Metabolic engineering of 2‐pentanone synthesis in Escherichia coli

Abstract

Expanding the chemical diversity of microbial fermentation products enables green production of fuel, chemicals, and pharmaceuticals. In recent years, coenzyme A (CoA) dependent chain elongation, resembling the reversed β‐oxidation pathway, has attracted interest for its use in producing higher alcohols, fatty acids, and polyhydroxyalkanoate. To expand the chemical diversity of this pathway, we metabolically engineered Escherichia coli to produce 2‐pentanone, which is not a natural fermentation product of E. coli. We describe the first demonstration of 2‐pentanone synthesis in E. coli by coupling the CoA‐dependent chain elongation with the acetone production pathway. By bioprospecting for enzymes capable of efficient hydrolysis of 3‐keto‐hexanoyl‐CoA, production of 2‐pentanone increased 20 fold, reaching a titer of 240 mg/L. © 2013 American Institute of Chemical Engineers AIChE J, 59: 3167–3175, 2013

Substantial improvements in methyl ketone production in E. coli and insights on the pathway from in vitro studies

“…More specifically, the pathway 4 involved a biosynthetic thiolase reaction catalyzed by BktB to condense acetyl-CoA with butyryl-CoA, forming -ketohexanoyl-CoA, which was converted by a PcaIJ-catalyzed CoAtransferase reaction to -ketohexanoic acid, which was then converted to 2-pentanone by acetoacetate decarboxylase (Adc). The best reported 2-pentanone titer in that study was 240 mg/L using 4% glucose in Terrific Broth (Lan et al, 2013). Another pathway for the synthesis of short-chain methyl ketones was recently reported by Yoneda et al (2014) for the production of 2-butanone in E. coli.…”

“…Unlike the pathway that we have developed, other recently reported heterologous pathways for methyl ketone production include a decarboxylase to catalyze the final step, namely, decarboxylation of a -keto acid to form a methyl ketone (Park et al, 2012;Lan et al, 2013). Previously, we hypothesized that the last (decarboxylation) step of our methyl ketone pathway occurs spontaneously (Goh et al, 2012).…”

“…R. eutropha was reported to produce up to 180 mg/L of methyl ketones under such chemolithoautotrophic growth conditions (Müller et al, 2013). Lan et al (2013) produced 2-pentanone (for possible biomedical application) in E. coli strain BW25113 by using a metabolic pathway that, unlike the other studies cited above, did not rely on fatty acid precursors. More specifically, the pathway 4 involved a biosynthetic thiolase reaction catalyzed by BktB to condense acetyl-CoA with butyryl-CoA, forming -ketohexanoyl-CoA, which was converted by a PcaIJ-catalyzed CoAtransferase reaction to -ketohexanoic acid, which was then converted to 2-pentanone by acetoacetate decarboxylase (Adc).…”

Escherichia coli as a host for metabolic engineering

“…Subsequently, E. coli based butanol production was improved in several aspects: lowering of nutrient demand in the culture media by co-cultivation of a butyrate producing strain and a butyrateto-butanol conversion strain (Saini et al, 2015), identification and mitigation of a CoA imbalance (Ohtake et al, 2017), construction of a self-inducible and stable strain by driving butanol biosynthetic genes under a promoter of native fermentative genes (Wen and Shen, 2016), development of a chromosomally stable butanol producing strain , and utilization of diverse substrates such as glycerol and xylose (Saini et al, 2017(Saini et al, , 2016. The Clostridal butanol pathway was also expanded via chain elongation with acetyl-CoA to produce hexanol (Dekishima et al, 2011) and 2-pentanone (Lan et al, 2013). In a similar work, E. coli was engineered to produce 2-butanone with a titer of 1.3 g/L through elongation of propionyl-CoA with acetyl-CoA followed by thioester hydrolysis and decarboxylation (Srirangan et al, 2016).…”

Natural products as biofuels and bio-based chemicals: fatty acids and isoprenoids

“…More specically, the pathway (Fig. 40 Another pathway for the synthesis of short-chain methyl ketones was recently reported by Yoneda et al 41 for the production of 2-butanone in E. coli (Fig. The best reported 2-pentanone titer in that study was 240 mg L À1 using 4% glucose in Terric Broth.…”