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JBEI scientists identify new biosynthetic alternative to diesel and engineer microbes to produce its precursor

Bisabolene-ncomms1494-f2
Chemical structures of fuels. Bisabolane (2); Hexadecane (3), a representative molecule for diesel; farnesane (4); and methyl palmiate (5), a representative molecule for fatty acid methyl esters. Source: Peralta-Yahya et al. Click to enlarge.

A team from the US Department of Energy (DOE) Joint BioEnergy Institute (JBEI) has identified a novel biosynthetic alternative to #2 diesel—bisabolane—and has engineered microbial platforms for higher volume production of its immediate precursor, bisabolene. They then chemically hydrogenated the biosynthetic bisabolene into bisabolane.

In a paper published in Nature Communications, they suggest that their work presents a framework for the identification of novel terpene-based advanced biofuels and the rapid engineering of microbial farnesyl diphosphate-overproducing platforms for the production of biofuels.

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. 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.

—Taek Soon Lee, corresponding author and director of JBEI’s metabolic engineering program

Co-authoring this paper were Pamela Peralta-Yahya, Mario Ouellet, Rossana Chan, Aindrila Mukhopadhyay and Jay Keasling.

JBEI is one of three Bioenergy Research Centers 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 a high-energy content and physicochemical properties similar to diesel and jet fuels. 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.

—Taek Soon Lee

Terpenes, which are traditionally used as fragrances and flavors, have the potential to serve as advanced biofuel precursors, the team notes. (Earlier post.) For example, Amyris is pursuing the fully reduced form of the linear terpene farnesene as an alternative biosynthetic diesel in the market. Terpenes are biosynthesized from the C5 universal precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Prenyltransferases assemble IPP and DMAPP into linear prenyl diphosphate precursors, such as farnesyl diphosphate (FPP, C15), which are rearranged by terpene synthases into a variety of different terpenes, such as sesquiterpenes (C15), they explain.

To our knowledge, there are no reports of bisabolane as a biosynthetic alternative to D2 diesel.
—Peralta-Yahya et al.

Although plants are natural sources of terpenes, engineered microbial platforms may be the most convenient and cost-effective approach for large-scale production of terpene-based advanced biofuels, they suggest.

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 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, the precursor to bisabolane.

We propose that the generality of the microbial FPP overproduction platforms should allow for the biosynthesis of sesquiterpenes to serve as advanced biofuel precursors by introduction of a sesquiterpene synthase producing the desired compound. The viability of this strategy hinges on identifying a sesquiterpene biofuel precursor, and a sesquiterpene synthase able to microbially produce the desired compound in high titers. Adding to the complexity of this proposition is the limited commercial availability of sesquiterpenes to derivatize and test for fuel properties, and the fact that most sesquiterpene synthases are of plant origin and thus potentially difficult to express in microbes.

Here we report the identification of a novel biosynthetic alternative to D2 diesel fuel, bisabolane, and the engineering of microbial platforms for the overproduction of its immediate precursor, bisabolene. Hydrogenation of commercially available bisabolene led to the identification of bisabolane as a biosynthetic alternative to D2 diesel. Then, we engineered microbial platforms to produce bisabolene (1), the immediate precursor to bisabolane (2).

First, using microbial platforms for the overproduction of FPP, we screened for sesquiterpene synthases able to convert FPP into bisabolene in high titers. Second, we optimized the mevalonate pathway in E. coli for increased bisabolene production by codon-optimization of heterologous pathway genes and introduction of extra promoters to increase expression of key enzymes in the pathway. Third, we produced bisabolene in S. cerevisiae, a widely used platform for the production of ethanol. Finally, we demonstrated the conversion of biosynthetic bisabolene into bisabolane using chemical hydrogenation. To our knowledge, this is the first report of a reduced mono-cyclic sesquiterpene, bisabolane, as a biosynthetic alternative to D2 diesel, and the first microbial overproduction of bisabolene in E. coil and S. cerevisiae at titers over 900 mgl−1 in shake flasks.

—Peralta-Yahya et al.

Bisabolene-ncomms1494-f1
Bisabolane from chemical hydrogenation of microbially produced bisabolene. The engineered microbe (yellow box) converts simple sugars into acetyl-CoA via primary metabolism. A combination of metabolic engineering of the heterologous mevalonate pathway to convert acetyl-CoA into FPP and enzyme screening to identify a terpene synthase to convert FPP into bisabolene (1) is used to produce bisabolene at high titers. Chemical hydrogenation of biosynthetic bisabolene leads to bisabolane (2), a biosynthetic alternative to D2 diesel. Source: Peralta-Yahya et al. Click to enlarge.

Bisabolane as a biosynthetic alternative to D2 diesel fuel. When they began this work, Lee and his colleagues did not know whether bisabolane could be used as a biofuel, but they targeted it on the basis of its chemical structure. D2 diesel is a mixture of linear, branched, and cyclic alkanes with an average carbon length of 16. Among other properties, biosynthetic alternatives to D2 diesel should have a similar cetane number and comparable cold properties.

Bisabolene-ncomms1494-f3
Gas chromatography analysis of commercial bisabolene and hydrogenated commercial bisabolene. Source: Peralta-Yahya et al. Click to enlarge.

Bisabolane has a carbon length (C15), close to the average carbon length of diesel (C16). Further, the branching found in the linear portion of bisabolane may result in beneficial fuel cold properties. Finally, the ring portion of bisabolane increases the density of the fuel, which will increase the energy density per volume of fuel.

Their first step was to perform fuel property tests on commercially available bisabolene, which comes as part of a mixture of compounds. The researchers then used biosynthesis to extract pure biosynthetic bisabolene from microbial cultures for hydrogenation into bisabolane. Subsequent fuel property tests on the bisabolane were again promising.

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. Once we confirmed that bisabolane could be a good fuel, we designed a mevalonate pathway to produce the precursor, bisabolene. This was basically the same platform used to produce the anti-malarial drug artemisinin except that we introduced a terpene synthase and further engineered the pathway to improve the bisabolene yield both in E. coli and yeast.

—Taek Soon Lee

table
Click to enlarge.

Economics. Lee and his colleagues are now preparing to make gallons of bisabolane for tests in actual diesel engines, using the new fermentation facilities at Berkeley Lab’s Advanced Biofuels Process Demonstration Unit (ABPDU). The ABPDU is a 15,000 square-foot facility

in Emeryville, California, designed to help expedite the commercialization of advanced next-generation biofuels by providing industry-scale test beds for discoveries made in the laboratory.

An economic analysis on the production of bisabolene takes into consideration many variables including the cost and type of feedstock, biomass depolymerization method, and the microbial yield of biofuel. Assuming a break-even price of sugar at the mill to be close to US $0.10/lb, which is lower than the current volatile spot price but closer to the long-term nominal price of the commodity, we can perform a rough calculation of the theoretical cost of bisabolene. On the basis of that number, the raw material cost of bisabolene production would be, ignoring non-sugar costs, approximately $0.88 per kg of bisabolene.

Assuming raw material costs to be only ~50% of the final cost, this would imply a final cost of $1.76 per kg, or $5.73 per gal of bisabolene on the basis of data for ethanol production. At an estimated ~$6 per gal, bisabolene is currently more expensive than current D2 diesel. However, it is still promising to investigate this emerging alternative biofuel when considering its superior properties and renewable nature.

—Peralta-Yahya et al.

In this work, the authors note, they resorted to chemical hydrogenation of bisabolene into the final product bisabolane. While this is industrially feasible, the ultimate goal is the complete microbial production of bisabolane—i.e., replacing 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.

The team will also be investigating the use of sugars from biomass as a source of carbon for bisabolene.

Resources

  • Pamela P. Peralta-Yahya, Mario Ouellet, Rossana Chan, Aindrila Mukhopadhyay, Jay D. Keasling & Taek Soon Lee (2011) Identification and microbial production of a terpene-based advanced biofuel. Nature Communications 2, Article number: 483 DOI: 10.1038/ncomms1494

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