the nutrients it needs to grow and make the protein of interest (fermentation, or upstream processing).
  3. Once the production host has made enough recombinant protein, we remove the protein from the bioreactor, separate it from the production host, purify it, and often dry it so that the final product is a fine powder (downstream processing).

A closer look at these three steps:

Step 1 (Strain Engineering): Strain engineering is the process of getting the microorganism ready to be the production host. Most microorganisms need to be modified slightly to produce a recombinant protein. In precision fermentation, yeast, filamentous fungi, or bacteria can be used as the production host.

The most important modification made to the microorganism is the one that enables it to produce the protein of interest. We start by creating a piece of DNA that encodes the instructions for how to make the product of interest. In the case of producing a dairy protein, we review the cow genetic sequence to find this information. Because this genetic info is already available in online databases, this is done without ever touching a cow!

Next, the strand of DNA that we’ve created to look like cow DNA is recombined with the DNA of the production host. By inserting this genetic sequence, the production host now has the “instructions” to make the recombinant protein.

Step 2 (Fermentation, or Upstream Processing): Now that the microorganism has been optimized to produce the recombinant protein, we enter the growing phase.

The production host is submerged in a broth that contains all the nutrients it needs to make the protein of interest and both are added to the bioreactor: a large steel vessel similar to those used to ferment beer.

This step presents one of the biggest differences between traditional and precision fermentation. Whereas in traditional fermentation the starting material becomes the final product, precision fermentation differs in that the production host uses the nutrient broth to make a brand-new product. In other words, the production host acts as a tiny cell factory and makes the recombinant protein!

Step 3 (Downstream Processing): Once the production host has made the recombinant protein, the downstream processing begins.

First, the recombinant protein is taken out of the bioreactor and isolated from the production host and the media. It then undergoes purification processes so that none of the production hosts or media exists in the final product. Drying is often performed to take the final product from a liquid to a solid powder state. Finally, we test the final product to make sure it meets our product specifications and does not contain any unwanted particles.

This process may vary depending on the production organism used, the desired product, or the scale at which the product is being produced. However, the basic steps of this process remain constant for producing any recombinant protein via precision fermentation.

What foods can you make with precision fermentation?

Precision fermentation is a great method for producing proteins and enzymes, especially those that are of animal origin. It can also produce flavoring agents, vitamins, natural pigments, and fats. The majority of vitamins and nutritional supplements on the market today are produced through fermentation.

Some newer ingredients produced with precision fermentation include Impossible Foods’ heme, which gives the plant-based company its ability to replicate blood;  Perfect Day’s whey protein, a major component of dairy; and the EVERY Company’s egg-white protein. Increasingly, companies are using precision fermentation to produce components of meat, seafood, egg, dairy, fat, collagen, and gelatin.

Precision fermentation also enables us to produce unique and exotic proteins never before available on the market. Take Geltor for example, which has found a way to create mastodon collagen with precision fermentation!

Are foods produced through precision fermentation considered genetically modified?

No. Recombinant dairy proteins made with precision fermentation are substantially equivalent to the dairy proteins derived from cow’s milk, meaning that they look, act, and taste the same.

The recombinant proteins themselves are not genetically modified. Only the production host is modified (as part of strain engineering) to enable it to produce the protein of interest. Because the production host is separated from the recombinant protein during downstream processing, no genetically modified components exist in the final recombinant protein product.

As such, while the microbes making the protein have been genetically altered, the final product is in no way genetically modified.

Are precision-fermentation-derived products safe?

Precision-fermentation-derived products have a long history of safe use even prior to their recent introduction in the realm of alternative proteins.

The first precision fermentation-derived product was approved for sale by the FDA in 1990. Just like for all food products, prior to commercialization, precision fermentation-derived products must undergo a complete safety assessment by the FDA to ensure they meet safety standards. Food is produced in controlled environments under standard Good Manufacturing Practices. Testing ensures that the final product is free from contaminants and bacteria, has been separated from the microbes that produced it, and is safe for consumption.

Recently, companies like Impossible Foods, Perfect Day, and Motif Bioworks have all received “No-Questions” letters from FDA for their precision fermentation-derived proteins, meaning that the FDA had no further questions regarding the safety assessment of their products.

If I have a dairy allergy, will I be allergic to precision-fermentation-derived dairy proteins?

The answer is tricky because it depends on what component of milk your body is sensitive to.

In general, those who have dairy allergies should still avoid precision fermentation-derived dairy products. That’s because the precision fermentation-derived dairy protein is still ultimately a dairy protein. If, for example, you are allergic to the whey protein in milk, then you’d still be allergic to Perfect Day’s precision fermentation-derived whey protein.

Allergenicity is one of the common tests performed as part of a food ingredient’s safety assessment for review by regulatory agencies. Perfect Day’s U.S. safety notice for β-lactoglobulin made with Trichoderma reesei, determined to be generally recognized as safe by FDA in March 2020, explicitly states that “because the notified substance is chemically identical to the protein found in bovine milk and isolated milk proteins, it will produce a milk protein allergy when consumed”.

Mass spectrometry is the gold-standard analytical tool for identifying allergens and contaminants in a food sample. This method works by firing light into the sample and measuring the wavelengths that bounce back. Each component in the sample emits a unique wavelength, and the molecular weights of the compounds can be determined based on the wavelengths. This information allows us to determine exactly what is present in the sample.

When evaluating the allergenicity of a recombinant protein, it is important to consider the potential for an allergic reaction to the production host. Mass spectrometry comes in handy in this situation, as it can detect any trace amounts of the production host that might still be present in the final product after downstream purification. If enough of the production host remains, there is a small chance of an allergic reaction.

To return to Perfect Day’s beta-lactoglobulin as an example, their mass spectrometry data showed that the amount of residual production host was not enough to cause allergenicity concerns. Thus, the only potential allergen present in Perfect Day’s whey protein is milk.

Will precision-fermentation-derived proteins cause issues if I’m lactose intolerant?

Lactose is a dairy sugar, not a dairy protein. This means that if you are lactose intolerant but are not allergic to any other dairy components, then it’s likely that you could consume precision-fermentation-derived dairy proteins.

Still, in the event of lactose intolerance or milk sensitivities, we suggest you work with your doctor to figure out exactly which components of milk your body disagrees with. This will help give you guidance as you explore the up-and-coming world of precision fermentation-derived dairy products.

Are recombinant proteins animal free?

Recombinant proteins produced via precision fermentation are animal-free by definition in the sense that no animals are involved in their production.

Even when creating a piece of DNA that encodes the instructions for producing said protein, no inputs (not even a biopsy of cells!) are needed from the dairy cow. Instead, we can reference existing genome databases to obtain the necessary information.

Are recombinant proteins considered vegan?

In short, recombinant proteins can be vegan, although it depends on the specific manufacturer whether their products are designed to meet the vegan criteria in practice.  According to Vegan.org, a vegan product must not contain any animal products or byproducts, must not use animal products or byproducts in the production process, and must not have undergone animal testing.

Until very recently, FDA required animal testing to be performed as part of a safety assessment for new ingredients to enter the market. This changed in December of 2022 when the U.S. government passed the FDA Modernization Act 2.0, which allows alternative nonclinical tests to be used in place of animal studies. This piece of legislation is a major step forward in biomedical research as it offers a way for manufacturers to demonstrate the safety of their novel, vegan-certified products.

Is precision fermentation sustainable?

The primary advantage of precision fermentation is that it eliminates the need for animals when producing animal-derived products.

USDA’s 2021 data shows that there are 9.4 million dairy cows in the U.S. alone. These cows require huge amounts of energy, land, and water but are highly inefficient at producing food. Dairy cows only convert 14% of the protein they consume into edible human protein. On top of that, these cows generate 13 times more bodily waste than the entire American population.

The bottom line is that animal agriculture is incredibly inefficient at producing food and it causes detrimental impacts to the planet in the process. Precision fermentation provides a way to create animal proteins without animals, thus improving the efficiency and sustainability of food production without compromising on taste or nutrition.

Can precision fermentation help solve food insecurity?

Precision fermentation can help improve food security by decreasing the global food system’s dependence on animal agriculture. As the global population grows to 8 billion people, we need to produce more food to feed everyone.

Using traditional agricultural methods, more food would require more animals, land, energy, and water. Using precision fermentation, however, the world can produce more animal products (like dairy proteins) without more animals on the planet.

Precision fermentation is an attractive solution for countries facing food security issues, such as those that rely on imports for sustenance. Singapore, for instance, has set a goal to sustainably produce 30% of their nutritional needs sustainably and locally by 2030. With its ability to produce food ingredients sustainably and at scale, precision fermentation could greatly alleviate potential issues with scarcity and reduce the country’s dependence on external sources of food.

Unlike traditional farming, precision fermentation facilities also take up significantly less space, making it a practical solution for urban and suburban areas. This is particularly advantageous for a small island country like Singapore, where land is limited. Precision fermentation can help countries overcome food security challenges by providing a means to produce nutritious food in areas not suitable for traditional farming methods.

Additionally, animal products like dairy and meat have key nutrients and health benefits that are very difficult to recreate in plant-based foods. By enabling the production of animal products without animals, precision fermentation can provide more nutritious ingredients to the world, especially when added to plant-based foods as an animal-free—yet nutritionally equivalent alternative—to animal products.

It will likely take time until precision fermentation improves food security in practice. As is the case for many novel technologies, precision fermentation products will likely be sold at a premium initially, given the costs associated with scaling and refining the technology.

As scaling costs decrease over time, however, these products are expected to become cheaper than their traditional counterparts. Think tank RethinkX predicts that precision-fermentation-based dairy proteins will be 5x cheaper than animal-based versions by 2030.

Once the costs associated with this technology drop, precision fermentation can have an even greater impact on food security as it will become more widely accessible.

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