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A new Bacillus megaterium whole-cell catalyst for the hydroxylation of the pentacyclic triterpene 11-keto-β-boswellic acid (KBA) based on a recombinant cytochrome P450 system

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Abstract

The use of cytochromes P450 for the regio- and stereoselective hydroxylation of non-activated carbon atoms in biotechnological applications reflects an efficient and cost-effective alternative in comparison to classical organic chemistry. The prokaryotic cytochrome P450 CYP106A2 from Bacillus megaterium ATCC 13368 hydroxylates a variety of 3-oxo-Δ4 steroids and recently it was identified to carry out a one-step regioselective allylic hydroxylation of the diterpene abietic acid. The anti-inflammatory pentacyclic triterpene 11-Keto-β-boswellic acid (KBA) was found to be a further substrate of CYP106A2, being the first report of a pentacyclic triterpene conversion by a prokaryotic P450. The reaction products were analyzed by HPLC and the corresponding kinetic parameters were investigated. Structure determination of the main product by NMR revealed a 15α-hydroxylation of this substrate. In order to overcome the inability of a recombinant P450 whole-cell system in E. coli for the uptake of acids with terpene structure, we developed for the first time an expression system for cytochromes P450 in B. megaterium (strains MS941 and ATCC 13368). Interestingly, CYP106A2 was only successfully expressed in the plasmid-less B. megaterium strain MS941 but not in ATCC13368. This recombinant system, with the co-expressed heterologous redox chain of the P450, bovine adrenodoxin reductase (AdR), and bovine adrenodoxin (Adx), was applied for the whole-cell conversion of KBA. The formation of 15α-hydroxy-KBA was increased 15-fold in comparison with the naturally CYP106A2-expressing B. megaterium strain ATCC 13368.

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Acknowledgment

The authors thank Prof. Dr. Dieter Jahn (TU Braunschweig) for the kind provision of the B. megaterium strain MS941 and the plasmid pKMBm4 and Dr. Simon Stammen (TU Braunschweig) for constructive discussions. We are thankful to Katharina Bompais and Wolfgang Reinle for the expression and excellent purification of CYP106A2, AdR, and Adx.

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Authors and Affiliations

  1. Department of Biochemistry, Saarland University, Campus, B 2.2, 66123, Saarbrücken, Germany

    Sabrina Bleif, Frank Hannemann & Rita Bernhardt

  2. Department of Pharmaceutical Biology, Saarland University, 66123, Saarbrücken, Germany

    Josef Zapp

  3. Department of Organic Chemistry II, Saarland University, 66123, Saarbrücken, Germany

    David Hartmann & Johann Jauch

Authors
  1. Sabrina Bleif
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  2. Frank Hannemann
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  3. Josef Zapp
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  4. David Hartmann
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  5. Johann Jauch
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  6. Rita Bernhardt
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Correspondence to Rita Bernhardt.

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Bleif, S., Hannemann, F., Zapp, J. et al. A new Bacillus megaterium whole-cell catalyst for the hydroxylation of the pentacyclic triterpene 11-keto-β-boswellic acid (KBA) based on a recombinant cytochrome P450 system. Appl Microbiol Biotechnol 93, 1135–1146 (2012). https://doi.org/10.1007/s00253-011-3467-0

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  • DOI: https://doi.org/10.1007/s00253-011-3467-0

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