E. Coli Bacteria Engineered to Make Transport Fuels

Researchers with the U.S. Department of Energy's Joint BioEnergy Institute have engineered the first strains of Escherichia coli bacteria to produce fuel substitute or precursor molecules suitable for gasoline, diesel and jet fuel.

While this is not the first demonstration of E. coli producing gasoline and diesel from sugars, it is the first demonstration of E. coli producing all three forms of transportation fuels.

E coli bacteria (Photo courtesy Berkeley Lab)

And, it was done, without any help from enzyme additives, using switchgrass, which is among the most promising of the potential feedstocks for advanced biofuels.

"This work shows that we can reduce one of the most expensive parts of the biofuel production process, the addition of enzymes to depolymerize cellulose and hemicellulose into fermentable sugars," says Jay Keasling, head of the Joint BioEnergy Institute and leader of this research.

"This will enable us to reduce fuel production costs by consolidating two steps - depolymerizing cellulose and hemicellulose into sugars, and fermenting the sugars into fuels - into a single step or one pot operation," explained Keasling.

Keasling, who also holds appointments with the Lawrence Berkeley National Laboratory and the University of California, Berkley, is the corresponding author of a paper in the current issue of the journal "Proceedings of the National Academy of Sciences" that describes this work.

Advanced biofuels made from the lignocellulosic biomass of non-food crops and agricultural waste are widely believed to represent the best source of renewable liquid transportation fuels.

Unlike ethanol, which in the United States is produced from corn starch, these advanced biofuels can replace gasoline on a gallon-for-gallon basis, and they can be used in today's engines and infrastructures.

The biggest problem in commercializing biofuels has been bringing down the cost of producing these fuels so that they are economically competitive with petroleum products.

Unlike the simple sugars in corn grain, the cellulose and hemicellulose in plant biomass are difficult to extract in part because they are embedded in a tough woody material called lignin.

Once extracted, these complex sugars must first be converted or hydrolyzed into simple sugars and then synthesized into fuels.

Gregory Bokinsky in his lab at the Joint BioEnergy Institute (Photo by Roy Kaltschmidt, Berkeley Lab)

At the Joint BioEnergy Institute, one approach has been to first pre-treat the biomass with the ionic liquid, molten salt, to dissolve it. Then the scientists engineered a single microorganism that can both digest the dissolved biomass and produce hydrocarbons that have the properties of petrochemical fuels.

"The magic is in the ionic liquid pre-treatment," says Gregory Bokinsky, a post-doctoral researcher with JBEI's synthetic biology group and lead author of the paper.

"I suspect you could use ionic liquid pre-treatment on any plant biomass and make it readily digestible by microbes," Bokinsky said. "For us it was the combination of biomass from the ionic liquid pretreatment with the engineered E. coli that enabled our success."

E. coli bacteria normally cannot grow on switchgrass, but JBEI researchers engineered strains of the bacteria to express several enzymes that enable them to digest cellulose and hemicellulose and use one or the other for growth.

These strains of E. coli, which can be combined as co-cultures on a sample of switchgrass, were further engineered with three metabolic pathways that enabled the bacteria to produce fuel substitute or precursor molecules suitable for gasoline, diesel and jet engines.

The techniques used in this switchgrass demonstration can be readily adapted to other microbes, the researchers say, opening the door to the production of advanced biofuels from feedstocks that are ecologically and economically appropriate to grow and harvest anywhere in the world.

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