WIsconsin Public Television University Place  Sustainability Via Synthetic Biology  Brian Pfleger  9/25/13 UP922  cc >> Welcome, everyone, to Wednesday Nite at the Lab. I'm Tom Zinnen. I work at the UW Madison Biotechnology Center. I also work for UW Extension Cooperative Extension, and on behalf and those folks and our other co-organizers, Wisconsin Public Television, the Wisconsin Alumni Association, and the UW Madison Science Alliance, thanks again for coming to Wednesday Nite at the Lab. We do this every Wednesday night, 50 times a year. It's your chance to participate in sharing in the discovery here at your public land-grant research university. Tonight it's my pleasure to be able to introduce to you Brian Pfleger. He received his bachelor's degree in Chemical Engineering from Cornell University in 2000, and he earned his PhD in Chemical Engineering in 2005 from the University of California at Berkeley. He worked under the supervision of Jay Keasling. Brian's thesis research focused on developing methods of controlling gene expression in bacteria that could be applied to enhancing the biosynthesis of pharmaceuticals. 

After graduating, he accepted a postdoctoral fellowship in the laboratory of David Sherman at the University of Michigan, where he studied how six Bacillus anthracis enzymes assemble a natural product essential for iron acquisition and pathogenesis. Brian is currently an Associate Professor of Chemical and Biological Engineering with appointments in Biomedical Engineering, the Microbiology Doctoral Training Program, and the graduate program in Cell and Molecular Biology. Brian's research has been recognized with young investigator awards from 3M, the National Science Foundation, specifically the Career Award which is the highest honor a young scientist can win from NSF, the DOE Early Career Award, and the Air Force Office of Scientific Research. Brian has also received the Benjamin Smith Reynolds teaching award from the UW-Madison College of Engineering for his efforts to introduce undergraduates to biotechnology. Tonight Brian will be talking with us about his work on sustainability by synthetic biology, a growing role for biotechnology in the chemical industry. Please join me in welcoming Brian to Wednesday Nite at the Lab. [APPLAUSE]  >> Thank you. Thank you, Tom, for the kind introduction and the opportunity to speak here on campus to a different audience. This is going to be a lot of fun for me to introduce you to some of the things I've been thinking about for a long time and then actively researching in the lab for the last six years on this campus. Hopefully I'll have a chance at the end for some questions. 

If there's something critical that I go over, by all means ask the question. I'll try to repeat it for the camera. So the topic for tonight is a topic I think that's important to everybody, and that's the topic of sustainability. So it's an issue that's going to touch many different facets of life and there are many different approaches to try to achieve sustainability in our society. The approach that I'm interested in is that of biotechnology. So what I want to try to introduce over the next 10 minutes is the field of synthetic biology, why it's such an exciting field at the moment, and the enabling technologies that have really allowed it to explode in the last five to 10 years or so. So to start, I need to acknowledge that synthetic biology is not a new field nor a new concept. This is something that dates back to even things on this campus from the mid to late '70s. This is the era of recombinant DNA technology, the idea that we could take genes out of organisms to make a particular protein, maybe for a therapeutic purpose, put it into another organism, and make that protein heterologously. And so when this was done successfully, a biochemist on this campus was able to say that, hey, this is the start of the era of synthetic biology. 

No longer will we have to worry about natural systems. We'll be able to start to engineer our own biological systems to study them as well as to use for industrial purposes. So from the 1970s, you saw the birth of companies like Amgen, Genentech, people who have made protein production in various microorganisms and mammalian organisms very profitable and very successful. So at that early stage we were talking about one piece of DNA, encoding one gene, becoming one protein. So that's a very simple story, and what you're going to see over the next few minutes is how it's evolved from that simple story to a system today where we can potentially synthesize entire genomes and put those into organisms. Once I have a chance to talk about synthetic biology, then we'll visit the idea of sustainability, why we want to talk about sustainability, and how synthetic biology in particular can be applied to improving the production of fuels and chemicals. And so if synthetic biology is not a new field, why are people so interested in saying that it is? And I think that the real change that's happened in the last decade is the influence or the inclusion of additional scientists to this field. So where recombinant DNA technology was classically the realm of microbiologists, molecular biologists, biochemists, now you're starting to see the entrance of engineers, and not just chemical engineers who have been doing fermentation science for a long time, but electrical engineers, mechanical engineers, and even people who think about things just inside the computer in terms of computer science and circuit design. So what these groups have seen is that there are natural examples of regulatory circuits that mimic the things that we would use in an electronic system. So if we can build these electronic systems, can we take the same components in a biological system, wire them up differently to create our own artificial circuits? So this has been done, and these are two examples here on the slide. 

The top talks about switches. You've probably seen these. These are things I'm not actually all that well qualified to talk about, but in biology you'll see switches to determine cell fate, to determine different times of turning on and off gene expression, and so you can design circuits to have a toggle functionality by using very similar proteins and put them into different artificial positions. The same thing has been done for oscillators and just about every other type of electronic circuit, gadget, logic function that you can imagine. So in the case of oscillators, you can look to the cyanobacteria or the photosynthetic organisms that have to deal with day and light cycles. So when there's no light around, you don't want the genes to harvest light turned on because you're going to waste those resources. So here they have a circadian rhythm to help them turn on the right genes under the right context. So this same architecture can be used with different proteins then to create synthetic oscillatory circuits or repressing circuits. And so these have been now used in many different applications but with the eye on trying to make a biological computer. Can we do computation in a living system. 

And so this is the new influence. Most of the scientists who will tell you that synthetic biology is a new field come from this perspective because it's new to them. So I think it's great that we're seeing the inclusion of these new disciplines into the field because it's giving us techniques to approach problems that we didn't have before. So to show you a couple of examples of how these types of circuits or systems can be used, I'm going to give you two examples of a simple concept. A biological system needs to understand its environment. What is the system that it's in? And then turn that signal or that piece of information into an action. And so on the top, I show you a circuit here where proteins are expressed inside a bacterial cell that sense red light. These proteins then transduce that signal, if it senses red light, into turning on expression of a protein that happens to just be a pigment. It's a dyed color. And so if you set up an artificial system where you have a mask where you cut out a shape for a person's face, as you see on the right, and you put that over top of a Petri dish that's covered with these cells and you shine light through that mask, the light hits the cells in certain places, and where the light is hit, you get the pigment produced, and so what you have is bacterial photography. 

And so this is the joke is this isn't what killed Kodak but certainly a technology that's very interesting and there have been lots of advancements on this in terms of biosensing capabilities. A more serious example was published a couple of years ago, more in a therapeutic approach. And so the idea here is to use a virus instead of a bacterial cell and have that virus have a circuit within its genome that senses whether it's inside a normal regular tissue or inside a cancerous tissue. And if it's in a cancerous tissue, turn on genes that induce apoptosis or cell death. So, ie, kill the cancer cell but leave the normal cell alone. And the mechanism here in the middle on the bottom is a complicated regulatory circuit that had to integrate at least eight different signals, and it's not something that's easy to go through. But in the end, it got three to one selective killing. So it killed three cancer cells for every normal cell that it killed. So this is a great start in trying to improve the specificity of these types of therapeutics. So there are lots of people in the synthetic biology community very interested in these sort of medical applications of treating disease, whether it be infectious disease, cancer, or other maladies. 

The field that I come from, though, is chemical engineering, and traditionally that's in the space of making the things that you use every day. And so one of the projects that I was fortunate to be a part of, and this is a fantastic story if you ever get to see Jay Keasling give this story, it comes from the world of malaria and how to fight that disease. And so the real issue with malaria is it's an economic problem. There are lots of different things that you can do to fight the disease once you catch it. If you went to the pharmacy you'd be able to purchase lots of drugs that are not that expensive. But in the parts of the world where this disease is most prevalent, that's just not the situation, right? People don't have the money to spend on antibiotics. And so the vision that we had in the Keasling lab was to take an anti-malarial compound that could be produced in a plant and purified and then used in the clinic, but that's fairly expensive due to the things that are associated with the agriculture, the harvesting, the purification. But we realized that those molecules that we wanted to make are just small molecules that are made by enzymes expressed inside the plant. So those enzymes are encoded in the genome of the plant. And just like the recombinant DNA case, we can take those genes out of the plant and put it in a different host. 

And so in this case we put it into a microorganism, a yeast commonly used to bake bread or brew beer, but in this case, when we took the genes out of the plant, put it into the microorganism, put them into a fermenter, those microorganisms produced this anti-malarial compound. So it can be purified and at the scales and things that you can do in a fermentation facility, you're cutting cost by roughly an order of magnitude. So this was work that was funded by the Bill and Melinda Gates Foundation which was a wonderful venture that started a company called Amherst Biotechnologies that is probably more famous today for its involvement in biofuels. But it took the technology that we had developed in the Keasling lab, made it commercially viable, and then used that technology, licensed it to Sanofi-Aventis, who worked with the Institute for OneWorld Health to commercialize and put this out into the market. So down here in the lower left is some of the first barrels of this Artemisinin that was produced in this way. So that was happening just earlier this year. And so this project is a banner case for how synthetic biology can be used to improve the production of molecules. So the rest of the stories I'll talk about later in the talk are going to be similar to this theme and hopefully will achieve this level of success. The thing I want to point out here is that this was no small venture. This was something that was roughly 10 years in the making. 

So it started in roughly 2001 and has just now been commercialized officially. And so it probably took somewhere in the order of 500 PhD years to get this project accomplished in addition to it being a 10-year project. So now what we're focusing on is cutting that development time significantly. So what are the technologies that are going to make that happen? But before I get there, let me give one other example, and this is the other benchmark case of commercial success. So Artemisinin is a very complicated molecule, very easy to see how that would be a profitable business, this is another molecule called 1,3-propanediol. It happens to go into nylon-like products that DuPont commercialized a number of years ago. And again, this is technology actually that started here on the UW Campus by Doug Cameron in chemical engineering in the early '90s. And so this is a technology where the plant sugars that are found in corn can be fermented instead of to ethanol to this molecule 1,3-propanediol. And so when DuPont uses that fermentation process, it's replacing petroleum so it no longer is getting 1,3-propanediol from petroleum, but it's getting it, rather, from a renewable source, a plant. It also has superior properties, technically, to a lot of the nylons that it's now being used to replace. 

And so it's using less oil, basically less energy, it has superior properties, and also, because it's a green process, it's going to have lower greenhouse gas emissions. And so you hit three of the major points to making a process like this successful. And so this is, again, something we're trying to emulate across the board. And so why is this technology possible? In the last two decades we've seen an explosion in genome sequencing capabilities. So this building houses a lot of instruments that are capable of taking a microorganism out of my lab that I might have isolated from an exotic location or the bottom of my shoe, and sequencing its genome in an afternoon for less than a thousand dollars. So this technology is amazing. So what comes out of that are amazing amounts of data that we have now at our disposal. And so what is that data? It's the building blocks of life. It's the genes. It's the sequences that encode proteins that do all sorts of amazing biochemistry, regulatory functions, etc. 

So since it's so cheap we don't have to worry about collecting this information anymore. We now have to start worrying about how to organize it, quality control it, and make sure that it's in some sort of scannable or searchable function. So you see a large number of databases out there that are based off of either protein structure, protein function, DNA sequence, organismal, genome-based, even metabolic models and things of that nature. And so this information now is the parts list. It's the cast of characters that a synthetic biologist has to work with in the laboratory. And so when we used to take that piece of information, say we wanted a particular gene in our organism, we'd have to then go physically get that piece of DNA, the actual chemical entity. And so we used to have to call up a friend who might have that in another lab. We used to have to clone the gene. Sometimes you'd have to go get the source material. This could be really labor intensive and oftentimes failed. 

But the other technology that's emerged in the last five years or so is the ability to synthesize DNA, not just read the DNA but to write DNA at similar prices. And so we're seeing things now below $.25 a base to synthesize a gene. And so I don't clone genes in the lab anymore. My students don't clone genes anymore in the lab. They go to commercial sources, purchase the gene for the first time, and then we work with it right away. So this is amazing, and in addition to being able to synthesize small things, genes, we're now able to start synthesizing things on genome scale. So the work of Craig Venter, a few years back, showed that we can take genomic DNA from one organism, purify it, put it into a protoplast, that's basically a cell without a genome, and when that genome is transplanted into the protoplast, the cell recovers and takes on the phenotype of the genome, not of the protoplast. And so this is a genome transplantation idea. So the concept here, where synthetic biology is headed in the next five to 10 years, is being able to synthesize our own genomes, from scratch, put them into the protoplast, and be able to make some sort of super bug. So bugs that would be really good at making 1,3-propanediol, Artemisinin, solving the energy crisis, sequestering carbon dioxide, all of the challenges that we're going to talk about here in the next few minutes. 

But with that promise also comes a great peril. This is a technology that could also be used for great harm, and so there's been a lot of discussion at the national level and in the scientific community about the ethics of synthetic biology. And so to give you another reason why I don't think synthetic biology is a new field, the President's bioethics commission released a report back a couple years ago saying that synthetic biology didn't need to be regulated in any way differently than that of recombinant DNA technology. So those regulations and those procedures were established after a wonderful conference called the Asilomar Conference back in the late 1970s where the recombinant DNA community, the scientists doing the work, came together and regulated themselves, and because that happened, we didn't have a slow down in the development in this type of technology. So where this is going in the future is going to be using these types of abilities to make organisms, make synthetic circuits, enhance the production capabilities of different organisms to solve a large number of problems. So the former dean here, Dean Peercy, used to like to use the grand challenges of engineering to motivate students. If you look at this slide, there's a lot of things that are really important, and I'm sure there's something that resonates with you. But for me, when I was looking at this, there was something that was missing and, conveniently, a really nice blank spot right there in the bottom of the slide. So I put in this one, maybe it's another grand challenge for the next generation, and that's to establish a sustainable chemical economy. And that's really important because we have cheap materials, cheap plastics, that enable a lot of the lifestyle, the things that we do on a daily basis. 

Without that, things change dramatically. So we need to become more sustainable. So as a chemical engineer, we looked to this scheme as one of our crowning achievements of the 20th century. So chemical engineering is roughly a hundred years old. It emerged because there were these source materials called petroleum or oil that was coming out of the ground, and some of the molecules in there had uses. But there was a large investment to get this stuff out of the ground, so we needed to do something with all of the rest of the molecules that happen to be in a barrel of oil. So what did we do? We separated the various fractions based on physical properties for the things that didn't have a good end market. We reacted them and converted them into other molecules that did and used that process to essentially subsidize the production of cheap fuels. And so what you see is the production of a very small volume of five to seven core commodity chemicals that then are the basis of the entire organic synthesis and chemical industry. Because those small volume are so much more valuable than fuels, it can subsidize the production of those fuels and therefore makes energy for transportation fuels very, very inexpensive. 

So this is a great achievement, and what the next generation of chemical engineers is going have to try to do is figure out how we're going to do similar processing but from a different feedstock. And so the reasons for this could be many, and I'm sure that several people would love to argue about this and we can talk about it afterward, but I think there are four main motivations for thinking about sustainability. Whether it's the supply of oil and fossil fuels, whether that's peaked or not is an open debate, but the fact that it's a finite resource is a fact, and it's going to run out at some point. So we need to worry about that. Security is a big issue. We import more oil that we produce, and that means that we're going to have to worry about things like what happens when our oil supply runs out. Costs, we've all seen the impact that oil has on our every day economy. So when there are spikes or when a hurricane is barreling down on a refinery, you see all sorts of things change in the marketplace. And then the gorilla in the room is obviously the amount of carbon dioxide we're taking from inside the planet and putting up into the atmosphere and what that's doing to the planet and to the climate around us. And so, for those reasons, we're very interested in trying to find alternatives. 

So the idea of sustainability is to have a closed cycle. The closed cycle we envision for chemicals is using carbon dioxide as your starting material. That's a very oxidized molecule. Most of the things we're going to want to work with are reduced. So to reduce it and make these chemicals, it takes energy. So where does that energy come from? It comes from the sun. And so the sun, through Mother Nature's wonderful evolution of photosynthesis, we can harness CO2 and reduce it into organic chemicals. Those organic chemicals are stored right now in the form of sugar polymers or oils in the form of algae. And then we can use chemical processes or biological processes to upgrade those intermediates to various products that we might want to use, refine them further, use them, burn them, release the CO2 back up into the atmosphere, and use the energy from that to either drive, transportation, power the lights, etc. And so I think this is a fabulous vision, and this is where we're trying to work, but I need to give you some context as to the order of magnitude, the scale of this problem. 

So it comes back to one of the most important energy concepts that we teach in our thermodynamics classes. Anybody remember what the first law of thermodynamics is? >> [INAUDIBLE] >> That's not the first law, but that is one of the laws of thermodynamics. Anybody remember what the first law is? Maybe I'll give you a hint here with Homer. Does anybody know what this is or remember this episode? >> [INAUDIBLE] >> Correct. Right. There's no free lunch in the energy world. You can't create energy out of nothing. Energy can only be transferred between forms. So that's a major point to remember. We have a finite source of energy that is the sun. 

It's a pretty large source of energy, so we're going to have to use that in order to get the things that we need. So the second major concept I want to point out here is the scale of this problem. So this is some data that's actually getting a little bit old now from a report from the United Nations in 2007. And so the little numbers, if you can actually make these out, in the brackets, are the number of exajoules per year that are consumed worldwide. And these are the rough categories on where they end up going. So the red numbers represent the percentage. So the thing I always think about in terms of exajoules, I'm not an astrophysicist so these exa prefixes doesn't make a lot of sense to me, so I always want to put these things in context. So what would you do with one exajoule, remembering that we used 500 exajoules in 2007. So you can take the lake out there and boil it three and a half times. That's amazing. 

Several nuclear bomb explosions. 8,000 space shuttle launches. My personal favorite is 2.5 million semi trucks of Big Macs in terms of the caloric content. [LAUGHTER] So this is a huge problem. And so there is nobody who's going to solve it. There's not going to be a single silver bullet unless some sort of science fiction-like solution comes into play. So everybody's going to have to do their part. We're going to have to find ways to be more efficient. We're going to have to find new sources. We're going to have to make sure that we don't waste anything. 

So that's another great way to think about this. Energy forms are not all created equal. So there's lots of things, there are lots of places where we can get energy from the market. We can get them from renewables, which you'll note are less than 1% back then. It's probably doubled since 2007, maybe tripled. You can use hydroelectric power in certain parts of the world. So if you're up near Niagara Falls, that's a great place to get your electricity. It's why electricity is pretty cheap up there. We have coal. We have biomass, but that biomass usage right there is not for ethanol production. 

That's wood-burning stoves in parts of the world where that's the dominate form of heat. We have petroleum, we have natural gas supplies, and so what you see with everything else in between is how it goes to either buildings, so that's the heat and electricity that we use, industry, which means either the heat and power that we use there plus the raw materials, and the transportation sector. But the biggest slice of this pie is the losses. And so these are things that are thermodynamic losses where we have no choice. If we're running an internal combustion engine, you're going to lose some energy. You're not going to recover all of it in terms of locomotion. But there are other areas, like the electrical grid, where we're losing a lot of the energy, so there's going to need to be a lot of investment into how to optimize and be as efficient as possible in terms of using energy. But from my perspective, the thing that stuck out to me was the fact that down here in the lower right corner, the transportation sector has no other bars except petroleum. So we don't really use enough liquified natural gas or ethanol or any sort of other electric vehicles to even put a line on this chart. And so that tells you how far we have to go. 

There's a big challenge. If petroleum were to run out or to become scarce or a security concern, how we get to grandma's house every year is going to become a challenge. And so one of the things then that we're interested in is trying to find ways to use biomass in the sustainability cycle to replace petroleum or jet fuels or diesel fuels and things of that nature. So first thing you want to do as a biotechnologist is say, what are the molecules I'm interested in making? And so most fuels that you would use are mixtures. So jet fuel here, I show you on the left, is a set of molecules that are both linear, cyclic, aromatic, branched, so they are typically medium chained in size. And so when you try to look for metabolites, things that are made in organisms that look like those molecules, you'll note that there aren't that many. Things over here on the right are loaded with oxygen, they're loaded with other types of atoms, and so what we really need to do is make some of these molecules and then get rid of the oxygen. So to give you that example, we'll take glucose and hexane, to just show you some physical properties. These are both six carbon molecules, for those of you who don't remember their chemistry. Glucose, of course, is a solid. 

Very water soluble. You're very familiar with that. It doesn't boil. It has a fairly high melting point, and it has a reasonable heat of combustion. If you lose the oxygens off of that molecule, it's now a liquid instead of being a solid. It's density is drastically different. It's melting point has dropped tremendously. It now can be vaporized. It has a very, very low solubility in water. And, voila, the energy has gone way up in terms of the heat of  combustion potential. 

And so this is what we have to do. We have to find molecules in biology that we can then deoxygenate to produce the things that are going to be good fuels. And so what else makes a good fuel? One of the things that people talk about is something called a neat fuel. That's a unimolecular or maybe a small mixture of two or three chemicals. Most of those are not going to work because fuels need a mixture of properties. So here, for jet A, the jet A standards you can see on the left. If you wanted to use ethanol in jet A, the biggest problem it has is it's not nearly dense enough in terms of energy to fly a plane. So you would need to have the same energy content. You would need to have a lot more weight in fuel, so that's not going to be physically possible. So you can move to things like dodecane, which is just a 12-carbon alkane which is one of the molecules that we saw in jet A. 

It has the right energy content, but then you start looking at its density is too low, and the real issue in jets then is that it's freezing point is roughly minus 10, where if you're up in the upper atmosphere, that's going to be a problem too. So as a neat fuel, that's going to be an issue. But there are molecules sometimes, like this one called --, which is something you would find in orange paint stripper. It comes from the citrus peels of different citrus fruits. It seems to have a really nice mix of all of these properties. And so this is a target that a lot of groups are now going after, trying to produce organisms to make that molecule as a source of jet fuel. So how do we do this? Why does an organism make anything? Why are we going to get this to happen? Well, organisms, like you and me, eat something. So most organisms like to eat sugar, so that's why sugar is the thing that everybody wants to use as a feedstock. And so from that sugar, the organism has life. It's allowed to survive. 

It gets motion if it needs to move. And it is allowed to then reproduce. It has the energy and the resources to make another microorganism. It also then makes waste products, just like you and me. Whether they're CO2, ethanol, lactic acid, or other organic molecules. So the first law of microbiology, to counter that of thermodynamics, is the purpose of a microorganism is to make more microorganisms. If you give an organism sugar, the thing it's going to want to do with that sugar is make another microorganism. So how do we get it to make ethanol, or how do we get it to make any of these other fuels? Well, there's no single chemical reaction that says glucose goes to new cell. That's just not physically possible. So what organisms have done is decided to structure or optimize a structure of metabolism that looks like two separate stages. 

The first, on the left, takes the carbon and other substrates that organism needs to grow and it breaks it down. We call that catabolism. And so those molecules are converted into smaller, sometimes oxidized, sometimes reduced molecules, reducing power in the form of electron carriers and energy in the form of ATP. And so those molecules are used then in the synthesis of new cells. And so the intermediates are converted into proteins, DNA, membranes, etc. And in the process, this reducing power, if oxygen is present, is used to make lots of energy through respiration. If you're familiar with that. And so the trick that comes in for fermentation is if oxygen is not present, it can't get the energy it needs through respiration and it has an excess of reducing power. So what it does is it says, okay, I'm going to dump these electrons on to some of these other intermediates. So in the case of yeast, it dumps it onto acetyl-CoA or pyruvate and becomes ethanol. 

And so, for that reason, to get rid of the extra electrons and balance its stoichiometry, it produces this molecule that you want. And so that's great if you can make fermentation products so things like butanol, isobutanol, a lot of the medium chain alcohols, lactic acid, acetic acid, these are all things that we've seen biotechnology succeed in producing for the last 20-30 years. The challenge, though, is when you want to start making some of these things, like the hydrocarbons for jet fuels or for biodiesel, is that they're not fermentation products. And so that means you have to have the organism engineered very precisely to also make an organism but on the side make some of these products that we want as well. So this is what I want to talk a little bit about now. The overall picture, though, is that we're going to take things that are human wastes, so maybe we can get resources like municipal landfill, maybe it's compost, maybe it's yard waste, maybe it's not human, and can use that as bacterial food to turn bacterial wastes into a human resource. And so while I drew that scheme of catabolism and anabolism very simply, what it really looks like is this. And it's an amazing mesh of reactions. But you can kind of see the structure. Down the center is the central metabolism, the central metabolic pathways of glycolysis, the TCA cycle. 

And so, essentially, what happens is any feedstock or any resource is fed into those pathways, and then there are nodes from those pathways that branch out to the synthesis of the other molecules. So it's an amazingly complex system that we can try to optimize, and there are lots of techniques being developed here on this campus and abroad to try to understand this. It's a whole field called systems biology. And so what we do as metabolic engineers or as synthetic biologists is try to engineer that metabolism to direct the flow of carbon to where we want it to go. And so one simple example is if you're in an organism like E. coli or sometimes in other species that make acetate instead of ethanol, this is something that can make vinegar out of your wine instead of a tasty alcohol content in the wine, what we do is if we don't want the acetate, we can block that particular reaction. How do we do that? Well, any reaction that you're working with is performed typically in a cell by an enzyme. It's catalyzed by an enzyme. That enzyme is typically a protein. That protein may have had to been modified in some way, dimerized, but in reality it's just a protein. 

That protein is encoded by a messenger RNA, and that messenger RNA is in the genome in the form of a gene, so gene A. So if we want to block a particular step in a pathway, all we need to do is either block the conversion of protein to active enzyme, the translation of messenger RNA into protein, or the transcription of the gene into messenger RNA, or just remove the gene entirely from the genome. So we have technologies to do all of these things. The harder part is when you start thinking about trying to add functionality. So say we want to make butanol. Butanol is another fuel that several companies are very interested in. It has properties like ethanol and some superior properties for other reasons. And so the pathway to make ethanol, which is down here, acetate, which is down here, all come from pyruvate. Pyruvate is one of the central molecules in metabolism. So, if we wanted to try to look at this pathway, the enzymes that are encoding the various reactions or catalyzing the various reactions can be encoded in the genome in various places. 

If you look at an organism like E. coli, E. coli has the genes to convert pyruvate to acetyl-acetyl-CoA, but it doesn't have any of these black genes. So it can't convert acetyl-acetyl-CoA to butanol. So what we can do, though, is take those genes and add them to E. coli using a recombinant DNA technology or a synthetic biology technology, and, at the same time, delete the genes that would lead the flow of carbon to these other products. So, in this sense, we can direct the flow of carbon to the molecules that we're interested in. And so who do we do this? If we want to look at the project, it's much more complex. So the Artemisinin project, for instance, isn't just adding one gene to an organism. It ended up being something about 10 genes that needed to be added and others that had to be optimized in terms of the amount of protein they were making. So it wasn't a simple task. The structure of a synthetic biology project is basically outlined on the left-hand side of this. You have to understand what players you need, what's the cast of characters, how do you find those, and how do you get them to work in the organism that you want to work in. 

So until we have these designer organisms that are probably still a decade or so away, we need to have the ability to put different types of genes into organisms that work really well. So we have model organisms like E. coli, like Saccharomyces that work really well, but it's not straightforward to just take a piece of DNA and make a functional protein in another organism. So there's a lot of optimization that goes into that. We then use those pieces of DNA, we combine them with other pieces, and we design different circuits. I'm not going to spend a lot of time going into that, but usually we have some knowledge, and at the end we've run into a problem where we don't know what we actually want to design and so we design many. And we test many samples and then we look for the best one. And if the best one happens to meet our objectives, we've solved the problem, we move onto the next challenge. But most of the time, that first construct stinks. So you're going to have to go back to the drawing board, evaluate what's wrong with it, redesign it, rebuild it, and it's a traditional engineering cycle. And so what I want to do now is talk a little bit about some of the work that my group's done in this field to show you and highlight some of those different features. 

So one of the things that my group has been really interested in, in terms of the chemicals that we've been looking at, are things that look like biodiesels or look like jet fuels. And so the closest molecule in biology to that is the fatty acid. Fatty acids make up your membranes, and so the membranes of cells can be also used as substrates to produce a wide range of what we call oleo chemicals. So these are things that are typically made from plant oils. And so you can make things like methyl ketones, biodiesel which is methyl or ethyl esters. You make straight chain alkanes. You can make fatty alcohols. You can make bioplastics that are very, very interesting. Our group's done a lot of work in that area. As well as, then, these molecules, alpha olefins, which probably have some of the highest value because they're used in producing plastics. 

And so when we started this project, the genes for making those types of molecules were unknown. So we knew that these molecules existed in biology. So, for instance, this organism here that is now a workhorse in our lab is a cyanobacteria. It was isolated from fish pens in Puerto Rico. It's an amazing organism for a number of reasons that I am happy to talk about afterward, but the real thing we were interested in was it produced these two molecules up here, two alpha olefins. And so this has been known since the 1960s that this organism makes it, but when we started this project in 2008, the genes responsible for that biochemistry were still unknown. So how do you figure out an unknown gene from a known biochemistry? Well, one of the advantages of this genome era or genomic sciences era is you have infinite, almost infinite, DNA sequence. So, Mother Nature, if it's done it once, is going to find other places to use similar implementations of biology. And so one of the things I studied as a postdoc was this molecule called curacin A. And so this is a natural product that David Sherman is very interested in for a number of reasons. 

And this molecule has several fascinating functional groups, but the one that I focused in on is this terminal double bond, or the alpha olefin character. And so what David's group had discovered was that the biosynthesis of this molecule was done by this enzyme cure M. So cure M was a very large protein with multiple domains that did several types of chemistry all in an assembly line fashion, so the KS did a reaction that handed the product off to the AT that handed it off to the KR, so on and so forth. And so it was these two blue domains, the STTE, that were responsible for doing this olefin chemistry. And so when we looked into the 7,002, our cyanobacterial genome, we could use that gene sequence or that protein sequence as a probe. And we say, is there a sequence in this organism's genome that looks like that protein? And we found one. So it was probably the obvious. It was the biggest gene in the organism, and it had nearly 50% identity to the probe sequence that we were using. So when we looked at it, what was interesting is it also had these two additional domains out here in the front. And so what these two do is take a fatty acid and then load them into this module, and so instead of having something that's upstream of the curacin cluster making that complex molecule, you just have a long chain fatty acid that goes through the same biochemistry and can produce these molecules. 

And so what we're now doing in the lab is trying to engineer E. coli in the cyanobacteria to produce lots of these. We're trying to alter the substrate specificity of this particular domain so that it can start with different molecules and then lead to the production of different alpha olefins. So that's not quite far enough along to talk about. And so what I want to do is then transition to another molecule we're really interested in, we just finished this study, and this is on fatty alcohols. Fatty alcohols are produced from intermediates and fatty acid metabolism. So I'm giving you a simplified version of that really complex diagram that focuses just on the anabolic pathways for fatty acids. So the trick to making fatty acids is three steps. The first is you need to block their consumption. So organisms eat these molecules all the time so you need to remove the genes that's going to eat your product. 

That's a simple step. The second is looking at what's the major regulatory player in the cell. So if you take this enzyme called an acyl ACP thioesterase, it provides the link from the central metabolism of fatty acid biosynthesis to this molecule of free fatty acid. So this is an interesting product. It can then be used to make lots of these other oleo chemicals. What's interesting, though, is it also depletes the key regulatory signal. So if you have this enzyme present, it decreases the abundance of this molecule, its substrate, that substrate happens to be the regulatory signal for all of the genes that precede it. And so if the enzymes in the cell sense that there's not enough fatty acids to make their membranes because there's not enough of this molecule around, then all of the sudden it says, hey, we need to up the activity of all of the genes preceding it and make more fatty acids. So you end up getting a large amplification of fatty acid production just by adding this one particular protein. So when you take that and you couple it by blocking the consumption of our products, adding in additional enzymes that we've optimized different ways, you construct this pathway to produce fatty alcohols from any type of carbon source because acetyl-CoA is a central metabolic entity. 

Just about any carbon source that an organism can grow on will be converted to acetyl-CoA at some point. So we can basically connect any carbon source, biomass, or glucose or what you like to making these fatty alcohols. So when we do that in a bioreactor, we can produce a pretty large amount. So these are fed batch studies. So we're continuously producing our product over about a hundred-hour window here. We're consuming glucose at a very similar rate. We are producing some acetate. So that's a byproduct that we now need to go in and get rid of the genes. So again, that concept of removing competing pathways, we need to block that. And one of the things that we discovered is really important is providing a sink for your product. 

So ethanol, if it builds up to too high a concentration in a fermentation, will kill your cells. That's why you don't see many wines that are above a certain percent alcohol. The same thing is true for lots of these other products. They could be very toxic to the organism that we're trying to engineer. And so what we did in this particular case was added a 10% volume of an alkane. That alkane is nontoxic to the organism, and it provides a sink to deposit these fatty alcohols. And so, in addition, we turn off the fermentation device, all the alkane floats to the surface, it has all of our product, we skim that off the top, and it's a much easier purification. And so it's a wonderful thing that, in addition to those advantages, upped our productivity roughly 50%. So these are all wonderful things that we've been able to do, but I want to point out a couple of other, the downside of this. So our yields are roughly .12 grams per gram glucose. 

That probably doesn't mean a lot to you. The theoretical conversion for glucose to one of these molecules is roughly .3. So we're just below 50% of the theoretical yield. So how good is that? So I want to use, you probably don't understand what the price of fatty alcohol is, I don't actually know what today's price is, but it's roughly three times the price of biodiesel. So this slide is going to look at, biodiesel is something you probably have seen at the pump. So this graph on the left represents the contribution of the feedstock, so the sugars going into this process, at the theoretical yield. So remember, we're below 50% of the theoretical yield. The green line happens to represent ethanol. So if you're at 20 cents a pound sugar, which is probably a little bit high on the end of today's market, you're just below $3 a gallon ethanol. So that's probably why you see $3 or so a gallon of gasoline. 

The ethanol that we have to put into gasoline today is not increasing the price. But if you go up to different biodiesels, so things that we can make from fatty acids in our organisms, you'll see that  they're $4 to $5 a gallon at theoretical yield. So remember, if we're only at 50%, you need to double these numbers, so we're really at somewhere between $8 and $10 a gallon of these biodiesels. And so that tells you we have two remaining challenges. One, we have to increase the yields of our strain to approach theoretical. So how do we do that? This is where we're using systems biology, tools, and techniques to try to dissect our organism. As I said, the first time you build an organism, you may not meet that industrial objective. And that's where we are. So what do you do? How do you dissect that complex metabolism? You have to use models. You have to use systems biology where you're looking at things from the global context, not taking things out in isolation. 

So one of the things you can do is look at where is the rest of your carbon going. So if we take a look in our bioreactor systems across a range of different experimental conditions, the majority of our carbon is going off as CO2, and that's because these are aerobic fermentations. So there is respiration taking place. That's because we need extra energy for this pathway to work. If that's the case, we're producing too much carbon dioxide, how is that? In terms of an overall sense, we can use a metabolic model that my colleague Jennifer Reed curates and develops in chemical engineering, and so these triangles represent the theoretical space that we could be performing. All of the data points represent where we are in our different experimental conditions. So without going through all the details, you can see in certain cases the blue represents the fatty acids, we're doing pretty well. We're pretty close to the theoretical yield, just happens to be at a very low yield point. In other situations where our highest productivity samples come from, we're right around the middle of this particular range. So the vertical axis, again, is kind of the goodness of the performance. 

And so where's the CO2? That's the red dots. You'll see it's also right in the 50% range. So we need to drop these and increase these. So we're looking for the sources of this carbon dioxide, and can we decrease the respiration rate or use energy more efficiently to direct more of that carbon into our products. The second thing that we have to worry is driving sugar prices down. We can't be working with 20 cents a pound sugar and making biodiesel at today's prices. It's just not going to happen. And so the big challenge there is looking for alternative sources of sugars, and so we don't have sugarcane in the same abundance that Brazil has. We can use corn sugar but that's a debate for another night over ethanol. There are lots of issues there. 

What we have to do is look for sources that are not going to compete with food, not going to cause other economic problems. And so that's why biomass, or this sort of cell wall material of plants, has become such an attractive source. There is one downside to that. Where sugarcane can be processed almost instantaneously from sugar juice to ethanol and corn sugar can just be treated with a simple cocktail of enzymes to get you a nice fermentation broth, biomass is recalcitrant. So to get at the sugar polymers, you have to remove this sheath that protects it, and that's the lignin or the lignocellulosic components of this. And so people have developed different technologies for approaching this problem. So the first thing is to break the sheath and allow access for enzymes to break them down. You can also just use chemical treatments that destroy the lignin sheath and break down the sugar polymers into sugars. A lot of those in the ionic liquids class are great, they technically look fantastic, but the cost of the ionic liquid or the solvent is cost prohibitive. And so what we've been doing with Jim Dumesic here on campus is studying this fantastic solvent that he's developed. 

And so this is called gamma valerolactone. It's a very interesting molecule for a lot of different reasons, but it can be made from cellulose. And the yields here, they've done better since their publication. So what we can do with this GVL is stuff a tube full of biomass. So it could be your grasses. It could be wood chips. It could be hardwood, softwood. It could be corn stover. It doesn't matter what we've thrown at this so far. You stuff a tube with it, and you start flowing this GVL, which is just an organic solvent, through the biomass tube. 

And so as that happens, you get release of different fractions of the biomass. And so initially you dissolve the lignin, it goes away. When the lignin is gone, if you have a little bit of acid around in this reaction, it breaks the sugar polymers. So you get the hemicellulose fraction coming off next and then the cellulose fraction. So this is fantastic. We can do this type of treatment with a biomass-derived solvent, so that means it's still potentially sustainable, we can do this without needing any sort of enzymes, and it dissolves everything. So it's not leaving some sort of solid behind that we have to deal with. And solid processing is a major issue in a lot of the other competing technologies. So what's fantastic also is we're able to recover close to 90% of the sugar polymers in some sort of fermentable, usable form. And so we've been using this technology either through catalytic upgrading, taking these molecules and making fuels out of them just with traditional catalysts, or finding ways to convert those sugars out of the solvent and put them into fermentation broth, and then using those sugars to produce ethanol or other things that I've already talked about. 

So that sounds fantastic. So I need to present a little bit of a balance to that optimism. So everybody's hoping that I was going to come into the room and tell you I've got the silver bullet for sustainability. That we're going to cure all the problems. Biomass can't do that. And so what this study on the graphic here shows is a study from the Department of Energy and the Department of Agriculture, and they asked how much biomass is out there. If we wanted to do this, the technologies that I've been talking about, how much of an impact can we have? So they looked at things that come from forests or paper pulp mills, things that are left over on farms and crops that we would grow specifically for this purpose, and whether you're conservative or optimistic in your numbers, it's somewhere between one and 1.5 billion tons. So that's a lot of biomass. But how much energy is that? So you can take a rough estimate for what's in a ton of biomass, you can take a rough estimate of what's in a barrel of oil, and use those numbers and you get roughly one to three billion barrels of oil. That's a lot. 

That's a big impact. But then look at what we used in 2010. We used over five billion. So we're talking about 30% of our transportation fuel, not heating and electricity, just transportation fuels. So it's going to be a big challenge in the future, but what we want to present is that this is something that could be a huge impact. It's not like biomass can only contribute 5% or 1% to the problem. 30% is a big deal, and that's actually bigger than the percentage of petroleum that goes into petrochemicals. So the argument can be made that we should be using biomass or these resources to make chemicals and the things that we need every day, maybe the things like jet fuel where there's really no alternative and finding alternative sources for the personal transportation and heavy transport, either through trains, public transit, etc. And so this is a little bit of a downer. The other thing we can do is start looking for other sources of biomass. 

This brings up the other side of the coin, which is algae. Algae has been used in the past to say, hey, let's make oils out of it, but algae also have cell walls. A lot of eukaryotic algae are basically just plants that grow in the water. So you've probably encountered seaweed. People have looked at seaweed as resource. It could be converted just as easily as biomass that's grown on a field. The issue with seaweed is how do you cultivate and harvest it in a way that doesn't spend more energy than you're going to get out of that seaweed? And so we've looked at this locally. This is me with Linda Graham, a friend in botany. She's an expert in this particular species called cladophora. So usually right around the end of July if you're down at the terrace, you've seen the sloughing of all of this stuff coming off the rocks and ending up on the steps. 

It usually stinks pretty bad after a couple of days too. It's actually a wonderful species for a lot of reasons. It's a really exciting microbial community that lives on this species, and Linda has a lot of fun research talking about this stuff. But what's really exciting about it is it's an aquatic plant, and so since it doesn't have to fight gravity, it's buoyant and in the water, it doesn't need all the rigidity in the cell wall. So that lignin stuff I was talking about, about the recalcitrance of biomass, throw that out the window. And so you throw in a little bit of acid or base after you bleach a sample of this and you get a piece of paper. So you get this little papyrus-looking mixture. So this is a wonderful substrate for producing biofuels. So we're working with Linda in trying to find ways to cultivate this in things that aren't Lake Mendota. Because, again, obviously the harvesting from Lake Mendota would be difficult. 

So that brings up the last or second to last major point I want to make, and that is what we've been doing is using photosynthesis to make large volume, low value intermediates. So that's either cellulose in the form of plants, terrestrial plants. Typically algae are thought of more for the lipids or oils. And then we're talking about big scale refineries to convert those raw materials into the things that we're interested in. Why do we have to do that? Photosynthetic organisms are just like the bacteria I was talking about before. They have the same types of metabolism, just different extra enzymes to do photosynthesis. They can be engineered as well. So could we construct small scale facilities to, instead of using photosynthesis to make these low value intermediates, make small volume, high value products directly? And so we're doing a lot of work now with funding from the National Science Foundation and the Department of Energy to investigate this idea of photosynthetic biorefining. And so what are the products that we need to make? How do we do this? How do we engineer these phototrophs? So the interesting process question here is you go from a situation where glucose or your raw materials are you most expensive component to now a system where those things are free, because you're dealing with carbon dioxide which we have lots of, wastewater which can provide you with your nitrogen and phosphorus, and light, which, if you're using the sun, is also free. So you have very little operating costs, but the reactors that you need to build in order to make this happen are incredibly expensive. 

So how do we develop these things and motivate investors to pursue a very risky technology, particularly in a marketplace where this type of biotech is kind of waned in terms of excitement from the venture capital funds. And so what we're interested in is, to make the economics work, linking with environmental engineering objectives. And so can you kill two birds with one stone? Can you clean up wastewater, reduce the manure runoff, and produce some sort of organic chemical? So we're interested in lots of things in this particular space. I want to close with kind of the larger landscape. So biomass isn't the only game in town. Biotechnology is not the only route for using biomass. We're going to have to compete with technologies like pyrolysis and gasification, and they're going to have their role to play. In may respects, those chemicals technologies may be superior in terms of making fuels because they'll make mixtures. They're not going to be the same mixture that you would get in you gasoline tank, but they would be mixtures that would probably have the right balance of properties. Versus biotechnology, which is very good at making very precise molecules. 

So we'll be able to make the chemicals that we're interested in. We could always just burn the biomass, put it into a power plant and get electricity out of it. There's lots of arguments that say that's more efficient and a more efficient use of the biomass than making ethanol. We're going to discover new petroleum discoveries, and that's going to slow this whole process down. We're entering a natural gas economy, whether we like it or not, and it's going to be a source of a large raw material that biotechnology can also play a role in. There are organisms that eat methane, so we can engineer those to convert methane to other produces. There are lots of research going on now at national labs and campuses to pursue that. And then all sorts of other things that are also going to come into play. So to close, I want to acknowledge the group. I haven't done research in six years. 

It's kind of sad, actually. But my students are absolutely fantastic. The infrastructure that we have on this campus for doing this type of research is one of a kind. To have a facility that's got a fantastic engineering school, a world-class biochemistry, the agricultural school to talk about biomass and cultivation issues, the botanists that have helped us with algae and cyanobacteria, and then the microbiologists here who have been incredibly welcoming to work with them within the Great Lakes Bioenergy Research Center as well as in one on one projects. So I think I have time for a few questions. I'm happy to stick around if we want to kind of do a public session, and then I'm happy to stick around for anybody who wants to ask more in depth questions. [APPLAUSE]