Bonding Over Science, Episode 9—How Circadian Rhythm Affects Cellulase Production for Biofuels

Feeling exhausted after the Daylight Savings time change?

Your circadian rhythm might be the reason.

Circadian rhythms are the 24-hour cycles that regulate the natural processes in all organisms, including humans, plants, and fungi. 

On this episode of the Bonding Over Science podcast, molecular biologist Jennifer Hurley talks about her circadian research looking at clock regulation over metabolism and how this understanding could help cellulase production for biofuel manufacturing. She also shares how circadian rhythms in humans are affected by shift work and Daylight Savings Time. Read more about Hurley's research.

 

Transcript:

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Dawn Stringer: How is your sleep schedule? I’m going to take a wild guess and say not as consistent as you’d like. Today’s guest may have you re-thinking your bedtime, and how you manage your daily schedule based on your natural circadian clock. 

I’m Dawn Stringer with Bonding Over Science, join me in learning how the circadian rhythm works in our bodies, and the organisms around us that maintain our environment. 

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Dawn Stringer: Associate Professor Doctor Jennifer Hurley has been studying this concept for several years. 

Although her research is helping her understand the effect of the circadian rhythm on the world and organisms around us, she has some personal experience as to what disrupting our circadian clock can do to us as humans. 

But first, let’s learn the fundamentals of her research. 

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Jennifer Hurley: My research is great in that almost anyone can connect to it because we all live on the planet and on this planet we can see that we have a night and a day and really don't think about this very often. But that's very stressful for organisms who live on the planet, right? If you're walking around at night and you live in a place where there's a panther, the panther is going to eat you at night and not during the day. 

That's the thing for humans. But for other organisms, like trees and algae and fungi, the amount of DNA damage that is done by direct sunlight is really significant. So just looking at the sun and being in the sun's purview is very damaging to organisms. And that is also the time of day, right, where plants want to make energy because they're getting energy from the sun and at night it's a waste of their energy to try and make energy because the sun's not available. 

So these regular stresses that we face are consistent, and biology is nothing if not adaptive, and what we have done is to adapt what we call circadian rhythms, which allow us to time our physiology so that things that should happen in the daytime happen in the daytime and things that should happen at night, happen at night. And this has been, you know, we've been revolving on this planet for billions of years. 

And so if we didn't really incorporate these circadian rhythms deeply into our systems, we'd be in a lot of trouble. And we found prior to my work on this particular project, but in part because of the research that the DOE [Department of Energy] collaborated with us on, we found that anywhere from 40 to 80% of the things that are in our systems and in fact, if you look at algae, it's as many as 100% in cyanobacteria, of genes and proteins all fall under circadian timing, which means that we have a whole lot of our physiology timed by the circadian clock. Now, there's two sides to this coin, right? In terms of what it does for us, it's great and it's really productive and it's really helpful. But if we're trying to live in an environment that's not a perfect 24-hour environment, we find ourselves out of sync.  

And this is problematic for humans because when we shift our day/night cycles, because we're traveling through the air, or we're going to Daylight Savings Time, or whatever else we're doing, shift work is another big problem, so much of our physiology is timed by circadian rhythms that when it gets out of sync, it causes health problems. And in fact, shift work over a life span is pretty profoundly impactful. 

So much so that the CDC classifies shift work as a class 2A carcinogen, which is on the same level as breathing in diesel fuel for your entire life. So it's pretty bad, if you think about organisms that the DOE cares about, not that they don't care about humans, but we're talking about algae and plants and fungi. If you desynchronize our clocks in these organisms, it can also have really negative effects. 

About 15 years ago now, there was a really landmark paper that took plants with two different circadian rhythms and it put them into opposite rhythms so that when it matched the circadian rhythm of the plants, the plant thrived. And when you took a plant that didn't match its circadian system, the plant tended to die. So we have really profound effects when we don't live within our circadian environment. 

And so it's important to understand not only why things go right, but what happens when things go wrong, and my lab does a whole bunch of that. We work on the immune system in humans because if you disrupt your immune system, which is heavily timed by the circadian clock, you can have increased risk for Alzheimer's disease and diabetes and all kinds of other things [to] go wrong. 

We also work on fungi called Neurospora crassa, and it's a great model system to work with because the clock in fungi is very much like the clock in humans. And so we can learn a lot about fungal systems as well as human systems by studying this clock. And what's important for the DOE, especially in terms of this project, is that neuroscience is a great model for organisms and fungi that produce biofuel components. 

And so there are, we have shown, good times of day to make cellulase and times of day when neurons don’t really want to make cellulase. And for a group of people that want to make the most cellulase, knowing that there's a time of day to harvest the organism to make the cellulase versus a different time of day to not make the cellulase. 

It's really important to understand that and then perhaps be able to manipulate it to increase the overall level of cellulase production. So there's lots of different reasons why we study what we study. For this particular project, it does get a little bit into the biochemistry in biophysics, but I'll keep it light for you. So the way that this clock works is it's a two-component system. 

Something turns on and it makes something that then turns the thing off. And that's called a negative feedback loop. And it's really just two components, something to activate and something that represses. For a very long time, we assumed that the activating part was the part that regulated physiology, and the repressing part just turned off the activating part. But about 10 years ago, our work and some other work in the clock community showed that the activating part was not the only part that was responsible for controlling what was happening in a cell to the circadian time, but the repressing part was doing that as well, and that was really exciting for us because the repressing part is made up of these very unusual proteins. And if you've got any idea of what proteins look like or what scientists think about proteins, for a long time we thought the proteins exist in this really structured and regular conformation. 

That's why we can crystallize a protein because it forms regular structures. Well, the kinds of proteins that are in the repressing part of the clock are like wet spaghetti noodles. They just flop around. Right. And this sloppiness we thought couldn't exist in a cell. But now that we started to learn more about them, we found that this floppiness not only exists but is important for the function. 

And so this project is about understanding how that wet spaghetti noodle acts like kind of a sticky hub, where at one time of day it reaches out and grabs one protein, another protein, and it brings them together so they can interact. And this interaction can cause the production of cellulase. And at another time of day, it releases those proteins and goes to a different sticky noodle conformation and those interactions don't occur, and so cellulase are not produced. And the amazing capabilities that the DOE has is they can look at these really unusual proteins and understand how they flop around and how that floppiness can actually lead to bringing proteins together or keeping them apart. So that's what, in basic words, that the project is all about. 

Dawn Stringer: What instrumentation are you using for your research at EMSL? 

Jennifer Hurley: So we're doing a couple of different things. To understand this protein, we first have to make it in isolation. And so with the JGI [Joint Genome Institute] who are producing the vectors for us, JGI’s vectors will be used in the expression system that EMSL has to mass produce lots of different types of proteins and lots of different mutations of proteins and then once we have all those mutants, we're going to do cryo-EM [cryo electron microscopy] on them, because cryo-EM is one of the few ways that we can deal with these floppy sticky proteins that are there. 

The unusual thing about them is not only are they floppy and sticky, but they're also really big. And there's good solutions out there for dealing with floppy and sticky proteins if they're small, but when they're really big, like the proteins we work with, cryo-EM is one of the few ways that we can handle these big floppy sticky proteins. 

And so that's why we wanted to work with EMSL—they were a great team to do this. 

Dawn Stringer: I want to back up a little and learn how you initially became interested in this research. 

Jennifer Hurley: It actually goes way back. So when I was a little kid, my parents were incredibly strict and we couldn't have sugar and sweets, no nothing. And when I would go to my grandfather's house, he and I would wake up at 4:30 in the morning and he would have hidden in the microwave these amazing cheese danishes and they were a huge package. 

And we between the hours of about 4:30 and 6:30, would eat all of them and have time to throw things away before anybody else woke up. So no one ever knew. I told my parents after he passed away, they were appalled, but I thought that was normal behavior as a 10 year old, that was what I just did. 

And it wasn't until later on when I grew up and I had sleepovers, nobody got up at 4:30 in the morning. That was odd. And so when I went to school, I realized that this was kind of a unique way to live your life and that I thought it was a sleep disorder. And I got to college and I had wanted to go to med school because I grew up in a very small town, and the only thing I knew about being able to do science was to be a teacher like my parents were or to be a physician. 

And so I didn’t want to be a teacher because I was rebellious. I didn't want to be my parents and I wanted to go to med school and I wanted to study sleep. And I quickly realized that I didn't like needles. So I denied all my medical applications and went to grad school instead and ended up at Rutgers. And there I met a man named Isaac Edery who was a postdoc with Michael Rosbash who won the Nobel Prize in circadian rhythm. 

And I rotated in Isaac's lab and he was doing circadian research and it was amazing and exciting. And he told me that if I wanted to be a circadian researcher, it was best to do something else as my graduate work and come back to it as a postdoc. So I went off and joined Nancy Woychik and Masayori Inouye, checking my theory in a way, who were doing biochemistry and protein structure in toxins. 

And I learned all about biochemistry and biophysics and how the movements in proteins can actually affect biology. And then when I was done, I was still interested in figuring out what was wrong with me in terms of my sleep. And so I went back to Isaac and I said, Where do I go? And he gave me this list of amazing biochemists. 

And really there were only two or three working in circadian biology, and one of them was Jay Dunlop, and Jennifer Loros who are at Dartmouth. And I joined their lab, and they were interested in Neurospora crassa, which is a filamentous fungus that grows on burned trees after forest fires. And it's an amazing model for lots and lots of different things, but one of those things is the circadian clock. 

And so I got into their lab with the idea that I was going to solve these structures of these proteins in the clock and, you know, 15 years ago that was easy to do. You expressed a protein, you crystallized it, and you were done. And about three years into my postdoc, I realized these things were never going to crystallize because they were these big floppy proteins. 

And it really kind of got the ball rolling on what half of my lab now does. And that is trying to understand how timing is, how really structured and organized timing can come out of chaotic and floppy things. And that's how I got into the research. How I got into this particular level of interacting with the DOE was actually I was at a conference with Scott Baker and I was talking about the things we were doing. 

So we were connecting protein motion to physiology and we were doing it on sort of a 1-to-1 basis. Right? You can understand this protein touches that protein and it affects this other protein. And Scott said to me, Well, you realize that we have all these capabilities to do instead of a 1-to-1 protein. You could do this on every single protein within the cell. 

And I said, I like your ideas. And so I wrote my first grant. I think I was still a postdoc back in 2013 and he funded it and I have been funded by the DOE ever since. 

Dawn Stringer: I have to rewind to your personal experiences. It begs the question, have you solved your sleep issues? 

Jennifer Hurley: So I in the process had two children and both of those children have ensured that I will never sleep past 5:00 again. My poor spouse, who is a late sleeper, had basically given up on life because if it's not my sons, it's myself. And we are always up early in the morning and running around like maniacs. We are early risers and that's the way it's going to be. 

Dawn Stringer: I'm sure the moms out there can relate. So what happens when you're done with your research? What's next for this science? 

Jennifer Hurley: Every time I think I finish something, something else comes up. It's never done. What we're trying to understand here is what the structure is and how it changes over the day. And hopefully that will give us information on to what parts of the sticky protein are available at one time of day versus another. We have information from my lab already about what binds to the protein when. 

So if we can take the structure and the things that are available and combine it with the information that we already have about what is binding it at what time of day, what I really want to do is be able to create a model where you can understand this protein binds in this particular location. And then once you know that, you can start predicting how you could affect that interaction to either increase it, tighten it, make it happen more often or decrease it, loosen it and make it happen less often. 

And there's all kinds of really interesting research going on in the field about how you can modulate those sticky regions in order to mess with them, as well as how you can use small molecules to actually just give it a small molecule to the organism and change that interaction to have it occur differently. Another thing that we would like to do with this is one of the things that affects the circadian clock is light, right? 

So if we can understand exactly how these protein motions occur and what time of day things happen and when they should happen, we can actually use light to modulate the behavior of the organism once we know the behavior itself. And so in theory, we could use a zero-cost, zero-energy input to change what's coming out of the fungi in order to produce more biofuels. 

And so long-term goals are to do cheap and easy increases in cellulase that can then be used downstream to make cheap and easy and more sustainable fuels. 

Dawn Stringer: Is there information out there already that shows what time of day these things are happening? 

Jennifer Hurley: We have some data that have come out of my lab as well as Jay Dunlop's back when I was still a postdoc, to say at this time of day we predict there's a lot more cellulase being produced, but [not] at other times of day. There's been some work from Louise Glass as well, who's now retired, but was an expert in biofuel. 

But there's also other projects going on in other fungi, like Monica Small, [who] is also funded by the Department of Energy and she's looking at a wide range of fungi in order to understand how we can make more biofuels from those fungi. And probably there's going to be certain fungi that make certain cellulase and other fungi that make other cellulase. 

But if we go beyond that, we can even go to the algae that are being used to make biofuels themselves. They have really profound circadian rhythms. And I know that there's some projects that are funded at the DOE that are looking at these algae and how they make biofuels at certain times of day. So there's lots of information out there to give us good predictive measures about when we should be seeing these cellulase and other biofuel components being produced. 

Dawn Stringer: Hearing all this information, I'm curious to hear what you think of Daylight Savings Time, and is it troublesome to our natural circadian rhythm? 

Jennifer Hurley: So there is a very strong point of view from the circadian community on Daylight Savings Time. So there's several different levels. One, shifting your clock in general is bad. So we prefer never to shift your clock if you don't have to. So there's that aspect of it. But there's a really big push in the circadian community to get off Daylight Savings Time permanently. 

And that is because if you think about Daylight Savings Time and especially where you guys are too, when you wake up on a morning with Daylight Savings Time, it's generally dark and you're getting out of bed in the dark. Now your brain visualizes morning as when the blue light comes up. So when sunrise happens and so when you get out of bed before the sun comes up, you're forcing your body to go out of rhythm with its natural cycle. 

And we've been able to show that even though small differences, one hour here, one hour there has some really profound effects on human health. There's a paper that came out recently wheat they did is they took information about where you lived in your time zone. So if you lived on the front edge of the time zone, when you saw the sun come up earliest versus the back edge of the time zone, people living on the front edge of the time zone have lower cancer rates, lower rates of heart attack, lower rates of diabetes, lower rates of obesity, all just because they see the sun come up right around the time they're waking up versus they tend to get up a little bit later in their solar cycle. So the circadian community in general wants everybody to shift to permanent standard time, no Daylight Savings Time. 

Dawn Stringer: I think a lot of people would agree with you. 

Jennifer Hurley: I think most people agree with that. There's some business interests that really push for Daylight Savings Time because you don't feel so bad working late if you've got the afternoon and sunshine in the afternoon to hang out. And I understand that. But, you know, human health, I can still eat my ice cream in the dark right. And go out go out for dinner when it's dark outside. I want to wake up when the sun comes up. 

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Dawn Stringer: Who knew daylight could affect not only our lives so much, but the ability to generate environmentally friendly and cost-effective biofuels! I’m sure after listening today, we’ll all reconsider how we manage our time during the day and night. 

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Dawn Stringer: Thank you for listening to Bonding Over Science, I’m Dawn Stringer for the Environmental Molecular Sciences Laboratory.  

We don’t have time to cover it all, so don’t forget to check out EMSL-DOT-PNNL-DOT-GOV for a full article on this topic featuring who I spoke with today. And don’t forget to follow us on all social media platforms for the latest and greatest news coming from EMSL! 

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Dawn Stringer: EMSL is a Department of Energy, Office of Science national user facility that accelerates scientific discovery and pioneers new capabilities to understand biological and environmental processes across temporal and spatial scales. EMSL leads the scientific community toward a predictive understanding of complex biological and environmental systems to enable sustainable solutions to the nation’s energy and environmental challenges. If you’re interested in working with EMSL, learn more at emsl.pnnl.gov, that’s E-M-S-L-DOT-P-N-N-L-DOT-G-O-V. 

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