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A Vat of Biofuels and Bacteria

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The United States alone consumes close to 20 million barrels of oil a day, roughly half of which is imported. So it's no wonder that the US Department of Energy has allocated millions of grant dollars to finding alternatives to fossil fuels. Even current biofuel production methods that aim to be both green and cost-effective still rely on plant material created through photosynthesis, which produces energy that can only be extracted through intensive processes requiring additional materials and resources.

To make better use of the molecular machinery contained in some micro-organisms, synthetic biologists are attempting to hack the genomes of well-studied — and some not-so-well-studied — bacteria to create liquid fuels called electrofuels, which use energy from electricity and chemical compounds. By engineering these bacteria to ramp up their ability to produce fuels from carbon dioxide with increased efficiency, massive amounts of water and land can be spared.

A Boston-based synthetic biology startup company called Ginkgo Bioworks is using Escherichia coli to produce electrofuels as part of a project supported by the DOE's Advanced Research Projects Agency-Energy electrofuels research initiative. The ARPA-E program was formed by the Obama administration in 2009 and awards annual grants to a range of alternative-energy projects. So far, ARPA-E has awarded a total of roughly $20 million over three years to 13 academic institutions and startup companies.

While E. coli does not naturally metabolize carbon dioxide, researchers at Ginkgo Bioworks have engineered a strain of the bacteria that contains the genes responsible for carbon dioxide metabolism. By delivering carbon dioxide to E. coli in the form of formic acid salts — a commonly used industrial chemical — the team is attempting to refine its genetically modified E. coli to have an accelerated rate of carbon dioxide consumption for ramped-up liquid biofuel production.

"While there are plenty of organisms that naturally grow on carbon dioxide as a carbon source, E. coli isn't one of those. So right away we're giving ourselves a big challenge," says Curt Fischer, cofounder of Ginkgo Bioworks. "The reason we like that is that E. coli is a model organism that has been widely engineered, so it lets us build in the systems that we want to have from the ground up. We can look at what pathway for carbon fixation is the most efficient and work on building those into the bacteria rather than relying on some existing pathway which, although it works, might not be possible from an energy-efficiency standpoint."

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Fischer's project, funded by a three-year ARPA-E grant totaling $6.6 million, is currently focused on building a set of metabolic modules capable of converting formate salts into liquid biofuels. In addition to E. coli, they are also expanding the implementation of these modules in host organisms such as alphaproteo-bacteria. "This bacteria simplifies our work because we don't have to engineer in metabolic pathways for CO2 and formate uptake," Fischer says. "The tradeoff is decreased control over pathway function, which is why we continue to work with E. coli, an organism where we are building in all the pathways from the ground up."

Exploring other alternatives to E. coli is the focus of a Harvard University team that is genetically engineering a deep-sea bacteria called Shewanella oneidensis — often referred to as an extremophile because of its ability to grow in harsh environments — to act as a battery for electrofuel production. The group, led by Harvard Medical School professor Pamela Silver, has engineered a strain of Shewanella that can sit directly on electrical conductors and soak up current supplied by solar panels. When the right amounts of carbon dioxide, water, and other nutrients are added to their environment the bacteria can produce liquid biofuel. The biofuel molecules produced by the bacteria are designed to spontaneously separate from their water-based culture so that they can be siphoned out and used as they are, without further chemical processing.

"We chose Shewanella because we thought that the electron conducting part would be done by the organism. But part of our project was also to engineer carbon dioxide fixation in these organisms," Silver says. "The ability to engineer carbon dioxide fixation is very challenging and also very important in and of itself. If everything else failed, that would be a high success — and that part of the project is going really well." Silver's project is supported by a three-year, $4.1 million grant from ARPA-E.

While ARPA-E's mandate is to fund projects aimed at producing biofuel solutions that could be implemented into the existing energy infrastructure, Silver says a lot of these projects tend to be a bit "blue sky" in nature.

But that's OK with her. "I'm happy to make fuel or fuel-like molecules because you learn a lot from trying to have cells make them and you get interesting intermediates as well as a general strategy of having cells use CO2," she says. "Ultimately you want to program them to make anything."

Brainy biofuel

In addition to skipping past photosynthesis, synthetic biologists are also harnessing the power of bacteria by making them smarter. So-called "intelligent" or "self-aware" bacteria are being engineered with genetic sensors that can be tripped when certain levels of intermediates used for bio-diesel production are present, resulting in a change in gene expression.

Jay Keasling, a professor at the University of California, Berkeley, is currently working on exploiting such a sensor system in a previously engineered strain of E. coli that he says could eventually facilitate cheaper biofuel production by making bacteria up to three times more efficient.

Unlike some of his colleagues who are exploring biofuel production synthesis methods that may not be ready for immediate use, Keasling says the focal point of his research is to produce solutions compatible with today's fuel infrastructure.

"There's a lot known about these organisms and a lot of tools available so we don't have to spend our time learning how to transform them with genes," Keasling says. "They are already used industrially for production of drugs and chemicals, and when we go to large biofuel production, those will probably be hosts that are industrially viable."

In a Nature paper published in early April, Keasling and his collaborators described a dynamic sensor-regulator system called DSRS that produces fatty acid-based precursors in E. coli using a transcription factor capable of sensing key intermediates. Keasling and his colleagues engineered fatty acid biosensors based on FadR, a naturally occurring fatty acid-sensing protein, and its cognate regulator. They reported that their DSRS not only improved the stability and yield of biodiesel-producing strains, but could also be applied to other synthetic pathways for metabolic balance.

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"The whole mantra of our work in the biofuel area is engineering microbes to produce fuels that are as close as possible — if not identical — to existing transportation fuels," he says. "So this particular microbe was producing fatty acids and ethanol and, by putting in this control system, we're able to balance the production of all these components so that we can get the fuel produced optimally and at a level that will be nontoxic to the cell."

Questioning what's known

Despite meaningful grant dollars being awarded to synthetic biology research teams to devise the perfect bacteria-powered electrofuel solution, however, sometimes long-held assumptions need to be updated before this research can advance. One example is the 44-year-old idea that cyano-bacteria — a photosynthetic -algae that can be grown in large ponds — contain an incomplete tricarboxylic acid cycle. The TCA cycle, which eventually leads to ATP production, is an important energy-making cycle present in the metabolisms of virtually all forms of bacteria, mold, protozoa, and animals.

Donald Bryant, a professor of biotechnology at Penn State, dispelled this notion when he demonstrated last December that a form of cyano-bacteria called Synechococcus sp. PCC 7002 has genes that encode for 2-oxoglutarate decarboxylase and succinic semialdehyde dehydrogenase. These enzymes work together to complete a non-canonical TCA cycle.

"This was a case where not having this information might have led one to think you had to install steps that you in fact don't have to install to build a pathway," Bryant says. "On the other hand, when you're building a de novo pathway in an organism, as long as you know you have the starting material, you can say it doesn't matter. It only becomes a problem if you're trying to build metabolic models when you're missing such basic information about the central metabolic pathways in the cell."

Bryant's discovery means that this form of cyanobacteria could be genetically engineered to synthesize -1,4-butanediol, an organic precursor compound for both biofuels and plastics. In addition, this data provides another alternative for researchers aiming to harness solar energy to create biofuels. "I think if synthetic biology is going to succeed in converting solar energy into products, then it has to be done in cyanobacteria or algae. However we don't have the predictive models that E. coli has," Bryant says. "So it's not as simple to make knockouts and push carbon around as it is in E. coli, and in the case of this particular pathway, you couldn't have even predicted how the carbon was going to flow anyway because the pathway wasn't understood properly."

Tools of the trade

Robust mass spectrometry platforms and the latest next-generation sequencing technologies are playing a crucial role in helping synthetic biologists to understand how to manipulate metabolic pathways.

Berkeley's Keasling uses mass spectrometry platforms extensively to measure metabolite levels inside the cell and the intermediates in the metabolic pathways to find out where the bottlenecks might be. "I think as the tool sets that we now have available to use to engineer microbes get better and better, then turnaround times for doing these engineering projects will be shorter and shorter, which then translates into less cost — but the bottom line is that we can do a lot more with engineering microbes than we could a decade ago," he adds.

Next-generation sequencing is also providing biofuel researchers with data on the adaptive evolution of microbial genomes to improve growth in environments of interest. "We have actually built out some long-term microbial cultures and if we impose an environment that's of interest to us and we want to be able to watch not just one or two genomes to see if they're growing or not, but to put in a diverse collection of genomes and look to see which ones stick around," Fischer says. "For applications like that, tracking adaptive evolution in laboratory environments at the population level, we think next-generation sequencing could really be useful."

Both Silver and Bryant are routinely in the position of having to develop techniques and tools in-house, and rarely use prepackaged or open source standardized biological parts, like those provided by MIT's BioBricks project.

"We have limited experience with the BioBrick system. It doesn't always work for us, so we end up using components of our own design and desire more often," Bryant says. "It's not always true that you can just move something from E. coli into a cyanobacteria and expect it to work, so we've had mixed success at best in using other people's components."

Scaling up

Moving synthetic biofuel production methods and innovations from the lab to the existing transportation and manufacturing infrastructure remains a formidable — and expensive — endeavor. Despite the nature of many of these projects, researchers are aware that it will take a lot of capital to create a regulatory and technical framework for scaling up biofuel production.

"It's very costly, we're talking about new fuels that you are going put into the infrastructure — things like jet fuels, which have to be tested extensively. And then there's engineering microbes to produce all the chemicals we use on a day-to-day basis that would otherwise come from petroleum, like plastic and nylon," Keasling says. "There is still a lot of work to be done in engineering crops to be better biomass crops that would be the substrate for producing all of these fuels and chemicals that we would want to produce."

Despite these hurdles, synthetic bio-logists like Ginko's Fischer say that sticking to the path of least resistance is the key to making electrofuel--producing bacteria on a mass scale. This path is comprised of choosing to develop biofuel solutions that work with existing industrial fuel production components as well as bacteria that are already well studied.
"That's the reason we use E. coli," Fischer says. "There would be no need to design new bioreactors."

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