Engineering of bacterial methyl ketone synthesis for biofuels.
We have engineered Escherichia coli to overproduce saturated and monounsaturated aliphatic methyl ketones in the C₁₁ to C₁₅ (diesel) range; this group of methyl ketones includes 2-undecanone and 2-tridecanone, which are of importance to the flavor and fragrance industry and also have favorable cetane numbers (as we report here). We describe specific improvements that resulted in a 700-fold enhancement in methyl ketone titer relative to that of a fatty acid-overproducing E. coli strain, including the following: (i) overproduction of β-ketoacyl coenzyme A (CoA) thioesters achieved by modification of the β-oxidation pathway (specifically, overexpression of a heterologous acyl-CoA oxidase and native FadB and chromosomal deletion of fadA) and (ii) overexpression of a native thioesterase (FadM). FadM was previously associated with oleic acid degradation, not methyl ketone synthesis, but outperformed a recently identified methyl ketone synthase (Solanum habrochaites MKS2 [ShMKS2], a thioesterase from wild tomato) in β-ketoacyl-CoA-overproducing strains tested. Whole-genome transcriptional (microarray) studies led to the discovery that FadM is a valuable catalyst for enhancing methyl ketone production. The use of a two-phase system with decane enhanced methyl ketone production by 4- to 7-fold in addition to increases from genetic modifications.
Joint BioEnergy Institute, Emeryville, California, USA.
Ee-Been
EB
Goh
Edward E K
EE
Baidoo
Jay D
JD
Keasling
Harry R
HR
Beller
D000214:Acyl Coenzyme A / Q000378:metabolism; D056804:Biofuels; D002853:Chromatography, Liquid; D003066:Coenzyme A Ligases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D007659:Ketones / Q000302:isolation & purification / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D012329:RNA, Bacterial / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013869:Thiolester Hydrolases / Q000378:metabolism
D000214:Acyl Coenzyme A; D056804:Biofuels; D029968:Escherichia coli Proteins; D007659:Ketones; D012329:RNA, Bacterial; C009541:2-tridecanone; C544474:FadM protein, E coli; D013869:Thiolester Hydrolases; D003066:Coenzyme A Ligases; C085934:acyl-coenzyme A synthetase, E coli; C526928:undecan-2-one