Applied Microbiology and Biotechnology

, Volume 83, Issue 5, pp 849–857 | Cite as

P450BM-3-catalyzed whole-cell biotransformation of α-pinene with recombinant Escherichia coli in an aqueous–organic two-phase system

Biotechnological Products and Process Engineering

Abstract

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 l Aq −1 , respectively. The total product yields Y P/P450 (µmol µmol P450 −1 ) were 80% higher when the strain with the cofactor regeneration system was used. A total product concentration of over 1 g l Aq −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.

Keywords

Aqueous–organic two-phase bioprocess P450BM-3 Whole-cell Biotransformation Cofactor regeneration Pinene Phthalate 

Notes

Acknowledgments

This research was financially supported by the German Federal Ministry of Economics and Technology via the AiF (projects no. 119 ZN and no. 15089 N). We thank H. Sahm, Research Centre Juelich for plasmid pET24glcdh and pZY507glf, U. Schwaneberg, Jacobs University Bremen for the supply with 12-pNCA, and R.D. Schmid, Institute of Technical Biochemistry, Stuttgart for providing pET28bm-3 qm.

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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  1. 1.Biochemical Engineering Group, DECHEMA e.V.Karl-Winnacker-InstitutFrankfurtGermany

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