Regenesis

PROLOGUE

FROM BIOPLASTICS TO
H. SAPIENS 2.0

In December 2009, patrons of the John F. Kennedy Center for the Performing Arts in Washington, DC, experienced a mild jolt of biological future shock when their pre-performance and intermission drinks—their beers, wines, and sodas—were served to them in a new type of clear plastic cup. The cups looked exactly like any other transparent plastic cup produced from petrochemicals, except for a single telling difference: each one bore the legend, “Plastic made 100% from plants.”

Plants?

Indeed. The plastic, known as Mirel, was the product of a joint venture between Metabolix, a Cambridge, Massachusetts, bioengineering firm, and Archer Daniels Midland, the giant food processing company that had recently constructed a bioplastics production plant in Clinton, Iowa. The plant had been designed to churn out Mirel at the rate of 110 million pounds per year.

Chemically, Mirel was a substance known as polyhydroxybutyrate (PHB), which was normally made from the hydrocarbons found in petroleum. But starting in the early 1990s, Oliver Peoples, a molecular biologist who was a cofounder of Metabolix, began looking for ways to produce polymers like PHB by fermentation, by the action of genetically altered microbes on a feedstock mixture.

After seventeen years of research and experimentation (and having been laughed out the doors of several chemical companies), Peoples had developed an industrial strain of a proprietary microbe that turned corn sugar into the PHB plastic polymer. In its broadest outlines, the process was not all that different from brewing beer, which was also accomplished by fermentation: microorganisms (yeast cells) acted on malt and hops to produce ethanol. In the case of Mirel, the microbial fermentation system consisted of a large vat that combined the engineered microbes with corn sugar and other biochemical herbs and spices. The microbes metabolized the corn sugar and turned it into bioplastic, which was then separated from the organisms and formed into pellets of Mirel. Ethanol was a chemical, and so was PHB, but in both cases microbes effected the transformation of organic raw material into a wholly different kind of finished product.

The microbial-based PHB had some key environmental advantages over the petrochemical-derived version. For one thing, since it wasn’t made from petroleum, it lessened our dependence on fossil fuels. For another, its chief feedstock material, corn, was an agriculturally renewable and sustainable resource, not something we were going to run out of any time soon. For a third, Mirel bioplastic resins were the only nonstarch bioplastics certified by Vinçotte, an independent inspection and certification organization, for biodegradability in natural soil and water environments, such as seawater. If any of the plastic cups used at the Kennedy Center ended up in the Potomac River, they would break down and be gone forever in a matter of months. (Biodegradation is not necessarily the panacea it was once thought to be, since it releases greenhouse gases, while non-degradation, ironically, sequesters carbon.)

Constructing a microbe that would convert corn into plastic, in a process akin to beer brewing, was just one example of the transformations made possible by the emerging discipline of synthetic biology—the science of selectively altering the genes of organisms to make them do things that they wouldn’t do in their original, natural, untouched state.

But the feat of turning corn into plastic was merely the tip of the synthetic biology iceberg. By the first decade of the twenty-first century microbe-made commodities were yielding up products that nobody would have guessed were manufactured by bacteria in three-story-high industrial vats. Carpet fibers, for example.

In 2005 Mohawk Industries introduced its new SmartStrand carpet line. It was based on the DuPont fiber Sorona, which was made out of “naturally occurring sugars from readily available and renewable crops.” The Sorona fiber had a unique, semicrystalline molecular structure that made it especially suitable for clothing, automobile upholstery, and carpets. The fiber had a pronounced kink in the middle, and the shape acted as a molecular spring, allowing the strands to stretch or deform and then automatically snap back into their original shape. That attribute was perfect for preventing baggy knees or elbows, or for making carpets that were highly resilient, comfortable, and supportive.

Sorona’s main ingredient was a chemical known as 1,3-propanediol (PDO), which was classically derived from petrochemicals and other ingredients that included ether, rhodium, cobalt, and nickel. In 1995 DuPont had teamed up with Genencor International, a genetic engineering firm with principal offices in Palo Alto, to research the possibility of producing PDO biologically. Scientists from the two companies took DNA from three different microorganisms and stitched them together in a way that resulted in a new industrial strain of the bacterium Escherichia coli. Specifically, they programmed twenty-six genetic changes into the microbe enabling it to convert glucose from corn directly into PDO in a fermenter vat, like beer and Mirel.

In 2003 DuPont trademarked the name Bio-PDO and started producing the substance in quantity. The company claimed that this was the first time a genetically engineered organism had been utilized to transform a naturally occurring renewable resource into an industrial chemical at high volumes. The US Environmental Protection Agency, which regarded Bio-PDO as a triumph of green chemistry, gave DuPont the 2003 Greener Reaction Conditions Award (a part of the Presidential Green Chemistry Challenge). And why not? The biofiber used greener feedstocks and reagents, and its synthesis required fewer and less expensive process steps than were involved in manufacturing other fibers. The production of Sorona consumed 30 percent less energy than was used to produce an equal amount of nylon, for example, and reduced greenhouse gas emissions by 63 percent. For its part, Mohawk touted its Sorona carpeting as environmentally friendly: “Every seven yards of SmartStrand with DuPont Sorona saves enough energy and resources to equal one gallon of gasoline—that’s 10 million gallons of gasoline a year!” Here it was, finally: the politically correct carpet.

What these examples hinted at, however, was something far more important than mere political correctness, namely, that biological organisms could be viewed as a kind of high technology, as nature’s own versatile engines of creation. Just as computers were universal machines in the sense that given the appropriate programming they could simulate the activities of any other machine, so biological organisms approached the condition of being universal constructors in the sense that with appropriate changes to their genetic programming, they could be made to produce practically any imaginable artifact. A living organism, after all, was a ready-made, prefabricated production system that, like a computer, was governed by a program, its genome. Synthetic biology and synthetic genomics, the large-scale remaking of a genome, were attempts to capitalize on the facts that biological organisms are programmable manufacturing systems, and that by making small changes in their genetic software a bioengineer can effect big changes in their output. Of course, organisms cannot manufacture just anything, for like all material objects and processes they are limited and circumscribed by the laws of nature. Microbes cannot convert lead into gold, for example. But they can convert sewage into electricity.

This astonishing capacity was first demonstrated in 2003 by a Penn State team headed by researcher Bruce Logan. He knew that in the United States alone, more than 126 billion liters of wastewater was treated every day at an annual cost of $25 billion, much of it spent on energy. Such costs, he thought, “cannot be borne by a global population of six billion people, particularly in developing countries.” It was widely known that bacteria could treat wastewater. Separately, microbiologists had known for years that bacteria could also generate electricity. So far, nobody had put those two talents together. But what if microbes could be made to do both things simultaneously, treating wastewater while producing electrical energy?

Key to the enterprise would be the microbial fuel cell—a sort of biological battery. In ordinary metabolism, bacteria produce free electrons. A microbial fuel cell (MFC) consists of two electrodes—an anode and a cathode. A current is set up between them by the release of electrons from bacteria in a liquid medium. Electrons pass from the bacteria to the anode, which is connected to the cathode by a wire.

Logan and his colleagues constructed a cylindrical microbial fuel cell, filled it with wastewater from the Penn State water treatment plant, and then inoculated it with a pure culture of the bacterium Geobacter metal-lireducens. Lo and behold, in a matter of hours the microbe had begun purifying the sewage while at the same time producing measurable amounts of electricity. These results “demonstrate for the first time electricity generation accompanied by wastewater treatment,” Logan said. “If power generation in these systems can be increased, MFC technology may provide a new method to offset wastewater treatment operating costs, making advanced wastewater treatment more affordable for both developing and industrialized nations.”

The general setup wasn’t difficult to replicate and within a few years a sophomore at Stuyvesant High School in New York City, Timothy Z. Chang, was designing, building, and operating microbial fuel cells at home and in his high school lab. He had experimented with some forty different strains of bacteria to discover which was best suited to maximum electricity production. “It may be possible to achieve even higher power yields through active manipulation of the microbial population,” he wrote in a formal report on the project.

By 2010 several teams of researchers were working on scaling up bacterial electricity production from sewage to make it into a practical, working, real-world option. By this time, synthetic biologists had gotten microbes to perform so many different feats of creation that it was clear that many of nature’s basic units of life—microbes—were undergoing an extreme DNA makeover, a major course of redesign from the ground up. Engineered microbes produced diesel oil, gasoline, and jet fuel. Microbes were made to detect arsenic in drinking water at extremely low concentrations (as low as 5 parts per billion) and report the fact by changing color. There were microbes that could be spread out into a biofilm. By producing a black pigment in response to selective illumination, they could copy superimposed patterns and projected images—in effect, microbial Xerox machines.

A student project reprogrammed E. coli bacteria to produce hemoglobin (“bactoblood”), which could be freeze-dried and then reconstituted in the field and used for emergency blood transfusions. In 2006, just for fun, five MIT undergrads successfully reprogrammed E. coli (which as a resident of the intestinal tract smelled like human waste) to smell like either bananas or wintergreen.

E. coli was so supple, pliable, and yielding that it seemed to be the perfect biological platform for countless bioengineering applications. One of its greatest virtues was that the E. coli bacterium (and cousins, the Vibrio) are the world’s fastest machines at doubling, small or large.* It reproduced itself every twenty minutes, so that theoretically, given enough simple food and stirring, a single particle of E. coli could multiply itself exponentially into a mass greater than the earth in less than two days.

Still, as malleable as it was, University of Wisconsin geneticist Fred Blattner decided he could materially improve the workhouse K-12 strain of the microbe to make it an even better chassis for synthetic biology engineering projects. The microbe had some 4,000 genes; many had no known function, while others were nonessential, redundant, or toxic. So Blattner stripped 15 percent of its natural genes from the K-12 genome, making it a sort of reduced instruction set organism, a streamlined, purer version of the microbe. Blattner described it as “rationally designed” and said that his genetic reduction “optimizes the E. coli strain as a biological factory, providing enhanced genetic stability and improved metabolic efficiency.” With forty genome changes, he had pre-engineered the microbe in order to make it easier to engineer.

In 2002 Blattner founded Scarab Genomics to sell his new and improved organism, now billing it as “Clean Genome E. coli” and marketing it under the slogan “Less is better and safer!” Researchers can buy quantities of the microbe, online or by fax, for as little as $89 a shot (plus a $50 shipping fee).

The upshot of all this is that, at least at the microbial level, nature has been redesigned and recoded in significant ways. Genomic engineering will become more common, less expensive, and more ambitious and radical in the future as we become more adept at reprogramming living organisms, as the cost of the lab machinery drops while its efficiency rises, and as we are motivated to maximize the use of green technologies.

Given the profusion and variety of biological organisms, plus the ability to reengineer them for a multiplicity of purposes, the question was not so much what they can be made to do but what they can’t be made to do, in principle. After all, tiny life forms, driven solely by their own natural DNA, have, just by themselves, produced large, complex objects: elephants, whales, dinosaurs. A minuscule fertilized whale egg produces an object as big as a house. So maybe one day we can program an organism, or a batch of them, to produce not the whale but the actual house. We already have bio-plastics that can be made into PVC plumbing pipes; biofibers for carpeting; lumber, nature’s own building material; microbe-made electricity to provide power and lighting; biodiesel to power the construction machinery. Why can’t other microbes be made to produce whatever else we need?

In 2009 Sidney Perkowitz, a physicist at Emory University in Atlanta with a special interest in materials science, was asked to speculate about the future of building materials. “Think about the science-fictionish possibility of bioengineering plants to produce plastic exactly in a desired shape, from a drinking cup to a house,” he said. “Current biotechnology is far short of this possibility, but science fiction has a way of pointing to the future. If bioplastics are the materials breakthrough of the 21st century, houses grown from seeds may be the breakthrough of the 22nd.”

Similar proposals have been made by others, and they may be much closer than the twenty-second century; for example, using modified gourds and trees to grow a primitive, arboreal house (inhabitat.com/grow-your-own-treehouse). The technology of determining the shape and chemical properties of plants by making them sensitive to simple cues of light and scaffolding is improving rapidly.

This focus on microbes and plants—especially on the overworked E. coli bacterium—may give rise to the impression that synthetic biology and genomic engineering have little to offer the charismatic megafauna—the higher organisms such as people. Nothing could be further from the truth. In fact these technologies have the power to improve human and animal health, extend our life span, increase our intelligence, and enhance our memory, among other things.

The idea of improving the human species has always had an enormously bad press, stemming largely from the errors and excesses associated with the eugenics movements of the past. Historically, eugenics has covered everything from selective breeding for the purpose of upgrading the human gene pool to massive human rights violations against classes of people regarded as undesirable, degenerate, or unfit because of traits such as religion, sexual preference, handicap, and so on, culminating, in the extreme case, in the Nazi extermination program.

Some proposals for enhancing the human body have had a harebrained ring to them, as for example the idea of equipping people with gills so that they could live in the sea alongside sharks. Burdened with past evils and silliness, any new proposal for changing human beings through genomic engineering faces an uphill battle. But consider this modest proposal: What if it were possible to make human beings immune to all viruses, known or unknown, natural or artificial? No more viral epidemics, influenza pandemics, or AIDS infections.

Viruses do their damage by entering the cells of the host organism and then using the cellular machinery to replicate themselves, often killing the host cells in the process. This leads to the release of new viruses that proceed to infect other cells, which in turn produce yet more virus particles, and so on. Viruses can take control of a cell’s genetic machinery because both the virus and the cell share the same genetic code. However, changing the genetic code of the host cell, as well as that of the cellular machinery that reads and expresses the viral genome, could thwart the virus’s ability to infect cells (see Chapter 5).

All this may sound wildly ambitious, but there is little doubt that the technology of genome engineering is in principle up to the task. An additional benefit of engineering a sweeping multivirus resistance into the body is that it would alleviate a common fear concerning synthetic biology—the accidental creation of an artificial supervirus to which humans would have no natural immunity.

Genomic technologies can actually allow us to raise the dead. Back in 1996, when the sheep Dolly was the first mammal cloned into existence, she was not cloned from the cells of a live animal. Instead, she was produced from the frozen udder cell of a six-year-old ewe that had died some three years prior to Dolly’s birth. Dolly was a product of nuclear transfer cloning, a process in which a cell nucleus of the animal to be cloned is physically transferred into an egg cell whose nucleus had previously been removed. The new egg cell is then implanted into the uterus of an animal of the same species, where it gestates and develops into the fully formed, live clone.

Although Dolly’s genetic parent had not been taken from the grave and magically resurrected, Dolly was nevertheless probably a nearly exact genetic duplicate of the deceased ewe from which she had been cloned, and so in that sense Dolly had indeed been “raised from the dead.” (Dolly was certainly different in the details of how the genome played out developmentally [a.k.a. epigenetically] but not so different as to discourage subsequent success in a variety of agricultural and research species.)

But even better things were in the offing. A few years after Dolly, a group of Spanish and French scientists brought to life a member of an extinct animal species—the Pyrenean ibex, or bucardo, a subspecies of wild mountain goat whose few remaining members had been confined to a national park in northern Spain. The species had become extinct in January 2000, when the very last living member, a thirteen-year-old female named Celia, was crushed to death by a falling tree. Consequently the International Union for the Conservation of Nature (IUCN) formally changed the conservation status of the species from EW, which meant “extinct in the wild,” to EX, which meant “extinct,” period.

Extinction, supposedly, was forever.

But in the spring of 1999, Dr. Jose Folch, a biologist working for the Aragon regional government, had taken skin scrapings from Celia’s ears and stored the tissue samples in liquid nitrogen in order to preserve the bucardo’s genetic line. A few years later, in 2003, Folch and his group removed the nucleus from one of Celia’s ear cells, transferred it into an egg cell of a domestic goat, and implanted it into a surrogate mother in a procedure called interspecies nuclear transfer cloning.

After a gestation period of five months, the surrogate mother gave birth to a live Pyrenean ibex. By any standard, this was an astonishing event. After being officially, literally, and totally extinct for more than two years, a new example of the vanished species was suddenly alive and breathing.

Not for long, however. The baby ibex lived for only a few minutes before dying of a lung condition. Still, those scant minutes of life were proof positive that an extinct species could be resurrected, not by magic or miracles but by science.

“Nuclear DNA confirmed that the clone was genetically identical to the bucardo’s donor cells,” the group wrote in its report on the project. “To our knowledge, this is the first animal born from an extinct subspecies.”

Almost certainly, it will not be the last. The bucardo’s birth involved a bit of genomic reprogramming because the egg cell that developed into the baby ibex had not been fertilized by a sperm cell but rather by the nucleus of a somatic (body) cell. The nucleus and the egg cell had to be jump-started into becoming an embryo in a process known as electrofusion, which melds the two together.

A later technique under development in my Harvard lab will allow us to resurrect practically any extinct animal whose genome is known or can be reconstructed from fossil remains, up to and including the woolly mammoth, the passenger pigeon, and even Neanderthal man. One of the obstacles to resurrecting those and other long extinct species is that intact cell nuclei of these animals no longer exist, which means that there is no nucleus available for nuclear transfer cloning. Nevertheless, the genome sequences of both the wooly mammoth and Neanderthal man have been substantially reconstructed; the genetic information that defines those animals exists, is known, and is stored in computer databases. The problem is to convert that information—those abstract sequences of letters—into actual strings of nucleotides that constitute the genes and genomes of the animals in question.

This could be done by means of MAGE technology—multiplex automated genome engineering. MAGE is sort of a mass-scale, accelerated version of genetic engineering. Whereas genetic engineering works by making genetic changes manually on a few nucleotides at a time, MAGE introduces them on a wholesale basis in automated fashion. It would allow researchers to start with an intact genome of one animal and, by making the necessary changes, convert it into a functional genome of another animal entirely.

You could start, for example, with an elephant’s genome and change it into a mammoth’s. First you would break up the elephant genome into about 30,000 chunks, each about 100,000 DNA units in length. Then, by using the mammoth’s reconstructed genome sequence as a template, you would selectively introduce the molecular changes necessary to make the elephant genome look like that of the mammoth. All of the revised chunks would then be reassembled to constitute a newly engineered mammoth genome, and the animal itself would then be cloned into existence by conventional interspecies nuclear transfer cloning (or perhaps by another method, the blastocyst injection of whole cells).

The same technique would work for the Neanderthal, except that you’d start with a stem cell genome from a human adult and gradually reverse-engineer it into the Neanderthal genome or a reasonably close equivalent. These stem cells can produce tissues and organs. If society becomes comfortable with cloning and sees value in true human diversity, then the whole Neanderthal creature itself could be cloned by a surrogate mother chimp—or by an extremely adventurous female human.

Any technology that can accomplish such feats—taking us back into a primeval era when mammoths and Neanderthals roamed the earth—is one of unprecedented power. Genomic technologies will permit us to replay scenes from our evolutionary past and take evolution to places where it has never gone, and where it would probably never go if left to its own devices.

Today we are at the point in science and technology where we humans can reduplicate and then improve what nature has already accomplished. We too can turn the inorganic into the organic. We too can read and interpret genomes—as well as modify them. And we too can create genetic diversity, adding to the considerable sum of it that nature has already produced.

In 1903 German naturalist Ernst Haeckel stated the pithy dictum “Ontogeny recapitulates phylogeny.” By this he meant that the development of an individual organism (ontogeny) goes through the major evolutionary stages of its ancestors (phylogeny). He based this aphorism on observations that the early embryos of different animals resembled each other and that, as they grew, each one seemed to pass through, or recapitulate, the evolutionary history of its species. (For example, the human embryo at one point has gill slits, thus replicating an evolutionary stage of our piscine past.)

While it is clear that embryos develop primitive characteristics that are subsequently lost in adults, Haeckel’s so-called biogenetic law is an overstatement and was not universally true when first proposed or today. However, I hereby propose a biogenetic law of my own, one that describes the current situation in molecular engineering and biotechnology: “Engineering recapitulates evolution.” Through human ingenuity, and by using the knowledge of physics and chemistry gained over the course of six industrial revolutions, we have developed the ability to manipulate and engineer matter, and by doing so we have rediscovered and harnessed the results of six similar revolutions that occurred during billions of years of biological evolution.

Using nanobiotechnology, we stand at the door of manipulating genomes in a way that reflects the progress of evolutionary history: starting with the simplest organisms and ending, most portentously, by being able to alter our own genetic makeup. Synthetic genomics has the potential to recapitulate the course of natural genomic evolution, with the difference that the course of synthetic genomics will be under our own conscious deliberation and control instead of being directed by the blind and opportunistic processes of natural selection.

We are already remaking ourselves and our world, retracing the steps of the original synthesis—redesigning, recoding, and reinventing nature itself in the process.

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* Bacteria called Clostridium perfringens and Vibrio natriegens seem to be the world’s fastest doublers, reproducing in seven to ten minutes respectively.

CHAPTER 1

-3,800 MYR, L