Precision Fermentation Perfected: Strain Engineering

Precision Fermentation

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As you may already know, TurtleTree is creating sustainable lactoferrin with precision fermentation technology—a promising innovation that enables us to create animal products using microorganisms and a computer database.

Our Precision Fermentation Perfected series explores how each step of this technology works and how these methodologies can revolutionize food production and more.

If you’re new to precision fermentation and are looking for an introduction, first check out our part 1 FAQ to ease into the topic.

In this part, we will be discussing the strain engineering step with help from our wonderful scientists. Find out what makes them tick and why they believe strain engineering and precision fermentation are important for our mission to make food for good.

Now to our Strain Engineering Scientists: Dr. Shannon Ceballos, Dr. Amanda Fischer, and Nina Jorgensen!

TurtleTree’s Strain Engineering team includes Dr. Amanda Fischer, Dr. Shannon Ceballos, and Nina Jorgensen.

How did you become interested in precision fermentation?

Dr. Amanda Fischer: In an undergraduate microbiology class I was excited to learn how precision fermentation can use microbes to make industrial enzymes like the ones found in the Tide detergent I used. As a graduate student, I engineered a protein that glowed due to its pigment. 

Nina Jorgensen: As a kid, I played a lot of Pokemon and really, really wanted to make a Pokemon in real life. As I learned more about genetic engineering as an undergraduate student, it became apparent that making a Pokemon through genetic engineering isn’t possible just yet. Despite this, I still appreciate precision fermentation because of its ability to contribute to safe and sustainable agricultural practices. 

What’s your favorite part of your job?

Dr. Shannon Ceballos: I love to solve problems and there are plenty of problems to solve in this job. Science never works in real life as easily as it does on paper. We are constantly making experiments to try to solve problems, but then complications come up that need to be worked through. When you finally do make that breakthrough, though, it’s a great feeling!

AF: In the long run: using biotechnology to bring sustainable products to the world.  On the daily: working with people who are passionate, engaged, and excited to try the next thing. 

What’s your favorite piece of lab equipment or lab method to use?

AF: I like to use any modular robot or lab automation tool that can remove the manual aspects from my work. 

SC: Streaking strains to plates.  So much of my work feels like moving clear liquid from one tube to another that it’s very satisfying to actually see the results.  

What are you most likely to be listening to while working in the lab?

AF: Last year it was Jane’s Addiction, this year it’s Dirty Heads. I get a lot of Spotify listening awards. 

NJ: Peach Pit!

SC: Anything upbeat that I can move to.

Now that you’ve been introduced, read on as our strain engineering scientists break down the basics of strain engineering, the first step in the precision fermentation process.

What is strain engineering in microbiology and how does it relate to genetic engineering?

Genetic engineering is the broad field of modifying an organism’s DNA. Theoretically, any organism could undergo genetic modification to produce new versions of that organism through genetic engineering.

Genetic engineering has been applied to plants and crops to create genetically modified food like apples that don’t brown when cut or crops that are resistant to bacterial infection.

A few have made genetically modified (GM) animals, like AquaBounty’s GM salmon that’s engineered to grow faster than traditional salmon.

Microorganisms too can be genetically engineered and in most cases they are engineered to optimize a strain for fermentation.

strain is defined as “a group of organisms that belong to the same species but share certain genetic characteristics not found in other members of the species”. When strains are genetically modified to increase their usefulness in microbiology and fermentation, we call this strain engineering. Strain engineering and precision fermentation go hand in hand.

Without strain engineering, only traditional fermentation can occur: the process that creates beer or wine. With strain engineering, precision fermentation can create new products from modified microorganisms.

Strain engineering* is an incredible tool that allows scientists to improve a strain’s overall usefulness for industrial fermentation applications. Various improvements can be made to any strain: from encouraging faster growth, to making growth more efficient at a certain temperature or pH, to reducing the amount of native protein that’s produced.

In precision fermentation, strain engineering is used to make organisms produce new outputs that they wouldn’t naturally make, such as using a microbe to produce dairy proteins.

In our previous post, we explained how strain engineering is the first of the three major steps of precision fermentation.

In its simplest explanation, strain engineering allows scientists to give genetic instructions to the organism on how to produce the product of interest.

*A note from our very own Dr. Amanda Fischer about terminology: While “genetic engineering” and “strain engineering” are common terminology in the field, “engineering” is really a misnomer here.

These technologies more squarely fall under biology than engineering. Engineering is static in the sense that established, well-understood, models are applied to various systems and are expected to work. As a field, biology is the complete opposite of engineering. B

iology is a cycle of constant discovery, followed by evolving ones practices to accommodate new understanding. Strain engineering is a tool of synthetic biology, and like many biotechnological innovations, requires constant trial and error to arrive at the best possible version of the strain.

Rather than following a comprehensive road map, instead scientists make educated guesses on what to do next based on institutional knowledge or academic literature.

The difference between strain engineering and genetic engineering

The biggest difference between strain engineering and other genetically engineered products, like a GM barley crop, for example, is what exactly receives genetic modification. In strain engineering, the strain is GM, but not the final product.

The compounds that the strain produces, which become the commercialized product, are not genetically modified.

For example, TurtleTree’s LF+ is not genetically modified. Only our strain is. On the flipside, genetic modification of a barley crop creates a genetically modified barley crop (a “GMO” barley).

Another difference between genetically engineering a barley crop and engineering a strain: the modified barley plant produces an entire barley plant.

Strain engineering can produce new strains of microbes, but when used with precision fermentation, those strains produce single compounds or ingredients.

Take TurtleTree for example, we use microbes to produce a single protein of cow milk – not a whole cow or even whole milk. This is in line with our broader, long term goal of bringing cell-based milk to the world.

While genetic modification (whether to a microbe or a food crop) might feel futuristic and scary, it’s actually a process as old as the earth itself, having happened since the origin of life via evolution and adaptation. Genetic engineering is a new biotechnology only in its ability to accelerate the natural genetic modification process and its ability to make very specific, intended, changes instead of global, random changes.

Remember Gregor Mendel’s pea plant experiment you learned about in high school biology class? You might call this “V1” of genetic modification.

Mendel succeeded in altering the pea plants’ genetics, but crossbreeding could only lead to modifications inherent to pea plants.

Moreover, the pea plant offspring could only carry a combination of the genes of the parent plants, not some entirely new DNA.

Genetic engineering, on the other hand, is a much more advanced version of genetic modification because genes from one species can be introduced into genes of another species.

For instance, with genetic engineering, Mendel might have been able to produce a pea plant that could glow in the dark like an underwater fish! Had Gregor Mendel been alive in the era of strain engineering, he might have used this technology to create a new strain of yeast that produces an enzyme typically produced by pea plants!

So now that we’ve delineated the differences between strain engineering and genetic engineering, let’s delve further into strain engineering and how it can be used to improve our food system when coupled with precision fermentation.

The evolution of precision fermentation and the addition of strain engineering

As we’ve covered in our FAQ, fermentation technology has been used for centuries to make alcohol and preserve foods.

In the last half-century, precision fermentation has combined fermentation and strain engineering techniques to make pharmaceuticals,  industrial enzymes, and more recently, food ingredients.

But this change from traditional fermentation to precision fermentation didn’t come about overnight as a radically new method. Instead, across fermentation’s long history in making food, techniques have evolved to improve the final product.

At first, these modifications were simply improvements to traditional fermentation.

However, starting in the 1980s, strain engineering was implemented to improve starter cultures.

Since then, the marriage of strain engineering and fermentation techniques has drastically changed the power of fermentation and created the precision fermentation technology we know today.

The evolution of fermentation and the addition of strain engineering can be broken down into 5 stages:

The evolution of fermentation: from spontaneous fermentation to microbiome engineering.

  1. Spontaneous fermentation: Traditional fermentation that has been around for centuries to create foods like sauerkraut, kimchi, or pickles.
  2. Back-slopping: This technique takes a small amount of previously fermented product and adds it to the new batch. This increases the concentration of microbes that are best adapted to the conditions to improve the fermentation and reduce the risk of failure. In kefir production, kefir from a previous batch is often added to a new batch as a back-slopping practice.
  3. Starter cultures: Using a starter culture is a step above back-slopping that uses an optimized starter culture to begin the fermentation. Where back-slopping takes a small amount of the ferment to transfer the microbes in the ferment, using a starter culture ensures that specifically-tailored microbes are used for fermentation. An example of this is the SCOBY (symbiotic culture of bacteria and yeast) used to make kombucha.
  4. Genetic improvement of starter cultures: This began the use of strain engineering to improve fermentation processes. Strain engineering was first applied to fermentation in 1982 to modify Streptococcus lactis for dairy fermentation. 
  5. Microbiome engineering: Microbiome engineering using technologies like CRISPR/Cas9 is an exciting development in strain engineering that enables very specific modifications. More on this later…

How does strain engineering work?

There are many techniques to engineer a strain. One possible sequence for modifying a strain is outlined as follows:

  1. First, scientists will search through online databases to find their DNA sequence of interest – this DNA sequence will code for their protein or product of interest. Then they can order this DNA fragment from a company and combine this fragment with other DNA pieces that code for proteins that ensure the gene of interest is expressed.
  2. Getting the new DNA to enter the cell can be challenging. Scientists have devised a number of tricks to encourage this process, from delivering an electroshock or a temperature shock, to completely removing the cell wall.
  3. The production host then integrates the new piece of DNA into its genome and begins expressing the protein coded in the DNA. Since the gene is integrated into the genome, it is replicated and passed-on like an endogenous gene.
  4. Sometimes the new DNA helps the strain grow better during fermentation and other times the new DNA allows the organism to produce a new compound or protein.
  5. Scientists test the production host and the secreted compounds to verify if they’ve made what they intended to. From there, it’s a process of trial and error to figure out what changes should be made to arrive at the optimal production host and secreted compound.

It’s important to understand that strain engineering is a constantly evolving process with continuous feedback and iteration to make the best possible strain.

As one of our strain engineering scientists said, “Strain development involves both the initial “Hooray it works!” and the subsequent, “Okay, now let’s make it even better!”.

The strain development process.

The benefits of strain engineering

You might not know it, but you’ve likely already used products made with strain engineering.

In the last century, strain engineering coupled with precision fermentation has produced novel pharmaceuticals, industrial enzymes, vitamins, and more recently, food. Many of the products have become household staples, like laundry detergent!

In the coming decades, strain engineering will be a vital tool to recreating a sustainable food system filled with healthy and nutritious options for all.

Currently, many strain modifications are designed to benefit the farmer or food producer, but this is just the beginning of what strain engineering can do. Already, genetic modifications improve food yield and quality, extend shelf life, and help organisms adapt to industrial conditions.

The AquaBounty salmon, for example, is designed to bring mature salmon to market faster so that salmon farmers can save time and money growing the fish to maturity. In fermentation, strain engineering can produce microbial strains that are resistant to bacteriophages: viruses that often infect starter cultures.

These infections pose a huge issue to dairy fermentation and food production in general because they lead to food spoilage and economic losses.

Until now, strain engineering has been mostly used to make modifications that benefit the food producers, but this technology has huge potential to benefit the consumer and the planet by making healthier, more nutritious, and sustainable foods.

In the last five years, we’ve already seen an uptick in strain engineering and precision fermentation being used to produce ingredients that are traditionally animal-derived: like animal-free milk and egg proteins. These ingredients provide all the nutritional and health benefits of animal-derived foods without the animal suffering and resource intensity associated with traditional agriculture.

At TurtleTree, we’re focused on bringing these better-for-you ingredients to all your favorite foods, and even to plant-based foods, where they can improve the food’s sensory quality and nutritional content. For expensive ingredients like lactoferrin, precision fermentation and strain engineering can also help bring down costs and increase access by enabling scalable manufacturing of animal proteins without the animal.

Still curious how precision fermentation and TurtleTree can improve the food system? Check out our recent blog post for more on how TurtleTree is uniquely using precision fermentation to produce a valuable milk bioactive protein that can enhance plant-based foods.

What types of strains are used for precision fermentation? What are the advantages and disadvantages of each?

Microbes are great production hosts for a number of reasons: they grow quickly and produce large amounts of product per cell, many genetic R&D tools already exist specific to these organisms, and many production hosts have a history of use in secreting industrial enzymes or food grade products.

The microbes used are well characterized, meaning that their allergenicity and toxicity profiles are well-established, and we can be certain that they do not carry viruses or diseases that can be transmitted to humans.

For these reasons, microbes are a fabulous and safe means of making products for human consumption.

In general, there are three categories of microbes: bacteria, yeast, and fungi. Each of these have unique advantages and disadvantages which make them more or less suitable for any given application.

The following are some generalizations that are true for many strains.

Of course, every strain is different and should be taken on a case by case basis.

Yeast make for great production hosts because they secrete extremely clean products that require little downstream processing. Some drawbacks may include that they produce lower titers and require more expensive raw materials as inputs.

Many times fungi, on the other hand, are the opposite. They often produce high titers and can use cheap raw materials like waste products, but their products are less pure and thus require much more downstream processing to arrive at the final product.

See the table below to understand how bacteria, yeast, and fungi stack up as production hosts.

Table 1: Differences in bacteria, yeast, and fungi as production hosts for precision fermentation

As for how to choose which strain to work with for a given project, unfortunately, it’s hard to know.

Instead of plugging numbers into an equation or referring to a gold standard guide to figure out the best microbe to use, scientists often resort to an empirical, trial and error process that’s hard to predict.

What are the current limitations of strain engineering and what’s next?

The major limitation of using strain engineering in food production is that the field is in its infancy. We have all of the tools and have been using them for years to make pharmaceuticals and commercial enzymes, but the food space is new.

The last five years have seen numerous precision fermentation ingredients enter the market and we believe that trend will only continue.

It’s an exciting time as we and the rest of the industry are tasked with paving the way in how to appropriately regulate and commercialize food products made with strain engineering.

Another challenge is using precision fermentation to produce food ingredients at scale. For instance, some methods or materials might work well in small batches, but experience challenges at a larger scale.

As for what’s next, there are always new technologies to incorporate and develop. One that has the potential to change food production and has already disrupted strain engineering is CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9).

CRISPR/Cas9 takes strain engineering to the next level ( “microbial engineering” in the 5 steps above) because it enables extremely precise gene editing.

This precision is exactly what makes the technology so useful. CRISPR/Cas9 can select for and target genes that improve the fermentation process and the final product and eliminate genes that hinder the process.

CRISP/Cas9 was first applied to strain engineering in 2013 when used to modify Saccharomyces cerevisiae to reduce urea production in the wine-making process.

 Since then, it’s also been used to improve a variety of starter culture strains: like those used in traditional Japanese and Korean fermentation and other strains used for Kimchi production.

And now, a few, final thoughts from our Strain Engineering Scientists.

What’s something you want more people to know about strain engineering?

AF: Engineered strains can be used to produce safe food products, and cultivation of these strains is safe for the environment. Biotechnology and strain engineering can help solve some of the world’s big environmental challenges.  

NJ: Modern medicine would not be the marvel it is without strain engineering and precision fermentation. This technology has already made an impact in your life without you even knowing it! With strain engineering and precision fermentation, we can revolutionize food availability, sustainability, and safety – all we need to do is give it a try.

SC: Strain engineering is an amazing tool to provide people with safer, potentially healthier products. There is a lot more care and control over the products than some traditional methods. 

 

We’re ecstatic that our brilliant science team could contribute to this educational article and hope you learned a little bit about this crucial step in the precision fermentation process. Stay tuned for the next part of our series!

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