July 2011

Pouring energy into biofuels

Many biochemists are working on alternatives to corn-derived fuel ethanol. 


That line in “America the Beautiful” about amber waves of grain was written as a testamony to our country’s abundance and ready opportunity to feed the hungry masses. But increasingly, America’s grains are feeding masses of hungry cars, not people. Nearly all gas in the U.S. contains 10 percent fuel ethanol, a product currently made by using yeast to ferment sugar derived from cornstarch. America produced about 13.2 billion gallons of fuel ethanol last year, making this the most common biofuel— fuel metabolically derived from living organisms as opposed to fossil fuels produced over hundreds of millions of years from long-dead organisms— in this country.

But while the corn lobby probably would be thrilled to keep ethanol made from their grain in the top spot, biofuel researchers have other ideas. They’re working toward new advances aimed at moving away from corn-derived fuel ethanol, such as engineering bigger and better grasses to pull more fuel from their vegetative tissues rather than their seeds and genetically modifying plants to make removing the sugar polymers that serve as a feedstock for fuels faster and easier. Others are working on modifying plants to produce energy rich oils preferentially instead of starch or teaching biofuel processing bacteria new tricks, such as making longer-chain alcohols that store more energy than ethanol, synthesizing biofuels out of proteins instead of sugars, or digesting sugar polymers directly and pumping out biofuels at the same time.

So instead of those amber waves of grain, America may eventually have green waves of switchgrass or miscanthus— or even waving cilia from fuel-making bacteria.

Going green

Although biofuel might seem like a hot topic at the moment, it’s really an old idea, explains Daniel Bush, professor and chair of the department of biology at Colorado State University.

“It’s just another way of transforming sunlight into a useful form of energy,” Bush says.

Plants do much of the work for us, he explains, by creating oils, simple sugars and sugar polymers such as starch and cellulose as products of photosynthesis. We can then process these products into ethanol, biodiesel (diesel fuel made from vegetable oil or animal fat) or other fuels. Though biofuels often have faded into the background during periods with low gas prices, Bush adds, they become more popular with every gas crisis.

Though biodiesel is more common in Europe, ethanol is king in the U.S. Fuel ethanol certainly has its benefits: it adds oxygen to gas, leading to a cleaner burn that produces less pollution, and it increases octane.

Learning about biofuels in Brazil

The American Society for Biochemistry and Molecular Biology is playing a vital role in creating the next generation of biofuels. Last fall, the society co-sponsored a week-long advanced course aimed at inspiring interested graduate students and postdoctoral fellows to join the biofuels revolution. At a small resort in the lush coastal city of Ubatuba, Brazil, 40-odd international participants gathered to attend lectures and participate in intense roundtable discussions.

The aim wasn’t to have attendees listen to endless talks, says Bettie Sue Masters, past president of ASBMB and principle organizer of the school. “It was really interactive,” she says. In the daily roundtable sessions, participants had the chance to discuss their own work or research aspirations or to solicit lecturers’ career advice.

Besides being a terrific chance for young researchers to learn about this burgeoning field, it also proved to be a great way to forge a strong partnership between ASBMB and colleagues in the Brazilian Society for Biochemistry and Molecular Biology and the International Union of Biochemistry and Molecular Biology. These groups are planning to cosponsor future meetings, including one in the fall of 2012 on protein folding and protein-protein interactions.

“It was much better than we ever thought it would be – a valuable experience for everyone involved,” Masters says.

However, ethanol also has a number of drawbacks. Crops used most often to produce it can be finicky about where they’ll grow. For example, sugarcane, another common source for ethanol, thrives in Florida but not in Michigan, and corn needs rich, pampered soil and not rocky, arid land. Additionally, since the most common sources of ethanol also are food for people, it sets up a competition over the best land between food and fuel.

“It could lead to an unstable market,” says Dominique Loque, a research scientist at the Joint BioEnergy Institute in Emeryville, Calif. “Only rich people will be able to drive and eat.”

Consequently, many researchers have suggested gathering energy from the vegetative tissues of plants instead of the parts we use for food. Stems, branches, and leaves contain cellulose, a polymer of glucose in the cell wall that holds ample energy for conversion to biofuels. Indeed, potential energy in cellulose is often more than 10 times that available in starch from a given plant. Moreover, these plant organs are frequently a throw-away byproduct of the food industry, so conversion to biofuels could prevent waste.

However, notes Bush, switching from corn kernels to foliage isn’t so simple. Though researchers have actively worked on improving corn and other food plants for hundreds of years, the focus has been on the seed, not the greenery. As a result, about half of corn’s above-ground biomass is in its ears. If the new biofuel focus is the rest of the plant, Bush says, researchers better get cracking on making new energy crops, such as grasses— significantly bigger.

That’s one of his lab’s projects. With colleagues at Colorado State University and the International Rice Research Institute in the Philippines, Bush is working on identifying genes that are responsible for making the most of rice’s green biomass. Rice is a good model for improving other grasses’ biomass, he says, since the genomes of all 20 rice varieties have been sequenced. Bush notes that in this work, rice is an experimental model and not a target as a biofuel crop.

“A long time ago, many breeders learned that if you see a very large plant, 50 percent larger than the others, to just ignore it— they put most of their carbon into vegetative growth and have lower seed production,” he says. But those big plants are just what he and his colleagues are looking for. The researchers have spent many days walking through rice fields searching for the largest plants produced either through hybrid crosses or mutagenesis. Using modern deep sequencing approaches, Bush and his colleagues can then locate the gene responsible for the plants’ extraordinary size. The team is now close to identifying the first promising gene from that approach.

Bush’s lab also is working on another way to make more greenery through bypassing the feedback system that controls a plant’s photosynthesis rate. Leaves are the hotbed for photosynthesis, and as plants spin sunlight into sucrose, that product is transported to non-photosynthetic tissues in the plant’s vascular system. If production exceeds export, Bush explains, plants shut off photosynthesis until the sweet stuff can distribute to other parts of the plant through its vascular system. Using sugarbeet as a model system, he and his colleagues have engineered plants whose cells have a sucrose transport gene placed behind a constitutively active promoter. Consequently, the leaves are constantly pumping out sucrose— and thus, keeping low sucrose in the leaves and preventing negative feedback on photosynthesis. Over a season, he hypothesizes, this furious activity translates into significantly more biomass per plant.

Another drawback researchers will need to overcome before vegetation rules the biomass roost is that in most plants, energy-rich cellulose is bound up with significant amounts of lignin, the cell wall component that provides mechanical strength. Currently, biofuel producers separate cellulose from lignin with harsh, expensive chemicals and high temperatures. Several researchers, including Loque, are looking for ways to avoid these.

Loque explains that altering lignin content is tricky. Remove too little, and deriving cellulose remains difficult; remove too much, and the plant has no support to grow.

He and his colleagues currently are working on two strategies to surmount the lignin problem. In the first, the researchers are tinkering with where plants deposit lignin. Loque notes that the entire lignin pathway is known and highly conserved. By using promoters throughout the pathway that produce different expression of lignin genes relative to the native ones, the researchers have successfully reduced lignin in undesirable areas while keeping it in necessary places, such as the vessels plants use for nutrient transport.

“In the end, we got plants that look like wild-type, but contain much less lignin,” he says.

He and his team also are working on engineering plants that make weaker lignin through genetic modifications that insert ester or amide bonds into the native structure, which has only carbon-carbon or carbon-oxygen bonds. These weak links eventually could reduce the amount of chemicals and lessen the temperatures needed to pretreat cellulosic feedstocks.

Escaping from ethanol

Another drawback of fuel ethanol is that researchers have calculated that, in many cases, it’s actually an energy sink rather than a source; the amount of petroleum used to plow and fertilize a cornfield, then transport and process the corn before fermentation, often contains more energy than the resulting ethanol. It’s also tremendous waste of the carbon atoms plants work so hard to fix. Only two thirds of a feedstock’s carbons are used in ethanol production, explains Katie Dehesh, a professor of plant biology at the University of California, Davis. The other one-third ends up as food for the fermenting yeast and in the air as carbon dioxide.

  
Katie Dehesh, a professor of plant biology at the University of California, Davis, is coaxing oats to make more oil than starch.

A possible solution is coaxing plants to make more oil than starch. Indeed, many plants already produce significant quantities of oil; it’s what fills the frying vats for much-loved fast-food fries. However, using these food crops for fuel oil has the same competitive disadvantages as creating ethanol from corn. Additionally, Dehesh points out, oil is only a minor component of most plants’ seeds and is even less abundant in their vegetative parts.

She and her colleagues recently published new research that could offer a possible solution to this problem by redirecting carbon flux toward oils and away from carbohydrates. The researchers used oat as their model organism, since this grain is a rare example of a plant that produces significant amounts of oil in its endosperm at the cost of carbohydrates. Using two varieties of oats— one that produced much more oil than the other— Dehesh’s team compared gene activity between the plants during seed development. Surprisingly, the fatty acid pathway that they expected to see upregulated in the high oil producer was actually the same between the two plants. However, the researchers found a variety of differences in the cofactors involved in respiratory metabolism. These cofactors, says Dehesh, appear to be the answer for determining carbon flux.

“I strongly believe that modification of these specific cofactors will provide us with the global key for conversion of starch to oil in any organism,” she says. In principle, she adds, there’s no need to switch starch for oil in seeds. Rather, genetic engineering could put the activity of these key cofactors in a plant’s vegetative tissues, or even in algae or bacteria, changing their metabolisms to spit out more oil.

James Liao, chancellor’s professor and vice-chairman of the department of chemical and biomolecular engineering at the University of California, Los Angeles, also is working on moving away from ethanol by using synthetic biology to engineer bacteria that churn out longer-chain alcohols with significantly higher energy density.

  
James Liao of the University of California, Los Angeles has engineered photosynthetic cyanobacteria that produce a variety of higher alcohols. Photo credit: Yixin Huo and Xiaoqian Li.

Using E. coli as their model organism, Liao and his colleagues leaned on this organism’s native amino acid biosynthesis pathways pathways to create starter molecules for various alcohols. They then strung together genes from various other organisms, including Sacchromyces, Lactococcus and Clostridium, for enzymes to convert these molecules into the desired product. Using this method, the researchers engineered E. coli that produced a variety of higher alcohols, including isobutanol, 1-butanol, 3-methyl-1-butanol, and 2-methyl-1-butanol, from glucose.

Not ones to rest on their laurels, Liao’s team followed this research up with another paper, published the next year, that used parts of the same pathway in photosynthetic cyanobacteria. The resulting organism produces isobutyraldehyde and isobutanol by pulling carbon directly from carbon dioxide in air.

In a recent paper, Liao’s lab detailed their synthesis of E. coli that produce alcohols from protein – thus far, an unutilized feedstock – by redirecting this organism’s metabolic flow of nitrogen.

“We like to keep pushing things further and further,” he says.

Jay Keasling, a professor in the departments of chemical and biomolecular engineering and bioengineering at the University of California, Berkeley, also is harnessing the power of synthetic biology for biofuels, both higher-chain alcohols and biodiesel from fatty acids.

In one recent paper, Keasling and his colleagues engineered yeast that make n-butanol, a far cry from the ethanol this organism usually makes. Rather than rely on the amino acid biosynthesis pathway that Liao’s team used, the researchers instead modified the acetyl-CoA pathway using genes from five other organisms. The team mixed combinations of individual genes, eventually producing seven different modified strains. One of these successfully produced significant quantities of n-butanol. This year, Keasling’s former postdoctoral fellow Michelle Chang, now an assistant professor of chemistry at University of California, Berkeley, significantly improved these yields with some of these same non-native components in E. coli.

 
A protein-rich algal species from James Liao's lab. Credit: Hidevaldo Machado and Yi-Xin Huo from Liao group.

Seeking to pack even more energy into their fuel molecules, Keasling’s group engineered another set of bacteria to generate biodiesel using a reaction similar to how biodiesel enthusiasts make their own homebrew. First, the researchers tricked E. coli into overproducing the fatty acids that make up its membrane, adding in a plant gene that prevented these hydrocarbons from becoming part of the phospholipid bilayer. A series of non-native genes attached ethanol to the structure, esterifying it much like a home biodiesel maker would. The resulting fuel can be skimmed off the top of the tank and go directly into a diesel engine, Keasling says.

Taking the research one step further, he made another tweak in these bacteria that allowed them to digest hemicellulose, using it as a feedstock for biodiesel production.

Keasling notes that it’s still very early times in the biofuel field. His and other academic labs energetically continue to churn out fresh ideas and research, which fuel companies – from big giants to tiny startups – are eyeing with interest. One of these ideas, he says, might eventually end up in the engine of your car.

“We’re fortunate, because there’s a lot of interest right now,” he says. “It’s a really great time to be working in this area.”

 

Christen Brownlee (christenbrownlee@gmail.com) is a freelance science writer based in Baltimore, Md.


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