Researchers with the US Department of Energy’s Joint BioEnergy Institute (JBEI) have identified a potential new advanced biofuel that could replace standard diesel fuel and could be produced in the US.
A JBEI research team engineered strains of two microbes, a bacteria and a yeast, to produce a precursor to bisabolane, a member of the terpene class of chemical compounds that are found in plants and used in fragrances and flavourings. Preliminary tests showed bisabolane to be a promising biosynthetic alternative to D2 diesel fuel.
“This is the first report of bisabolane as a biosynthetic alternative to D2 diesel, and the first microbial overproduction of bisabolene in Escherichia coli and Saccharomyces cerevisiae,” says Taek Soon Lee, who directs JBEI’s metabolic engineering programme and is a project scientist with Lawrence Berkeley National Laboratory (Berkeley Lab)’s Physical Biosciences Division. “This work is also a proof-of-principle for advanced biofuels research in that we’ve shown that we can design a biofuel target, evaluate this fuel target, and produce the fuel with microbes that we’ve engineered.”
Lee is the corresponding author of a paper reporting this research in the journal Nature Communications entitled “Identification and microbial production of a terpene-based advanced biofuel.” Co-authors were Pamela Peralta-Yahya, Mario Ouellet, Rossana Chan, Aindrila Mukhopadhyay and Jay Keasling.
JBEI is one of three bioenergy research centres established by the DOE’s Office of Science in 2007. Researchers at JBEI are pursuing the fundamental science needed to make production of advanced biofuels cost-effective on a national scale. One of the avenues being explored is sesquiterpenes, terpene compounds that contain 15 carbon atoms (diesel fuel typically contains 10 to 24 carbon atoms).
“Sesquiterpenes have high-energy content and physical-chemical properties similar to diesel and jet fuels,” Lee says. “Although plants are the natural source of terpene compounds, engineered microbial platforms would be the most convenient and cost-effective approach for large-scale production of advanced biofuels.”
In earlier work, Lee and his group engineered a new mevalonate pathway (a metabolic reaction critical to biosynthesis) in both E. coli and S. cerevisiae that resulted in these two microorganisms over-producing a chemical compound called farnesyl diphosphate (FPP), which can be treated with enzymes to synthesize a desired terpene. In this latest work, Lee and his group used that mevalonate pathway to create bisabolene, which is a precursor to bisabolane.
“We proposed that the generality of the microbial FPP overproduction platforms would allow for the biosynthesis of sesquiterpenes,” Lee says. “Through multiple rounds of large-scale preparation in shake flasks, we were able to prepare approximately 20ml of biosynthetic bisabolene, which we then hydrogenated to produce bisabolane.”
“Bisabolane has properties almost identical to D2 diesel but its branched and cyclic chemical structure gives it much lower freezing and cloud points, which should be advantageous for use as a fuel,” Lee says. He and his colleagues are now preparing to make gallons of bisabolane for tests in diesel engines, using the new fermentation facilities at Berkeley Lab’s Advanced Biofuels Process Demonstration Unit.
“Once the complete fuel properties of hydrogenated biosynthetic bisabolene can be obtained, we’ll be able to do an economic analysis that takes into consideration production variables such as the cost and type of feedstock, biomass depolymerisation method, and the microbial yield of biofuel,” Lee says. “We will also be able to estimate the impact of byproducts present in the hydrogenated commercial bisabolene, such as farnesane and aromatized bisabolene.”
Ultimately, Lee and his colleagues would like to replace the chemical processing step of bisabolene hydrogenation with an alkene reductase enzyme engineered into the E.coli and yeast so that all of the chemistry is performed within the microbes. “Enzymatic hydrogenation of this type of molecule is a very challenging project and will be a long-term goal,” Lee says. “Our near-term goal is to develop strains of E.coli and yeast for use in commercial-scale fermenters. Also, we will be investigating the use of sugars from biomass as a source of carbon for producing bisabolene.”
JBEI is a scientific partnership led by Lawrence Berkeley National Laboratory (Berkeley Lab) and including the Sandia National Laboratories, the UC campuses of Berkeley and Davis, the Carnegie Institution for Science, and the Lawrence Livermore National Laboratory.
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