A QUICK NOTE

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ABSTRACT

Good bacteria, designed to work for you.

Humans are walking ecosystems, carrying tens of trillions of bacteria with millions of different genes and expressing a hundred-fold larger diversity of traits than the human body itself.

These traits the bacteria express in and on us help us function better in the world: they help us digest our food, protect us from infection, affect our circadian rhythm and psychology, and even likely influence our neurological and immunological development.

These same bacteria naturally edit their DNA constantly, taking up new DNA using processes evolved over billions of years.

At ZBiotics we leverage these good bacteria and their naturally evolved methods of DNA editing to engineer new strains to address specific problems affecting people in their normal lives. In our first strain – B. subtilis ZB183™ – we transferred a trait for acetaldehyde breakdown from the liver into a probiotic bacteria for the purpose of helping you feel better after drinking alcohol.

We tested the strain extensively to demonstrate safety and functionality, validating that we edited ZB183 the way we intended to, and not in any unintended ways.

This is our first proof of concept on a bigger journey to use the immense potential of microbes to benefit humanity in a way that works with nature, not against it.

INTRODUCTION

Harnessing the power of microbes

Science at ZBiotics takes advantage of three fundamental facts:

1. Humans and microbes work really well together

As humans, our bodies are constantly interacting with microbes (microscopic organisms like bacteria and yeast). Most of these interactions are good for our health, and some are quite necessary.

2. There is an immense variety of ways that microbes can benefit us

These microbes are cells, each of which contains DNA. This DNA determines how each microbe behaves – its genetic traits – when interacting with our bodies. This is just like how our own DNA determines the traits of our human cells, but with a key difference….

In a single human, every human cell contains the same DNA, manifesting in about 25,000 different traits our cells can express. But in the microbes living on that human, each microbial cell contains different DNA encoding different traits, resulting in 3.3 million traits our microbes collectively can express (citation). This means that the microbes living in and on us are capable of >100-fold more functions than we are, presenting a massive variety of ways microbes can behave that could benefit our health.

3. Microbes have a natural desire to adopt new traits, making them easy to design

Microbes evolved over billions of years to naturally look for and adopt new traits that are useful for survival. They do so by continuously looking for new DNA and incorporating it into their own cells. This natural system has been studied and understood for years, and in the past few decades science has become efficient and precise at leveraging this process for purposes useful to humanity. This, in essence, is genetic engineering.

At ZBiotics, we combine these three facts to unlock new benefits for your health and lifestyle. We take a probiotic bacteria that works well with your body, and we use the natural ability of the bacteria to take up new DNA to give it a desired trait that helps your body in a new and compelling way.

In our first product, that desired trait is the production of an enzyme you don’t find much of in your gut – an enzyme that helps break down acetaldehyde. When you eat a ZBiotics™ probiotic, it’s this new trait that does the work for you.

This strategy isn’t new. Scientists have been giving bacteria new traits for decades in research, clinical, and industrial settings. And bacteria have been exchanging traits amongst themselves for eons.

ZBiotics is simply the first to use this strategy with probiotic bacteria – to design new, enhanced probiotics that help people face the common problems of everyday life.

Our process is simple:

  • Step 1: We pick a problem we want to solve.
  • Step 2: We identify a trait we think can solve it (our hypothesis).
  • Step 3: We build a new probiotic that has that trait.
  • Step 4: We test our new probiotic to show that it is safe and solves the problem.

If testing doesn’t go well, we go back to the drawing board and repeat each step until we have something we know is both safe and functional.

ZBiotics™ – our first product – is the result of this process.

STEP 1: THE PROBLEM

Acetaldehyde: an unwanted byproduct of alcohol

When you drink, alcohol produces a variety of small molecules and cellular responses that create a symphony (or really, a cacophony) of issues for your body. These issues can culminate in a pretty miserable next day. In brief, alcohol causes:

  • 1. Poor sleep (citation);
  • 2. Mild dehydration (citation) (*NOTE: contrary to popular belief dehydration has very little to do with how you feel the day after drinking (citation));
  • 3. Microbiome perturbation and gut irritation (citation); and most importantly‍
  • 4. Acetaldehyde buildup.

We can summarize acetaldehyde by saying: it’s no good! This toxic byproduct of alcohol metabolism wreaks havoc on your body. (citation, citation, citation)

Where does acetaldehyde come from? Your liver digests alcohol using a two-step pathway: First, it converts alcohol to acetaldehyde. Second, it converts acetaldehyde to another chemical called acetate, which is benign (it’s essentially vinegar). Each step requires a different enzyme – a functional molecule produced by your liver.

Via this pathway, nearly all the alcohol you drink is absorbed into your bloodstream and metabolized by your liver into harmless acetate. However, some of the alcohol you drink never reaches your liver.

Instead, it is metabolized in your gut, in large part by the microbes that reside there in your gut microbiome. Those microbes convert alcohol into acetaldehyde, but, in contrast to your liver, convert far less of this acetaldehyde into acetate. This is the major source of acetaldehyde buildup in your body. (citation)

STEP 2: THE HYPOTHESIS

A probiotic that breaks down acetaldehyde

Thus arises a clear hypothesis: by promoting the metabolism of acetaldehyde to acetate in the gut, we can potentially augment the microbiome’s ability to break down acetaldehyde – a result that would be very beneficial if you planned on having a drink or two.

The obvious way to do this would be to just deliver the same type of enzyme your liver uses into the gut, mimicking exactly what your body already does so effectively. Unfortunately, in most cases you can’t just make an enzyme and eat it. An enzyme is a protein, and most proteins you eat get broken down very quickly as food for your body, thus losing their function.

But using the ZBiotics’ strategy provides a solution. Rather than manufacture the enzyme separately, we can engineer a probiotic bacteria to produce the enzyme de novo inside your gut. Put another way, why not transfer the trait for acetaldehyde breakdown from the liver to a probiotic bacteria?

STEP 3: BUILD IT

A genetically engineered solution

Transferring a trait from the liver to a probiotic bacteria may sound daunting, but thanks to billions of years of bacterial evolution and scientists’ ever-advancing understanding of bacterial genetics, it’s actually relatively straightforward.

THE TECHNIQUE: HOMOLOGOUS RECOMBINATION

Bacteria have naturally honed the ability to take up DNA from the environment and precisely insert that DNA into their genome (the string of a bacteria’s own DNA). They do so by first identifying homologies – stretches of DNA that are identical in both a piece of environmental DNA and their own genome. When a homology is identified, the bacteria will swap in the new environmental DNA and swap out the homologous stretch on their genome. It basically functions like a super precise “find and replace” function in a word-processing program: the identical stretch of DNA is the “find,” and whatever is next to that stretch in the environmental DNA replaces whatever is next to the same stretch in the bacterial genome.

So if a scientist wants to leverage this natural process, all he or she needs to do is to choose a desired trait they want to add to a bacteria (e.g. an enzyme to break down acetaldehyde) and design a piece of DNA encoding that desired trait but flanked by stretches of DNA that are identical to stretches on the bacteria’s own chromosome.

This process is called homologous recombination, and bacteria have been doing it longer than most other life on this planet have even existed! It’s one of the most important and powerful tools of genetic engineering.




THE APPLICATION: BUILDING ZBIOTICS™

Homologous recombination is also how we built B. subtilis ZB183, the probiotic bacteria in ZBiotics™. Though the actual building and testing behind ZB183 took years, here is what we did on a basic level:

We started with a natural probiotic bacteria called B. subtilis. Humans have been intentionally consuming B. subtilis as a probiotic in supplements and fermented foods (e.g. natto, kombucha, etc.) for centuries. And B. subtilis has the natural ability to pass through your stomach acid unharmed and make enzymes in your gut (citation), making it ideal for our purposes.

We then used homologous recombination to transfer the trait for constitutive and robust acetaldehyde breakdown into a precise desired spot on the bacterial chromosome. The result was ZB183 – a probiotic strain of B. subtilis that is identical in every way to the one humans already consume, but with one additional trait: the ability to break down acetaldehyde.

STEP 4: TEST IT

Proving safety and functionality

Once ZB183 was built, we needed to ensure that it was safe, and we had to make sure it worked the way we intended it.

SAFETY

To confirm safety, we subjected ZB183 to years of laboratory testing and review by America’s top food toxicologists, the results of which confirmed it to be completely safe.

The DNA of ZB183 was fully sequenced to ensure that no unintentional edits were made. All sequencing confirmed that the ZB183 strain was essentially identical to the parent B. subtilis strain except for the traits we intentionally transferred. In addition, sequencing confirmed that ZB183 contains no antibiotic resistance cassettes, horizontally transferable genetic elements, toxins, or allergens.

Furthermore, ZB183 was subjected to a variety of laboratory safety tests, which it passed with flying colors. The data from these tests is out for publication in the peer-reviewed Journal of Toxicology. In the meantime, you can read the Preprint on bioRxiv.

Following all this testing, a cohesive dossier containing all the safety information about ZB183 was reviewed by leading food toxicologists, who determined ZB183 to be generally recognized as safe (GRAS).

The result is that the safety and marketability of ZB183 are fully FDA-compliant. Furthermore, just to make sure, ZB183 was sampled by thousands of people, including each team member of ZBiotics (some over 100 times), without issue.

FUNCTIONALITY

To confirm that ZB183 was working as intended, we tested its ability to make the enzyme that breaks down acetaldehyde. Over a series of tests, we saw that it was very effective at producing this enzyme.

This figure is a coomassie stain; essentially, each horizontal band represents a protein, and the darker the band, the more protein is present. In the lane on the right is all the proteins in our B. subtilis ZB183 strain compared to all the proteins in the unedited parent strain on the left. The carat indicates a very dark band in the right lane that is not present in the left lane, demonstrating that ZB183 produces a significant amount of our enzyme.

But we also tested to make sure that the enzyme was active. To do that, we tested the amount of acetaldehyde ZB183 could break down in a half-hour compared to the unedited parent strain. In that test, we started with two test tubes, each containing the same known concentration of acetaldehyde, and we added ZB183 to one and the unedited parent strain (“control”) to the other. We saw that while the parent strain broke down almost no acetaldehyde, ZB183 broke down a huge amount – far more acetaldehyde than you would ever encounter due to a night of drinking.

While these tests make it clear that ZB183 is working as intended, we want to learn more – and to develop a greater understanding of our strategy, acetaldehyde, and the interaction of alcohol with our microbiome!

That’s why we are in the process of drafting additional protocols to better understand the specifics of ZB183’s activity in the human body. We are most interested in testing the interaction of our probiotic with the microbiome, characterizing what genes are being turned on and when, and understanding just how good our probiotic is at breaking down acetaldehyde in the human gut.

Gathering this data not only will be invaluable to our understanding of the function of our platform but also will further human understanding of acetaldehyde as it relates to the microbiome and drinking alcohol. It’s pretty exciting, and we can’t wait to see how they turn out!

To dive deeper into our safety data, click here.

The Team

The people behind the probiotic

Our desire to learn more comes from a commitment to scientific rigor and discovery that stems from who we are as scientists. Here is a little background on us: the people who performed this work and are continuing to build new ZBiotics products every day.

DR. ZACK ABBOTT
CEO and Co-founder
PhD, Microbiology & Immunology, University of Michigan

Zack’s path to ZBiotics started in earnest while he was a research assistant at an HIV lab at UC Davis, where he spent a year and a half working on two projects: one exploring different routes of administration of a known effective HIV therapy, and the other examining the efficacy of a novel HIV vaccine. Absolutely loving the work, Zack decided to pursue a doctoral degree in the field.

Zack decided to attend the University of Michigan for a PhD in microbiology and immunology. While he expected to be studying HIV, fate took a turn, and he landed in a rotation studying a bacteria called Legionella pneumophila, which causes a rare form of pneumonia called Legionnaires’ Disease. Zack planned to go back to the HIV lab for his second rotation and settle in there for the rest of his PhD, but he fell in love with the lab and the project, sparking a passion and interest in bacterial genetics.

He learned all about how amazing bacteria are – how elegantly they regulate their genomes and adapt to all kinds of different environments. His mind was blown by all the amazing things that bacteria can do, and how they ultimately have a hand in the function of all life on the planet. He became obsessed with the incredible versatility and power of bacteria and never looked back.

After getting his PhD, Zack moved down to Miami to work at a contract research organization (CRO) that specializes in designing and managing clinical trials for biotech and pharmaceutical companies. During the year he spent working at the CRO, he designed dozens of clinical trials for companies ranging from small biotech startups to huge global pharmaceutical companies. He designed trials for drugs for a variety of indications, including chronic pain, psychological disorders, dermatological diseases, infections, and gastrointestinal disorders.

While working at the CRO, two of Zack's colleagues left to start their own company and told Zack about an accelerator called Y Combinator. They encouraged him to apply as well with his startup idea, which he did, and that is how ZBiotics was begun.

DR. JOHN W. K. OLIVER
VP of Research & Development
PhD, Organic Chemistry with a Designated Emphasis in Biotechnology, UC Davis
Postdoctoral Scholar, Harvard Medical School / Wyss Institute

John's path to ZBiotics also began, interestingly enough, with HIV research. John's pursuit of a graduate degree in chemistry at UC Davis began with him working for a year in the lab of Jacquelyn Gervay-Hague, developing synthesis routes to complex sugar molecules important to HIV research.

It was at this time that John discovered Metabolic Engineering and transferred to the lab of Shota Atsumi, where he joined a team led by Iara Machado. John credits Iara and Shota with teaching him from square one the rules and the magic behind biology: from cloning his first E. coli, to designing and building strains of Cyanobacteria that maximized and eventually broke the flux of carbon possible from living bacterial photosynthesis.

After receiving his doctorate in 2014, John secured a postdoctoral research position with Pamela Silver at Harvard Medical School and the Wyss Institute for Biologically Inspired Engineering, joining multidisciplinary teams working on microbiome research. At the Wyss, John apprenticed in development of the Gut-on-a-chip human cell culturing system and designed multiplexed qPCR assays to monitor growth patterns of engineered microbiomes. He learned extensively about the structure of the human gut and it's interactions with the immune system, while broadening his understanding of engineering across different microbiome cell-types. Under Jeff C. Way's mentorship, he apprenticed in a deep base-pair level understanding of DNA design and the use of small highly-effective search spaces.

John left the Wyss in 2016 to pursue his own startup developing DNA design as a service – Syntrophix Organism Engineering Technologies. He and his wife, who is also a scientist, married in 2017 and moved back to California, where he connected with Zack around the idea of setting up R&D for ZBiotics. John closed down Syntrophix OET, transferring these tools to building the world's first engineered probiotics.

For more information, check out our Team page

CONCLUSION

Laying groundwork for an amazing future

The science at the heart of ZBiotics is new and exciting. But ultimately, ZBiotics™ is about innovation, not disruption.

Honestly, the most amazing technology we use is the one nature made and refined over the last few billion years! We are just lucky enough to live in a time when science has progressed enough to be able to use it.

Nature is really the innovator here. We believe in a future where humanity stops struggling to dominate nature, but instead uses technology to thrive with nature.

A probiotic with a trait transferred from our liver is a simple first step. But it is only the beginning. There is so much we can do, and we’re excited for the opportunity to build new and incredible things!

Check out more about our vision for the future here

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