path: root/transcripts/hgp-write/2016-05-10/synthesizing-a-prototrophic-human-genome.mdwn

Synthesizing a prototrophic human genome

Harris Wang

2016-05-10

video: <https://www.youtube.com/watch?v=ZF2wFdMFn9Q&list=PLHpV_30XFQ8R2Kpcc1pwwXsFnJOCQVndt&index=3&t=19m22s>

<https://twitter.com/kanzure/status/833824575272210432>

I'd like to thank everyone for being here. I want to tell you about our idea in terms of a 1% project. I'd like to acknowledge Robbie, a graduate student in my lab, who has helped craft this project and prepared some of the slides here. The aspect of this project is engineering metabolism in humans. I'll try to motivate why this is potentially important in the large time-scale and the possible impact of doing this in multiple cell lines and potentially into whole organisms obviously. Maybe also in the context of other mammals too.

As you know, chronic malnutrition is a global health problem. 45% of all children under 5 years old that have died are dying from chronic malnutrition, especially in underdeveloped countries. These lead to a variety of problematic health outcomes both acutely and chronically. In the absence of central protein and necessary vitamins during development, you can have a variety of diseases that inflicts an individual throughout the course of their lifetime.

We're going to have to feed more people in the future. So we need to be thinking about food security and the rise in food demand. We need to think about transformative potential of some of this. And really this builds on the idea that food is so important for us; we've been experimenting with food since antiquity especially as to how it contributes to health.

We've been experimenting with growing human cells in terms of specific medium that allows us to do in vitro cell culture. You need to add specific amino acids and vitamins in order for cells to grow in vitro. This was pioneered by Harry Eagle in 1955. Kind of the famous Eagle medium that we're all familiar with for tissue culture.

* Nutrition requirements of mammlian cells in tissue culture. Harry Eagle. 1955.

There's amino acids, salts, vitamins, but also things like serum that are needed. In stem cells, they also require more defined things for keeping the stem cells safe. These are emerging as to how we perceive or think about engineering stem cells in the in vitro setting, and how metabolism works, and understanding differentiation.

As you may know, humans and basically all mammals lack the ability to synthesize 9 essential amino acids. These are outlined here. They are really linked with the essentially the cause of biosynthesis. The more steps required to synthesize those different amino acids, basically the less likely that humans are able to synthesize them. We're able to synthesize the simple amino acids, but not the more complex ones. If you go backwards and say what does that mean in evolutionary terms, these are the non-essential amino acids which can be synthesized by almost all organisms on the planet, compared to the essential amino acids which are only typically synthesized by plants... almost all amino acids that are generated on earth are being produced by ... and the entire food pyramid is predicated on the consumption of those essential resources.

Obviously, ecoli we are all familiar with, is able to synthesize simple and complex amino acids and also the essential vitamins, from simple carbon sources. When we think about developing a human cell line, we can start to think about using only carbon elemental building blocks instead of more complex types of molecules.

There's precedent in thinking about this and even implementing some of this. We've engineered rice, "golden rice", where we have incorporated pathways for synthesizing beta-caretin, a precursor for vitamins, like vitamin A deficiency is a really big problem in many parts of the world where it may not be part of their daily diet.

The general idea is to synthesize a prototrophic human genome... we could do this in human cells as well as microbiome like gut bacteria. The idea would be to incorporate pathways from bacteria and plants, developing the specific pathways for human cells, synthesizing them, optimizing them and inserting them into the human genome, and test them relatively easily.

We've compiled a list of some of these metabolic networks that might be interesting.

Essential amino acids

<table>
<tr><td>Compound</td><td>Steps</td><td>Input</td></tr>
<tr><td>Histidine</td><td>10</td><td>Ribose-5-phosphate</td></tr>
<tr><td>Isoleucine</td><td>8</td><td>Pyruvate</td></tr>
<tr><td>Leucine</td><td>9</td><td>Pyruvate</td></tr>
<tr><td>Lysine</td><td>9</td><td>L-aspartate</td></tr>
<tr><td>Methionine</td><td>7</td><td>L-aspartate</td></tr>
<tr><td>Phenylalanine</td><td>13</td><td>L-aspartate</td></tr>
<tr><td>Threonine</td><td>5</td><td>L-aspartate</td></tr>
<tr><td>Trpytophan</td><td>16</td><td>Ribose-5-phosphate</td></tr>
<tr><td>Valine</td><td>4</td><td>Pyruvate</td></tr>
</table>

Essential fatty acids

<table>
<tr><td>Compound</td><td>Steps</td><td>Input</td></tr>
<tr><td>alpha-linolenic acid</td><td>4</td><td>oleate</td></tr>
<tr><td>linoleic acid</td><td>3</td><td>oleate</td></tr>
</table>

Essential vitamins

<table>
<tr><td>Compound</td><td>Steps</td><td>Input</td></tr>
<tr><td>vitamin A (retinoate)</td><td>22</td><td>IPP</td></tr>
<tr><td>vitamin B1 (thiamine)</td><td>7</td><td>5-amino-1-(5-phospho-beta-D-ribosyl)imidazole / NAD+ / glycine</td></tr>
<tr><td>vitamin B2 (riboflavin)</td><td>7</td><td>ribulose-5-phosphate / GTP</td></tr>
<tr><td>vitamin B5 (pantothenate)</td><td>2</td><td>3-methyl-2-oxobulanoate</td></tr>
<tr><td>vitamin B6 (pyridioxal-5'phosphate)</td><td>8</td><td>D-erthyrose-4-phosphate / D-glyceraldehyde 3-phosphate</td></tr>
<tr><td>vitamin B7 (biotin)</td><td>17</td><td>acetyl-CoA</td></tr>
<tr><td>vitamin B9 (folate)</td><td>9</td><td>GTP / glutamate / chorismate</td></tr>
<tr><td>choline</td><td>6</td><td>serine</td></tr>
<tr><td>vitamin B12 (cobalemin)</td><td>32</td><td>glutamate / L-threonine / riboflavin</td></tr>
<tr><td>vitamin E (a-tocopherol)</td><td>5</td><td>4-hydroxyphenylpyruvate / phytyl diphosphate</td></tr>
<tr><td>vitamin K1 (phylloquinol)</td><td>13</td><td>chorismate / phytyl diphosphate</td></tr>
<tr><td>vitamin K2 (menaquinone)</td><td>11</td><td>chorismate</td></tr>
</table>

Approximately 230 genes would be required to allow human cells to produce all essential amino acids, fatty acids and vitamins.

Really it would require engineering metabolism on many different scales. The essential amino acids and fatty acids are potentially quite interesting. Obviously there are some pathways that require many different steps which will be more challenging for synthetic biology. If you add up the total number of genes required, it's probably north of 230 to 250 genes to produce all the essential amino acids, fatty acids and vitamins necessary to make a prototrophic cells.

One of the key advantages of this is that this could be parallelized. Many different people could be working on pathways in parallel and test them in their own setup and then we can all put them together, in a similar way to what the yeast genome project has done.

This will also require foundational technologies which are hopefully being developed through this project. Things like stable integration into human cell lines, pathway optimization, as well as all the other things associated with the microbiome manipulation.

The practical application with regards to industry is that human cell lines and mammalian cell lines are used for biopharmaceutical production and this could be quite important in terms of defined medium for those.

Other things like insight into human metabolism, in terms of cancer and development, will also be important. The long-term consequence of htis is really just thinking about making things more efficient in vitro. Cultured food, combatting malnutrition and thinking about more autotrophic human opportunities such as space exploration where nutrition is at a premium.

I think there's an interesting opportunity here to engineer human metabolism and augment it with pathways that for one reason or another we have lost through evolution. Hopefully this will let us understand the basic blueprints for what it takes to make a viable cell. I'd love to hear your thoughts and comments, so I'll take questions now.

Q: If you're over-producing, then it might turn out to be detrimental. How do you want to control the dosage?

A: Great question. How do we determine the right dosage of these things? I think this is part of the technology that would hopefully come out of this. How do we build sensors, control elements to modulate these micronutrients, both at transcription or translation levels, to give us the optimal cell growth. That's an open question. I think that is something we would love to consider more.

Q: Thank you for the talk. The type of synthesis of a prototrophic human being is provocative because the ultimate downstream thinking is that you will engineer humans that will have this, right? So what is the safety and public perception of this project? is this a good pilot project to convince the public?

A: That's a good point. This is a much larger discussion, so I think we'll hold that until the break.

31m 20sec