Synthesis of ω‐hydroxy dodecanoic acid based on an engineered CYP153A fusion construct

Abstract

A bacterial P450 monooxygenase-based whole cell biocatalyst using Escherichia coli has been applied in the production of ω-hydroxy dodecanoic acid from dodecanoic acid (C12-FA) or the corresponding methyl ester. We have constructed and purified a chimeric protein where the fusion of the monooxygenase CYP153A from Marinobacter aquaeloei to the reductase domain of P450 BM3 from Bacillus megaterium ensures optimal protein expression and efficient electron coupling. The chimera was demonstrated to be functional and three times more efficient than other sets of redox components evaluated. The established fusion protein (CYP153AM. aq.-CPR) was used for the hydroxylation of C12-FA in in vivo studies. These experiments yielded 1.2 g l–1 ω-hydroxy dodecanoic from 10 g l–1 C12-FA with high regioselectivity (> 95%) for the terminal position. As a second strategy, we utilized C12-FA methyl ester as substrate in a two-phase system (5:1 aqueous/organic phase) configuration to overcome low substrate solubility and product toxicity by continuous extraction. The biocatalytic system was further improved with the coexpression of an additional outer membrane transport system (AlkL) to increase the substrate transfer into the cell, resulting in the production of 4 g l–1 ω-hydroxy dodecanoic acid. We further summarized the most important aspects of the whole-cell process and thereupon discuss the limits of the applied oxygenation reactions referring to hydrogen peroxide, acetate and P450 concentrations that impact the efficiency of the production host negatively.

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

“…Whereas self-sufficient P450s show the highest turnover numbers and coupling efficiency up to now, fusion to redox partners or modern electron donor systems allow also other CYPs to run with increased efficiency [94,125,131,132]. Drawbacks of the CYP family are poor (regio-)selectivities, low productivities, uncoupling, and the dependence on NADPH as reducing agent [14,17,18].…”

“…The fusion-protein of CYP153A33 G307A variant with the diflavin-reductase domain of CYM102A1 BM3 was demonstrated to be superior to other redox-systems and allowed 12% conversion of C12:0 at 10 g L −1 employing E. coli cells (Table 8, entry 4). Productivity was enhanced when the corresponding methyl ester was supplied as substrate (4 g L −1 ) [125]. A G307A/S233G double substitution identified by semi-rational design allowed to double the overall activity for medium-chain fatty acids [129].…”

Production of medium-chain, a, omega-bifunctional monomers from fatty acids and n-alkanes

“…Most successful was the conversion of 1 mM nonanoic acid by E. coli pBGTHJKL-eeb1, which produced 0.75 mM of mono-ethyl azelate. These titers are in the same order of magnitude as medium-chain dicarboxylic acid production from fatty acids in shake flask experiments reported before 19,67,122 . To our knowledge however, this is the first report of the combination of ester biosynthesis and ω-oxidation.…”

Combinatorial Engineering of Yarrowia lipolytica as a Promising Cell Biorefinery Platform for the de novo Production of Multi-Purpose Long Chain Dicarboxylic Acids

“…This can explain the stimulatory effect of ω-oxidation upregulation on biomass proliferation in the F strain with deficient FAA1p activity. In fact, the natural enzymatic machinery of the host cell plays an influential role in fatty acid over-oxidation [18].…”