Designed Pathways And Microbes

SYNTHETIC BIOLOGY. It sounds big and sweeping like synthetic chemistry, and it is. Yet it refers to a rapidly growing scientific field that the general public and even many scientists remain unfamiliar with.

"I don't know what the term 'synthetic biology' means, but I can tell you what people outside think it might mean—which is the creation of monsters," said genetics and molecular biology pioneer Sydney Brenner of the Salk Institute for Biological Studies, in La Jolla, Calif., in a recent talk.

Brenner, a Nobel Laureate, was referring obliquely to the fact that some people are concerned about the societal implications of synthetic biology—a field not actually dedicated to the creation of monsters but instead to the engineering of new biological pathways in cells and to the creation of wholly new organisms from scratch.

Polish geneticist Wacław Szybalski was one of the first people to recognize the potential utility of synthetic biology. In a 1974 paper, Szybalski wrote: "The real challenge will start when we enter the synthetic biology phase of research in our field. We will then devise new control elements and add these new modules to ... existing genomes or build up wholly new genomes. This would be a field with ... unlimited expansion potential."

"Some thought I was right," Szybalski said in Hong Kong last month at Synthetic Biology 4.0, a conference at which he, Brenner, and many other synthetic biologists discussed their relatively new field.

As they describe it, synthetic biology is like genetic engineering on steroids: Synthetic biologists focus primarily on engineering microorganisms, not with single genes, as genetic engineers typically do, but with sets of coordinated genes, entire biosynthetic pathways, and even whole chromosomes. The aim is to use these organisms to generate useful new products, such as drugs, biofuels, and new types of enzymes.

Another branch of synthetic biology, synthetic genomics, aims to create microorganisms never seen before. Whether or not some of these turn out to be Brenner's monsters remains to be seen.

Scientists are also beginning to envision the use of modified organisms as drugs—not merely as means for producing drugs. These therapeutic microbes would be delivered into patients as live agents designed to fight disease. This idea is not as far-fetched as it seems. Live bacterial cultures in dairy products and nutraceuticals are touted as digestive aids, and one live bacterium is already a Food & Drug Administration-approved medication.

THE POTENTIAL for synthetic biology to do harm as well as good has not gone unnoticed, and the field has its critics. But many synthetic biologists argue that the criticism their field receives is unwarranted.

In genetic engineering (also known as recombinant DNA technology), researchers generally identify an existing gene or create a new one that encodes a useful protein. They put the gene into cells that express the protein and then harvest the protein for use as a research tool or as a medication that can be sold for billions of dollars, such as the antianemia drug erythropoietin.

Synthetic biology is now beginning to usher the strategy of genetic modification into applications that traditional genetic engineering can't touch.

For example, chemical engineering professor Kristala Jones Prather and coworkers at Massachusetts Institute of Technology are using synthetic biology to create novel compounds by incorporating nonnative biosynthetic pathways into microorganisms. If we can engineer enzymes capable of converting nonnative substrates into novel compounds, then we open the door to a huge new range of potentially useful products, Prather said.

Geneticist Daphne Preuss and coworkers at the University of Chicago have developed minichromosomes that make it possible to transfer multiple genes into plant cells in a controllable way, and Preuss moved to industry to develop the concept commercially. Now cofounder and chief executive officer of Chromatin, in Chicago, Preuss explained that traditional one-gene-at-a-time recombinant DNA technology can insert up to only about five genes into a plant; that the genes are integrated into host cells at random genomic sites, some good, some bad; and that these problems can make it time-consuming, inefficient, and costly to engineer plants with multiple genes.

"We wanted to solve this—make it faster, more efficient, and more affordable," she said. To do this, she and her colleagues design chromosomes from scratch.

They use natural plant centromeres (a key part of chromosomes needed for their inheritance), promoters (gene activation sites), and gene-termination sequences to assemble linear or circular minichromosomes that contain at least a dozen genes that can improve crops by promoting traits like pest and disease resistance. They then insert these minichromosomes into plant cells, making the cells capable of expressing a bunch of new proteins.

Chromatin is currently using minichromosomes "to design biofuel crops that give more yield, are lower cost, and reduce processing costs for cellulose," Preuss said. She estimates that engineering sugarcane this way could save 26 cents per gal in the production of sugarcane-based ethanol, which translates to about $8 billion in savings for a decade of ethanol use. "It's those kinds of numbers that are going to push industry to do this sort of thing and adopt it," she said.

RATHER THAN relying on plants, Emeryville, Calif.-based Amyris Biotechnologies is engineering microbes to express new biochemical pathways that yield biofuels. The kind of diesel fuel the firm's microbes make "is going to be the most cost-effective fuel you can buy," Amyris researcher Zach Serber said. "It can be used in existing engines and transported in existing pipelines," whereas some current biofuels, such as ethanol, do not fit as well into today's commercial fuel infrastructure, he said.

Amyris already has experience using engineered microbes to make useful chemicals. In collaboration with chemical engineer and bioengineer Jay D. Keasling and coworkers of the University of California, Berkeley, and Lawrence Berkeley National Laboratory, the firm has refined a method for producing the antimalaria drug artemisinin from a precursor made in engineered bacteria and yeast. The researchers hope the process, which is now being scaled up by Sanofi-Aventis on a nonprofit basis, will improve the efficiency and lower the cost of production of artemisinin, which is currently extracted from the bark of wormwood trees.

Clyde A. Hutchison III and coworkers at the J. Craig Venter Institute, both in San Diego and in Rockville, Md., are also using synthetic biology to construct new biosynthetic pathways and chromosomes, but they want to go further and create an entire designed genome by chemical synthesis. No one has achieved such a feat.

"We like to talk about a minimal cell, in which all the genes are essential," Hutchison said. "Interrupting any one of the genes would lead to a nonliving cell." He and his coworkers believe their work will eventually ease the computer design of wholly new genomes and organisms.

These researchers have adopted two approaches to synthetic genomes. In a top-down approach, they start with Mycoplasma genitalium, which has the smallest genome of any cell grown in pure culture, and systematically reduce its approximately 500 genes to only those needed to run a living cell. And in a bottom-up approach, they build up a genome starting from oligonucleotides made with a DNA synthesizer. In time, they plan to take a lab-constructed genome and use it to replace the genome of a recipient cell.

Researchers hope synthetic biology will also provide pathways to engineered viruses, bacteria, and human cells that work as living drugs. "We would like to develop drugs that are able to do things chemicals can't do," said Jeffrey Way, a former biopharmaceutical researcher who is now starting a bioenergy company. "That's the whole point of synthetic biology."

A key potential advantage of live-cell medications is that they can be "smarter" than chemical compounds. For example, their surface receptors might enable them to be targeted more easily to specific cells and tissues, whereas chemical compounds tend to have broader systemic activity that can more readily cause side effects. And live medications might be capable of responding to different localized conditions by making "decisions" about what actions to take, such as whether or not to deliver a payload.

"WE IMAGINE that we could engineer cells that could read signatures associated with tumors and then bind, label, and perhaps kill metastases," said cell-signaling specialist Wendell Lim of UC San Francisco. He and his coworkers also envision live cells that would secrete anti-inflammatory cytokines in a localized manner to suppress flare-ups in autoimmune diseases; engineered live cells that would provide rapid, controlled wound healing; and stem cells that could be guided to sites where they might trigger regeneration processes.

For their part, synthetic biologist J. Christopher Anderson and coworkers at UC Berkeley are developing tumor-killing bacteria. "For some reason, the idea of using bacteria as therapeutics elicits a 'yuck' factor," Anderson said. "But there's a long history of using bacteria as therapeutic agents."

For example, people buy the yogurt product Activia and the nutraceutical product Culturelle to infect themselves with bacteria that they believe will improve their digestion. And a primary treatment for bladder cancer is a live bacterium, TICE BCG (Bacillus Calmette-Guérin). When introduced into the bladder, TICE BCG elicits a specific immune response that fights the tumor.

Anderson said the major challenge to his group's tumor-killing bacteria project is that injected bacteria are generally cleared from blood within five minutes. "A major hurdle in this work is getting them to last long enough that they get to the tumor," he said.

His group's long-term goal is to engineer live bacteria to be smart therapeutics. "You can have binding specificity by putting things on the surface" of the bacteria, he said, such as molecular-scale sensors and actuating elements that would detect and respond to chemical cues such as the presence of toxic proteins. "And bacteria can swim, which is something you just don't get out of a chemical species," Anderson said. "So a lot of intrinsic functionality is possible" with live bacterial therapeutics, he added.

Other researchers are investigating live bacteria as microbicides for fighting HIV, the cause of AIDS. Geneticist Dean H. Hamer and coworkers at the National Cancer Institute are trying to develop a live bacterial topical microbicide that would stop HIV from getting into the body and replicating.

They would like to take a microbicide, which has transient infection-blocking activity, and endow it with long-lasting action. "Our idea is to take bacteria that normally live in the vagina or rectum and reengineer them so they now become anti-HIV microbes," Hamer said. He and his coworkers, in collaboration with researchers at Osel, a biotech firm in Santa Clara, Calif., have engineered an HIV-blocking bacterial protein into Lactobacillus species and are currently testing the efficacy of the engineered bacteria.

Osel is one of a few biotech companies that are pursuing biomedical applications of synthetic biology. Large pharmaceutical companies don't appear to be interested yet. "There's institutional resistance," Way said. "It's very difficult to get synthetic biology projects going in traditional big pharma. I think synthetic biology really does offer an opportunity to rescue the pharmaceutical industry," which is threatened by financial problems from patent expirations and relatively dry drug pipelines. "But it's not clear that existing companies are really going to take advantage of it," he said.

Despite the promise of synthetic biology for applications such as efficient biofuels production and novel disease treatments, the field has attracted criticism from people who believe it could cause profound problems and has so far had inadequate regulatory oversight.

"Synthetic biology as a field is no longer being driven by a bunch of well-meaning individuals," said Research Program Manager Jim Thomas of ETC Group, a sustainability and human rights organization in Ottawa. "It's being driven by a different set of necessities—the bottom line, basically."

At the Hong Kong meeting he noted that the business plan of many current synthetic biology projects involves biorefining—the conversion of glucose, corn sugar, sugarcane, and cellulose into commercial products such as biofuels and drugs. Thomas showed a biofactory cartoon captioned: "Welcome to the new global sugar economy, where syn-biotech pillages forests, farmlands, and all of biodiversity to make plastics, fuels, drugs, and huge corporate profits."

"WE HAVE A global food crisis that has not been resolved, where millions of people are being pushed into starvation because the price of food is going up???in large part because those sugars are now as valuable or maybe more valuable for making fuels," Thomas said. "Increasingly, that will be true for making plastics or making chemicals" with synthetic biology, he noted, adding, "That's not acceptable."

Thomas noted that the commercialization of synthetic biology "is going to enable a massive grab on plant matter that we haven't seen before. To replace petroleum in fuels, plastics, and chemicals, we're going to dig into massive deforestation, massive impacts on agricultural soils, [and greater amounts of] greenhouse gases going into the atmosphere."

In addition, it's difficult to assess the biosafety implications of microbes with new synthetic pathways, he said. "If you're developing microbes that can turn sugars, and in a short while cellulose, into jet fuel, that's an organism I would be very concerned about getting out into the environment. And we know microorganisms constantly escape into the environment from labs and facilities that work with genetically engineered microorganisms," Thomas said.

"I think there is a role for understanding life and nature better by rebuilding it," he added. "But my recommendation would be that synthetic biology shouldn't be pursued in a commercial environment."

No session at Synthetic Biology 4.0 was dedicated specifically to a dialogue between synthetic biology critics and proponents. But comments made by synthetic biologists at the conference suggest that many doubt that the field will have a negative effect on crop availability or that it will adversely affect food supplies. And they noted that the use of genetically engineered microorganisms during recent decades has yet to result in earthshaking problems.

At the meeting, which was overshadowed by news of the world financial crisis, Szybalski, the man who helped coin the term "synthetic biology," expressed his own view about criticism of the field. "There is one thing we do that I think is wrong—we worry too much," he said. "We worry about dangers of genetic engineering, dangers of synthetic biology, and that's nonsense. Because they're not real dangers. It's a very safe science. What we should be worried about is bankers! We should watch them and not scientists."