Natural Prodcast Episode 19: Bill Fenical

DAN UDWARY: Hey, everyone. I’ve got a great episode for you here today. We got the opportunity to talk to one of the real legends of natural products, Professor Bill Fenical from Scripps Institution of Oceanography in San Diego. Bill is a pioneer of marine natural products, getting into the ocean and looking for molecules from the very beginning of the field. I learned a lot so much history from this conversation about why the ocean was basically unexplored until the 70s, and how diving was the catalyst of that. We also talk about his move to microbiology, about drug discovery and why it’s so hard to succeed, and we talk about an ongoing project I’ve been working with him on as an exploration into the secondary metabolism of some more novel marine bacteria. You know, I first met Bill as a postdoc working under Brad Moore on the Salinispora project,  so this is pretty comfortable territory for us, and I’m grateful to be working with him, and so happy we got the chance to record this conversation.

I also want to note that this episode will be monumental as the last episode with Alison as my co-host. We recorded this an awfully long time ago, and she’s been in her new job for a long time now, but I guess you all might not know that. So, retroactively, please wish her all the best of luck in her new-now-old endeavors! I have one more episode that will be out next where I’m co-host-less, and then I’ll be introducing my new partner, who, if you’re listening carefully, you might already know about. More on that later.

But, now, here’s the great Bill Fenical.

DAN UDWARY: Hey, Alison!

ALISON TAKEMURA: Hey, Dan.

DAN UDWARY: So Alison, I’m really excited today. We have, by far, I think, one of the most decorated and legendary natural products people– he’s making a face. Talking to us today is Bill Fenical from Scripps Institution of Oceanography.

So really happy to have you here today. There’s a couple of things I want to make sure that we talk about. You have a JGI project going with us that we want to talk a little bit about. It’s sort of early stages. But I think that is emblematic of some of the research that you’ve been doing the last couple of years. And I really want to make sure that we talk about your place in the field, because you’ve been at this for a while and I think you’re definitely one of the people that shaped the field early on.

So here we go. So maybe we start with what’s your origin story in natural products? How did you get started in this area?

BILL FENICAL: Yeah, well, thank you, Dan. I was actually trained as a synthetic organic chemist. My PhD is in synthesis. And I, of course, love organic molecules and get excited about chemistry. But at some point in time, being a Californian, I looked out and saw the ocean and thought to myself, what’s going on with the organic chemistry of the ocean? And I was shocked to find that there was almost nothing. Now, this is back in the 1970s.

So, I thought to myself, maybe I can use my background in chemistry and start to do something to discover what’s going on with the organic chemistry in the ocean. And that was the beginning of what I wanted to do with my career. In order to really make that happen, I kept looking for open jobs that said, we want a chemist to study the ocean. And I didn’t find a single job advertisement.

But one day, I decided to really be more aggressive. And I came down to Scripps Institution of Oceanography, which of course is part of the University of California San Diego. I walked into the director’s office, asked him if I could speak to him. And I said, you know, I’m here to find a job. I would like to do something completely new. And that is to begin to study the organic chemistry of life in the sea.

And he looked at me and he said, you know, this isn’t how it works. We decide what we want to do, not you. So but I said, you know what, I want to do something you don’t even know about. So after thinking about it for a long time, he said, guess what, I’m going to give you nine months of salary and a lousy job with no future. And let’s see what you can do.

So with that nine months of salary, I really got busy and I started to create a program that involves researching the local marine environment. I went out and picked up marine plants, I picked up animals. And I asked the simple question, are these plants and animals making chemical compounds in a way that’s similar or analogous to what’s going on in terrestrial systems with plants making toxins and so on and so forth.

And in the first nine months of my job, we started to find some interesting things. And so I managed to get some grants moving and got some things happening. And we began to outline what was, at that time, an unprecedented presence in the ocean, of plants and animals making weird chemical compounds, the kinds of chemical compounds that people never thought they would ever see from a natural source. They contain bromine, they contain chlorine.

And we saw this and started publishing papers. And one thing led to another. And we started increasing our ability. And ultimately we went through a whole lot of different processes with plants and animals, really, all over the world.

We used ships. We used land-based marine labs. We wrote cooperative grants with people in other countries. And it became quite clear that this was an important field evolving, not only through me, but as other people started to see this.

And one of the things that was clear in the beginning was that it couldn’t be anybody doing this. Because one of the things you had to do, in the beginning at least, you had to be a scuba diver. You had to be willing to jump into the ocean, and go look at what’s there, and bring it into the lab. And I think that criterion explains why the natural products in the ocean weren’t studied up until the 1970s.

You know, I mean, people knew about plants’ chemistry ever since the isolation of morphine, 1870 or something like that. And so people were well aware of natural products that were used as drugs and so on. But the ocean was something that was unfamiliar, foreign, required a degree of adventure that was different from wandering through a rainforest and picking up a mushroom. It required, I must say, a couple of times, risking your life in order to find something to work on and bringing those kinds of things into the lab.

DAN UDWARY: Were you a big diver at the time?

BILL FENICAL: I had begun diving when I was a teenager, about 15. I grew up in Northern California. And we’d go down to Monterey, in the Bay Area, and go diving there. So, this was always in my background.

I never thought that I could combine a  training in chemistry with a hobby of putting your head underwater. But thankfully that worked out. And I spent 30, 40 years diving all over the world and bringing samples into the lab for study.

DAN UDWARY: Where were you on the timing of that compared to some of the other guys that are no longer with us, like Dick Moore and Paul Scheuer, John Faulkner?

BILL FENICAL: Yeah, we were all contemporaries. I think Paul Scheuer was a little ahead of most of us in that he came from Europe, and went to the University of Hawaii, and immediately started thinking about marine toxins and things like that. There were people there working on toxic marine life. And so he began to think about things. And of course Dick Moore became a postdoc with him, and then ultimately settled there as well, as a faculty member. But we were all together. There was even a degree of, I would say, competition between all of us about who could really do something exciting. Who could find the big, interesting things in the ocean.

And I would point out there was almost nothing known, absolutely nothing known, which was exciting but also somewhat frightening. Because when you begin to dedicate your entire career to do something, you’d like to be sure that something’s going to work. And fortunately it did. And of course, we now know that the ocean is as complex or even more complex, chemically, than any of the rainforests that have been described in such detail.

DAN UDWARY: Alison, feel free to jump in with questions. But otherwise I’m going to ask things all day.

ALISON TAKEMURA: I wanted to ask, did scientists at that time just think that the ocean was kind of like a desert? Or that certainly there was marine life, there were fishes, coral, et cetera, but they just didn’t make interesting chemistry? It seems hard to believe in retrospect.

BILL FENICAL: Well, in 2021, absolutely hard to believe. But when you go way back, you think about the history, the capacity for diving only evolved in World War II with the invention of the demand regulator by a guy named Jacques Cousteau and Émile Gagnan. And that ultimately– not in the ’40s, but really in the ’50s– led to a little more common use of scuba. And very crude early equipment was produced that sometimes failed while you’re trying to dive and that sort of thing.

So it’s not unsurprising this took a while for things to happen. You know, there were opportunities to collect marine animals, for example by dredging and all these kinds of techniques. But the ocean was just not considered to be of any significance. And you know, it was because people didn’t understand the ocean very well.

And oceanographers never had the kind of interest that we had. When you talk about marine chemistry, people talk about what are the elements in seawater, what’s the concentration of calcium, and so on. And what difference does it make? The idea that you would say marine life make organic molecules for survival, the idea you would say that and then try to prove it, was just not known. And the first time I made that comment here at Scripps to our very, very well-known and accomplished ecologists, they said, oh, come on. What are you talking about?

Now we know, after years of young students who can use chemistry and can also study ecology of the ocean, we now know that organic molecules in the ocean are the foundation of communication by chemical means, of survival by providing deterrence and rendering some organisms just simply unpalatable. And we know that this is one of the major adaptations in the ocean. So it took a while to convince those people of that fact, however, a long time, 10 years!

ALISON TAKEMURA: Wow. It’s all happened so fast. I mean, in terms of human evolution and knowledge, like discovering that the ocean is a really vibrant, vital place in just, I don’t know, 70 years.

BILL FENICAL: I don’t know if it’s that fast, Alison.

ALISON TAKEMURA: Right, right, right. Not compared to a lifetime.

BILL FENICAL: I was there, banging on everybody, to try and make that point. I think it took 15 to 18 years before we were able to convince the diversity of marine ecologists that they needed to consider chemical impact on species interactions, communication, that kind of thing. But ultimately, they bought on. So now we have The Journal of Chemical Ecology that has a marine component. And really the community out there now understands this fact very well.

So I would like me to continue about this sort of historical overview?

DAN UDWARY: Yeah, whatever you have to say. I wanted to maybe touch on something you said earlier. You talked about competition in the field. And my understanding is that, well, it was very competitive when I was in graduate school too. And I think some of that’s changed now. My perception is that a lot of that competition was driven by drug discovery and the money that’s available there when you find something.

I wanted to get a sense from you if that’s true. But also, in your research, I think I’ve always had the perception that you’re as interested in the chemical ecology side of things as you are in the drug discovery, though you’ve had lots of success in both. And where do you see that balance in your research?

BILL FENICAL: Well, I think you’re right. I mean, that too was an issue. What function, what utility, what kind of applications might molecules from the ocean have? In the beginning, there was absolutely no focus on anything other than what’s out there. So we and almost everybody else started looking at things and doing chemical structures, learning a little bit about things. And that was the excitement. Because we saw things that could never be produced by any life on land. And that was the excitement.

Well, OK, then we looked at those things and we said, why are they being produced anyway? It seems like it takes a lot of energy to do that. And many times we would see 5% of the weight of these plants and animals being these molecules. And so a couple of my early students were interested, you know, what are they doing in the ocean?

So, we started looking at the impact of those molecules on survival. We started doing laboratory experiments with fish, trying to see if, first o f all, were they toxic to fish? Did they then have an impact on the fish in terms of sensors? Were they sensing molecules in the ocean like this? And we started to gather evidence that truly showed the importance of the molecules in ecology.

And that took another 10 years at least. But I had several students. And I worked with several other ecology students who, today, run full-fledged marine chemical ecology programs. Two of the great students are in Georgia, in Atlanta, at Georgia Tech. Another great student is in Florida continuing to do this kind of work.

So that was how that started. Students got interested that had the ability to understand chemistry and to really put the chemistry into the ecological experimental protocols. So that took about 15 years.

Of course, at some point in time, everybody said, if these are bioactive, why couldn’t they be utilized as drugs? If they have an impact on another organism, maybe we need to find out what that impact is.

So, we started finding that the molecules out there would be toxic to cancer cells. And that started something big. Because at that time, nobody knew anything about a cancer drug from a marine organism. And so, we were able to then switch from financial support from the National Science Foundation to financial support from the National Cancer Institute. And that was lovely. Because they really saw the potential. We started showing them molecules that would impact tumors. We started working with industries, with Bristol Myers Squibb, with Novartis, with others.

And in collaborative programs, with money from NCI and other programs, we started showing real drug quality in [our] molecules. And that continued on for a long, long time. And I’m happy to say that we now have two drugs in phase III clinical trials for cancer, two different kinds of cancers. We have another drug that’s in development here, not yet professionally transmitted to the pharmaceutical industry, but being developed here, for melanoma. So, cancer has been the first application, the most effective application, of marine molecules in general.

DAN UDWARY: Maybe can you explain to our audience who might not be knowledgeable, why would marine organisms be making something that kills cancer?

BILL FENICAL: Sure. Yeah, everybody asks that question– why would you predict that a molecule from the ocean might have an impact on cancer? Well, first of all, we don’t know how to find things in the ocean. But it is of considerable understanding, when you think about the fact that, when plants and animals and microbes evolve, they evolve to make molecules that improve their survival. Those molecules, over evolutionary time, become targeted to certain biological systems in the target organism.

So, if you want to restrict a fish from eating you, and you make a compound, you want that to be effective by hitting a target in a fish. Well, what is that target? We didn’t know, but it made sense that the molecules we were isolating did have an impact on biological targets. So, what are the targets? Well, they could be a whole bunch of things. When we started testing our molecules, we found the targets to be diverse but effective. For example, we found molecules that bound inside the cells to tubulin, these proteins that allow the cells to divide. We found all sorts of molecules with specific targets.

Did we know how to find those? Not at all. We just tested. And we tested more and more. Mainly we asked the question, does this chemical compound from a marine organism kill cancer cells? If it does, we want to know why. We want to know, does it kill cancer cells better than normal cells? We want to know, does it kill only certain cancer cells? And so we worked with the National Cancer Institute who had a panel of 60 different cancer cell lines. And we would send them these compounds. We would test them here in our lab first to see that they were cytotoxic. And then we’d send them to NCI. And they would test them broadly. And they would come back and say, these look interesting. Because we didn’t know anything about cancer, the exact details of cancer, but they did. And they would say to us, this is really interesting.

So well, OK, so what do we do with that? We start to develop it. We find out how much we can get– we want to get them into toxicity tests to see if they’re good, if they can be used.

The process of drug discovery, of course, is very complicated, and sort of by definition has to involve big companies with lots of money. Because ultimately, you’re going to take your drug candidate, and they’re going to say to you, we want to test that in humans with cancer. And that means that you have to provide up to maybe 100 grams of the natural product. And that was, for us, very difficult in the beginning. Because we couldn’t get a lot of chemical compound from a beautiful coral reef animal. We didn’t want to collect all of the animals from these beautiful coral reefs. And nor could you. You weren’t given permits to do that.

So this changed our style completely. It changed how we do research. And it moved me to ask a simple question. In 1929, Alexander Fleming discovered a molecule called penicillin. And this was– maybe still is– perhaps the most important drug ever discovered. Maybe not so much, maybe morphine. But the point I’m making is that penicillin was a product of a microorganism.

So around 1985, I said, why aren’t we studying microbes in the ocean? Why don’t we do that?

DAN UDWARY: What were you doing before bacteria then?

BILL FENICAL: Well, we were studying marine animals, marine plants, things like sponges and things you could collect. But when we’d find these wonderful compounds, we couldn’t produce enough to put them into human trials. Even couldn’t produce enough for early, what’s called preclinical development.

So, we started asking questions about microbiology of the ocean. And it was amazing what was not known, amazing! Everybody, all the terrestrial microbiologists, said, “there’s nothing new in the ocean. Everything that’s in there is rinsed into the ocean through rivers and rainwater and spores flying into the ocean. So, there’s nothing new.”

OK, that’s pretty ominous. Because these were powerful people. These were people that really controlled microbial drug discovery. And a guy like me, with no background in microbiology, said, I think we should do this. They said, “eh, you’re kind of crazy.”

But there was a guy, a very, very experienced man, named Arnold Demain. Arnold Demain had run the microbiology program at Merck for 40 years, then retired. And I went to see him. And I said, “You know, Arnie, are we dumb? I mean, are we wasting our time? Are we going to find zero?” And he said, “Bill, go take a look.”

So that’s what we did. We started. We went out, and we did not know what to do. We did not know how to culture marine isolates from the ocean. We didn’t know whether to study bacteria, fungi, archaea, the whole range of microbial life.

So, we just started out. And we started sampling. And NCI said, with regard to our grant, “OK, we’ll let you go. Go ahead, find out.” So we started looking. And we started looking in the ocean in terms of what these naysayers had said– (MOCK TONE) “nothing new there!” Well, we started finding things that were really new.

First of all, we found that bacteria isolated from the ocean required salt. Oh, interesting. What are they doing with the salt? Well, maybe they’re just balancing cells against seawater.

Then we found out that, when you ask that question about what they might be making, that we found molecules again that contain chlorine, contain bromine, which were characteristic of elements in the ocean. And so we started finding chemical compounds, again, that were different, different structure types. We started amassing a collection of marine bacteria. We also studied fungi. We studied a few archaea. But you know, those were not very prolific. We have a collection now of about 17,000 organisms we’ve isolated from sediments, bottom mud, all over the world. We created ways to go out, 20 miles offshore, and sample at 4,000 meters depth, get samples of the bottom, and bring it to the surface, bring it to the lab.

And you know, I worked with a man named Paul Jensen here who’s done a terrific job. He’s a microbiologist. And together we created a program that really began to bear fruit.

So,  whereas working with an animal to develop a drug was really difficult, working with microbes to develop a drug was not difficult at all. And that’s because the whole history of the pharmaceutical industry beginning in 1950– or really 1940– was based on microbial cultivation, microbial fermentation in huge quantities, 50,000 liter fermenters, to produce a kilogram of a drug, for example.

So we saw the option to now work on marine organisms to be able to solve the problem of supply. And this changed everything for us. We soon began finding things that we could culture. We learned how to culture. We cultured everything in seawater media. We found out all kinds of weird things. Glucose is toxic to a large number of marine bacteria. Do you believe that? I mean, glucose is the carbohydrate component of media.

So we went back and said, I don’t think we can use standard media. I think we have to devise our own culture media. So we started using a whole bunch of polysaccharides. We cultured things with fish meal, seaweed paste. I mean, we tried really hard to think about what nutrients were available the ocean, translate that into a laboratory setting, and get things growing that maybe wouldn’t have grown before.

Now, I should mention that there was a serious limit in culturing that was called the Great Plate Anomaly. And this is that if you take a sample of, let’s say, water, and you look at it under a microscope, you can probably 2,000, 5,000 different cells, different colors, different sizes, different shapes, motility, all this stuff. But when you take that sample and put it onto a culture plate, you don’t see hardly anything growing. And so people said, well, we’ve done this experiment. And most, 95% to 98% or more of everything in the ocean, will not grow.

DAN UDWARY: On that one specific plate type, yeah.

BILL FENICAL: Talk about telling everybody to quit working on marine microbes. They won’t grow, well, they won’t grow because everyone tried to grow them using Pasteur’s media, same old, same old. And of course they don’t grow.

Plus, what people found was that nothing grew in three days. And this is the standard protocol. Hey, look, we inoculated the plate. Look at this, nothing grew. Three days, five days, nothing’s growing.

Unfortunately, you have to wait a month. And then they grow. So, everybody threw out everything. And this led to this negativism that bacteria, microbes in the ocean, are not culturable, which is completely wrong. But that’s what we were faced with. When we write a grant, someone would say, what are you doing? You’re wasting your time. Well, finally we convince people that we could get cool things.

And so about the same time– this was around 1990– we made some discoveries that really looked promising. We found that there were new genera, these basic taxonomic group called the genus. We found there were new genera in the ocean that no one had seen before. One of the important ones was something that we named called Salinispora. And it won’t grow without salt water. There are others. There are many others, including Streptomycetes, which are common soil bacteria.

DAN UDWARY: Yeah, we talk about them a lot here.

BILL FENICAL: Yeah, Streptomycetes is a very horrible genus. Because first of all, it’s not a fungus, so why would you have m-y-c-e-s as a suffix? But that’s historical.

But the point I’m making is, even things that key out to what you would say are classic soil microorganisms from the ocean, half or more require salt. So how do you define a marine bacterium? I think you define it by perhaps having a salt requirement, a salinity requirement, for culturing, and then of course using modern phylogenetic tools measuring genes, the 16S gene, and comparing it with everything that’s known.

So Salinispora, if I can go back to that for a moment, Salinispora is an amazingly complex marine bacterium. There are seven species known now, three very common ones. And they’re unequally distributed around the world.

Salinispora tropica, for example, is only found in the tropical Atlantic. Why that should be is a microbiological puzzle, but it’s the case. This was one of the first ones that we worked on, first species, Salinispora tropica. And we isolated a strain that produced something extremely potent in killing cancer cells.

Well, chemically, we found out what it was pretty soon. We started testing it in collaboration with the National Cancer Institute. It became clear this was something very extraordinary.

We named the compound salinosporamide A, after the genus Salinispora. And we began to find out that this was something that was in need of being developed. In the beginning, we didn’t know how to do that. I mean, we’re not a pharmaceutical company. So, Paul Jensen and I started a company, here in La Jolla, called Nereus Pharmaceuticals, small biotech. And that’s a whole other story, biotechs.

But they, Nereus licensed the compound, salinosporamide A, from the University of California, and began to develop it. And it went smoothly. Boy oh boy, it was great. They developed it. They got it into phase I human clinical trials. And it showed cures in some lymphomas even in phase I, which is just a toxicity evaluation. And so this became an incredibly important discovery for us.

Around the same time, we found another molecule. This time it was from a fungal strain collected from sediments in the ocean. No, sorry, collected from the surface of a marine plant. And it too had something unusual that we began thinking about developing. And the company, Nereus, of course, which we didn’t control but which we wanted to see develop our discoveries, took two compounds from us and began developing it.

And along the way, the biotech, as biotechs go, they ended up folding. But the compounds that we had developed with them were then moved on to different companies. So our important compounds, both for cancer, both were important in cancer, both of those compounds were developed by different pharmaceutical companies, large companies that could afford to do all of the complex biology and production that we couldn’t do.

So 2021, we have two drugs in late phase III, both for cancer. The one from Salinispora has a drug name called marizomib, m-a-r-i-z-o-m-i-b, which is called the drug name. And it is in phase III clinical trials for glioblastoma brain cancer, which is a very tough cancer to cure. Doing well. So we have our fingers crossed. It’s exciting.

And what’s unusual about that drug, marizomib, it that it is capable of passing through what’s called the blood-brain barrier. And most chemical compounds do not. So, if you have a brain tumor, you need a drug that’s going to go into that tumor. So, we were very fortunate that this drug would do that. And we’re excited about the progress.

The other drug, from the fungal strain–

ALISON TAKEMURA: Oh Bill, maybe before you go on to the next drug, can I ask you, what is the mechanism of action of the first drug?

BILL FENICAL: Yeah, of course. We found out the mechanism of action well before it went into the Nereus Pharmaceutical company. And it is an inhibitor of what’s called the 20S proteasome. The proteasome in the cell is a bundle of enzymes that is used to degrade proteins, peptides, that are signaling, normally doing some signaling activities in the cell.

But you don’t want them to be there forever. Or the cell gets oversignaled. So, the proteasome basically incorporates those spent proteins and degrades them. And what was clear was the proteasome inhibitor was an area of cancer drug discovery of some utility. But this was like a superinhibitor. It was picomolar active. And it was active by oral administration as well as intravenous and so on.

So, this was picked up right away. It was picked up by Celgene. And then Celgene was purchased by Bristol Myers Squibb. And it is now in phase III in multiple sites, phase III human clinical trials.

ALISON TAKEMURA: Does inhibiting this proteasome damage healthy cells as well? I mean, I guess, how does that affect cancer cells more than healthy cells?

BILL FENICAL: Yeah. That’s the massive question in cancer chemotherapy, is how do you find a target in a cancer cell that is not next door in a normal cell? And the answer to that is, most of the time, you don’t. Most of the time, the target is a common target. After all, cancer cells are your cells. So, what’s in those cells is the same, perhaps, mostly, that is in an adjacent cell that is also your cell.

But what makes chemotherapy work is not that the target is unique, it’s that cancer cells are metabolizing at an extremely heavy rate such that they preferentially get impacted by something that is toxic to them. So, when you talk about chemotherapy, of course people suffer in chemotherapy. Because 99.9% of cancer drugs are not so selective that they don’t have an impact on normal cells, particularly your hair cells, which are also rapidly growing, the cells in the mucosa lining of your mouth are rapidly growing.

So, cancer cells, however, are preferentially affected.

ALISON TAKEMURA: I see thank you. And so you were talking also about this second drug that’s in development from a fungus.

BILL FENICAL: Yeah. This drug is called plinabulan. And it is also quite active. It was discovered at Nereus, and then ultimately passed on to another company for clinical trials. It is a selective inhibitor of tubulin, which is a protein in the cells, which is responsible for the division of the cell. You can see cartoons where you’ll have the chromosomes pulling apart during cell division. And that’s because they’re attached to tubulin. And tubulin is responsible for effective cell division.

It has a novel target on tubulin. It is effective in a whole different group of cancers and is in phase III for non-small-cell lung cancer. And I know a little bit more about it because it’s being tested right here at the UC San Diego Cancer Center. So, I’m a member of that center. So, I know a little bit what’s going on. And it’s also showing promise.

DAN UDWARY: Are these the closest things you’ve had to getting to the clinic?

BILL FENICAL: Oh yeah. I would have loved to have taken 20 drugs to the clinic. But there are all kinds of practical limitations to that. One practical limitation is time. Another practical limitation is $1.2 billion in investment. I discover things, boom, they’re off my plate. I can’t impact anything unless I would be the one providing for financing clinical trials, that kind of thing, which is impossible.

So, the average time to develop a drug, more than 15 years. So, we’re pretty happy that we discovered this and it got developed in somewhere around 2004, I guess, something like that, and now, 2021, it doesn’t have very far to go. We’re very happy with the progress.

So, one of the things I want to mention, Dan, is that we’ve never thought that you can’t culture 99.5% of what’s in the ocean. We never believed it for a minute because we saw the limits of people devising how to grow bacteria from the ocean. They weren’t using nutrients from the ocean. They weren’t doing environmental evaluations of what nutrients are there.

So we asked a couple of questions. One time we spent about a year looking at samples and revising all the media and revising methods. We didn’t spend a lot of time on this because people didn’t want to give us any money to do that. But what we decided to do was to use bizarre media, even no media. We would take isolates from the ocean and put them onto agar surfaces with no added nutrients. There’s enough nutrients in agar that you don’t need to add anything else.

So we started a program. We took about 50 different inoculum, different kinds of samples, and we went through a process of using diverse media, sometimes no nutrients. And we followed cultivations. We set up a library of Petri plates to watch what happens over time. And in the high-nutrient media, we saw all the same stuff, which are called the copiatrophic. Copious nutrients.

DAN UDWARY: Never heard that word. OK, cool.

BILL FENICAL: Copiatrophic organisms, which are minor in the ocean. And they grow like crazy when you do huge amounts of nitrogen, carbohydrates, all that stuff. And then we got to these media which were much more low-nutrient media. And we watched what would happen. First of all, nothing happened in the first 10 days of inoculating the plates. Nothing happened at all. So we watched it again. We would check off the time. And here’s all these plates in this library, growing. Watching what would happen over time.

And after one month, we saw a little red colony on one of the plates. One month. Whoa, was that a contaminant? Or what happened? It wasn’t. After two months, we saw two yellow colonies come up on a different nutrient media– or low-nutrient. And over a six-month period, we found 20 strains that became visible. We took off the plates. We started to grow them. We found out that you could grow them in liquid media and that the limitation was their adaptation to grow on agar. They had to sit there for months before they figured out how to grow. And once they did grow, we could culture them.

So of course, we decided, what are these? And we did the ribosomal taxonomic approach, looking at that, which allows you to compare the ribosomal gene, 16S gene, to all other bacteria known. And these, all 20, were unknown, completely unknown. They were part of the comb the “unculturable” microbes in the ocean.

So we looked at them carefully. By looking at where their taxonomic positions were, we saw that many were new genera, very different things. Some were new orders, certainly new families. And we saw exactly how they fit into the scheme of life. It was shocking.

And we were able to grow them. Once we had them growing, we could grow them on a variety of different media. We could get high cell densities in liquid culture. And of course, this is when we started thinking about, are these a resource for drug discovery that nobody knew about? And the answer was, you bet.

We didn’t study them all, because I ran out of personnel. But we studied two strains. One was a new order, and one was a new family. And these two microbes produced new alkaloids, different chemical types of alkaloids that we were pretty excited to see. Nobody knew about this. Nobody had ever cultured or even seen this organism before. So, this was exciting for us.

So, this was when we started saying, we have to have the genomes. We have to look at the full picture of these. And so, we selected eight of these weird bacteria. And we proposed to the Joint Genome Institute.

DAN UDWARY: Oh, that’s where we come in.

BILL FENICAL: Yeah, you know something about that.

DAN UDWARY: Little bit.

BILL FENICAL: We proposed that these are so novel, so unique, that we needed to ask whether they would do the full genomes for us. So fortunately, the secondary metabolite component of the JGI, people are interested in this area.

DAN UDWARY: I know about that too.

BILL FENICAL: Yeah, you know that too, don’t you?

And so, we’re very happy. We’re in the process. We now have cell pellets. And we’re pulling genomic DNA out of those cell pellets. So, we’re about ready to send those for genomic analysis.

And of course, we don’t know anything about the genome composition of these kinds of things. There could be some real surprises. I mean, they’re probably smaller genomes. They’re gram-negative bacteria. But we want to see those biosynthetic gene clusters that are making these alkaloids. And we want to know if any other clusters are in the six others that we’re getting genomes of that we haven’t chemically studied.

So, you know, microbiology, in historical terms, has avoided looking at some of the most rare and non-obvious microbes. The pharmaceutical industry studied actinomycetes for 50, 60, 70 years. And of course, they found a lot. But they didn’t study many other things– very well anyway, at least from what I know. So, we’re excited about doing a little bit more of this in due time.

DAN UDWARY: Well, I can say that we’re all excited about this project too. I’m really looking forward to seeing the data when it finally gets off the sequencers. The pandemic has obviously slowed JGI down a little bit. But I know these things are underway. And I hope that once we do get some results, it’ll be really fun to have you on to talk about them again.

BILL FENICAL: I’d be happy to do so. And by that time, we’ve probably got some other exciting things too, Dan.

DAN UDWARY: Always, right? Yeah. Well, Bill, it’s been really great talking to you today. Thanks so much for your time. And yeah, looking forward to keeping the conversation going with you.

BILL FENICAL: Pleasure speaking to you. Alison, thank you very much.

ALISON TAKEMURA: Oh, thank you. Thank you also for your historical perspective. It’s so interesting to see, from a relatively young scientist or “trained as a scientist person” point of view, just how much the field has progressed. So yeah, thank you so much for sharing that.

BILL FENICAL: I was trained in synthetic chemistry. And I’ve studied microbiology for about 25, 30 years. And I can actually go give a talk at ASM [The American Society for Microbiology] and those kinds of places. I can fake it.

DAN UDWARY: I mean, yeah, that’s the fun about natural products. We get to do all kinds of crazy things. I started in synthetic chemistry also. And that didn’t go anywhere. But I found my niche, and get to do all kinds of fun things. Yep.

DAN UDWARY: Always good to talk to you. Thank you so much.

BILL FENICAL: Bye bye.

ALISON TAKEMURA: Bye!