JBEI researchers engineer E. Coli to produce gasoline, diesel and jet fuel substitutes or precursors directly from switchgrass without external enzyme assistance
Researchers with the US Department of Energy (DOE)’s Joint BioEnergy Institute (JBEI) have engineered the first strains of Escherichia coli bacteria that can utilize both the cellulose and hemicellulose fractions of switchgrass pre-treated with ionic liquids to produce gasoline, diesel and jet fuel substitutes or precursors without any assistance from enzyme additives.
While this is not the first demonstration of E. coli producing fuel molecules from sugars, it is the first demonstration of E. coli producing molecules suited for all three major forms of transportation fuels. Furthermore, it was done using switchgrass, which is among the most highly touted of the potential feedstocks for advanced biofuels.
|Our goal has been to put as much chemistry as we can into microbes.|
Jay Keasling, CEO of JBEI who also holds appointments with the Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California (UC) Berkley, is the corresponding author of an open access paper describing the work in the Proceedings of the National Academy of Sciences (PNAS).
The biggest roadblock to advanced cellulosic biofuels is reducing the cost of production. 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 lignin. Once extracted, these complex sugars must first be converted (hydrolyzed) into simple sugars and then synthesized into fuels.
One approach to reducing the costs of advanced biofuel production from cellulosic biomass is to engineer a single microorganism to both digest plant biomass and produce hydrocarbons that have the properties of petrochemical fuels. Such an organism would require pathways for hydrocarbon production and the capacity to secrete sufficient enzymes to efficiently hydrolyze cellulose and hemicellulose. To demonstrate how one might engineer and coordinate all of the necessary components for a biomass-degrading, hydrocarbon-producing microorganism, we engineered a microorganism naïve to both processes, Escherichia coli, to grow using both the cellulose and hemicellulose fractions of several types of plant biomass pretreated with ionic liquids. Our engineered strains express cellulase, xylanase, beta-glucosidase, and xylobiosidase enzymes under control of native E. coli promoters selected to optimize growth on model cellulosic and hemicellulosic substrates.
Furthermore, our strains grow using either the cellulose or hemicellulose components of ionic liquid-pretreated biomass or on both components when combined as a co-culture. Both cellulolytic and hemicellulolytic strains were further engineered with three biofuel synthesis pathways to demonstrate the production of fuel substitutes or precursors suitable for gasoline, diesel, and jet engines directly from ionic liquid-treated switchgrass without externally supplied hydrolase enzymes.
This demonstration represents a major advance toward realizing a consolidated bioprocess. With improvements in both biofuel synthesis pathways and biomass digestion capabilities, our approach could provide an economical route to production of advanced biofuels.—Bokinsky et al.
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 the fuel substitute or precursor molecules.
The JEBI team chose to implement pathways that produce alcohols, linear hydrocarbons, or branched-chain hydrocarbons to test the integration of the biomass-consumption pathways with the “extensive” biosynthesis capabilities of E. coli. The team chose:
Biodiesel. Biodiesel can be made by E. coli in vivo in the form of fatty-acid ethyl esters (FAEE). They encoded a six-gene FAEE production pathway on a single plasmid and introduced the construct into a strain of E. coli. Using a co-culture of two strains grown in minimal medium containing 5.5% w∕vol IL-treated switchgrass, they produced 71 ± 43 mg∕L of FAEE. This corresponds to 80% of the estimated yield obtainable with this pathway from the amount of sugars anticipated to be released from 5.5% switchgrass by the Cel and Xyn10B enzymes.
Butanol. Butanol has been proposed as a gasoline replacement because it is fully compatible with existing internal combustion engines. Based in part on previous work, they constructed a heterologous butanol pathway encoded on a single plasmid. A co-culture yielded 28 ± 5 mg∕L butanol from defined rich medium containing 3.3% w∕vol IL-treated switchgrass. A control strain produced 8 ± 2 mg∕L butanol from pretreated switchgrass.
Pinene. The monoterpene pinene is an immediate chemical precursor to a potential jet fuel. The pinene synthesis pathway was encoded on a single plasmid. A co-culture yielded 1.7 ± 0.6 mg∕L pinene from pretreated switchgrass.
The pre-treatment of the switchgrass with ionic liquids was essential to this demonstration, according to Gregory Bokinsky, a post-doctoral researcher with JBEI’s synthetic biology group and lead author of the PNAS paper.
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.—Gregory Bokinsky
The JBEI researchers also attribute the success of this work to the “unparalleled genetic and metabolic tractability” of E. coli, which over the years has been engineered to produce a wide range of chemical products. However, the researchers believe that the techniques used in this demonstration should also be readily adapted to other microbes. This would open the door to the production of advanced biofuels from lignocellulosic feedstocks that are ecologically and economically appropriate to grow and harvest anywhere in the world. For the JBEI researchers, however, the next step is to increase the yields of the fuels they can synthesize from switchgrass.
We already have hydrocarbon fuel production pathways that give far better yields than what we obtained with this demonstration. And these other pathways are very likely to be compatible with the biomass-consumption pathways we’ve engineered into our E. coli. However, we need to find enzymes that can both digest more of the ionic liquid pre-treated biomass and be secreted by E coli. We also need to work on optimizing the ionic liquid pre-treatment steps to yield biomass that is even easier for the microbes to digest.—Gregory Bokinsky
Co-authoring the PNAS paper with Keasling and Bokinsky were Pamela Peralta-Yahya, Anthe George, Bradley Holmes, Eric Steen, Jeffrey Dietrich, Taek Soon Lee, Danielle Tullman-Ercek, Christopher Voigt and Blake Simmons.
This research was supported in part by the DOE Office of Science and a UC Discovery Grant.
Gregory Bokinsky, Pamela P. Peralta-Yahya, Anthe George, Bradley M. Holmes, Eric J. Steen, Jeffrey Dietrich, Taek Soon Lee, Danielle Tullman-Ercek, Christopher A. Voigt, Blake A. Simmons, and Jay D. Keasling (2011) Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli. PNAS doi: 10.1073/pnas.1106958108