Improving methyl ketone production in Escherichia coli by heterologous expression of NADH‐dependent FabG

Abstract

We previously engineered Escherichia coli to overproduce medium- to long-chain saturated and monounsaturated methyl ketones, which could potentially be applied as diesel fuel blending agents or in the flavor and fragrance industry. Recent efforts at strain optimization have focused on cofactor balance, as fatty acid-derived pathways face the systematic metabolic challenge of net NADPH consumption (in large part, resulting from the key fatty acid biosynthetic enzyme FabG [β-ketoacyl-ACP reductase]) and net NADH production. In this study, we attempted to mitigate cofactor imbalance by heterologously expressing NADH-dependent, rather than NADPH-dependent, versions of FabG identified in previous studies. Of the four NADH-dependent versions of FabG tested in our previously best-reported methyl ketone-producing strain (EGS1895), the version from Acholeplasma laidlawii (Al_FabG) showed the greatest increase in methyl ketone yield in shake flasks (35-75% higher than for an RFP negative-control strain, depending on sugar loading). An improved strain (EGS2920) attained methyl ketone titers during fed-batch fermentation of 5.4 ± 0.5 g/L, which were, on average, ca. 40% greater than those for the base strain (EGS1895) under fermentation conditions optimized in this study. Shotgun proteomic data for strains EGS2920 and EGS1895 during fed-batch fermentation were consistent with the goal of alleviating NADPH limitation through expression of Al_FabG. For example, relative to strain EGS1895, strain EGS2920 significantly upregulated glucose-6-phosphate isomerase (directing flux into glycolysis rather than the NADPH-producing pentose phosphate pathway) and downregulated MaeB (a NADP -dependent malate dehydrogenase). Overall, the results suggest that heterologous expression of NADH-dependent FabG in E. coli may improve sustained production of fatty acid-derived renewable fuels and chemicals.

Efficient production of an ezetimibe intermediate using carbonyl reductase coupled with glucose dehydrogenase

“…13 CRs have been successfully applied industrially for the synthesis of various chiral alcohols. However, the industrial potential of CRs was largely restricted by the cofactor regeneration efficiency and the expensive price of the cofactor NAD(P)H. [14][15][16] In situ cofactor regeneration is considered essential for the economic viability of industrial scale biotransformation. Kizaki In our previous study, a recombinant carbonyl reductase CR125 was obtained from Lactobacillus kefiri to catalyze 150 g/L ET-4 to (S)-ET-5 with the conversion of 99.1% and the e.e.…”

“…CRs have been successfully applied industrially for the synthesis of various chiral alcohols. However, the industrial potential of CRs was largely restricted by the cofactor regeneration efficiency and the expensive price of the cofactor NAD(P)H 14‐16 . In situ cofactor regeneration is considered essential for the economic viability of industrial scale biotransformation.…”

High titer methyl ketone production with tailoredPseudomonas taiwanensisVLB120

“…Microbes do not naturally synthesize methyl ketones, but recombinant production of medium-chain length congeners has been achieved in several species (Dong et al, 2019;Goh et al, 2012;Hanko et al, 2018;Müller et al, 2013;Park et al, 2012). However, most reported titers are in the mg L -1 range, and lower gram-scale production has only been reported for an intensively engineered E. coli in a 200-h fed-batch cultivation (Goh et al, 2018). Besides overexpression of the product biosynthesis pathway, a typical strategy to boost the synthesis of fatty acid derivatives is engineering the supply of the precursors acetyl-CoA and malonyl-CoA.…”

Methyl ketone production by Pseudomonas putida is enhanced by plant‐derived amino acids

“…Unlike free fatty acids, methyl ketones do not require additional processing steps to be blended into conventional diesel fuels. Methyl ketone production has been most intensively studied in E. coli , for which a titer of 3.4–5.4 g/L was achieved by fed‐batch glucose fermentation (Goh et al, ; Goh, Chen, Petzold, Keasling, & Beller, ). Methyl ketone production has also been demonstrated under fed‐batch glucose fermentation in oleaginous yeast Yarrowia lipolytica (315 mg/L; Hanko et al, ) and under autotrophic conditions from H 2 /CO 2 in Ralstonia eutropha (180 mg/L; Müller et al, ).…”