P450BM-3-catalyzed whole-cell biotransformation of α-pinene with recombinant Escherichia coli in an aqueous–organic two-phase system
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A recombinant Escherichia coli BL21 (DE3) strain overexpressing a variant of P450BM-3 (V26T/R47F/A74G/F87V/L188K; abbreviated: BL21 (P450BM-3 QM)) oxyfunctionalizes the bicyclic monoterpene α-pinene to α-pinene oxide, verbenol, and myrtenol. To address the low water solubility and the toxicity of terpenoids, an aqueous–organic two-phase bioprocess was developed. Diisononyl phthalate was selected as a biocompatible organic carrier solvent capable of masking the toxic effects mediated by α-pinene and of efficiently extracting the products enabling scale-up to the bioreactor. With an aqueous to organic phase ratio of 3:2 and 30% (v/v) of α-pinene in the organic phase, a biocatalytic product formation period of more than 4 h was achieved. A comparison of the biotransformation performance of BL21 (P450BM-3 QM) and a strain with an additional heterologous NADPH regeneration system comprising glucose facilitator and dehydrogenase, but only expressing half the amount of P450BM-3 QM, shows comparable product concentrations of 1,020 ± 144 and 800 ± 61 mg lAq−1, respectively. The total product yields YP/P450 (µmol µmolP450−1) were 80% higher when the strain with the cofactor regeneration system was used. A total product concentration of over 1 g lAq−1, corresponding to the highest value reported for microbial α-pinene oxyfunctionalization so far, marks a promising step forward toward a future application of recombinant microorganisms for the selective oxidation of terpenoids to value-added products.