Applied Microbiology and Biotechnology

, Volume 97, Issue 17, pp 7741–7754 | Cite as

Application of a new versatile electron transfer system for cytochrome P450-based Escherichia coli whole-cell bioconversions

  • Michael Ringle
  • Yogan Khatri
  • Josef Zapp
  • Frank Hannemann
  • Rita Bernhardt
Biotechnologically relevant enzymes and proteins

Abstract

Cytochromes P450 monooxygenases are highly interesting biocatalysts for biotechnological applications, since they perform a diversity of reactions on a broad range of organic molecules. Nevertheless, the application of cytochromes P450 is limited compared to other enzymes mainly because of the necessity of a functional redox chain to transfer electrons from NAD(P)H to the monooxygenase. In this study, we established a novel robust redox chain based on adrenodoxin, which can deliver electrons to mitochondrial, bacterial and microsomal P450s. The natural membrane-associated reductase of adrenodoxin was replaced by the soluble Escherichia coli reductase. We could demonstrate for the first time that this reductase can transfer electrons to adrenodoxin. In the first step, the electron transfer properties and the potential of this new system were investigated in vitro, and in the second step, an efficient E. coli whole-cell system using CYP264A1 from Sorangium cellulosum So ce56 was developed. It could be demonstrated that this novel redox chain leads to an initial conversion rate of 55 μM/h, which was 52 % higher compared to the 36 μM/h of the redox chain containing adrenodoxin reductase. Moreover, we optimized the whole-cell biotransformation system by a detailed investigation of the effects of different media. Finally, we are able to demonstrate that the new system is generally applicable to other cytochromes P450 by combining it with the biotechnologically important steroid hydroxylase CYP106A2 from Bacillus megaterium.

Keywords

Cytochromes P450 Biotechnology Adrenodoxin Escherichia coli reductase Redox chain Whole-cell system CYP264A1 CYP106A1 

Notes

Acknowledgments

This work was supported by a fellowship awarded by the Scholarship Program of the German Federal Environmental Foundation [Deutsche Bundesstiftung Umwelt (DBU)] to Michael Ringle. We are also thankful to Wolfgang Reinle for the expression and purification of Adx and AdR.

Supplementary material

253_2012_4612_MOESM1_ESM.pdf (1.6 mb)
ESM 1 (PDF 1599 kb)

References

  1. Agematu H, Matsumoto N, Fujii Y, Kabumoto H, Doi S, Machida K, Ishikawa J, Arisawa A (2006) Hydroxylation of testosterone by bacterial cytochromes P450 using the Escherichia coli expression system. Biosci Biotechnol Biochem 70:307–311PubMedCrossRefGoogle Scholar
  2. Bell SG, Harford-Cross CF, Wong L-L (2001) Engineering the CYP101 system for in vivo oxidation of unnatural substrates. Protein Eng 14:797–802PubMedCrossRefGoogle Scholar
  3. Bell SG, Hoskins N, Xu F, Caprotti D, Rao Z, Wong LL (2006) Cytochrome P450 enzymes from the metabolically diverse bacterium Rhodopseudomonas palustris. Biochem Biophys Res Commun 342:191–196PubMedCrossRefGoogle Scholar
  4. Bell S, Dale A, Rees N, Wong L-L (2010) A cytochrome P450 class I electron transfer system from Novosphingobium aromaticivorans. Appl Microbiol Biotechnol 86:163–175PubMedCrossRefGoogle Scholar
  5. Berg A, Gustafsson JA, Ingelman-Sundberg M (1976) Characterization of a cytochrome P-450-dependent steroid hydroxylase system present in Bacillus megaterium. J Biol Chem 251:2831–2838PubMedGoogle Scholar
  6. Berg A, Ingelman-Sundberg M, Gustafsson JA (1979) Isolation and characterization of cytochrome P-450meg. Acta Biol Med Ger 38:333–344PubMedGoogle Scholar
  7. Bernhardt R (1996) Cytochrome P450: structure, function, and generation of reactive oxygen species. Rev Physiol Biochem Pharmacol 127:137–221PubMedCrossRefGoogle Scholar
  8. Bernhardt R (2006) Cytochromes P450 as versatile biocatalysts. J Biotechnol 124:128–145PubMedCrossRefGoogle Scholar
  9. Bleif S, Hannemann F, Lisurek M, von Kries JP, Zapp J, Dietzen M, Antes I, Bernhardt R (2011) Identification of CYP106A2 as a regioselective allylic bacterial diterpene hydroxylase. ChemBioChem 12:576–582PubMedCrossRefGoogle Scholar
  10. Bleif S, Hannemann F, Zapp J, Hartmann D, Jauch J, Bernhardt R (2012) A new Bacillus megaterium whole-cell catalyst for the hydroxylation of the pentacyclic triterpene 11-keto-beta-boswellic acid (KBA) based on a recombinant cytochrome P450 system. Appl Microbiol Biotechnol 93:1135–1146PubMedCrossRefGoogle Scholar
  11. Botsford JL, DeMoss RD (1971) Catabolite repression of tryptophanase in Escherichia coli. J Bacteriol 105:303–312PubMedGoogle Scholar
  12. Cao PR, Bulow H, Dumas B, Bernhardt R (2000) Construction and characterization of a catalytic fusion protein system: P-450(11beta)-adrenodoxin reductase-adrenodoxin. Biochim Biophys Acta 1476:253–264PubMedCrossRefGoogle Scholar
  13. Chefson A, Auclair K (2006) Progress towards the easier use of P450 enzymes. Mol Biosyst 2:462–469PubMedCrossRefGoogle Scholar
  14. Chun YJ, Shimada T, Waterman MR, Guengerich FP (2006) Understanding electron transport systems of Streptomyces cytochrome P450. Biochem Soc Trans 34:1183–1185PubMedCrossRefGoogle Scholar
  15. Denisov IG, Makris TM, Sligar SG, Schlichting I (2005) Structure and chemistry of cytochrome P450. Chem Rev 105:2253–2278PubMedCrossRefGoogle Scholar
  16. Ensley BD, Ratzkin BJ, Osslund TD, Simon MJ, Wackett LP, Gibson DT (1983) Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo. Science 222:167–169PubMedCrossRefGoogle Scholar
  17. Ewen KM, Schiffler B, Uhlmann-Schiffler H, Bernhardt R, Hannemann F (2008) The endogenous adrenodoxin reductase-like flavoprotein arh1 supports heterologous cytochrome P450-dependent substrate conversions in Schizosaccharomyces pombe. FEMS Yeast Res 8:432–441PubMedCrossRefGoogle Scholar
  18. Ewen KM, Hannemann F, Khatri Y, Perlova O, Kappl R, Krug D, Hüttermann J, Müller R, Bernhardt R (2009) Genome mining in Sorangium cellulosum So ce56—identification and characterization of the homologous electron transfer proteins of a myxobacterial cytochrome P450. J Biol Chem 284:28590–28598PubMedCrossRefGoogle Scholar
  19. Ewen KM, Hannemann F, Iametti S, Morleo A, Bernhardt R (2011) Functional characterization of Fdx1: evidence for an evolutionary relationship between P450-type and ISC-type ferredoxins. J Mol Biol 413:940–951PubMedCrossRefGoogle Scholar
  20. Ewen KM, Ringle M, Bernhardt R (2012) Adrenodoxin—a versatile ferredoxin. IUBMB Life 64:506–512PubMedCrossRefGoogle Scholar
  21. Faro M, Schiffler B, Heinz A, Nogues I, Medina M, Bernhardt R, Gomez-Moreno C (2003) Insights into the design of a hybrid system between Anabaena ferredoxin-NADP+ reductase and bovine adrenodoxin. Eur J Biochem 270:726–735PubMedCrossRefGoogle Scholar
  22. Fischer F, Raimondi D, Aliverti A, Zanetti G (2002) Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase. Eur J Biochem 269:3005–3013PubMedCrossRefGoogle Scholar
  23. Frank LH, Demoss RD (1957) Specific enzymic method for the estimation of l-tryptophan. Arch Biochem Biophys 67:387–397PubMedCrossRefGoogle Scholar
  24. Gillam EMJ, Guengerich FP (2001) Exploiting the versatility of human cytochrome P450 enzymes: the promise of blue roses from biotechnology. IUBMB Life 52:271–277PubMedCrossRefGoogle Scholar
  25. Gillam EMJ, Notley LM, Cai H, De Voss JJ, Guengerich FP (2000) Oxidation of indole by cytochrome P450 enzymes. Biochemistry (Mosc) 39:13817–13824CrossRefGoogle Scholar
  26. Girhard M, Machida K, Itoh M, Schmid RD, Arisawa A, Urlacher VB (2009) Regioselective biooxidation of (+)-valencene by recombinant E. coli expressing CYP109B1 from Bacillus subtilis in a two-liquid-phase system. Microb Cell Fact 8:36PubMedCrossRefGoogle Scholar
  27. Girhard M, Klaus T, Khatri Y, Bernhardt R, Urlacher VB (2010) Characterization of the versatile monooxygenase CYP109B1 from Bacillus subtilis. Appl Microbiol Biotechnol 87:595–607PubMedCrossRefGoogle Scholar
  28. Gudiminchi R, Randall C, Opperman D, Olaofe O, Harrison SL, Albertyn J, Smit M (2012) Whole-cell hydroxylation of n-octane by Escherichia coli strains expressing the CYP153A6 operon. Appl Microbiol Biotechnol 96:1507–1516PubMedCrossRefGoogle Scholar
  29. Guengerich FP (2001) Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity. Chem Res Toxicol 14:611–650PubMedCrossRefGoogle Scholar
  30. Guzov VM, Unnithan GC, Chernogolov AA, Feyereisen R (1998) CYP12A1, a mitochondrial cytochrome P450 from the house fly. Arch Biochem Biophys 359:231–240PubMedCrossRefGoogle Scholar
  31. Hannemann F, Virus C, Bernhardt R (2006) Design of an Escherichia coli system for whole cell mediated steroid synthesis and molecular evolution of steroid hydroxylases. J Biotechnol 124:172–181PubMedCrossRefGoogle Scholar
  32. Hannemann F, Bichet A, Ewen KM, Bernhardt R (2007) Cytochrome P450 systems—biological variations of electron transport chains. Biochim Biophys Acta Gen Subj 1770:330–344CrossRefGoogle Scholar
  33. Hawkes DB, Slessor KE, Bernhardt PV, De Voss JJ (2010) Cloning, expression and purification of cindoxin, an unusual Fmn-containing cytochrome P450 redox partner. ChemBioChem 11:1107–1114PubMedCrossRefGoogle Scholar
  34. Isin EM, Guengerich FP (2007) Complex reactions catalyzed by cytochrome P450 enzymes. Biochim Biophys Acta Gen Subj 1770:314–329CrossRefGoogle Scholar
  35. Jenkins CM, Waterman MR (1994) Flavodoxin and NADPH-flavodoxin reductase from Escherichia coli support bovine cytochrome P450c17 hydroxylase activities. J Biol Chem 269:27401–27408PubMedGoogle Scholar
  36. Jenkins CM, Waterman MR (1998) NADPH–flavodoxin reductase and flavodoxin from Escherichia coli: characteristics as a soluble microsomal P450 reductase. Biochemistry (Mosc) 37:6106–6113CrossRefGoogle Scholar
  37. Khatri Y, Girhard M, Romankiewicz A, Ringle M, Hannemann F, Urlacher V, Hutter M, Bernhardt R (2010a) Regioselective hydroxylation of norisoprenoids by CYP109D1 from Sorangium cellulosum So ce56. Appl Microbiol Biotechnol 88:485–495PubMedCrossRefGoogle Scholar
  38. Khatri Y, Hannemann F, Ewen KM, Pistorius D, Perlova O, Kagawa N, Brachmann AO, Müller R, Bernhardt R (2010b) The CYPome of Sorangium cellulosum So ce56 and identification of CYP109D1 as a new fatty acid hydroxylase. Chem Biol 17:1295–1305PubMedCrossRefGoogle Scholar
  39. Kille S, Zilly FE, Acevedo JP, Reetz MT (2011) Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution. Nat Chem 3:738–743PubMedCrossRefGoogle Scholar
  40. Kyung-Hwan J (2006) Continuous production of recombinant interferon-α in Escherichia coli via the derepression of trp promoter using casamino acid. Process Biochem 41:809–814CrossRefGoogle Scholar
  41. Li HM, Mei LH, Urlacher V, Schmid R (2008) Cytochrome P450 BM-3 evolved by random and saturation mutagenesis as an effective indole-hydroxylating catalyst. Appl Biochem Biotechnol 144:27–36PubMedCrossRefGoogle Scholar
  42. Liao WL, Dodder NG, Mast N, Pikuleva IA, Turko IV (2009) Steroid and protein ligand binding to cytochrome P450 46A1 as assessed by hydrogen-deuterium exchange and mass spectrometry. Biochemistry 48:4150–4158PubMedCrossRefGoogle Scholar
  43. Lisurek M, Kang MJ, Hartmann RW, Bernhardt R (2004) Identification of monohydroxy progesterones produced by CYP106A2 using comparative HPLC and electrospray ionisation collision-induced dissociation mass spectrometry. Biochem Biophys Res Commun 319:677–682PubMedCrossRefGoogle Scholar
  44. Ly T, Khatri Y, Zapp J, Hutter M, Bernhardt R (2012) CYP264B1 from Sorangium cellulosum So ce56: a fascinating norisoprenoid and sesquiterpene hydroxylase. Appl Microbiol Biotechnol 95:123–133PubMedCrossRefGoogle Scholar
  45. Manna SK, Mazumdar S (2010) Tuning the substrate specificity by engineering the active site of cytochrome P450cam: a rational approach. Dalton Trans 39:3115–3123PubMedCrossRefGoogle Scholar
  46. Martinez-Gomez K, Flores N, Castaneda HM, Martinez-Batallar G, Hernandez-Chavez G, Ramirez OT, Gosset G, Encarnacion S, Bolivar F (2012) New insights into Escherichia coli metabolism: carbon scavenging, acetate metabolism and carbon recycling responses during growth on glycerol. Microb Cell Fact 11:46PubMedCrossRefGoogle Scholar
  47. Munro AW, Girvan HM, McLean KJ (2007) Variations on a (t)heme—novel mechanisms, redox partners and catalytic functions in the cytochrome P450 superfamily. Nat Prod Rep 24:585–609PubMedCrossRefGoogle Scholar
  48. Nelson DR (2006) Cytochrome P450 nomenclature, 2004. Methods Mol Biol 320:1–10PubMedGoogle Scholar
  49. Nguyen KT, Virus C, Günnewich N, Hannemann F, Bernhardt R (2012) Changing the regioselectivity of a P450 from C15 to C11 hydroxylation of progesterone. ChemBioChem 13:1161–1166PubMedCrossRefGoogle Scholar
  50. O'Keefe DP, Gibson KJ, Emptage MH, Lenstra R, Romesser JA, Litle PJ, Omer CA (1991) Ferredoxins from two sulfonylurea herbicide monooxygenase systems in Streptomyces griseolus. Biochemistry (Mosc) 30:447–455CrossRefGoogle Scholar
  51. Omura T, Sato R (1964) The carbon monoxide-binding pigment of liver microsomes. J Biol Chem 239:2370–2378PubMedGoogle Scholar
  52. O'Reilly E, Kohler V, Flitsch SL, Turner NJ (2011) Cytochromes P450 as useful biocatalysts: addressing the limitations. Chem Commun (Camb) 47:2490–2501CrossRefGoogle Scholar
  53. Ortiz de Montellano P, Voss J (2005) Substrate oxidation by cytochrome P450 enzymes. Springer, New York, pp 183–245CrossRefGoogle Scholar
  54. Pechurskaya TA, Harnastai IN, Grabovec IP, Gilep AA, Usanov SA (2007) Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s. Biochem Biophys Res Commun 353:598–604PubMedCrossRefGoogle Scholar
  55. Perlova O, Gerth K, Kaiser O, Hans A, Müller R (2006) Identification and analysis of the chivosazol biosynthetic gene cluster from the myxobacterial model strain Sorangium cellulosum So ce56. J Biotechnol 121:174–191PubMedCrossRefGoogle Scholar
  56. Robin M-A, Anandatheerthavarada HK, Fang J-K, Cudic M, Otvos L, Avadhani NG (2001) Mitochondrial targeted cytochrome P450 2E1 (P450 MT5) contains an intact N terminus and requires mitochondrial specific electron transfer proteins for activity. J Biol Chem 276:24680–24689PubMedCrossRefGoogle Scholar
  57. Rosic N (2009) Versatile capacity of shuffled cytochrome P450s for dye production. Appl Microbiol Biotechnol 82:203–210PubMedCrossRefGoogle Scholar
  58. Sagara Y, Wada A, Takata Y, Waterman MR, Sekimizu K, Horiuchi T (1993) Direct expression of adrenodoxin reductase in Escherichia coli and the functional characterization. Biol Pharm Bull 16:627–630PubMedCrossRefGoogle Scholar
  59. Sakaki T, Kagawa N, Yamamoto K, Inouye K (2005) Metabolism of vitamin D3 by cytochromes P450. Front Biosci 10:119–134PubMedCrossRefGoogle Scholar
  60. Salamanca-Pinzón SG, Guengerich FP (2011) A tricistronic human adrenodoxin reductase-adrenodoxin-cytochrome P450 27A1 vector system for substrate hydroxylation in Escherichia coli. Protein Expression Purif 79:231–236CrossRefGoogle Scholar
  61. Schmitz D, Zapp J, Bernhardt R (2012) Hydroxylation of the triterpenoid dipterocarpol with CYP106A2 from Bacillus megaterium. FEBS J 279:1663–1674PubMedCrossRefGoogle Scholar
  62. Schuster I (2011) Cytochromes P450 are essential players in the vitamin D signaling system. Biochim Biophys Acta 1814:186–199PubMedCrossRefGoogle Scholar
  63. Seifert A, Pleiss J (2012) Identification of selectivity determinants in CYP monooxygenases by modelling and systematic analysis of sequence and structure. Curr Drug Metab 13:197–202PubMedCrossRefGoogle Scholar
  64. Simgen B, Contzen J, Schwarzer R, Bernhardt R, Jung C (2000) Substrate binding to 15β-hydroxylase (CYP106A2) probed by FT infrared spectroscopic studies of the iron ligand CO stretch vibration. Biochem Biophys Res Commun 269:737–742PubMedCrossRefGoogle Scholar
  65. Sono M, Roach MP, Coulter ED, Dawson JH (1996) Heme-containing oxygenases. Chem Rev 96:2841–2888PubMedCrossRefGoogle Scholar
  66. Studier FW (2005) Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif 41:207–234PubMedCrossRefGoogle Scholar
  67. Uhlmann H, Kraft R, Bernhardt R (1994) C-terminal region of adrenodoxin affects its structural integrity and determines differences in its electron transfer function to cytochrome P-450. J Biol Chem 269:22557–22564PubMedGoogle Scholar
  68. Urlacher VB, Eiben S (2006) Cytochrome P450 monooxygenases: perspectives for synthetic application. Trends Biotechnol 24:324–330PubMedCrossRefGoogle Scholar
  69. Urlacher VB, Girhard M (2012) Cytochrome P450 monooxygenases: an update on perspectives for synthetic application. Trends Biotechnol 30:26–36PubMedCrossRefGoogle Scholar
  70. Virus C, Lisurek M, Simgen B, Hannemann F, Bernhardt R (2006) Function and engineering of the 15beta-hydroxylase CYP106A2. Biochem Soc Trans 34:1215–1218PubMedCrossRefGoogle Scholar
  71. Werck-Reichhart D, Feyereisen R (2000) Cytochromes P450: a success story. Genome Biol 1:REVIEWS3003Google Scholar
  72. Whitehouse CJC, Yang W, Yorke JA, Tufton HG, Ogilvie LCI, Bell SG, Zhou W, Bartlam M, Rao Z, Wong L-L (2011) Structure, electronic properties and catalytic behaviour of an activity-enhancing CYP102A1 (P450BM3) variant. Dalton Trans 40:10383–10396PubMedCrossRefGoogle Scholar
  73. Wong L-L (2011) P450BM3 on steroids: the Swiss army knife P450 enzyme just gets better. ChemBioChem 12:2537–2539PubMedCrossRefGoogle Scholar
  74. Zehentgruber D, Hannemann F, Bleif S, Bernhardt R, Lütz S (2010) Towards preparative scale steroid hydroxylation with cytochrome P450 monooxygenase CYP106A2. ChemBioChem 11:713–721PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Michael Ringle
    • 1
  • Yogan Khatri
    • 1
  • Josef Zapp
    • 2
  • Frank Hannemann
    • 1
  • Rita Bernhardt
    • 1
    • 3
  1. 1.Department of BiochemistrySaarland UniversitySaarbrueckenGermany
  2. 2.Department of Pharmaceutical BiologySaarland UniversitySaarbrueckenGermany
  3. 3.Institut für BiochemieUniversität des SaarlandesSaarbrueckenGermany

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