Natural Prodcast Episode 4: Nancy Keller

DAN: You’re listening to the US Department of Energy Joint Genome Institute’s “Natural Prodcast,” a podcast about the science and scientists of secondary metabolism. 

DAN: Welcome back to Natural Prodcast! This is episode 4! From here on out, for a while, we’re going to be presenting some conversations we had with scientists in the field of natural products. So, it’s a departure from the previous episodes where Alison [Takemura] and I chatted about science. This episode is our conversation with Nancy Keller, a fantastic fungal natural products researcher from the University of Wisconsin. Like we discussed in the primer podcasts (part 1 here, part 2 here, and part 3 here), which you should be able to find earlier in the feed, the secondary metabolism field has been dominated for … well, as long as I can remember, by bacterial research.  So it was great to hear her passion and enthusiasm for fungi. And we talk a lot about some of the technical challenges to that, most of which seem to be history.
 

I should mention that this is one of several interviews you’ll hear which we recorded at the Society for Industrial Microbiology’s Natural Products conference, which took place a few weeks ago, in January of 2020. So, you might hear us referencing a conference, and that’s what we’re talking about. Lots of other fun talks coming out of that conference, and there should be a few more in the feed, that I’m releasing along with this one, at the same time.
 

But now, here’s the conversation with one of my heroes, Dr. Nancy Keller. 
 

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DAN: Today we have Nancy Keller joining us from the University of Wisconsin. I wanted to say that you are the first person we’ve had on to talk about fungal natural products. I can say that I started in fungal natural products – I did my Ph.D. with Craig Townsend and worked on aflatoxin biosynthesis, aflatoxin being a secondary metabolite from an Aspergillus species that is pretty toxic – can cause liver cancer and infects peanuts and corn if they’re not stored properly. And so I read lots of your papers when I was in graduate school. 
 

NANCY: Forced to!
 

DAN: No, I think your stuff was really important to forming my concepts of what – at least outside of working directly with Craig – but forming those concepts of what natural product studies look like. And so, I enjoyed your talk today. It was another great example of probing fungi for natural products and finding new things. And so, really happy to have you here. And thanks so much for doing this.
 

NANCY: My pleasure.
 

DAN: I guess I wanted to start, maybe just by asking you how did you get into the field of natural products?
 

NANCY: Yeah, that’s a good question. Mine is probably a lesser-walked path. So my interest actually came from my Peace Corps experience. After I got my undergraduate degree, which was in general biology, I joined the Peace Corps. And when I was there in the Peace Corps – I was there for about three and a half years – I worked at a high school. And one of the things that I noticed is all the food that the students got was coming from other countries, and there would be episodes of food poisoning or the food was so lousy that the students would come to me and ask me if I had some food for them which of course, I couldn’t feed the whole school. 
 

ALISON: Where was this?
 

NANCY: This was in Lesotho. Lesotho is a little landlocked country, in southern Africa. So at the time, I recognized that some of the food was bad from mold, but that’s all I really knew. I didn’t really think about it a lot. And so after I was finished with the Peace Corps, I decided I wanted to go into international agriculture. I had really changed my mind. I was thinking of medical school first, but then I got interested in ag[riculture] and I really liked – I became interested in plant disease in part because of what happened in my Peace Corps days. So I got my degree at Cornell, and there was, really, a salient seminar I heard when I was in grad school that got me into natural products to be truthful. So, well, two things. My PhD work was with a fungal pathogen called Cochliobulis heterostrophis. This is a very well known fungus because it produces a secondary metabolite or natural product called T-toxin. And this kills corn plants. So I became very interested in toxins – phytotoxins – produced by fungi. But the key seminar that tied everything back to the Peace Corps is there was a visiting professor from Penn State. In this seminar this guy was talking about this mycotoxin, or secondary metabolite, produced by Fusarium species that was produced to very high levels in South Africa, including Lesotho where I done my Peace Corps volunteer stint, and that it was in all the maize. So the main food that you ate was called Papa, which is this maize meal. And he was talking about how all this – fumonisin was the natural product he was talking about. It was associated possibly with esophogeal cancer or a neurological defect. And I was just sitting there in the seminar wondering if I had eaten some of that myself during my Peace Corps days. So I became incredibly interested in what fungi could do and what their natural products could do to us. So I’d say my initial entry was more, sort of, in an agricultural sense in that both on what toxins/fungi could make that would kill plants but also very strongly mycotoxins. And as you know, Daniel, I started working with aflatoxin, my postdoc, you know, these very potent, nasty natural products. So that’s how I got my start.
 

DAN: A lot of the natural products field is based on, you know, in bacterial investigations. You’ve spent your whole career as far as I’m aware working on specifically fungal natural products and fungal biosynthesis. What’s kept you in that area?
 

NANCY: Oh, well, I love fungi, okay, this doesn’t even have anything to use natural products necessarily. Fungi are large. They’re microbes, but they’re large microbes. And they have all sorts of cool phenotypes that you can visualize by your eye. So one thing I like with fungi is, because they’re complex, they have different tissues, different structures, different spores. I like looking at that I’m a very visual person. So, when we make genetic mutants in a fungal isolate, I can see something right away, and that’s exciting to me. I also like it because of the complexity. They’re more complex than bacteria. Not that bacteria aren’t complex themselves. But a lot of these natural products are tissue-specific in fungi. Some might go be produced in a sexual spore, some might be in an asexual spore, some might be in these things we called sclerotia, which are overwintering bodies. So the complexity appealed to me. Also, I would say that when I first started out the natural product community was almost 100% bacterial. Only people working in mycotoxins worked with fungi. And I had some of the greats in the bacterial field say, “oh, Nancy, you know what, your career is going to be ruined. If you stick with fungi, you’re just not going to get anywhere. They’re too hard to work with.” And that made me more determined than ever to stick with fungi. And now I think we can see that. Wow, you can really work with fungi very well.
 

DAN: Do you want to tell us about some of the challenges to working with fungi at least compared to bacteria maybe?
 

ALISON: And how you overcame them? 
 

NANCY:  Yeah, okay. Of course. Fungi have a larger genome. The genetics was slower to begin with. So transformation, or manipulation of the DNA of genes and bacteria, occurred much faster earlier than they did with fungi. And it was actually when I was in graduate school that the first filamentous fungi were transformed. So I wasn’t even thinking of molecular genetics of finding genes when I first started out, because that wasn’t on the horizon. It just started. And I guess some of the challenges were associated with getting good transformation systems going. That has a lot to do with, well, the fact that it’s a eukaryotic genome, I guess, and the way DNA is activated. The promoter systems are different. We don’t have operons. It’s just getting that initial information, but honestly, it didn’t take that long, or I feel it didn’t. And I, you know, is this a mystery? This is a good question because to this day, fungi don’t get the credit they deserve. They are so exciting. I get frustrated. Like microbiomes  – you know how that’s a big deal now. You go to all these meetings on microbiomes they only talk about the bacteria. My god! 30% of our gut microbes are fungi. It’s really frustrating to me. Maybe bacteriologists are afraid of fungi? 
 

DAN: I think yes, I think that’s correct. I mean, you know, my experience with Aspergillus was pretty good, but, you know, just from a data standpoint, just figuring out introns and dealing with, you know, weirdness in the sequence. You know, some clusters are not clustered…
 

NANCY: Right. I think that put off people. Just having individual genes, for one, not being in an operon. And I still too many times when I’m at a mixed conference like here, where you – there’s still people who only work with bacteria and they’re still thinking there’s operons in fungi. I’ll say “No, each gene is transcribed individually”. And I think that just put people off. I don’t know, you know, if you take a basic microbiology – so I do teach classes too – not how I teach it, but if you hear how other professors teach microbiology – “microbiology” – it’s almost 95% bacteria! What happened to all the other microbes? So it’s maybe the culture. I’m a faculty member in two departments at [the University of Wisconsin-Madison] UW. I’m in Medical Microbiology and Immunology. That’s one department. And the other one is called Bacteriology. But it’s supposed to include all microbes in it. But yeah, it’s called Bacteriology.
 

DAN: Yeah. So do you think that’s really a mainly a perception issue? Or is… I’ve always perceived it just as a technical – as sort of technical hurdles, and people choosing the easier route. But do you think it’s mostly perception?
 

NANCY: I believe it’s both. The technical hurdles now are minimal. I tell anybody who comes into my lab who’s worked with bacteria, it’s essentially the same. And they agree, when they start working. So I think there’s still a perception. Yeah, but you know, it is changing. I look at now, some of the people working in natural products, a lot of people who used to be exclusively working with bacteria and Streptomyces are now doing quite a bit of work with fungi. So, I can see – and I don’t see it normally going the other way. 
 

DAN: No, yeah. Yeah. Okay. Okay. 
 

NANCY: For whatever that means.
 

ALISON: I was just thinking back to my own graduate school days, because I went to grad school in microbiology. And certainly, I mean, the majority of it was focused on bacteria. And this was at MIT. And I know we had some seminars that were at Harvard where I want to say it was – I saw a couple of fungal lectures. But what struck me, and in hearing you talk about this, is that I don’t have an awareness of how to go about studying them. Like to me, fungi are, you know, how do you culture them? Well, how do you how do you check if they’re there? Like we always talk about 16S for bacteria, but we don’t talk about it so easily with fungi. So…
 

NANCY: Yeah, I don’t know why. For example, with that – actually there’s a good talk today. So fungi, you look at the ITS region. That’s equivalent to the 16S. However, we did have a talk by Mark Stadler today who said “Oh, the ITS which we thought would identify to species”… he’s now found that some fungi have different ITS sequences in their genome, which might get you a little confused as to which species the fungus actually belongs to. I don’t know. It’s really interesting to me exactly in that point. We have some really great microbiome folks at UW and they collect all this DNA from various samples, patients, this and that. They’re doing all this 16S and I said, “Why don’t you just include the ITS too? It’s so easy to add to it!” And “yeah, yeah, well, yeah, that’s right. I should do that”. It’s crazy to me. And I think it’s how people were taught you know? If you had a microbiology [class] that was mostly bacteria, that’s where your mindset is. Is that what you’re saying, right now? So if you were teaching a class, you’d be following what you taught. So you actually have to have a conscious – you have to have another person who now starts talking about fungi for you to appreciate “Oh my gosh, I’ve been forgetting a lot about these larger eukaryotic microbes!” It’s one of my missions in life to get people to think more about fungi. 
 

ALISON: Yeah, it’s really nice to hear that it’s actually not that hard. I mean, technically, you know, it’s not very accessible. 
 

NANCY: They’re not at all. It’s not. It’s very accessible. Now, fungi and bacteria are like cats and dogs actually, one should think of, they’re always paired together. And maybe they’re not always – some like them, some don’t. So fungi and bacteria talk to each other all the time. And often, natural products and fungi are induced by growing beside a bacterium and I’m sure the opposite way around, because they’re sending messages to each other. Sometimes they’re “warfare” messages, or little chemicals to inhibit, usually, the growth of the other microbe. They really are paired. And it’s fascinating. It really is fascinating. You know, us, in the laboratory, humans, we started by isolating this fungus by itself or this bacterium by itself. In reality, we now know of course, that there’s a bunch of microbes, all talking to each other in every environment you can think about.
 

DAN: What kind of genomic resources are available these days for the natural product producers that you work with. So in, you know, bacterial natural products, usually, these days, you’re probably going to do a complete genome sequence before you know you do a whole lot of chemistry or anything with the bacteria that you’re working with. Is that the case for fungi?
 

NANCY: I think a lot of labs yet are doing that now, because it’s gotten so, so cheap to sequence. Right now, for our particular projects, we’re not doing that right now, but we might. I guess, actually, everything is funding, right, isn’t it? I thank JGI for sequencing a lot of fungi. But the thing with them – to get a lousy sequence is really cheap. I’ve got colleagues that are sequencing dozens and dozens of fungi, mostly coming out of China and India, very cheap for $100-$200. But you have so many contigs. This is a problem with natural product clusters. So, to sequence it more thoroughly, then, you have to go to PacBio or whatever you’re going to use. And that becomes pretty expensive. And because the average fungal genome, I don’t know, is 30 megabases? Basidiomyetes might get to 50. Some of them go to over 100. There. That can be the problem. Whereas the bacteria, of course, are much smaller. But I think a lot of groups are sequencing fungi. Just going and sequencing them. Yeah.
 

DAN: Yeah. Is that one of the technical hurdles. That’s going to help us get more access to fungal natural products, do you think?
 

NANCY: That’s one of them. Okay, other hurdles. And I think this is regardless whether it’s a bacterium or a fungus, but what we definitely see in the fungal world is, first of all, finding all the clusters. Yeah, you have to sequence the fungi. Then you have to maybe improve some of your bioinformatic analysis. You don’t want to get clusters that are encoding the same things. And so you have to have good algorithms. And one interesting thing is though, you might have the same backbone gene synthases, but having a different decoration enzyme actually could give you something that’s quite different with enhanced properties. So you still have to decide carefully which clusters you’re going to look at and how much difference you need to have on cluster sets that are sort of the same. You know, family clusters, like if you looked at the statins, you might find many fungi are making similar looking statins. But, this one could be really good because, hey, it had a methyltransferase that changed. And, you know, I wish we could predict. I wish we could look at these enzymes and predict, and really place it to structure. There’s still no way to do that. 
 

DAN: Yes. Still tricky. 
 

NANCY: Yeah. The other thing with fungi, and I assume bacteria, too, is just because you see a cluster – I mean, I talk with my friends all the time on this – is it expressed? What if it’s just a dud cluster? And then you take chances on these clusters that, you know, maybe are not expressed in the producing fungus. Yyou put them in a heterologous system, and you activate all the genes, and sometimes they still don’t make anything. Most researchers don’t talk about that. But that happens not infrequently. Why is that? It looks like it’s encoding an enzyme, there’s no stop. So finding out – expressing the quiet clusters and then trying to figure out why, even if you express them, you’re not getting anything. Those are challenges. Those remain challenges.
 

DAN: Sure. I know it’s early on, but you do have a project with the JGI. It’s doing some some fungal genome sequencing, I understand. Do you want to tell us about the origin of that, or?
 

NANCY: Well, so, I’m involved in two projects with JGI. The fungal sequencing one is with a host of many other fungal researchers. 
 

DAN: Got it. 
 

NANCY: And this is really to complete the genomes, and I’m really thankful for this. It sort of goes back to something that we were talking about earlier. In that it has become so cheap to do Illumina sequencing and we can get – thousands of fungal genomes are floating out there – some are private – that, you know, maybe you have 300 contigs. And so most fungi… Oh, the fungi vary in number of chromosomes. Like, ironically, yeast, which has a small genome has 15 chromosomes. Where Aspergillus has eight chromosomes. It goes by genera. Neurospora has four chromosomes. So if you have one of these fungi, can you imagine eight chromosomes and 300 contigs. So this is a project where those of us – it’s a big international program – where we have chosen honestly, some of the fungi that are most important. Either they’re agricultural, problematic fungi, or industrial workhorses, or representative of a taxon. So once you get a good sequence of even one fungus in a taxon, then you can sequence related fungi and have crappy – Well, you know, you can have contigs, but you can get sort of an idea from synteny how to place it together. So this is really just an effort to close those gaps. And I’m just one of many people where we provided DNA. Actually, we know that one of the fungi we’re working with we have a, I think, a perfect sequence for this. 
 

DAN: Very nice!
 

NANCY: Yeah. And then we have a little minor project. This is still in its infancy. This one is cool if it works out. So another type of microbe that nobody talks about that much are parasites, a general class. And parasites, we don’t think about as necessarily having natural products. But there is one Cryptosporidium that has one of the biggest polyketide synthases found in microbes. It’s an obligate parasite. This particular parasite you really can’t grow well. You have to have it in cell culture. So we are going to see if we can express that polyketide in Aspergillus.
 

DAN: What is it a parasitic to?
 

NANCY: Humans. It causes – it’s terrible – it’s mostly in Africa. You get severe diarrhea, and if you aren’t treated, people die. But why this polyketide is interesting… Actually, we don’t if it has anything to do with the disease, but it’s going to be a linear polyketide that’s of the sort that people are looking for, for biofuel production. We’ll see how that goes. 
 

ALISON: Is this the only class of organisms that makes this polyketide structure that we’re aware of?
 

NANCY: Yes, of this particular polyketide. Nobody’s found it in any other microbes. There are linear polyketides that fungi make and that bacteria make, but this is its own. It’s actually quite peculiar how this got into this parasite because they just don’t have them, you know, and the related parasites don’t have polyketides. So it’s quite unusual.
 

ALISON: Well, I was curious. So you mentioned that there are other linear polyketides in other organisms. So why is it particularly, you know, interesting to go after this one that hasn’t been expressed, or have they? Is it hard to get the others to express it as well? 
 

NANCY: Well, because it would be a first. Trying to even express a Cryptosporidium gene and get a protein product. It’s a very challenging organism to work with. And so, as I’m interested in natural products, in general, this is a much harder one. It’s a challenge, right? And to know if this actually has any impact on, let’s say cryptosporidiosis – they can’t genetically manipulate the organism. So if we could express this and get the product, one could – it won’t be something we would do – but one could then use that product and look in cell culture and see if this compound is having an impact. You know, maybe it is important to cryptosporidiosis, but we’re more interested just to see if we can use Aspergillus as a model for expressing hard to work with eukaryote [genes]. So parasites are eukaryotes. We thought it might be easier to express this gene and get a protein with a fungus than putting it into a bacterium. It would open the doors to be able to start looking at some of these parasite genes, and their proteins, and since this one happens to fall in my area of expertise, it seemed like a good one to start with. 
 

ALISON: Your area of expertise – Do you mean… 
 

NANCY: Natural products. Well, but also though I do work with infectious disease too. I’d probably be working with – if we can get it to be expressed I probably would be doing some work with – well know I would be – with a person who works on cryptosporidiosis. So I’m equally as interested in my other half. You know, one half of me is a natural product person, but the other half is a pathogenesis person, where I work with pathogens and always fungal pathogens of humans, and plants, and to some degree animals. So I’m always interested in what’s going on there.
 

ALISON: Yeah, it sounds like it’s very closely tied to your experiences in the Peace Corps. And then from there, you know, how are our crops and people – in particular in developing countries – being affected by parasites.
 

NANCY: Yeah very much so. I’m really – I’m always keeping involved in international development with diseases, or, for example, with aflatoxin. You know, I don’t know if you know this, Daniel, there’s a lot of worry about that with the climate change that’s occurring. There’s been a lot of modeling of mycotoxins, aflatoxin in particular, and it’s expected to become much worse. Yeah. And so that’s a scary thing too. Microbes produce natural products in time of stress. A lot of these natural products, they’re protections for the fungus of both abiotic or biotic stresses. I could imagine that we’re going to see more things. More disease development, actually, and I’m interested in seeing what’s happening in those in the area. More mycotoxin production. We still haven’t solved it. This is a funny thing. You know, actually, we know the aflatoxin genes – we’ve known them for ages. All of the major mycotoxins: we know the gene clusters. We still can’t stop it from being produced. Applied science is so much harder than basic research.
 

DAN: We know how to make it. We don’t know how to stop it. For sure.
 

ALISON: Any ideas for stopping it?
 

NANCY: Well, the thing is, what you have to think about with toxins that fungi or bacteria make – What we’ve come to realize over these decades is that they are fitness factors for the fungus. So it’s really difficult to interfere with a fitness factor of a microbe. I’ll give you an example of why it’s more difficult. So, we have a lot of fungi that cause plant disease. And it’s actually been pretty easy to breed plants with resistance to pathogenic fungi that invade or colonize their tissue. But we can’t breed against a natural product that a fungus makes. These mycotoxins normally don’t do anything to the plant. It’s us that eats the plant. So how do you breed for resistance to a compound that, with the exception of maybe the trichothecenes, does nothing to the plant? Maybe it actually is beneficial for a plant because a lot of insects are sensitive to mycotoxins and if you have a seed – I mean, this is bandied about – let’s say that has a lot of aflatoxin and the European corn borer chews that seed and it drops dead. Or birds. Birds are very sensitive to a lot of these mycotoxins and they won’t eat the corn crop because there’s a lot of mycotoxin. Well, that’s beneficial for the corn crop, right? It’s been really hard. There’s biocontrol, where people tried to – Well, I don’t know, I’m probably going on too much here now. But it’s a challenge for the mycotoxigenic fungi to stop them. It really isn’t, you know, as I was saying there, there’s some concern with climate change that we’re actually going to see an increase in the rise of mycotoxigenic fungi. It’s actually easier to find new compounds, natural products, in the lab, honestly, by activating these cryptic clusters, believe it or not, than solving a problem out in the world with mycotoxins.
 

DAN: Right, right. Yeah. Fungus want to grow!
 

ALISON: What’s next, for your work or your collaborations with JGI, what do you see on the horizon?
 

NANCY: I think the disease complex. The natural products that are produced by – for me, as I say, I like working with fungi and bacteria. I think some of these natural products which can impact the host occur in mixed infections. And I think that’s an area – well, certainly an area I’m interested in. And I think it’s one we really need to think about. Because no microbe alone – I’m interested in pathogens. It’s just never a single microbe. And we have several recent papers and are continuing to work on the area of: how does the metabolome change, especially the secondary metabolome, of fungi in inter-Kingdom interactions. So fungi, bacteria and their host. And I think that’s a really exciting area to look at. I’m also interested in – this is myself but I could see other people getting interested in this – on… There’s always been talk about horizontal transfer of some genes, from bacteria to bacteria, fungi to fungi, and fungi to bacteria. I think there are avenues now to understand that a little bit better, with some advances in evolution and cell biology. I’m interested in that. And we got some cool stuff going on now about bacteria that live inside fungi and that’s my next big one.
 

DAN: Dr. Keller, thank you very much. It’s been a pleasure talking to you and I hope you enjoy the rest of the conference.
 

NANCY: Well, thank you so much! It was a pleasure talking with you, too.
 

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DAN: I’m Dan Udwary, and you’ve been listening to Natural Prodcast, a podcast produced by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility located at Lawrence Berkeley National Lab. You can find links to transcripts, more information on this episode, and our other episodes at naturalprodcast.com
 

Special thanks, as always, to my co-host, Alison Takemura. <woohoo> If you like Alison, and want to hear more science from her, check out her podcast, Genome Insider. She talks to lots of great scientists outside of secondary metabolism, and if you like what we’re doing here, you’ll probably enjoy Genome Insider too. So, check it out.
 

My intro and outro music are by Jahzzar.
 

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