A team of researchers with the US Department of Energy (DOE)’s Joint BioEnergy Institute (JBEI) has engineered the bacterium Ralstonia eutropha—a microbe now used to produce biodegradable plastic—for the production of fatty acid-derived, diesel-range methyl ketones. A paper on their work is published in the journal Applied and Environmental Microbiology.
R. eutropha is a chemolithoautotroph (an organism that obtains its nutrition through the oxidation of non-organic compounds or other chemical processes) that can grow with organic substrates or H2 and CO2 under aerobic conditions. Under conditions of nutrient imbalance, R. eutropha produces “copious” amounts of polyhydroxybutyrate (PHB). Its ability to utilize CO2 as a sole carbon source renders it an interesting new candidate host for the production of renewable liquid transportation fuels, the team noted in their paper.
We’ve shown that the bacterium Ralstonia eutropha growing with carbon dioxide and hydrogen gas is able to generate significant quantities of diesel-range methyl ketones. This holds the promise of making carbon-neutral biofuels using non-photosynthetic, carbon-dioxide fixing bacteria as a less resource-intensive alternative to making these biofuels from cellulosic biomass.—Harry Beller, corresponding author
Beller, who directs the Biofuels Pathways department for JBEI’s Fuels Synthesis Division, and also is a Senior Scientist with Berkeley Lab’s Earth Sciences Division, led a previous study in which genetic engineering was used to develop a strain of the bacterium Escherichia coli that made methyl ketone compounds from the glucose in cellulosic biomass. (Earlier post.)
Methyl ketones are naturally occurring aliphatic compounds now used in fragrances and flavorings. Beller and his JBEI colleagues have demonstrated that methyl ketones also have high diesel fuel ratings (cetane numbers), making them strong candidates as advanced biofuels.
We’ve shown that, with the same set of genetic modifications, R. eutropha and E. coli can make comparable amounts of methyl ketones, but R. eutropha is making the ketones from carbon dioxide while E. coli is making them from glucose. This shows that the methyl ketone pathway that we’ve designed is versatile and able to function well in bacterial hosts with substantially different metabolic lifestyles.—Harry Beller
Modifications engineered in R. eutropha included overexpression of a cytoplasmic version of the TesA thioesterase, which led to a substantial (>150-fold) increase in fatty acid titer under certain conditions. In addition, deletion of two putative β-oxidation operons and heterologous expression of three genes (the acyl coenzyme A oxidase gene from Micrococcus luteus and fadB and fadM from Escherichia coli) led to the production of 50 to 65 mg/liter of diesel-range methyl ketones under heterotrophic growth conditions and 50 to 180 mg/liter under chemolithoautotrophic growth conditions (with CO2 and H2 as the sole carbon source and electron donor, respectively).
The induction of the methyl ketone pathway diverted substantial carbon flux away from PHB biosynthesis and appeared to enhance carbon flux through the pathway for biosynthesis of fatty acids, which are the precursors of methyl ketones, the team found.
Since our engineered strains of R. eutropha can use fixed carbon dioxide to make methyl ketones, its biofuels don’t require many of the steps needed to convert cellulosic biomass into fuels, such as growing and harvesting the biofuel crop, digesting the lignocellulosic biomass, and enzymatically saccharifying the digested biomass to produce fermentable sugars. The resources needed for these steps could therefore be eliminated if R. eutropha were used to make biofuels directly from carbon dioxide.—Harry Beller
The research was funded through DOE’s Advanced Research Projects Agency-Energy (ARPA-E) program.
Jana Müller, Daniel MacEachran, Helcio Burd, Noppadon Sathitsuksanoh, Changhao Bi, Yi-Chun Yeh, Taek Soon Lee, Nathan J. Hillson, Swapnil R. Chhabra, Steven W. Singer, and Harry R. Beller (2013) Engineering of Ralstonia eutropha H16 for Autotrophic and Heterotrophic Production of Methyl Ketones. Appl. Environ. Microbiol. 79:14 4433-4439 doi: 10.1128/AEM.00973-13