Strains of E. coli bacteria were engineered to digest switchgrass biomass and synthesize its sugars into gasoline, diesel, and jet fuel.
Researchers with DOE’s Joint BioEnergy Institute (JBEI) have engineered the first strains of Escherichia coli bacteria that can digest switchgrass biomass and synthesize its sugars into all three of those transportation fuels. The microbes are even able to do this without any help from enzyme additives.

“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, CEO of JBEI and leader of this research. Keasling also holds appointments with Berkeley Lab and UC Berkley. “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.”

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 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.

At JBEI, a DOE Bioenergy Research Center led by Berkeley Lab, one approach has been to pre-treat the biomass with an ionic liquid (molten salt) to dissolve it, then engineer a single microorganism that can both digest the dissolved biomass and produce hydrocarbons that have the properties of petrochemical fuels.

For the three fuels E. coli engineered at JBEI, cellulose and hemicellulose are hydrolyzed by cellulase and hemicellulose enzymes (blue) into oligosaccharides, which are further hydrolyzed by β-glucosidase enzymes (red) into monosaccharides that can be metabolized into biofuels.
“Our goal has been to put as much chemistry as we can into microbes,” Keasling says. “For advanced biofuels this requires a microbe with pathways for hydrocarbon production and the biomass-degrading capacity to secrete enzymes that efficiently hydrolyze cellulose and hemicellulose. We’ve now been able to engineer strains of Escherichia coli that can utilize both the cellulose and hemicellulose fractions of switchgrass that’s been pre-treated with ionic liquids.”

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 cellulolytic and hemicellulolytic 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 E. coli to produce fuel substitute or precursor molecules suitable for gasoline, diesel, and jet engines. It is the first demonstration of E. coli producing all three forms of transportation fuels.

Gregory Bokinsky, a post-doctoral researcher with JBEI’s synthetic biology group, explains that the pre-treatment of the switchgrass with ionic liquids was essential to this demonstration.

“The magic is in the ionic liquid pre-treatment,” Bokinsky says. “If properly optimized, I suspect you could use ionic liquid pre-treatment on any plant biomass and make it readily digestible by microbes. For us it was the combination of biomass from the ionic liquid pretreatment with the engineered E. coli that enabled our success.”

(Berkeley Lab)

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