In 2012, researchers at the US Department of Energy’s Joint BioEnergy Institute (JBEI) engineered Escherichia coli (E. coli) bacteria to overproduce from glucose 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. (Earlier post.)
Now, after further genetic modifications of the bacteria, they have managed to boost the E.coli’s methyl ketone production 160-fold. A paper describing this work is published in the journal Metabolic Engineering.
We previously reported development of a metabolic pathway in Escherichia coli for overproduction of medium-chain methyl ketones (MK), which are relevant to the biofuel and flavor-and-fragrance industries. This MK pathway was a re-engineered version of β-oxidation designed to overproduce β-ketoacyl-CoAs and involved overexpression of the fadM thioesterase gene.
Here, we document metabolic engineering modifications that have led to a MK titer of 3.4 g/L after ~45 h of fed-batch glucose fermentation and attainment of 40% of the maximum theoretical yield (the best values reported to date for MK). Modifications included balancing overexpression of fadR and fadD to increase fatty acid flux into the pathway, consolidation of the pathway from two plasmids into one, codon optimization, and knocking out key acetate production pathways. In vitro studies confirmed that a decarboxylase is not required to convert β-keto acids into MK and that FadM is promiscuous and can hydrolyze several CoA-thioester pathway intermediates.—Goh et al.
While the team is encouraged about such a large improvement in methyl ketone production with a relatively small number of genetic modifications, said Harry Beller, a JBEI microbiologist who led this and the earlier study, they believe they can further improve production using the knowledge gained from in vitro studies of the novel metabolic pathway.
Methyl ketones are naturally occurring compounds discovered more than a century ago in the aromatic evergreen plant known as rue. Since then they’ve been found to be common in tomatoes and other plants, as well as insects and microorganisms. Today they are used to provide scents in essential oils and flavoring in cheese and other dairy products. Although native E. coli make virtually undetectable quantities of methyl ketones, Beller, co-author Ee-Been Goh and their colleagues have been able to overcome this deficiency using the tools of synthetic biology.
Although the improved production is still not at a commercial level in the biofuel market, it is near a commercial level for use in flavor and fragrances, where certain methyl ketones are much more highly valued than they would be in the biofuel market. It may be possible for a company to sell a small percentage of methyl ketones in the flavor and fragrance market and use the profits to enhance the economic viability of the production of methyl ketones as biofuels.—Harry Beller
The in vitro studies carried out by Beller and Goh provided insights into the pathway, some of which point to even further production gains. One key finding was the confirmation that a decarboxylase enzyme is not required for this methyl ketone pathway.
Several different metabolic pathways have been developed in the past couple of years for methyl ketone production in E. coli, a couple of which use decarboxylase enzymes to catalyze the last step of the pathway. Our methyl ketone pathway is performing quite a bit better than these other pathways, but it does not include a native or added decarboxylase.—Harry Beller
The in vitro studies also addressed concerns about the FadM enzyme being somewhat “promiscuous” in its hydrolyzing (thioesterase) activities. Beller and Goh found that FadM can act on intermediates in the methyl ketone pathway and effectively reduce the flux of carbon to the final methyl ketone products. However, they say that with some informed metabolic engineering, this need not be a problem and knowledge of the phenomenon could even be used to enhance production.
In all likelihood, there is a sweet spot in the level of expression of the FadM enzyme that will allow for maximal production of methyl ketones without siphoning away metabolic intermediates.—Harry Beller
This research was supported by JBEI through the US Department of Energy’s Office of Science.
Ee-Been Goh, Edward E.K. Baidoo, Helcio Burd, Taek Soon Lee, Jay D. Keasling, Harry R. Beller (2014) “Substantial improvements in methyl ketone production in E. coli and insights on the pathway from in vitro studies,” Metabolic Engineering, Volume 26, Pages 67-76 doi: 10.1016/j.ymben.2014.09.003