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Design and characterization of an efficient CYP105A1-based whole-cell biocatalyst for the conversion of resin acid diterpenoids in permeabilized Escherichia coli

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Abstract

Cytochrome P450 enzymes exhibit a tremendous potential for biotechnological applications due to their ability to introduce oxygen into non-activated carbon atoms. Their catalytic diversity is complemented by a broad substrate range covering many natural compounds. Especially the functionalization of terpenoids by P450s becomes increasingly interesting due to the diverse biological effects of these compounds. The bacterial CYP105A1 from Streptomyces griseolus was recently identified to carry out a one-step hydroxylation of several abietane-type resin acids. In this work, a whole-cell system for CYP105A1 with its heterologous electron transfer proteins Arh1 and Etp1fd from Schizosaccharomyces pombe was designed in Escherichia coli JM109 cells. Additionally, an enzyme-coupled cofactor regeneration system was integrated by co-expression of alcohol dehydrogenase from Lactobacillus brevis. In order to overcome mass transfer limitations of substrate into the cell, different agents were tested towards their permeabilizing activity on the E. coli membrane. The peptide antibiotic polymyxin B proved to be the most effective permeabilizer. After optimising the expression and conversion conditions, the cells were able to completely convert 200 μM of abietic acid into 15-hydroxyabietic acid within 2 h, exhibiting an initial conversion rate of 125 μM/h. These results demonstrate the high potential of this whole-cell system for the synthesis of functionalized resin acid diterpenoids.

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Acknowledgments

This work was financially supported by the Deutsche Bundesstiftung Umwelt (DBU) (Osnabrück, Germany) and the INTERREG programme of the EU. Dr. Stephanie Bringer-Meyer and Dr. Stephan Lütz (Institute for Biotechnology, Research Centre Jülich) are gratefully acknowledged for kindly providing us the LbADH gene. We are also thankful to Tanja Sagadin for her assistance in the cloning experiments.

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  1. Department of Biochemistry, Saarland University, Saarbruecken, Germany

    Simon Janocha & Rita Bernhardt

  2. Institut für Biochemie, Universität des Saarlandes, Campus B2.2, 66123, Saarbrücken, Germany

    Rita Bernhardt

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  1. Simon Janocha
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  2. Rita Bernhardt
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Correspondence to Rita Bernhardt.

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Janocha, S., Bernhardt, R. Design and characterization of an efficient CYP105A1-based whole-cell biocatalyst for the conversion of resin acid diterpenoids in permeabilized Escherichia coli . Appl Microbiol Biotechnol 97, 7639–7649 (2013). https://doi.org/10.1007/s00253-013-5008-5

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  • DOI: https://doi.org/10.1007/s00253-013-5008-5

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