Joint BioEnergy Institute team engineers E. coli to overproduce diesel-range methyl ketones; may be appropriate for blendstock
15 March 2012
Researchers at the US Department of Energy’s (DOE) Joint BioEnergy Institute (JBEI) have engineered Escherichia coli bacteria to overproduce saturated and monounsaturated aliphatic methyl ketones in the C11 to C15 (diesel) range from glucose. In subsequent tests, these methyl ketones yielded high cetane numbers, making them promising candidates for the production of advanced biofuels or blendstocks.
The team, led by Dr. Harry Beller, found that it was possible to increase the methyl ketone titer production of E. coli more than 4,500-fold relative to that of a fatty acid-overproducing E. coli strain by using a relatively small number of genetic modifications. Methyl ketone titers in the best producing strains were in the range of 380 mg/L.
A paper describing this work was published in the journal Applied and Environmental Microbiology. Co-authoring this paper were Ee-Been Goh, who is the first author on the paper, plus Edward Baidoo and Jay Keasling.
Aliphatic methyl ketones are naturally occurring compounds first discovered more than a century ago and that have been commonly found in microorganisms, plants, insects, and mammalian cells. They play a variety of roles, including acting as pheromones and natural insecticides in plants, or providing scents in essential oils and flavoring in cheese and other dairy products.
In previous research, Beller and his colleagues engineered E. coli to synthesize from fatty acids long-chain alkene hydrocarbons that can be turned into diesel fuel. In those studies, said Beller, the team noticed that bacteria engineered to produce unnaturally high levels of fatty acids also produced some methyl ketones.
When we tested the cetane numbers of these ketones and saw that they were quite favorable, we were prompted to look more closely at developing methyl ketones as biofuels.—Harry Beller
Although native E. coli make virtually undetectable quantities of methyl ketones, Beller and his colleagues were able to overcome this deficiency using the same tools of synthetic biology they used to engineer high fatty acid-producing E.coli. They first modified specific steps in β-oxidation, the metabolic pathway that E. coli uses to break down fatty acids, and then increased the expression of a native E. coli protein called FadM. These two modifications combined to greatly enhance the production of methyl ketones.
Beller and his colleagues tested two methyl ketones—undecanone and tridecanone—for cetane numbers. (In the United States, diesel fuel must have a minimum cetane number of 40.) The cetane number for undecanone was 56.6. The number for a 50/50 mix of undecanone and tridecanone was 58.4.
As is the case for other fatty acid-derived biofuels, such as fatty acid ethyl esters, saturated, medium-chain methyl ketones addressed in this article have favorable cetane numbers (CN). A less favorable property of the saturated methyl ketones addressed in this article is relatively high melting point (e.g., 30.5 °C for 2-tridecanone), which is related to cold-temperature diesel fuel properties such as cloud point. This disadvantage could be significantly mitigated by the prominent monounsaturated methyl ketones observed in the best producing strains (monounsaturated methyl ketones account for ~40% of total methyl ketones in strain EGS895).
Melting point depression caused by monounsaturation in fatty acid methyl esters illustrates this point. For example, for C16 and C18 fatty acid methyl esters, the cis-Δ9 monounsaturated homologs have melting points approximately 60 °C lower than those of their saturated counterparts [the melting point of methyl palmitoleate (16:1) is -33.9 °C whereas that of methyl palmitate (16:0) is 30 °C; the melting point of methyl oleate (18:1) is -19.5 °C whereas that of methyl stearate (18:0) is 39 °C]. However, unsaturation can also be expected to decrease CN (e.g., a decrease of ~30 in CN applies to C16 fatty acid methyl esters).
In addition to degree of unsaturation, chain length will also affect fuel properties (increasing chain length increases CN and melting point). The ensemble of saturated and unsaturated methyl ketones generated by strain EGS895 (and related strains) may have sufficiently favorable collective fuel properties to be appropriate for blending with petroleum-based diesel. Nonetheless, future efforts will be directed at enhancing methyl ketone production (e.g., by enhancing intracellular malonyl-CoA levels) and modulating the methyl ketone composition to optimize diesel fuel properties.—Goh et al.
This research was supported by JBEI through the DOE Office of Science.
Ee-Been Goh, Edward E. K. Baidoo, Jay D. Keasling, and Harry R. Beller (2012) Engineering of Bacterial Methyl Ketone Synthesis for Biofuels. Appl. Environ. Microbiol. 78:70-80; published ahead of print 28 October 2011, doi: 10.1128/AEM.06785-11
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