Natural Prodcast Episode 23 – Aaron Puri

Natural Prodcast talks to Aaron Puri, from the University of Utah’s Chemistry Department. We talked about carbon-fixing methylotrophs, quorum sensing, and inverse stable isotopic labeling.

Articles referenced may be found below in the Show Notes – you can jump to them here. Catch up on previous Natural Prodcast episodes here.

Transcript

DAN: Hey, everybody. Welcome back for another episode of Natural Prodcast. Right now I’m working on this the afternoon of the day I’m due to release it, which is not ideal for me keeping myself on my self-imposed schedule. So, we’ll have to see if I’m going to slip a day on releasing it into the feeds, but I’m trying, you guys. Schedules are hard.

But, I’m working on it because I really do want you all to hear this great conversation with Aaron Puri. He is an Assistant Professor from the Chemistry department at the University of Utah, so that makes him one of Jackie’s colleagues. Aaron is doing some cool stuff with cool bacteria that I’m fascinated to learn more about. He works with methylotrophs, which are a group of carbon-fixing bacteria that I would say are are still fairly unexplored in terms of their natural products chemistry, and he has a project with the JGI to synthesize the genes for and explore the chemistry of quorum sensing systems, specifically the acyl homoserine lactones.  If you listened to Betsy Parkinson’s recent episode, you’ve heard a little about these, and Aaron and JGI are working together to try to understand the enzymology of their synthases so that we can start to use the DNA sequence to predict or just understand better the language of microbial cell-cell communication. And he told us about his work in inverse stable isotopic labeling, an interesting way to explore biosynthetic chemistry. It’s some fun topics, and I think you’ll enjoy it!

So, that’s this episode. I still have more stuff in the pipeline, including another recording made at SIMB, and any minute now you’ll hear about something I’ve been working on for what feels like forever, and we also recorded a proper introduction with Jackie and her work. So, more Natural Prodcast to come. Thanks for listening, and enjoy our talk with Aaron Puri.

DAN: Jackie, we have one of your colleagues here today!

JACKIE: We do. It is very exciting to have Aaron Puri here with us who is an assistant professor at the University of Utah in the Department of Chemistry. So welcome.

DAN: How do we introduce Aaron?

JACKIE: How would you like to be introduced, Aaron? I guess because you’ve had a different– I mean, a little bit different introduction in natural products, so I might just start out by asking you know how did you get started. I know you’re not classically trained originally in natural products. And so do you want to give us a little intro into just what sparked your interest in getting into natural products.

DAN: Why are you here at SIMB?

AARON PURI: Yeah. Thanks Jackie. And thanks Dan. I really enjoyed listening to this podcast. And Alison has done a really nice job of providing that general scientific audience whereas now I’m sitting here with two people who know way more about natural products than I do. So, it’s a little intimidating.

DAN: Unfortunately Alison is no longer with us… She’s alive! She just doesn’t work for JGI anymore!

[LAUGHTER]

AARON PURI: Yeah. That got way more serious than I thought all of a sudden!

DAN: No, no, no. Alison is alive and well and doing very well. She’s a real journalist now. Just not working with me.

AARON PURI: No, it’s great. Yeah. So, thanks a lot for having me.

So, my backgrounds. I started out doing I think what a lot of people would consider more straightforward chemical biology, a lot of which people classify as tool building. So I worked with a really great guy named Matt Bogyo in grad school synthesizing activity based probes which are basically molecules that will covalently attach through a more permanent bond with their target. And so, for example, you could think of designing one of those that can trick an enzyme into thinking that that’s its substrate. And then when the enzyme tries to turn it over it’ll become covalently bound to it and maybe there will be some a affinity tag or a fluorescent tag, and so you can use that as a readout of activity.

And I was really fascinated by that. And really more generally from undergrad the intersection of chemistry and biology. But that wasn’t quite where I could see myself going in the future.

So, then we were using these probes to look at host pathogen interactions mostly with like enteric bacteria. And I really fell in love with the bacteria part of that. And so I decided to completely change gears and go and do a postdoc with a woman named Mary Lidstrom at the University of Washington in Seattle. And she studies bacteria that grow on one-carbon compounds like methanol and methane. And I really dove into microbial physiology and microbial genetics, and really away from chemistry and synthesizing these molecules.

And then what actually brought me to natural products was reaching back into that love of the intersection of chemistry and biology. And it actually was jumping onto the Integrated Microbial Genomes and Metagenomes, the IMG platform that JGI has that really got me into that because we had these new genomes that JGI had sequenced that were of these bacteria that eat methane, so they can use methane gas as their sole source of carbon and energy.

And it was before group meeting and it was like a nice summer day in Seattle I think this was like 2013 or 2014. And antiSMASH — I know you’ve interviewed Marnix [Medema] — was out there. And so I had this new genome from JGI and this was before they were integrated.

So I thought, why not feed it in there. And I went to group meeting and came back and it was just so fun looking at this as someone who had a bit more of a chemistry background and thinking about what could be– when you’re doing this genome gazing and all these– maybe the charitable way to put it would be like you’re generating hypotheses — but it’s really I think just like a fantasy — looking at all of these different putative biosynthetic gene clusters in your organism that you love dearly and can genetically manipulate.

And yeah. And so that’s how I ended up here. So definitely no, I do feel like an outsider in this community although both of you have been so wonderful. I think it’s a really welcoming field. And I’ve really enjoyed that.

And this is yeah, one of only a handful of meetings especially with COVID that I’ve been able to go to in the community so far. But being here and then being at the Marine Natural Products meeting in Ventura where I got to meet you Dan. It’s been a really cool group.

DAN: Yeah. I like what you say about the natural products community being well welcoming because that’s my experience too. I mean, I think the really cool thing about the field is many people come at it from many different directions. And if somebody brings some new approach or some new way of thinking or technology, that’s usually very beneficial to the field. It’s very, very, very multidisciplinary, is the usual term for that. So yeah.

AARON PURI: Yeah. It can be very question-driven. It’s not a bunch of people who have hammers that are looking for nails. Everyone is gazing at these beautiful nails and trying to come up with different ways to–

DAN: Beautiful nails. [LAUGHTER]

JACKIE: Or chasing each other with hammers.

[LAUGHTER]

DAN: That happens sometimes too. But it’s not as bad as it used to be.

[LAUGHTER]

AARON PURI: Maybe I’m getting in at the right time.

DAN: Yeah. Yeah. Exactly. For sure. For sure.

JACKIE: So you mentioned briefly this word methanotrophs. Do you want to touch a little bit on that? What are these organisms?

AARON PURI: Yeah. Sure. So when we say a methanotroph it is just an organism that can use methane as its sole carbon and energy source. But generally we’re talking about aerobic methanotrophs.

So you think about these weird organisms, you think about methane gas. And oftentimes people’s mind go right to anaerobic ecosystems. But these are actually– so methane– biologically produced methane is produced from degradation of carbon in the absence of oxygen. But these are the next step in the food web.

So these are organisms– if you think about maybe you’re out kayaking and you drop your burrito and it drifts to the bottom of Mission Bay or something like that. And let’s say it eventually ends up in the sediment, there’s no oxygen and that a lot of that carbon ends up as methane gas. As it starts to go back up through the sediment it’ll get to this Goldilocks zone where there’s some oxygen available that has diffused down through the water column into the sediment and the same thing happens in terrestrial systems as well.

DAN: Sure. Sure.

AARON PURI: And so the methane is coming up from the anaerobic ecosystem. And you have some oxygen that is making its way down. And in that Goldilocks zone is where these aerobic methanotrophs would live.

JACKIE: So you mentioned briefly when you were looking at these genomes for the first time getting excited about this. And so you find these clusters. And one can think about when we talk about genome mining in organisms a lot of times they’re genetically tractable.

Are these organisms? Can you manipulate them? Or can you move the genes and heterologously express them? Is there like a great host that you could use if you can’t knock genes out?

AARON PURI: Yeah. It’s a great question. So that yeah– the short answer is yes, you can genetically manipulate them. Most of the aerobic methanotrophs that people work with are actually alpha or gammaproteobacteria. They just have this very restricted niche. And they do need broad host-range genetic tools so it’s more complicated than working with something like E. coli, but it’s doable. And that was a big part of my postdoc was trying to make that a little bit easier. And that’s something that we are trying to do in our independent lab now is use these as a resource for natural product discovery because we can genetically manipulate them.

I think that there’s no reason not to look at as many different ways as possible to access the chemical potential of these or any organisms. So developing good heterologous expression hosts like maybe in many fields now like in cyanobacteria or even [Streptomyces] coelicolor you’ll see it’s much easier to port a biosynthetic gene cluster or a gene or a group of genes into maybe the “easiest” organism to grow within that same genus. And so that’s what we’re trying to do right now is develop the chassis strains that then we can port things into as well.

DAN: Give us an example then. What’s your what’s your favorite of these organisms? What’s its name? And what kind of secondary metabolism has it got?

AARON PURI: Yeah. OK. So I think that my favorite one– and so this– a lot of this field started in England. And so from my postdoctoral advisor I’ll say instead of saying methyl [meh-thul] we’ll say methyl [muh-thile].

Well, so that’s weird, because it’s a bastardized version of the two because we’ll say methyl so we call them methylotrophs [muh-thigh-luh-trophs]. This is like, every square is a rectangle, but not every rectangle is a square, right? So methanotrophs are methylotrophs but methylotrophs more broadly can use other carbons like methanol and things like that.

DAN: Got it. OK.

AARON PURI: My favorite methanotroph is Methylobacter tundripaludum And yeah, so the tundripaludum part there comes– the type strain was isolated from Svalbard Norway so right there by the Arctic Circle. But the strain that I ended up working with and the one that I was just telling the story about that we put into antiSMASH after downloading it was isolated from Lake Washington. So it’s really interesting that it was there in Seattle as well, just on the bottom of the lake. OK.

And so this goes back to that one of the gene clusters that was in that organism identified or predicted by antiSMASH was this group of genes. It was actually originally predicted to be a nonribosomal peptide synthetase but then flanked by these two genes that were annotated as a quorum sensing system. Yeah. And so I definitely would love to talk more about quorum sensing but this is a way that bacteria communicate with each other and coordinate group behaviors.

And so immediately you know like I said, we’re making all of these hypotheses or you’re fantasizing about what could be going on here. And I knew very little about this field at that point. And the quorum sensing part was actually the easiest part for me to get a hook on because on campus at the University of Washington there’s a guy named Peter Greenberg, and in the medical school in the Department of Microbiology who’s an old colleague of Mary my original postdoctoral advisor as well. And he’s actually the one in a paper from the– I think when I was like six or eight or something like that who coined this term quorum sensing. And so it was really cool to have an expert on campus.

And so I emailed him and eventually got connected with a wonderful person who’s a Co-PI on a JGI project Amy Schaefer. So we’ve been friends ever since. She’s a senior scientist who works with Pete. And they were really interested in looking at this cluster with me because it turns out that oftentimes in bacterial genomes if you have a quorum sensing system or the genes that could encode for that right next to a larger biosynthetic gene cluster the products of the biosynthetic gene cluster is regulated.

DAN: By the quorum sensing system. Yeah.

AARON PURI: Sure. Exactly.

DAN: Sure. Sure. Sure.

AARON PURI: So this is a very long answer to your question but this gene cluster in this organism Methylobacterium tundripaludum is my favorite and is again, maybe highlighting the people-centric aspects of the broader field and just going from one question to another. That’s my favorite.

JACKIE: It’s evolved.

AARON PURI: Yes.

DAN: Very cool. Yeah. Yeah. Yeah. One of the things that I have observed in the past is that it seems like a lot of natural products are actually quorum sensing molecules, especially usually the simple more diffusible molecules. You suspect that they probably are used in cell-cell communication. It’s really, really important part of their ecology, are these cells communicating with one another. What can you tell us about quorum sensing?

AARON PURI: Yeah. So I definitely– I think that there’s a lot there and this actually a great microbiologist named Roberto Kolter had this piece talking about how he thinks quorum sensing has– it was a commentary about how quorum sensing has really captured scientists imaginations because it provides a hook into how to think about something. It’s anthropomorphizing a little bit here, but it allows us to think about what the quote unquote “purpose” is for some of these things right?

And you all know much better than me there’s a lot of people who talk about even antibiotics as signals versus growth inhibitory compounds or warfare agents. And there’s no reason they can’t be both depending on the concentration. So yeah, Dan, absolutely I think a lot of different types of molecules can be thought of as signals or have other functions depending on their concentration and their context.

When I refer to quorum sensing, I’m talking about probably one of the most well-studied forms which is the form that uses these compounds that are known as acyl homoserine lactones. So again, podcast format probably not the easiest to describe.

DAN: We’ll put a figure in the show notes.

AARON PURI: Awesome. Thank you.

DAN: No worries.

AARON PURI: But so these are compounds that are mostly used by gram negative bacteria. And the system is pretty elegant. So these bacteria don’t have eyes but it’s really nice to be able to– for them to be able to sense, I guess I’m anthropomorphizing a lot myself, for them to be able to sense how many of their kin are around them right at least from the canonical way that we think about these systems.

And there are many different group behaviors that would not be advantageous from a fitness perspective for these individual cells to be transcribing and doing unless there’s enough of them around to affect their environment. So I think the classical example yeah, is biofilm formation. So you’re producing different extra polymeric substances. But if there’s only a couple bacteria or a couple of your kin around, you’re not going to be able to make enough to adhere to your surface. And so you’re going to have a huge fitness disadvantage if you’re just constantly producing those things.

So what quorum sensing allows you to do — generally there’s just three components in these acyl homoserine lactone based systems. So you have these synthase enzymes that can make the signal. You have the signal itself, which again is the acyl homoserine lactone. And then you have a transcription factor that will bind to that. And, so, at high concentrations of the signal, you get more bound to the transcription factor. These can diffuse in and out of the cell to a first approximation.

DAN: Sure. Sure. Sure. Yeah.

AARON PURI: And then when you have enough bound to the transcription factors they’ll undergo a conformational change and go and sit down in the genome and then they’ll turn– usually, I think most examples show turning on groups of genes involved in behaviors although there are certainly examples of them inhibiting behaviors as well.

And they can be things like biofilm formation. They can also be production, as we were mentioning before, of more complex secondary metabolites that an organism might want to use to affect its environment. I think that you could imagine if you have two colonies of bacteria in the soil or something and they’re competing with each other, if one colony doesn’t have very many bacteria in it, it doesn’t really make a lot of sense to be spending a ton of energy producing some sort of–

DAN: Especially as a system like a nonribosomal peptide synthetase. That’s — just the sheer amount of ATP that it takes to actually transcribe and translate all of those genes and maintain them in the cells. That’s a huge amount of energy. So you don’t want to waste that energy, just willy-nilly transcribing all your stuff. [LAUGHTER]

AARON PURI: Yeah. No, you’re exactly right. So you can see why these are tightly regulated.

DAN: For sure. For sure. Yeah. Yeah. Yeah. So then they get expressed as the cells grow up in I don’t know– I don’t know what. Is it more density in the natural environment I guess? Or do these things grow as colonies or?

AARON PURI: So there– I think that–

DAN: I guess I’m trying to get at what is what is a methylotroph that’s eating something that nobody else seems to be eating… What does it need a active compounds for?

AARON PURI: Yeah. So I think about this question a lot. And so in– so my first part of the answer would be that there’s actually a great deal of diversity within these methanotrophs for example, that are using methane, which seems like a very specialized niche. So there’s certainly going to be some competition there.

The other thing I’m really fascinated by looking at the field of cyanobacterial chemical ecology and all of that. So again, maybe a somewhat restricted niche there. But oftentimes cyanobacteria are wonderful natural product producers. And they’re this linchpin right in this overall food web because they’re able to provide fixed carbon.

And we see the same thing with the methanotrophs. And with cyanobacteria, I know that there’s a lot to be said about for free living ones this idea that maybe they’re gardening who is– or who is allowed to take up that fixed carbon. And it forms this food web and this community that may be in some way selected by the cyanobacteria. And I think something very similar could be happening with methanotrophs.

The other thing is when you look at symbiotic cyanobacteria they’re providing things for their host. They’re providing fixed carbon. But sometimes they’re also providing bioactive compounds for defense or things like this. And again, I think that this might be something that we also see with methanotrophs, which are also known to be symbionts of things like invertebrates like mussels.

DAN: Oh, OK. Cool. Very cool.

JACKIE: Yeah, very cool. So I want to go back to these acyl homoserine lactones and these forms of communication. And so one can think of is that it’s not one compound. Right?

We have many languages we use to communicate with each other. And one would envision that microorganisms have evolved multiple variations of these compounds. And so if you’re trying to figure out– you have these clusters and maybe you have something that shows potentially it’s regulated by one of these small molecules, how would you go about finding what that small molecule is?

AARON PURI: Yeah. So this is– so first of all exactly right. One portion the quote unquote “tail of the acyl homoserine lactone” can have different structures, and that is akin to what you’re referring to Jackie, as different languages. And so it’s really important because that structural diversity it encodes for communication specificity. So it’s important to be able to tell what language is being spoken.

And when we look at the explosion of microbial genomes we can see all of these quorum sensing systems which again would just in many cases be these two genes for a synthase and a transcription factor receptor. And it’s surprising even though the first signal was characterized I think in the ’70s. It’s still almost impossible if there isn’t a closely defined homolog or sorry, a homolog that is very close that has been characterized to predict what molecule is being made.

So then you have to go in and tackle in different ways. So for your question if you don’t have access to the organism and to be able to culture the organism itself, we’ve been playing around as part of a project with the JGI with heterologously expressing those synthase genes in organisms like E. coli, and then being able to look using the metabolomics capabilities also at JGI — it’s really cool that it’s all integrated — to see what molecules are being produced by those synthases. And now by linking a particular signal with a particular synthase amino acid sequence we think that can be a great resource for the microbial ecology community to be able to better predict how their microbes are interacting with each other from sequence information.

DAN: Very cool. What have you learned?

AARON PURI: Yeah. So the first thing I’ve learned is that when JGI gives you a ton of data you better have someone really smart to be able to tackle it.

DAN: Uh Oh. [LAUGHTER]

AARON PURI: So which fortunately we’ve been able to get some– I mean, the best part of this job as an assistant professor is all the wonderful people you get to work with. Both people like at JGI but also people in the lab so. Yeah. So it’s been really fun to take– so I guess to maybe describe it in slightly more detail what the project that we are just wrapping up now with JGI is we identified using some different computational methods a bunch of different syntheses that make quorum sensing signals but we don’t know what signal is being made.

And so we picked a bunch of very diverse ones to try to learn as much about it as possible starting with we picked 183 of them. And we sent that list to the Joint Genome Institute, and they were able to synthesize the genes for every single one of those and put them in individual plasmids and put those plasmids in E. coli. We were then able to express them and extract the supernatant and send that to back to JGI and have them do untargeted metabolomics, which we can then using some tricks to try to identify this particular class of compounds, these acyl homoserine lactones.

We can try to identify what’s being made. In some cases it might be novel, in some cases it might be– might be a known structure but we couldn’t predict what was being made. And so now that’s what I’m joking about having a lot of data. It’s a lot of metabolomics data sets.

So what have we learned? So one thing that we’ve learned so far is that you can have very, very disparate amino acid sequences for synthases that produce the same signal. So that’s– so that could be an example of convergent evolution, but just speculating there. But it’s interesting because you may not always be finding new chemical diversity and new language but you still can’t predict what’s being made. Yeah. So that’s one thing.

DAN: You would think you’re probably limited somewhat by like just the fatty acids or whatever the precursors that are available to the cell, right?

AARON PURI: Yeah. So we can dive into the biosynthesis of these as much as you want but they– so there’s one class of the synthases that make these that you’re absolutely right. The tails are fatty acid derived from fatty acid biosynthesis. And so yeah, you basically have your chain length and then some sort of substituent at the third carbon where you start to do oxidation.

DAN: There’s another pathway? That’s the one I remember from grad school. [LAUGHTER]

AARON PURI: Yeah. Yeah. So the more recent one that they found, which has a lot more structural diversity — so that one uses acyl carrier proteins as co-substrate. So the other ones they use CoA substrates. So now you can use anything.

DAN: Oh, that’s right.

AARON PURI: Yeah. So those are the cool ones. Those are–

JACKIE: They’re all cool.

AARON PURI: Yes. Thank you.

[LAUGHTER]

That’s sure.

DAN: You got to Love all your kids.

JACKIE: You just don’t tell them that.

[LAUGHTER]

AARON PURI: You can imagine you can have the first one that was found there is this p-coumaroyl homoserine lactone. So that was pretty amazing right now. You have this aromatic ring totally different kind. And yeah, they found out there’s a CoA ligase that will take para-coumaric acid turn it into a CoA, and then that can interact with a particular synthase and staple on the homoserine lactone.

So the other thing about that class is that in phase one of the project that we’re doing with JGI is we’re going to miss the products of those synthases, a lot of them I think. Because E. coli does not have the substrate pool.

DAN: The weird precursors, yeah.

AARON PURI: So in some ways– so to go back you were asking what have we learned. One thing that we learned is this worked better than I think I maybe originally thought it would. So we were able to identify a lot of different primary signals and then we were lucky one of the co-PIs on the project his name is Dale Pelletier and he’s from Oak Ridge National Lab. He’s an amazing microbiologist. And some of the synthases that we chose for JGI to work on he has the native strain for. So we were able to take a bunch of native strains and we’re verifying that right now.

So the story is not over. But we’ve been pretty happy with what we’ve been able to do. But for a phase II of the project, you’re exactly right Dan.

These ones that quote unquote “failed” the first round here they may be the ones that have the most structural diversity in the signal but it’s just E.coli couldn’t make them. So we can maybe go back into a chassis strain or something else. This has been a really neat way — I think in some ways quite reductionistic — but to think about heterologous expression for natural product production. it’s like, one product, one gene, one enzyme here, which has been fun because then we’ve been able to blow out the scale a little bit for the project.

DAN: Do you see broader applications for understanding these systems?

AARON PURI: Yeah. Are you talking about in terms of being able to predict quorum sensing signals from the genes or are you talking about even more broadly than that?

DAN: Yeah. As broadly as you want to go. What’s the bigger– is there a bigger picture here towards this research? And what do you want to get out of it?

AARON PURI: One of the really exciting things about the platforms that JGI offers is this idea of being able to do heterologous expression and mass here. And I know that you all are working with a lot of people on a lot of different kinds of enzymes. So I think that just getting to be part of that kicking the tires for how does this all work, how does it integrate, how does gene synthesis integrate with metabolomics and bring it all together. I think that’s going to be really exciting and you’re going to be able to do a lot more gene to function in the future.

Specifically within what I’m passionate about here with quorum sensing, I just get really excited about microbial ecology and you have your favorite bacterial community. For us, it’s these methane oxidizing bacterial communities that we like to look through as a lens, and trying to be able to get to the point where people can predict what molecules are being used to communicate is really exciting to me, because maybe we could add some of those exogenously to change behaviors. Maybe we could target some specifically to degrade, using something like a specific lactonase and modify behaviors to try to change functions in some human-centric way.

DAN: Very cool. All right.

JACKIE: You learn how to communicate with the bacteria.

AARON PURI: Yeah. Right. And that’s you’ll find me in the woods then.

[LAUGHTER]

JACKIE: So we’re kind of across — covered quite a few different topics. And so right now what is an exciting project in your lab? So you’ve gone through like your history, chemical biology, and introducing genome mining, and then you’ve got chemical metabolomics, looking at new chemical language. And so I guess is there something really fun that you want to– a new area of your lab is exploring?

AARON PURI: Yeah. Thanks. So–

DAN: Almost like you know that there is.

AARON PURI: It’s a planted question Dan. Yeah. So overall I think what we’re trying to carve out as our niche here is a lot of natural products labs look at maybe geographical diversity to try to find new natural products and we’re certainly not the only people doing this but we are very interested in looking at metabolic diversity and new metabolic niches to try to find new natural products. So sticking with these organisms that grow on one carbon compounds I think that there’s an opportunity there to find natural products that may have been missed by companies like Pfizer or Wyeth or that because they probably weren’t isolating bacteria on methane gas during their time.

DAN: That’s virtually guaranteed.

AARON PURI: Sure. Great. I have a job for a couple of years.

But the other thing that we can do that we’ve been playing around with with that is we can take advantage of some of these metabolic idiosyncrasies that these bacteria have. And one thing that we’ve been having a lot of fun with is what we’ve been referring to as– actually this was a term that was first coined by Sean Brady and Jon Clardy, but this inverse stable isotopic labeling.

So in short, this is basically this idea that when natural products chemists are interested in how a compound is made, they will oftentimes have a hypothesis that there’s some precursor that’s incorporated and they can get some labeled version of that precursor if it’s available or synthesize it and feed it to the producing organism and then look for incorporation.

The problem is if that compound is not– that precursor is not available sometimes you can be out of luck right or it could be a whole grad student thesis to synthesize a 13C-labeled version of your compound. So people have been growing methanotrophs and methylotrophs on 13c labeled one carbon compound some of the cheapest 13C-labeled substrates you can buy, so 13C-labeled methane, 13C-labeled methanol for decades.

And so what we’ve been doing is growing up our strain collection on 13C-labeled methane or methanol, and now we have access to the whole sigma catalog of precursors in their 12C form. You can feed those and look for incorporation in that way. And we’ve been primarily doing this through mass spectrometry right now.

So this has been really fun. You could use this– you could imagine to answer biosynthetic questions. But there’s a lot of great groups that have also been using stable isotopic labeling to try to discover new compounds that incorporate a precursor of interest. Maybe you can find new biochemistry that way or new derivatives of compounds or you can use this method to link gene to compound as well.

And so the first way that we’ve been doing this goes back to quorum sensing, something that I like because we know a lot about that system and these acyl homoserine lactones I’ve brought up multiple times here. The homoserine lactone portion of those molecules always comes from methionine.

So, the first thing that we tried with this is we would grow up our bacteria on 13C methane or methanol, feed 12C methionine, and we were able to pull out the quorum sensing signals very quickly. So there’s four carbons that are derived from methionine, you run two parallel experiments, one with 13C carbon source and the other with 13C carbon source plus your 12C precursor. You look for a shift of four. Boom. It works surprisingly well.

So we can identify quorum sensing signals that way, including ones that you may not know what the structure of it is. But by looking at the mass you can imagine it would be a new structure. And so we’ve had some success doing that so far.

And then so 13C methionine is available. This is just our proof of concept. And where we’re taking this now is back to the way I started to explain this approach here is looking at precursors that are not available or in 13C-labeled form at least that we are aware of. And so we’ve been doing that a lot now with aromatic carboxylic acids, things like that. And I’m really excited about some stories that are going to hopefully come together … relatively soon since I’m an assistant professor.

JACKIE: Now that’s exciting. I mean, it’s a great way. And it’s really goes back to classical natural products before we even had access to genomes.

How you identify the pathways? You would do for gene labeling. You would do you know C14 in some cases. And so it’s going– I think it’s going back to the basics and really learning about basic biochemistry and what we can glean from that.

AARON PURI: Yeah. Thanks. It’s been really fun. And it’s fun– this isn’t something we were working on when I started. The job — it wasn’t in my job talk, but it’s fun to see where it goes.

DAN: Yeah. Yeah. No, it sounds awesome.

JACKIE: Very cool.

DAN: Aaron, thanks so much for talking to us today.

JACKIE: Thank you.

DAN: This is really, really interesting to hear about. So, so much fun.

AARON PURI: Thank you so much for the opportunity.

DAN: Yeah, Yeah, anytime. Thanks, man.

 

Show Notes

• Publication: Suo Z et al. A Mesorhizobium japonicum quorum sensing circuit that involves three linked genes and an unusual acyl-homoserine lactone signal. mBio. 2023 Aug 31;14(4):e0101023. doi: 10.1128/mbio.01010-23.
• Publication: Cummings DA Jr. Methylotroph Quorum Sensing Signal Identification by Inverse Stable Isotopic Labeling. ACS Chem Biol. 2021 Aug 20;16(8):1332-1338. doi: 10.1021/acschembio.1c00329.