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Cell physiology rather than enzyme kinetics can determine the efficiency of cytochrome P450-catalyzed C–H-oxyfunctionalization

  • Original Paper
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Journal of Industrial Microbiology & Biotechnology

Abstract

Cell physiology is a critical factor determining the efficiency of reactions performed by microbial biocatalysts. In order to develop an efficient biotransformation procedure for the hydroxylation of (S)-limonene to (S)-perillyl alcohol by recombinant Pseudomonas putida cells harboring the cytochrome P450 monooxygenase CYP153A6, physiological parameters were optimized. The previously reported synthesis of (S)-perillyl alcohol by P. putida GPo12 was based on complex and sensitive octane feeding strategies (van Beilen et al. in Appl Environ Microbiol 71:1737–1744, 2005), indicating the pivotal role of cell physiology. In contrast to previous findings, the screening of different carbon sources showed that glycerol and citrate are suitable alternatives to octane allowing high specific limonene hydroxylation activities. The use of P. putida KT2440 as an alternative host strain and citrate as the carbon source improved practical handling and allowed a 7.5-fold increase of the specific activity (to 22.6 U g −1CDW ). In two-liquid-phase biotransformations, 4.3 g of (S)-perillyl alcohol L −1tot were produced in 24 h, representing a sixfold improvement in productivity compared to previously reported results. It is concluded that, for selective cytochrome P450-based hydrocarbon oxyfunctionalizations by means of living microbial cells, the relationship between cell physiology and the target biotransformation is crucial, and that understanding the relationship should guide biocatalyst and bioprocess design.

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Acknowledgments

We gratefully thank Dr. J. B. van Beilen and Prof. Dr. B. Witholt for providing bacterial strains and plasmids. This project was co-financed by the Deutsche Bundesstiftung Umwelt (AZ 20006/855), the ERA-Net project PSYSMO, and the Ministry of Innovation, Science, Research and Technology of North Rhine-Westphalia, Germany.

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  1. Amit Tatyasaheb Deshmukh

    Present address: Bioprocess Technology Group, Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands

Authors and Affiliations

  1. Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, Emil-Figge-Strasse 66, 44227, Dortmund, Germany

    Sjef Cornelissen, Shanshan Liu, Amit Tatyasaheb Deshmukh, Andreas Schmid & Bruno Bühler

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  1. Sjef Cornelissen
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Correspondence to Andreas Schmid.

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Cornelissen, S., Liu, S., Deshmukh, A.T. et al. Cell physiology rather than enzyme kinetics can determine the efficiency of cytochrome P450-catalyzed C–H-oxyfunctionalization. J Ind Microbiol Biotechnol 38, 1359–1370 (2011). https://doi.org/10.1007/s10295-010-0919-y

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