The concept of genetically engineering E. coli to make biofuels directly is not new. In 1987, for example, a group of researchers from the University of Florida and Southern Illinois University managed to get E. coli to produce ethanol. They did this by taking some genes from the bacterium Zymomonas mobilis, which was known to produce ethanol as one of its principal fermentation products, and inserting those genes into E. coli. Using simple sugars as their feedstocks, the researchers found that the re-programmed E. coli turned out ethanol quickly in appreciable amounts. Although sugar, a foodstuff, was consumed in the process, the group pointed out that further engineering of the microbe ought to enable it to produce ethanol using hemicellulose (inedible plant parts) as feedstock materials.
Of course, ethanol is not petroleum. But in 2010, a group of LS9 researchers published in the journal Science that they had found the holy grail enzymes that make alkanes (real diesel, not “biodiesel” esters) from fats. The trick was to select the appropriate genetic structures from other organisms found in nature, which the researchers did by comparing DNA from ten species of cyanobacteria that made “trace amounts” of alkanes with one species that made “undetectable amounts.” To prove that these genes were correct they inserted them into E. coli which enabled the microbes to directly grow small research quantities of diesel oil. The next step was to scale up the process.
By this time, LS9 had a pilot plant going in South San Francisco. The heart of the operation was a 1,000-liter fermenter tank, which was soon producing larger, batch quantities of our Ultraclean Diesel, as we call it. The microbial fermentation took only three days from start to finish, and the end product, a synthetic biodiesel, was so chemically close to conventional diesel oil that it met the American Society for Testing and Materials (ASTM) standards for road use in the United States, and was found to be chemically equivalent to California clean diesel.
In January 2010, LS9 took a major step toward the mass production of diesel oil by purchasing a bankrupt biofuels production plant for pennies on the dollar in Okeechobee, Florida. The facility included four large, million-liter fermenter tanks, storage tanks, cooling tower, and a water treatment system. We aim to produce UltraClean Diesel at the rate of 50,000 to 100,000 gallons per year initially, and then to ramp up to 10 million gallons.
At first the plant will use sugar cane syrup as feedstock, but ultimately we want to use inedible hemicellulose in place of sugar, thereby enabling us to make biofuel without using any food sources. In 2010 Keasling, together with colleagues at Berkeley and LS9, published a piece in the journal Nature laying out the engineered metabolic pathways that would allow E. coli to do this. In recognition of its achievements, the Environmental Protection Agency awarded LS9 the 2010 Presidential Green Chemistry Award.
The current situation in biofuels is one of finding and then optimizing the major players: optimizing the microbes for high-yield, efficient production through genome engineering, matching the microbe with appropriate feedstock molecules or micronutrients, and then fine-tuning the entire production process for generating clean fuels that are drop-in ready for use in the gas tank at costs that are competitive with those of natural petroleum products. But the E. coli at LS9 is not photosynthetic, and anyway it’s not a good idea to put all of your eggs in one basket. Consequently in 2007 David Berry (who was one of LS9’s cofounders), venture capitalist Noubar Afeyan, and I formed another company, Joule Unlimited, to make fuels using what many regard as the most promising microbe family yet, cyanobacteria.
Cyanobacteria were once known as blue-green algae. They are not really algae, however, but a class of bacteria that derive their energy from photosynthesis, just as if they were plants. These photosynthetic bacteria happen to be one of the game-changing organisms in earth’s history, for they are thought to have been responsible for the “great oxygenation event,” a major environmental transformation that happened about 2.4 billion years ago, during the Archean. The predominant life forms at that time consisted of anaerobic organisms, which thrive in the absence of free oxygen. The metabolism of cyanobacteria, however, released free oxygen into the atmosphere, which had the dual result of wiping out most of the oxygen-intolerant organisms while simultaneously making possible the evolution of aerobic organisms (such as ourselves), which depend on free oxygen. So the fact that we exist at all is in large part attributable to the metabolic activities of cyanobacteria.
More than ten thousand varieties of cyanobacteria have been discovered. They are found in frigid Siberia and in fiery deserts; on shower curtains and in toilet tanks; in the world’s oceans, and in niche environments such as hot springs, salt works, and hypersaline bays. Indeed, some biologists regard cyanobacteria as the most successful group of microorganisms on earth.
Joule Unlimited expects these organisms to be equally successful at converting sunlight into diesel fuel. During the first two years of its existence, the company operated in relative obscurity (indeed in stealth mode) out of a nondescript building on Rogers Street in Cambridge, not far from MIT. News accounts in the Boston Globe often referred to Joule’s “secret ingredient,” an unknown, heavily engineered microbe. But the company was only protecting its intellectual property until the time the founders could patent their uniquely designed cyanobacteria. This it did in 2010, with a patent titled “Hyperphotosynthetic Organisms.”
These engineered cyanos, it turns out, have the ability to take sunlight, CO2, and brackish water, and then convert these ingredients into alkanes, the molecular constituents of diesel oil. Like LS9’s E. coli bacteria, Joule’s cyanobacteria secrete their end products into the surrounding watery medium. But these microbes have the additional advantage that they require no feedstock molecules as raw materials: no sugar, hemicellulose, salt or pepper, just some micronutrients that act more or less like fertilizer. Otherwise, it’s as if they run on sunlight alone (in a process that the company refers to as helioculture).
The result, according to company president Bill Sims, “is the world’s first platform for converting sunlight and waste CO2 directly into diesel, requiring no costly intermediates, no use of agricultural land or fresh water, and no downstream processing.”
Centerpiece of the system is Joule’s SolarConverter, which is essentially an inexpensive, flat, transparent solar panel through which circulate thin films of cyanobacteria suspended in a bath of water and micronutrients. CO2 bubbles in at the bottom, and the end product—alkanes—rises to the top. Powered by sunlight, the cyanos release oxygen, sugar, and clean-burning, fossil-free diesel oil.
The process has been demonstrated in the lab, as well as in a Joule pilot plant in Leander, Texas. The company calculates that an array of its Solar-Converter panels can crank out more than 13,000 gallons of diesel fuel per acre per year. Based on an industrial-scale plant, the firm expects to be able to deliver diesel at the cost of $50 per barrel (a barrel contains 42 US gallons = 159 liters). And in line with the current fashion in the biofuels business of equating delivered fuels with land use, the company estimates that it could supply all of the transportation fuel requirements of the United States from a land area the size of the Texas panhandle.
If anything’s clear from all this, it’s that we’re now in a transitional period, caught between the age of fossil fuels and the age of biofuels. Fossil fuels are (probably) a product of dead microbes, organisms that took millions of years to be converted from biological entities to the hydrocarbons of crude oil. Today, by contrast, we’re making basically the same thing happen, only faster, and with live microbes. These living biological microorganisms are creating those same hydrocarbons that the old dead ones did, and they’re doing it directly and in a matter of days, not millions of years, and they’re doing it right before our eyes, not deep down inside the ocean bedrock from which they’ve got to be laboriously pumped out.
Nobody knows yet which microbe, process, or company will be successful and which will fall by the wayside. The competitors are in a classic Darwinian struggle for exist
ence, and it’s not yet obvious who the winners will be . . . or even whether biofuels are truly the wave of the future.
Still, every gasoline price hike boosts the attractiveness of biofuels. Conversely, every drop in the cost of DNA sequencing and synthesis brings us closer to being able to genomically engineer the miracle microbe that will spout cheap diesel, while at the same time scrubbing CO2 from the atmosphere. So maybe this dream is not in fact too good to be true. Granted, you can’t get something for nothing. But gradually, over time, the biofuels development process will become more efficient and optimized, bringing us gasoline at prices if not too cheap to meter, then at least cheap enough not to fret about every time we face the pump.
The only question then remaining will be which land mass to sacrifice: Maryland, France times two, or the Texas panhandle (or maybe all three?). Me, I vote for Texas.
CHAPTER 5
-60 MYR, PALEOCENE
Emergence of Mammalian Immune System.
Solving the Health Care Crisis Through
Genome Engineering
On September 12, 2004, fifteen-year-old Jeanna Giese attended mass at St. Patrick Catholic Church in Fond du Lac, Wisconsin. During the service, a small bat flew down from the vaulted ceiling, struck one of the stained glass windows, and fell to the floor. Jeanna Giese loved animals. At home she had two dogs and three rabbits, and she hoped one day to become a veterinarian. She asked her mother if she could catch the bat, take it outdoors, and release it, and her mother said that she could.
Jeanna picked up the bat with her bare hands, carrying it by the wingtips, then went outside and tried to let go of it. But instead of flying off, the bat sank its teeth into the tip of Jeanna’s left index finger. The bite felt like a needle prick. Jeanna then grabbed the creature with her other hand and flung it away.
Back at home, Jeanna’s mother, Ann, washed the wound, which was less than a quarter inch (0.6 cm) long, with hydrogen peroxide solution. Her father, John, was a construction worker and a deer hunter and he was accustomed to all types of minor scrapes, cuts, and bruises. The bite didn’t look like much, and everybody forgot about it.
A month after the incident, however, Jeanna developed the symptoms of clinical rabies: fever, blurred vision, and nervous system abnormalities. Physicians at the Children’s Hospital of Wisconsin took blood, saliva, and cerebrospinal fluid samples, and sent them to the Centers for Disease Control and Prevention in Atlanta. The next day, the CDC confirmed the diagnosis of rabies.
Rabies is a disease with an almost 100 percent mortality rate for patients who are not vaccinated before symptoms appear. The human immune system is just about helpless against it, since the rabies virus kills before the body can generate a sufficient immune response. The virus replicates at very low levels, and this makes it hard for the immune system to detect its presence within the bloodstream, tissues, or cerebrospinal fluid. And so before the body can generate a stock of antibodies that would disable it, the virus has already done its work.
Which meant that if events followed their normal course in this case, Jeanna Giese would soon be dead.
The mammalian immune system evolved to a high degree of maturity in the Paleocene epoch, which lasted from about 65 million to 55 million years ago. The epoch is noted for having followed the mass extinction event that marked the end of the dinosaurs (the so-called K-T boundary, for Cretaceous [Kreidezeit]-Tertiary). During this period mammalian species diversified widely and spread out across the planet. The success of the mammals was in great measure attributable to the invention of the immune system and its ability to thwart a wide variety of pathogens: viruses, bacteria, fungi, and parasites.
The immune system’s ability to fight these invaders resulted from the fact that all mammals, including humans, evolved in a world full of microorganisms. This parallel coevolution of pathogen and host was a never-ending battle of wits as invading organisms continuously developed new strategies for infection while the immune system invented its own new countermeasures for defeating them. The same cat and mouse game continues to this day, and left to its own devices would presumably go on into the indefinite future. Synthetic genomics, however, offers the potential to tip the balance decidedly in our favor, outstripping any future evolutionary advances on the part of the microbes.
The human immune system is an amazing collection of biological mechanisms and devices that have evolved an almost magical ability to recognize a pathogen that it has never before encountered, mount a precise and targeted defense, and rid the body of it. But with some pathogens, such as the causative agents of rabies, AIDS, herpes, malaria, and tuberculosis, among others, the immune system offers only a weak defense. Even worse, sometimes the system is oversensitive, treating essentially harmless invaders as enemies, in what is known as an allergic reaction. Further, in autoimmune diseases the body becomes allergic to its own proteins. An immune reaction can also be too aggressive. In some types of hepatitis, for example, people die not from the hepatitis virus itself, but from the immune system’s destruction of the liver. Further, the immune system is responsible for organ rejection, and it also fails to protect us from most cancers. Decidedly, the human immune system, and the phenomenon of immunity itself, are mixed blessings.
It has been known since ancient times that if people contracted and recovered from certain diseases, they rarely suffered from them again. Nearly 2,500 years ago, in his History of the Peloponnesian War, Thucydides wrote about a plague epidemic: “No one caught the disease twice,” he said, “or if he did, the second attack was never fatal.”
The biological underpinnings of the immune response have been understood only recently. And not fully understood at that, because this is a system of enormous complexity, with many active agents and strategies. An account of it is not for anyone in a hurry, nor is it for those with delicate stomachs or weak nerves. First of all, when we speak of being immune to a pathogen we mean that the body has developed a mechanism for recognizing an invader as a threat, has activated a means for defeating the attacker’s mode of action, and has acquired the ability to respond more strongly to that same pathogen in the future. How all of this happens is a convoluted tale suggestive of major military engagements such as the Peloponnesian War itself.
The foreign object in question is called an antigen, a biological substance (a pathogen, toxin, or enzyme) that stimulates an immune response in the body. A canonical immune response to the appearance of an antigen within the body involves three main classes of disease-fighting agents: macrophages, antibodies, and T cells of various types. An antigen floating in the lymphatic system, for example, will sooner or later enter a lymph node, where it will meet up with a macrophage (literally, a “big eater”), one of the body’s natural scavengers and all-purpose trash disposal units. A macrophage gobbles up foreign, dead, deformed, or used-up body parts much as Pac-Man swallows a yellow pellet, except that when the macrophage spits out pieces of the semidigested object, the particles often adhere to its outer surface. This is a rather messy operation, with a definite resemblance to an infant’s dribbling excess baby food out of its mouth.
Roving bands of T cells continuously scan the exteriors of macrophages, as if searching for trace evidence of invasive forces. T cells, which were discovered in the 1960s, are lymphocytes (white blood cells) that have matured in the thymus. There are two major types of T cells, helper T cells, also known as CD4 T cells after the CD4 molecule attached to their outer surfaces (a CD4 molecule is a type of glycoprotein), and killer T cells, the top guns of the immune system. They are also called CD8 T cells because their outsides display an identifying trademark CD8 protein molecule.
When a helper T cell catches a macrophage dribbling out antigen fragments, it will report this fact by chemical messenger (a lymphokine, such as interferon) to another type of white blood cell, a B cell, also called a B lymphocyte (B for the bursa, an organ in birds in which B cells were first discovered in the 1950s). Upon receipt of the chemical message that antigens are
at large in the bloodstream and the lymphatic system, the B cell goes ballistic and essentially turns itself into an antibody factory.
If killer T cells are the body’s top guns, antibodies are its stun guns. They are little Y-shaped protein devices that attach themselves to (or “bind”) the antigen against which they are made. This has the effect of neutralizing it and rendering it hors de combat—out of action. The human body has the ability to churn out staggeringly huge numbers of antibodies, of at least 100 million different types, an attribute known as immune diversity. Each type of antibody, furthermore, has the special property of being able to bind to and neutralize primarily the specific antigen that triggered its formation; this is immune specificity. Taken together, these two features of the immune system, diversity and specificity, enable it to produce vast quantities of antibodies that are tailor-made to fight as many of the millions of different pathogens as it might one day encounter.
Antibodies are virtually the defining units of immunology. The question is, How is the human body able to produce such a great variety of antibodies without knowing in advance the nature of the specific microbial invader that it must defeat? (Among immunologists, this is known as the “generation of diversity,” or the GOD problem.) To begin with, the antigenic universe is not infinite. All pathogens are made from a few different types of atoms: sulfur, carbon, hydrogen, phosphorus, oxygen, nitrogen. These are enough to produce an extremely wide variety of pathogens, but that number is nevertheless finite and limited. Antibodies are proteins, and they too are composed of a few different kinds of atoms. The problem facing the immune system, then, is how to generate a sufficient variety of antibodies to bind to and disable any and all of the different types of antigens it might meet.
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