Hello, I'm Lauren Richardson, and this is the A16Z BioJournal Club.This is our podcast where we cover recent scientific advances, why they matter, and how to take them from proof of principle to practice.
In today's episode, I'm talking with BioDeal team partner, Judy Savitskaya, a resident expert in all things synthetic biology.
We cover recent research that seeks to improve the processes of photosynthesis and carbon fixation, and how these advances could one day be used to improve crop growth and carbon sequestration in plants.First, a quick biochem refresher.
During photosynthesis, also known as the light cycle, light energy is captured by chlorophyll and then passed through a series of reactions to the energy-rich chemical cofactors ATP and NADPH.
These cofactors are then used by the carbon fixation cycle, or dark cycle, to drive the capture and conversion of carbon dioxide into more complex carbon molecules like glucose.
Plants and other phototrophs use these two processes to turn sunlight and carbon dioxide from the air into organic matter.
These are hugely powerful processes that have generated essentially all the organic matter on Earth, from the wooden trees to our own bodies.
But these processes also aren't perfect, and scientists have, for decades, been trying to make them more efficient.
The two articles that we discussed today were both published in the journal Science and are both from the lab of Tobias Erb at the Max Planck Institute for Terrestrial Microbiology.
The first article, published in 2016, develops a synthetic pathway for the fixation of carbon dioxide in vitro.
The second article, which was published in May, combines this synthetic carbon fixation pathway with the natural photosynthetic pathway isolated from spinach to create a synthetic chloroplast.
This combination of natural and synthetic components to improve the efficiency of these pathways has a number of potential applications, including engineering our crops to grow faster.
Judy and I discussed these exciting applications, how evolution has restricted the efficiency of carbon fixation, and how these bioengineered solutions get around that problem, and the use of microfluidics for vastly improved experimental design.
But first, we start with a discussion of why the dark cycle, this process of carbon fixation, is not as efficient as it could be. The key thing here is that the dark phase has this great limiting step, which is this enzyme known as Rubisco.
It is just super slow.And that's the first enzyme in the pathway that binds carbon dioxide.
Poor old Rubisco, when I imagine it, it's like an old man enzyme with like a long white beard and it makes a lot of mistakes and it goes really slow.But it evolved really early on and then was a key requirement for these organisms to live.
Furthermore, Robisco makes a lot of mistakes, which is that it often subs in oxygen molecules for carbon dioxide molecules.So there's a huge body of work trying to evolve Robisco to be better.
But as it stands, our plants are stuck with this really old enzyme that is not as efficient as it could be.
Yeah, instead of evolving Rubisco, it seems like plants have evolved kind of everything around it.
So there's all different classes of plants that have modified to support the slow cycling of Rubisco and to be efficacious in different environments and to limit the error, as you call it, of Rubisco, which is also known as photorespiration.
It's kind of crazy that rather than this enzyme evolving to be better, there's entire mechanical systems evolved to open these pores in the plant cells to be able to let in more or less oxygen at different times of the day.
It's this highly complex thing that has evolved to make up for the just poor efficiency of one enzyme.
The Tobias Erb lab developed essentially a synthetic Calvin cycle, so it's a different method for fixing CO2 into some sort of carbon-containing substance.I say dark cycle, you say Calvin cycle.
Fun factoid is that it's actually the Calvin Benson Bassam cycle, but Bassam doesn't want his name included because he thinks it's a disservice to all the students that worked with him on the project.
So he has requested that it be called the Calvin-Benson cycle.
In the 2016 article that you mentioned, the authors developed this very cool synthetic pathway for CO2 fixation that did not use RuBisCO.
Instead, it used a combination of 17 different enzymes from 9 different organisms that could do this dark phase half of the reaction 10 times faster than the plant version that does rely on RuBisCO.
And they called this the catch cycle or the CETCH cycle.
In the previous paper, they sort of cheated by adding in these enzymes that would just produce NADPH and ATP as starting points for their synthetic carbon fixation cycle so that they can kickstart the part of the
experiment that they really cared about.In this new paper, what they're doing is adding in a module to create that NADPH and that ATP that is light-driven.
So it doesn't require the experimentalists to add in these enzymes or to add in the substrates for these enzymes.
Yeah, what they're doing here is they're linking the light cycle, so the photosynthetic element, to the dark cycle, the carbon fixation part.So the goal is to have this own self-sustaining reaction because that's what plants are.
So let's talk about the implications of this research.
The biggest and most interesting implication here is that you could use some of the insights from these papers to upgrade how plants perform.
And the idea is to basically counteract some of the evolutionary pressures that were present when we weren't using these plants for crops, or to sort of make up for some of the inefficiencies of natural selection, like for example, rubisco being a bad enzyme.
This entirely new cycle for doing carbon fixation could really dramatically increase the rate of carbon fixation and the rate of growth for plants that we use as crops.
These synthetic chloroplasts that they created are actually more efficient than natural chloroplasts.
And that's because they don't have rubisco, which is slow, and they also don't suffer from photorespiration, which is that wasteful process we were talking about where rubisco uses oxygen instead of carbon dioxide.
And in most plants, they waste about 25% of their energy from photosynthesis on photorespiration. So there's this way in which you could kind of get around the photorespiration problem with something like these synthetic chloroplasts.
When we think about, on a global scale, the carbon cycle, and if we're concerned about release of too much carbon into the atmosphere, there's sort of an interesting class of solutions here, which is to increase the rate at which our crops pull carbon dioxide out of the atmosphere, and that kills two birds with one stone.
One is that it increases your efficiency of food production and at the same time you're removing more carbon dioxide from the air.You're actually using it for something useful.
Yeah, that's possibly a very elegant solution.Let's dig into these methods and results now.So in plants photosynthesis happens in chloroplasts and
Chloroplasts contain an internal membrane structure called thylakoid membranes, which contain chlorophyll, the molecule that actually is able to capture light energy and convert it into energy that the plant can use, and all the other enzymes in the pathway that are needed to go from light energy to ATP and NADPH, which are these energy storing molecules.
So the way I see it, there were three key advances in this paper.
The first was extracting these membranes from spinach that contain the enzymes for the light cycle and getting that into a functional unit, then linking it to this synthetic catch cycle, this synthetic carbon fixation pathway that they'd created.
And then the third was to use microfluidics to really optimize and integrate these two cycles together so that there was this self-sustaining basically synthetic chloroplast.
I mean, I think it's cool that they're able to show you can get this thylakoid membrane module, separate it from the rest of a chloroplast, which is an integrated, complex, large organelle.
They can just take this one piece of it and then it works like the black box you would expect it to.There was one change they had to make, which was to add exogenous varidoxin, which is like
the one component of this sort of electron transfer process that is not attached to the thylakoid membrane.Other than that, it kind of just transferred wholesale into this in vitro context and worked.
So I'm sure there's like lots of experiments here that were failures that we're not seeing or that are like buried in the very, very large supplemental materials for this paper.
But it's really impressive that they're able to basically show the function of this module in vitro without all of the bells and whistles surrounding it from the natural organism.
So next, they linked these thylakoid membranes, the part that's performing photosynthesis, to the synthetic catch cycle. What do you think about this fusion of the natural and synthetic components?
Because that's what they're basically they're doing here.They've got the natural photosynthesis machinery and then they've got the synthetic dark cycle machinery.
Yeah, it's interesting because it's sort of like demonstrating that we understand half of it, right?So there's this, there's two approaches to understanding the parts of the system.There's the bottom up and the and top down.So
If you understand all of the components of some enzymatic pathway You should be able to add them all in one at a time purified and then recapitulate the behavior of the full pathway So that's sort of what they've done with their first paper with the catch cycle And then there's a different way that biologists understand Nature, which is by breaking it down So you start with like this is how the organism works and then take away pieces until you figure out what's like the set of things That is necessary to do a certain reaction
And this is kind of cool because it's the fusion of both of those worlds.
Yeah, I think there's something interesting and the rate limiting step is this rubisco, that's part of the dark phase.It makes sense to tinker with that element, but you don't have to reinvent the photosynthesis arm.
The part that is working, you can appreciate kind of the beauty that nature has already provided and use that in combination with the things you want to change.
Yeah, that's a really good point, actually.I hadn't thought of that.But this really suggests that you can move this catch cycle that they've engineered into an organism that already has that thylakoid membrane piece intact.
And you should expect them to just work together well.
So in the third aspect of the paper, they're using microfluidics to integrate the thylakoid membranes with the catch cycle and to create these basically artificial chloroplasts.
So talk to me about what they did with the microfluidics and what the benefits of using microfluidics for this approach are.
Yeah, the real benefits of droplet-based experiments is that you can do many of them at once.
So the idea here was to create lots of these little droplets so that each one can contain a different experiment with a slightly different version of the catch cycle or a different ratio of these components that they're putting together.
and they used color-based barcoding so they could tell what reaction was happening in a given droplet by changing the amount of these different dyes that they added in.
The idea is to basically be able to do many experiments in parallel and look at them in one go.So basically it's a way to multiplex. the experimental design.Yeah, that's a perfect way to say it.
There was this interesting figure at the end where they showed that they get more production of glycolase, so sort of like output of their process, in the droplets than they do in bulk solution, given the same amount of chlorophyll to start with.
My understanding was that it's all about the right amount of cofactor regeneration, so ATP and NADPH regeneration from the thylakoid membranes to support the optimal functioning of the catch cycle.
And then do you think the inherent next step is using microfluidics?Would they be able to kind of dose in the exact amount for optimal production?
Yeah, I mean they've got 17 enzymes to play with.So that's like a lot of parameters that you can modify and then you can change the levels of each of those enzymes.
So this microfluidic tool gives them the opportunity to test like at very high levels of multiplexing how to optimize this cycle and optimize its interaction with the thylakoid membrane.
I'm wondering how many steps do you think there are between this work, like what they've achieved now, and actually getting that into plants?
That's actually a really interesting question because they've shown that this synthetic like hodgepodge enzyme set works in vitro. that does not mean that it's going to work in vivo at all.
So the first thing is to put this into some really simple organism that's easy to engineer, like an algae.
And the idea here is that you would use the natural thylakoid membrane's activity from that organism, but then it would express the enzymes from this different catch cycle instead of the natural Calvin cycle.
And what you'd need to do is a ton of optimization.I'm not going to sugar coat it.So is this on the horizon?Probably not.
I think the microfluidic experiments that they have are going to be helpful, because if they can start with sort of extracts of this algae, put it into these microfluidic experiments, and then do their multiplexing there, they can do many more experiments at once.
But there's still going to be a big jump from that to the actual organism.
Yeah, it's kind of like the benefit of the catch cycle was that they could use all of these different enzymes from all of these different organisms and create this brand new pathway, which is so neat in vitro, but that creates a whole host of new problems for that in vitro to in vivo switch.
Yeah, absolutely.I mean, I think that's actually where a lot of the interesting insights into biology come from, is we understand how the system works in isolation.We put it into the context of the cell, and suddenly everything breaks.
And so now the question is, why did it break?So lots of cool biology coming from trying to transfer this work, but I would not expect next year to see a paper where this cycle is fully functioning.
The authors of this paper really blew my mind with the last paragraph of their discussion where they talk about using these synthetic chloroplasts in combination with other life characteristics such as self-repair and reproduction
in the idea of basically creating a fully artificial cell.
When you start thinking about fully artificial or synthetic cells you know that makes you think about fully artificial or synthetic tissues and that kind of scales up to a fully synthetic organism.
Having the ability to synthetically harness the light from the sun, carbon dioxide from the air, and turn this into you know a designer
metabolic pathway that could fuel a synthetic life force is very exciting to me and just kind of wild to think about.
I love the term synthetic life force.If you think about the cell and all of its functions as a graph, like in the classical computer science sense of the word graph,
It is like a super complex structure with many interacting nodes and it's very hard to get your head around it.How could you ever build that from scratch and make it self-sustaining?But this is a really big piece of that.
Generating energy, making it happen without an external agent putting in new molecules, that could handle a very large portion of the graph that is necessary to make life work.
I will say what this gets you is that you don't have to feed sugar, right?
There's definitely something about the, like, independence of it, though.Like, there's, you know, providing sugar or feeding it versus being able to create those energy storing molecules de novo, which can then be turned into mass.
or broken down again as sources of energy.
I think it's really interesting to think about what are the essential processes that you would need to create a fully self-sustaining, independent of human support system that is lifelike in this way, based on biology and not a robot that we build in the lab.
But also, how do we define lifelike?Just because we metabolize and the particular chemistry that we use to do that, that's just one instantiation.
It's kind of like what happened to result from evolution and then stick because it's really hard to evolve out of this, maybe even local minimum, maybe not global minimum in terms of how good the processes are.
Yeah, I think let's definitely push on synthetic cells.I think it makes a lot of sense to start with like things that look like existing biology, but like why stop there?
Why not go to something that's a sort of hybrid or exploits entirely new chemistry that we've never seen?
Yeah, and this kind of can even get us back to what we were talking about at the beginning, which was like how bad rubisco is as an enzyme. Rubisco originally evolved in environments where there was not a lot of oxygen.
So it was before the great oxygenation of the atmosphere.And so this problem with substituting oxygen for carbon dioxide just wasn't a thing when it first evolved.
And as oxygen increased in the atmosphere, it had to start making trade-offs between the specificity of whether it chose oxygen or carbon dioxide and its efficiency.
So it could be more efficient, but then it would incorporate oxygen more often, versus it could be more specific, but then it would be even slower.
So if you're designing a system de novo, is there a way to bypass some of the evolutionarily inherited trade-offs and make something that's just more finely tuned to the situation that you want to design?
So evolution is kind of always lagging behind how the world is changing, which is exactly why Rubisco is evolved for a world that we no longer live in.But humans can adapt much faster.
That's like, I guess, this interesting philosophical idea that people will say evolution has infinite creativity.Like we could never, you know, think up the things that evolution has created.
I think that's true to some extent, but evolution is fundamentally limited to the designs that are within a certain distance of the designs that are out there in nature today.
You're not going to get a really huge rapid change in an organism just because it wouldn't survive that sort of transition period.So there's all of these transitions that evolution can't pass through, but we can as humans.So I actually think like
in a lot of ways human creativity can go way beyond what evolution has made and I think there's like a ton of opportunity here.
Yeah it's I don't think it's necessarily about being better than evolution it's learning from evolution and seeing all the different ways that evolution has functioned and then kind of taking you know the best of the best and matching and our own yeah our own knowledge and you know
what AI will be able to provide to us is like, even beyond our own knowledge, is like new ways of looking at these problems and these solutions and saying, and like being able to input them in completely creative ways that, you know, evolution hasn't, hasn't found yet and neither have people.
Thank you, Judy, for joining me on Journal Club this week.To sum up, we are excited about this work as it demonstrates that you can improve the process of carbon fixation and link it to the natural photosynthesis machinery from plants.
This bioengineering solution could be applied to our crops to improve growth efficiency and carbon dioxide sequestration.That's it for Journal Club this week.You can find all these episodes at a16z.com forward slash journal club.Thanks for listening.
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