Subscribe
Share
Share
Embed
Talking Biotech is a weekly podcast that uncovers the stories, ideas and research of people at the frontier of biology and engineering.
Each episode explores how science and technology will transform agriculture, protect the environment, and feed 10 billion people by 2050.
Interviews are led by Dr. Kevin Folta, a professor of molecular biology and genomics.
Kevin Folta (00:21)
Hi, everybody, and welcome to this week's Talking Biotech Podcast. Now, a few weeks ago and over the course of the podcast, we've talked about the production of proteins and even other products in precision fermentation or in bacterial systems, going all the way back to insulin, right? We're talking about ways that we're making different proteins in heterologous systems.
But there's been more and more of a push lately to begin to construct proteins that don't naturally begin to come from microbes, maybe animal proteins or plant proteins in microbial systems because it would seem to work a little bit faster. Recently we've learned about production of casein in soybeans. So here a milk protein that could be built inside of a plant. So this kind of cross-kingdom
expression is oftentimes a good idea because it allows us to make new products and new proteins that may have important applications in different areas of the human existence. And today's a really nice example.
We're speaking with Dr. Alexia Groff. She's a PhD graduate from the Imperial College in London.
And we're going to talk about her research. So welcome to the podcast, Doctor Groff.
Alexia Groff (01:33)
Thank you. Thank you for having me. I'm really happy to be here.
Kevin Folta (01:36)
Yeah, I'm really glad to have you aboard because this really is, as I just mentioned, a a really nice example of an interesting way to express an important protein and do it in plants in a way that's a little bit more sophisticated than ways we've done this before. So people have been expressing these proteins in plants for a long time. You know, it's pretty easy to do. Whole companies have tried to make a business out of things like plant vaccines, things like this.
But what's the significance of myoglobin in particular? And and how does research support the larger questions of food and farming?
Alexia Groff (02:13)
So, myoglobin is a hemoprotein found in vertebrate, skeletal, and cardiac muscle. And it's significant in the food industry because it has this important role in the taste, colour, and nutritional properties of meat. Being a hemoprotein, heme iron is much more bioavailable to humans than non-heme iron.
In our research, we were asking whether plants can produce this functional mammalian protein which has that has this specific role in meat. And this is especially relevant at this time where we, you know, as a society, we are looking for ways to produce meat-like products without the damage of animal agriculture. As you mentioned, this is something.
This kind of approach is nothing new. it's an example of plant molecular farming, which is this practice of genetically engineering plants to act as living biofactories to produce variable, valuable commercial molecules.
Kevin Folta (03:26)
And this is really a little more interesting than normal plant transformation to make a protein. you specifically expressed this protein in the chloroplast.
What's the advantage of targeting the chloroplast genome rather than targeting the nucleus like we usually do with agrobacteria mediated transformation?
Alexia Groff (03:48)
Yes, so maybe just for some context we can remind everyone of the endosymbiotic theory. So this is a widely accepted theory in the field that states that the chloroplast evolved from an engulfment event of a phot photosynthetic prokaryote, bacteria like cell by a primitive cell.
And this is why we see certain features of the chloroplast. So, for example, the double membrane structure, and also the fact that it has its own DNA, similar to the mitochondria. And this ancestry is the reason why we see some inherent differences between nuclear and chloroplast gene expression, which leads to some advantages for expressing proteins in the chloroplast compared to the nucleus. So the main advantage is that you can achieve much higher.
Transgene expression in the chloroplast by chloroplast transformation. This is partly due to the much higher copy number of the chloroplast genome compared to the nuclear genome, because within each cell, within each plant cell, you have many chloroplasts, and then within each chloroplast, you have many copies of the chloroplast genome. In the chloroplasts, there is also a lack of gene silencing mechanisms that exist in the nucleus, so that's another.
Advantage, which leads to this high transgene expression. And chloroplast genomes are also maternally inherited in many crops, which can help reduce the spread of transgenes through the pollen into the environment and to other organisms. And there are quite a few advantages, but another would be
That expressing the protein directly in the chloroplast means that there's no need for a targeting sequence and imports, so you can express the native sequence without the need of an N-terminal signal peptide. And in our case, in particular, the myoglobin also has a heme cofactor, and heme is produced in the chloroplast. So using the chloroplast transformation, that allows us.
Allowed us to couple protein expression with the biosynthesis of the cofactor, which could favor the incorporation of heme into the myoglobin being.
Kevin Folta (06:13)
But there's a lot of advantages to to being able to transform a chloroplast. I mean it sounds like that's what we should be doing. So w what are some of the disadvantages that maybe are technical barriers because we all put genes in the nucleus and so you know, why are are we not always using a chloroplast transformation event?
Alexia Groff (06:33)
Yeah, so one of the main disadvantages of the chloroplast transformation is that it's only well established for a relatively limited number of plant species, while nuclear transformation is generally much much more broadly applicable. Chloroplast transformation is also very time-consuming compared to nuclear transformation, due to this phenomenon known as
Homoplasmy. So, as I mentioned before, there are many copies of the chloroplast genome within each cell, but during transformation, you are only transforming a few of those a few of those genome copies. So, in order to get a stable transformant in chloroplast transformation, you need to continuously apply a selection pressure.
Until all of the wild type genomes are replaced with the edited genome that you have transformed. Otherwise, you have a risk of reversion back to the wild type because it is not a stable transformer. And this can take many months of regeneration on selective media while nuclear transformation is usually much faster than that.
And another disadvantage for certain proteins is that the chloroplast is not always appropriate. For example, the chloroplast is unable to perform certain eukaryotic post-translational modifications, like in glycosylation, and it may not contain the cofactor of interest. In our case, heme is a cofactor that is produced in the chloroplast, but that is not the case for all cofactors of all proteins.
Kevin Folta (08:17)
I guess the other big part is kind of a technical one too. I mean nuclear is you incubate plant material with some bacteria that can do the genetic exchange. But how is chloroplast transformation different?
Alexia Groff (08:30)
Yes, so nuclear transformation is usually done using agrobacterium, a domesticated soil bacterium, that is, as you said, quite simple to apply. But the chloroplast, as we mentioned, it's a double membrane organelle which is intern inside of a plant cell that has its own membrane and its own cell wall, so it's quite inaccessible. So
In order to transform the chloroplast, we use this device called a gene gun or a biolistics device. What we first do is coat our DNA construct onto gold microcarriers. these kind of act like bullets, so to speak, for the gene gun. they are placed above your plant tissue.
And then using a burst of high-pressure gas, often helium, this accelerates the gold microcarriers coated with your DNA of interest towards the plant leaf tissue, allowing them to penetrate the plant cells such that the DNA can enter the chloroplasts. And from there, the DNA inserts itself by native homologous recombination processes that exist in the chloroplast.
and then as I mentioned, there's this whole process of reaching homoplasmy through various through multiple rounds of regeneration.
Kevin Folta (10:04)
Very cool. So it it is a little bit different process. You're essentially shooting this
Alexia Groff (10:08)
Yes.
Kevin Folta (10:09)
this little this little particles of gold into a cell, and then they're
Alexia Groff (10:12)
Yes.
Kevin Folta (10:12)
coated with DNA. And once in a while that DNA gets taken up and integrated. It's a pretty interesting process that it can work. Now, the ones that you've done, you've actually incorporated porcine myoglobin, so pig myoglobin, into tobacco and lettuce and
single celled alga called clamidomonas. And so they all give different levels of expression though. So you're incorporating it, but it's not the same in each. So are there specific host factors that really underlie this discrepancy and and why is lettuce such an exciting host for this?
Alexia Groff (10:47)
Yes, so we didn't directly investigate the reasons for this difference in expression, but we do think it's likely a reflection of the biology of each host. So things like chloroplast genome copy number, protein stability, and the capacity of each host to fold and accumulate myoglobin.
lettuce in particular has this advantage of obviously being an edible food crop. I'm sure everyone knows. And this facilitates purification and even potentially offers an opportunity for the leaf itself to be used as a heme iron biofortified food. that would obviously be subject to regulatory approval,
Yeah, lettuce slots easily into this existing fresh produce supply chains and consumer familiarity, which combines this promising yield that we've seen with a plausible path to play.
Kevin Folta (11:55)
Yeah, you you could putting it in the tobacco. That was done just because tobacco's an easy regeneration system or is it that you're trying to invent a way to make people want more
Alexia Groff (12:07)
no, yeah, it was more like a stepping stone towards the lettuce. tobacco is a model organism for plants and lettuce is definitely not. It took us much longer to achieve lettuce chloroplast transformation than it did for tobacco, hence why a lot of the experiments that we did were done in tobacco. yet lettuce obviously has that advantage of being an edible.
food crop that would rely on potentially less purification.
Kevin Folta (12:36)
Yeah, t tobacco could be used for smoked meats, right? All right, hit the
Alexia Groff (12:39)
Yeah.
Kevin Folta (12:40)
hit the the drum single symbol, but the the other big thing you mentioned earlier, and maybe I'd like you to dig a little deeper into it, is the idea of how plastic transformation relates to limiting gene flow and biosecurity and and maximizing biosecurity. So why is this an important step for regulatory clearance if you really wanted to scale this for commercial production?
Alexia Groff (13:03)
Yes, so from a biosecurity perspective there is this advantage of being genetically maternally inherited in many clo crop species. So the chloroplast encoding transgenes are much like much less likely to be transmitted through the pollen, which reduces the risk of gene flow from genetically modified crops into neighboring organisms.
However, this is not absolute, so it would not by itself guarantee any kind of regulatory approval, but it is an important biosafety advantage in this case.
Kevin Folta (13:47)
The other thing that was interesting about this was that mass spec, you could look at the protein and the recombinant myoglobin that was made in plant leaves, it undergoes the proper initiation, the proper cleavages and correct folding, all the things as though it was in a pig, right? So were you surprised to see a mammalian like post-translational processing happen so perfectly inside a plant chloroplast?
Alexia Groff (14:12)
We were actually not too surprised to see correct post-translational processing. Partly because the initiative methionine cleavage is one of the most highly conserved steps in protein synthesis across all three domains of life. And the protein folding also we thought this was quite promising from the beginning because.
My globin is quite a simple protein. It's all alpha helical, no disulfide bonds or glycosylation needed. And it was already it was already known to fold correctly in bacterial systems like E. coli. So yeah, we were we extrapolated that kind of bacterial folding to to the chloroplast, but of course it was important to show
with our data that the chloroplast environment was compatible with producing a correctly folded heterologous heterologous chemoprotein.
Kevin Folta (15:19)
Yeah, one of the other big parts of this though is that you have to have association with heme if myoglobin is going to be myoglobin and you know, functional.
Alexia Groff (15:26)
Yes.
Kevin Folta (15:27)
and so the saturation in things like tobacco were only like thirty five percent compared to if you did it in E. coli and precision fermentation, you could get much higher, like eighty percent or whatever. And so is the limitation really just the endogenous plant tetraparole biosynthesis? Like you can't produce enough of the heme to
satisfy the production of the protein. And how could you maybe engineer that to create higher heme availability down the road?
Alexia Groff (15:55)
Yeah, so that's lower heme saturation that we observed, it does suggest that the chloroplast may not be able to supply sufficient heme to fully load the recombinant myoglobin that that is being produced. of course we think that endogenous heme availability is likely to be one of the most important limitations, but we were not able to conclude from our results that tetrabio.
Tetrapyrol biosynthesis itself is the single rate limiting step. But as we described in the paper, we think there are some interesting strategies to explore in order to investigate this. So these strategies could include engineering plant lines for increased heme accumulation by overexpressing heme biosynthesis enzymes.
for example, or supplying heme precursors such as ALA to increase the flux through the chloroplast tetrapyrol pathway.
Kevin Folta (17:01)
Yeah, that last part should work, shouldn't it? Because you could you could I remember the experiments where they used to use things like bilirubin and and other types of chemistries to supplement the production of tetraperoles or be able to bypass genetic blocks in tetraperyl
biosynthesis and heme oxygen heme oxygenase mutants. Kind of dating myself there a little bit, but that's a but it seems like that should be able to work and serve as the substrate that could potentially bypass that problem. I'm having have you looked at any of those other mutants or tried this in a rapidopsis where maybe you have a better collection of potential mutants that could potentially shunt off more substrate that may eventually end up as heme?
Alexia Groff (17:48)
yeah, unfortunately we didn't have time to get around to doing that, but I think it would be a logical next step. in particular, we were interested in overexpressing ferroculitase one, which catalytes catalyzes the final step of heme beast biosynthesis. So yeah, it was on our to-do list, but unfortunately we didn't get around to it.
Kevin Folta (18:13)
Yeah, as a professor it's always easy to suggest more experiments. So so Yeah, so we're
Alexia Groff (18:17)
Yeah, unfortunately, yeah. So many experiments, so little time.
Kevin Folta (18:21)
Yeah, I know. You know the feeling. I was speaking with Doctor Alexia Groff. She's a PhD graduate of Imperial College of London. And this is the Talking Biotech Podcast and we'll be back in just a minute.
And now we're back on the Talking Biotech podcast. We're speaking with Alexia Groff. She's a graduate of the a PhD graduate.
Of the Imperial College London. And we're speaking about making myoglobin inside plants. And a lot of folks would say, well, why do you bother using plants to do it? And there's a lot of good reasons for that that we've already discussed. But going forward, when you're accumulating heme binding proteins in a photosynthetic tissue and in a photosynthetic compartment, you could
theoretically compete with chlorophyll synthesis, right? And or electrode on transport. So did expressing high levels of myoglobin cause a decrease in photosynthesis or some other kinds of penalty that either in growth or in chloroplast function?
Alexia Groff (19:24)
we did worry about that as heme and chlorophyll biosynthesis share the same upstream pathway in the chloroplast. as you said, heme is also itself involved in photosynthetic electron transport, which is another reason why draining heme availability could have adverse effects on photosynthesis. However,
We did not observe a significant penalty on photosynthesis. We looked at several parameters, so effective quantum yield of PS2, for example, also electron transport rate and non-photochemical quenching, and we found that they were similar between all of the tobacco plants, myoglobin producing and the controls.
Which suggested that the expression did not significantly compromise synthetic function. We also looked at plant growth and we did not see any drastic effect. We did see that the myoglobin-expressing tobacco plant had a growth delay, but we also saw this in the empty vector control. So this suggests that.
the growth delay is more likely due to the transformation or the insertion site itself rather than the expression of myoglobin.
and we also saw a significant increase in the total chlorophyll among the tobacco transformants and an increase in total heme in the myoglobin expressing plants, which suggests to us that the plants were able to accommodate the additional heme demand without obvious disruptions of photosynthesis. of course, the
the main drawback of this was that we still did not see sufficient heme occupancy in the myoglobe.
Kevin Folta (21:23)
Yeah, so let's turn our attention away from plants and turn it over to veggie burgers. You know, myoglobin, this is the protein which is critical for delivering that meat light red color, metallic
Alexia Groff (21:34)
Yes.
Kevin Folta (21:35)
notes, and and just being making iron bioavailable, right? So this is a really important part of why meat is meat. And have you begun testing the sensory or nutritional properties such as you know, iron availability,
Maybe the heat induced shifts that may happen with oxidation or color or that kind of thing. And and have you actually tested this on humans who can say this is the most meaty lettuce I've ever had? Or you know, how like do can people see a sensory difference between, say, this and maybe a you know, typical veggie burger or textured vegetable protein?
Alexia Groff (22:15)
no, unfortunately we haven't got that far. For now, we are still looking to improve the heme loading, which is obviously a very important prerequisite step for achieving those meat-like characteristics. so that is our main priority alongside increasing the myoglobin protein yield, but absolutely ultimately we want to
assess the properties such as colour changes during processing and the bioavailability of the heme ion to determine how closely the plant produced myoglobin compares to the animal derived myoglobin. But unfortunately not yet.
Kevin Folta (22:59)
Not yeah. Well that's it's it's kind of an exciting thing because that's always been, you know, as others who who have investigated this like in the Beyond series and other meats that are you know, meat substitutes, that's always been a major step, like the leg hemoglobin and some of those other steps. So it it'll be exciting when that is tested. the other major driver is the precision fermentation.
And how do you isolate the proteins and purify them from that? So with an edible host like lettuce, how realistic is it to use a minimally processed whole plant tissue directly in meat substitute formulations rather than having this intermediate step of making a protein isolate?
Alexia Groff (23:39)
Yes, so that is one of the advantages of using edible crop like lettuce is that you reduce the need for the downstream protein extraction, which can help save on energy use and costs. however we haven't tested whether the lettuce tissue can be incorporated directly into a meat-like analogue, but
So we can't really say how realistic that approach is. As far as I know, lettuce is not really currently found in plant-based formulations. That may be because it hasn't been explored, or more likely, the lettuce has some adverse effect on the texture, flavoring, or processing. Of course, we know that lettuce doesn't do very well when it's heated. But
Another possibility for the lettuce is that as I mentioned before, it could also possibly be used as a nutritionally enhanced lettuce in its own right, subject to regulatory approval. but this would again require the myoglobin and heme concentrations to still be optimized, but who knows, maybe in the future.
Kevin Folta (25:01)
Yeah, it's a really it's a really good question because we really start looking at the impacts of farming and and I'm pro-farmer and I'm pro-rancher. I have no problem with people doing it. But as we have larger and larger populations and the cost of of meat goes up, as we're seeing, it may be really beneficial to have these types of alternative sources for things like iron. And that's why this is such a brilliant idea. But how does it compare? So on a freshway basis
you reported that yields reached roughly forty-eight milligrams per kilogram in lettuce and ninety-four in tobacco and and and so while that's than what you see like in beef or any in muscle tissue, how does it compare to conventional livestock farming or even microbial fermentation on a per hectare energy use?
Alexia Groff (25:51)
So the honest answer is that we haven't measured that directly. We don't have a per hectare energy figure yet. we do believe that the technoeconomic analysis will be vital to determine how competitive this approach is compared to current technologies such as precision fermentation. but
we believe there are reasons to think that it is promising as plant cultivation is much more resource efficient. plants are extremely scalable, they don't rely on sterile conditions to grow, nor do they require dedicated infrastructure such as bioreactors, which is the case for microorganisms. but again, unfortunately we haven't
proven this quantitatively yet, which is as I say an important next step, but I think especially when we have optimized our myoglobin and heme occupancy, I think that will give us a better idea of of how this compares to current techniques.
Kevin Folta (27:04)
Yeah, think one of the things that people don't appreciate with the idea of expressing things in heterologous systems is that correct processing and decoration, modification in ways that make it do what it's supposed to do. And and now that this has been achieved with myoglobin and you have this functional hemoprotein in edible plants, what other complex muscle proteins or enzymes are on the radar for chloroplast based transformation?
Alexia Groff (27:35)
there are some interesting animal proteins to be expressed. for example, collagen for gelatin, casein and whey for cheese and dairy analogs and egg proteins. However, these are more complicated to express in the chloroplasts due to the protein requirements. we were
of course lucky that myoglobin is a very simple protein that doesn't require much modification. There are some non-animal proteins that may be worth exploring as well. For example, sweet proteins having some interest right now. but in our case there's still some way to go with myoglobin, so we're currently focusing focusing on that.
Kevin Folta (28:27)
Very good. you had mentioned that earlier that your PhD career was sponsored by a company that's in this space. And could you tell me a little more about them and what their motivations are?
Alexia Groff (28:40)
Yes, so Kiome is the name of the company that's a next generation food ingredient startup company focusing on biotechnology approaches. they are based in Cambridge here in the UK and they were behind this project and they sponsored this project and partly my PhD studies as well.
so they that is Kiyome spelt KYO E I. Their website is kyome.co dot uk and they're also active on LinkedIn if if there's anyone that would like to follow them. And their next activities.
Kevin Folta (29:28)
Perfect, you read my mind. That's what I was planning on asking you. Very good. So, Alexia Gruff, thank you so much for joining me on the podcast today. This is really exciting work in in that we're making more steps to make these kinds of important proteins and ultimately improve foods to make them more exciting for people. So thank you for joining me today.
Alexia Groff (29:49)
Thank you so much.
Kevin Folta (29:51)
Yeah, and this is just another great example of how we can use these types of heterologist systems.
To not just learn more about how to manufacture proteins outside of their native context, but maybe ways that we can do it with even more efficiency than we can grow them in the animal itself. And I know a lot of people say, well, lab grown meat, we'd never use it. You know, you don't want to get too down on these types of innovations that maybe could help somebody down the road. And I think it's a good time for us to lean into this a little bit more. Lean, the lean into the muscle proteins, right? And
and really explore how we can make these critical proteins that ferry products like iron into the human system up for optimal nutrition. So this is another step in that direction. And this is the Talking Biotech podcast and we thank you for listening and talk to you again next week.