(191bg) Photocatalytic Production of a Jet Fuel Precursor Limonene

With the increased interest in developing more sustainable sources of fuels and chemicals to limit our carbon footprint, cyanobacteria are attractive platform strains for metabolic engineering efforts. Terpenoids are a class of chemicals that have received considerable attention for use as renewable drop-in biofuels ¹; they are attractive for fuel applications because they have a greater energy density and lower hygroscopicity than short chain alcohols, and the structural diversity to mimic the alkanes and aromatics of desired chain lengths for gasoline, diesel and jet fuels. Importantly, this means that terpenoids can be blended with petroleum-based fuels, and are compatible with existing transport fuel infrastructure. Limonene (C₁₀H₁₆) is a monocyclic terpenoid that has properties suitable as a precursor for next generation jet fuels, and as a fuel additive that enhances cold-weather performance ^2,3. Currently, limonene is extracted from citrus fruit peel as a byproduct of juice processing, and used in the flavor/fragrance industry and as an environmentally-friendly solvent and degreaser. The worldwide production of limonene in 2013 was over 70,000 tons ⁴. However, the extraction methods from citrus peel are energy-intensive, and the seasonal and environmental factors that affect citrus fruit crops make the pricing and supply of limonene extremely volatile. The largest potential market for limonene is for jet fuel, which has a current demand of 1.53 million barrels per day in the United State (U.S. Energy Information Administration http://www.eia.gov/forecasts/steo/report/us_oil.cfm); which translates to an almost $60 billion market for jet fuel alone. We have chosen to engineer the fast growing cyanobacterium, Synechococcus sp. PCC 7002 (Synechococcus hereafter) as a platform for photosynthetic limonene production. Synechococcus has been identified as an excellent platform for biotechnological applications because of its fast growth and ability to thrive in salt water and changing light conditions. We have engineered Synechococcus to yield 4 mg limonene per L culture through heterologous expression of the Mentha spicata L-limonene synthase ⁵. To date, our limonene-producing (LS) strain has the highest reported limonene yield among photosynthetic engineered organisms with comparable genetic manipulation. Further, we have ensured a high purity of limonene through careful selection of the M. spicata LS, which has high product specificity ^6,7 We have also overcome another important challenge of working with cyanobacteria: stable and homoplastic integration of the LS gene into the Synechococcus genome. Cyanobacteria have several to hundreds of copies of their chromosome depending on the species and/or growth condition ⁸, therefore, a long selection and outgrowth period are required to ensure that all copies of the chromosome are homogenous. The next step of our research is to use ¹³C-MFA to help develop a rationale engineering approach to further increase limonene titers. We will discuss the results of our flux analysis and compare how the MEP pathway in cyanobacteria differs from heterotrophic bacteria.

References

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2 Renninger, N. S., Ryder, J. A. & Fisher, K. J. Jet fuel compositions and methods of making and using same. (2011).

3 Tracy, N. I., Chen, D., Crunkleton, D. W. & Price, G. L. Hydrogenated monoterpenes as diesel fuel additives. Fuel 88, 2238-2240, doi:10.1016/j.fuel.2009.02.002 (2009).

4 Ciriminna, R., Lomeli-Rodriguez, M., Cara, P. D., Lopez-Sanchez, J. A. & Pagliaro, M. Limonene: a versatile chemical of the bioeconomy. Chemical Communications 50, 15288-15296, doi:10.1039/c4cc06147k (2014).

5 Davies, F. K., Work, V. H., Beliaev, A. S. & Posewitz, M. C. Engineering limonene and bisabolene production in wild type and a glycogen-deficient mutant of Synechococcus sp. PCC 7002. Front Bioeng Biotechnol., doi:10.3389/fbioe.2014.00021 (2014).

6 Alonso, W. R., Rajaonarivony, J. I. M., Gershenzon, J. & Croteau, R. Purification of 4S-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha x piperita) and spearmint (Mentha spicata). Journal of Biological Chemistry 267, 7582-7587 (1992).

7 Rajaonarivony, J. I. M., Gershenzon, J. & Croteau, R. Characterization and mechanism of (4S)-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha X piperita). Archives of Biochemistry and Biophysics 296, 49-57, doi:10.1016/0003-9861(92)90543-6 (1992).

8 Griese, M., Lange, C. & Soppa, J. Ploidy in cyanobacteria. FEMS Microbiology Letters 323, 124-131, doi:10.1111/j.1574-6968.2011.02368.x (2011).