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Published on: March 9, 2017
Synthesis of methyl ketones by metabolically engineered Escherichia coli
John Park1, María Rodríguez-Moyá, Mai Li
1Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX 77005, USA.
Abstract:
Methyl ketones are a group of highly reduced platform chemicals with widespread applications in the fragrance, flavor and pharmacological industries. Current methods for the industrial production of methyl ketones include oxidation of hydrocarbons, but recent advances in the characterization of methyl ketone synthases from wild tomato have sparked interest towards the development of microbial platforms for the industrial production of methyl ketones. A functional methyl ketone biosynthetic pathway was constructed in Escherichia coli by over-expressing two genes from Solanum habrochaites: shmks2, encoding a 3-ketoacyl-ACP thioesterase, and shmks1, encoding a beta-decarboxylase. These enzymes enabled methyl ketone synthesis from 3-ketoacyl-ACP, an intermediate in the fatty acid biosynthetic cycle. The production of 2-nonanone, 2-undecanone, and 2-tridecanone by MG1655 pTH-shmks2-shmks1 was initially detected by nuclear magnetic resonance and gas chromatography-mass spectrometry analyses at levels close to 6 mg/L. The deletion of major fermentative pathways leading to ethanol (adhE), lactate (ldhA), and acetate (pta, poxB) production allowed for the carbon flux to be redirected towards methyl ketone production, doubling total methyl ketone concentration. Variations in methyl ketone production observed under different working volumes in flask experiments led to a more detailed analysis of the effects of oxygen availability on methyl ketone concentration in order to determine optimal levels of oxygen. The methyl ketone concentration achieved with MG1655 ∆adhE ∆ldhA ∆poxB ∆pta pTrcHis2A-shmks2-shmks1, the best performer strain in this study, was approximately 500 mg/L, the highest reported for an engineered microorganism. Through the establishment of optimal operating conditions and by executing rational metabolic engineering strategies, we were able to increase methyl ketone concentrations by almost 75-fold from the initial confirmatory levels.
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