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References

  1. Bernhardt R. 1996. Cytochrome P450: structure, function, and generation of reactive oxygen species. Rev. Physiol. Biochem. Pharmacol. 127: 137-221.
    CrossRef
  2. Nelson DR, Koymans L, Kamataki T, Stegeman JJ, Feyereisen R, Waxman DJ, et al. 1996. P450 superfamily:update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6: 1-42.
    Pubmed CrossRef
  3. Urlacher VB, Eiben S. 2006. Cytochrome P450 monooxygenases:perspectives for synthetic application. Trends Biotechnol. 24:324-330.
    Pubmed CrossRef
  4. Milhim M, Gerber A, Neunzig J, Hannemann F, Bernhardt R. 2016. A novel NADPH-dependent flavoprotein reductase from Bacillus megaterium acts as an efficient cytochrome P450 reductase. J. Biotechnol. 231: 83-94.
    Pubmed CrossRef
  5. Ortiz de Montellano PR. 2005. Cytochrome P450 Structure, Mechanism, and Biochemistry, 3rd Ed. Kluwer Academic/Plenum Publishers, New York, USA.
  6. Zhang A, Zhang T, Hall EA, Hutchinson S, Cryle MJ, Wong LL, et al. 2015. The crystal structure of the versatile cytochrome P450 enzyme CYP109B1 Bacillus subtilis. Mol. Biosyst. 11:869-881.
    Pubmed CrossRef
  7. Holland HL. 1999. Recent advances in applied and mechanistic aspects of the enzymatic hydroxylation of steroids by whole-cell biocatalysts. Steroids 64: 178-186.
    CrossRef
  8. Bureik M, Lisurek M, Bernhardt R. 2002. The human steroid hydroxlases CYP1B1 and CYP11B2. Biol. Chem. 383: 1537-1551.
    Pubmed CrossRef
  9. Lee GY, Kim DH, Kim D, Ahn T, Yun CH. 2015. Functional characterization of steroid hydroxylase CYP106A1 derived from Bacillus megaterium. Arch. Pharm. Res. 38: 98-107.
    Pubmed CrossRef
  10. 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: 2831-2838.
    CrossRef
  11. Jozwik IK, Kiss FM, Gricman L, Abdulmughni A, Brill E, Zapp J, et al. 2016. Structural basis of steroid binding and oxidation by the cytochrome P450 CYP109E1 from Bacillus megaterium. FEBS J. 283: 4128-4148.
    Pubmed PMC CrossRef
  12. Makino T, Katsuyama Y, Otomatsu T, Misawa N, Ohnishi Y. 2014. Regio- and stereospecific hydroxylation of various steroids at the 16α position of the D ring by the Streptomyces griseus cytochrome P450 CYP154C3. Appl. Environ. Microbiol.80: 1371-1379.
    Pubmed PMC CrossRef
  13. Bracco P, Janssen DB, Schallmey A. 2013. Selective steroid oxyfunctionalisation by CYP154C5, a bacterial cytochrome P450. Microb. Cell Fact. 12: 95.
    Pubmed PMC CrossRef
  14. Khatri Y, Ringle M, Lisurek M, von Kries JP, Zapp J, Bernhardt R. 2016. Substrate hunting for the myxobacterial CYP260A1 revealed new 1α-hydroxylated products from C-19 steroids. Chembiochem 17: 90-101.
    Pubmed CrossRef
  15. Salamanca-Pinzon SG, Khatri Y, Carius Y, Keller L, Muller R, Lancaster CR, et al. 2016. Structure-function analysis for the hydroxylation of Δ4 C21-steroids by the myxobacterial CYP260B1. FEBS Lett. 590: 1838-1851.
    Pubmed CrossRef
  16. Kiss FM, Schmitz D, Zapp J, Dier TK, Volmer DA, Bernhardt R. 2015. Comparison of CYP106A1 and CYP106A2 from Bacillus megaterium - identification of a novel 11-oxidase activity. Appl. Microbiol. Biotechnol. 99: 8495-8514.
    Pubmed CrossRef
  17. Nguyen KT, Virus C, Gunnewich N, Hannemann F, Bernhardt R. 2012. Changing the regioselectivity of a P450 from C15 to C11 hydroxylation of progesterone. Chembiochem 13: 1161-1166.
    Pubmed CrossRef
  18. Nikolaus J, Nguyen KT, Virus C, Riehm JL, Hutter M, Bernhardt R. 2017. Engineering of CYP106A2 for steroid 9α-and 6β-hydroxylation. Steroids 120: 41-48.
    Pubmed CrossRef
  19. Janocha S, Carius Y, Hutter M, Lancaster CR, Bernhardt R. 2016. Crystal structure of CYP106A2 in substrate-free and substrate-bound form. Chembiochem 17: 852-860.
    Pubmed CrossRef
  20. Omura T, Sato R. 1964. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239: 2370-2378.
  21. Johnston JB, Ouellet H, Ortiz de Montellano PR. 2010. Functional redundancy of steroid C26-monooxygenase activity in Mycobacterium tuberculosis revealed by biochemical and genetic analyses. J. Biol. Chem. 285: 36352-36360.
    Pubmed PMC CrossRef
  22. Minor W, Otwinowski Z. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276:307-326.
    CrossRef
  23. Vagin A, Teplyakov A. 1997. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30:1022-1025.
    CrossRef
  24. Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, et al. 2011. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67: 235-242.
    Pubmed PMC CrossRef
  25. Murshudov GN, Skubák P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, et al. 2011. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67:355-367.
    Pubmed PMC CrossRef
  26. Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, et al. 2010. PHENIX: a comprehensive pythonbased system for macromolecular structure solution. Acta Crystallogr. D 66: 213-221.
    Pubmed PMC CrossRef
  27. Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, et al. 2010. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66: 12-21.
    Pubmed PMC CrossRef
  28. DeLano WL. 2002. The PyMOL molecular graphics system. CCP4 Newsletter on Protein Crystallography 40: 82-92.
  29. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, et al. 2006. The Protein Data Bank, 1999, pp. 675-684. International Tables for Crystallography, Vol. F. John Wiley & Sons, IN.
    CrossRef
  30. Rauschenbach R, Isernhagen M, Noeske-Jungblut C, Boidol W, Siewert G. 1993. Cloning sequencing and expression of the gene for cytochrome P450meg, the steroid-15 betamonooxygenase from Bacillus megaterium ATCC 13368. Mol. Gen. Genet. 241: 170-176.
    CrossRef
  31. Lee CW, L ee J H, R imal H , Park H , Lee J H, Oh TJ . 2016. Crystal structure of cytochrome P450 (CYP105P2) from Streptomyces peucetius and its conformational changes in response to substrate binding. Int. J. Mol. Sci. 17: 813.
    Pubmed PMC CrossRef
  32. Lee CW, Yu SC, Lee JH, Park SH, Park H, Oh TJ, et al. 2016. Crystal structure of a putative cytochrome P450 alkane hydroxylase (CYP153D17) from Sphingomonas sp. PAMC 26605 and its conformational substrate binding. Int. J. Mol. Sci. 17: 2067.
    Pubmed PMC CrossRef
  33. Schenkman JB, Sligar SG, Cinti DL. 1981. Substrate interaction with cytochrome P-450. Pharmacol. Ther. 12: 43-71.
    CrossRef
  34. Brill E. 2013. Identifizierung und charakterisierung neuer cytochrom P450 systeme aus Bacillus megaterium DSM319 (Doctoral dissertation).
  35. Schmitz D, Zapp J, Bernhardt R. 2014. Steroid conversion with CYP106A2 – production of pharmaceutically interesting DHEA metabolites. Microb. Cell Fact. 13: 81.
    Pubmed PMC CrossRef
  36. Holm L. 2010. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38: W545-W549.
    Pubmed PMC CrossRef
  37. Savino C, Montemiglio LC, Sciara G, Miele AE, Kendrew SG, Jemth P, et al. 2009. Investigating the structural plasticity of a cytochrome P450: three-dimensional structures of P450 EryK and binding to its physiological substrate. J. Biol. Chem. 284: 29170-29179.
    Pubmed PMC CrossRef
  38. Zhang A, Zhang T, Hall EA, Hutchinson S, Cryle MJ, Wong LL, et al. 2015. The crystal structure of the versatile cytochrome P450 enzyme CYP109B1 from Bacillus subtilis. Mol. Biosyst. 11: 869-881.
    Pubmed CrossRef
  39. Montemiglio LC, Parisi G, Scaglione A, Sciara G, Savino C, Vallone B. 2016. Functional analysis and crystallographic structure of clotrimazole bound OleP, a cytochrome P450 epoxidase from Streptomyces antibioticus involved in oleandomycin biosynthesis. Biochim. Biophys. Acta 1860: 465-475.
    Pubmed CrossRef
  40. Li S, Tietz DR, Rutaganira FU, Kells PM, Anzai Y, Kato F, et al. 2012. Substrate recognition by the multifunctional cytochrome P450 MycG in mycinamicin hydroxylation and epoxidation reactions. J. Biol. Chem. 287: 37880-37890.
    Pubmed PMC CrossRef
  41. Yasutake Y, Fujii Y, Nishioka T, Cheon WK, Arisawa A, Tamura T. 2010. Structural evidence for enhancement of sequential vitamin D3 hydroxylation activities by directed evolution of cytochrome P450 vitamin D3 hydroxylase. J. Biol. Chem. 285: 31193-31201.
    Pubmed PMC CrossRef
  42. Brill E, Hannemann F, Zapp J, Bruning G, Jauch J, Bernhardt R. 2014. A new cytochrome P450 system from Bacillus megaterium DSM319 for the hydroxylation of 11-keto-β-boswellic acid (KBA). Appl. Microbiol. Biotechnol. 98:1701-1717.
    Pubmed CrossRef
  43. Donova MV, Egorova OV. 2012. Microbial steroid transformations: current state and prospects. Appl. Microbiol. Biotechnol. 94: 1423-1447.
    Pubmed CrossRef
  44. Wang R, S ui P , Hou X, C ao T , J ia L, Lu F , et al. 2017. Cloning and identification of a novel steroid 1α-hydroxylase gene from Absidia coerulea. J. Steroid Biochem. Mol. Biol. DOI:10.1016/j.jsbmb.2017.04.006.
    Pubmed CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2017; 27(8): 1472-1482

Published online August 28, 2017 https://doi.org/10.4014/jmb.1706.06013

Copyright © The Korean Society for Microbiology and Biotechnology.

Crystal Structure and Functional Characterization of a Cytochrome P450 (BaCYP106A2) from Bacillus sp. PAMC 23377

Ki-Hwa Kim 1, Chang Woo Lee 2, 3, Bikash Dangi 1, Sun-Ha Park 2, Hyun Park 2, 3, Tae-Jin Oh 1, 4* and Jun Hyuck Lee 2, 3*

Department of Life Science and Biochemical Engineering, Sunmoon University, Asan 31460, Republic of Korea, 1Unit of Polar Genomics, Korea Polar Research Institute, Incheon 21990, Republic of Korea, 2Department of Polar Sciences, University of Science and Technology, Incheon 21990, Republic of Korea, 3Department of BT-Convergent Pharmaceutical Engineering, Sunmoon University, Asan 31460, Republic of Korea

Received: June 7, 2017; Accepted: June 19, 2017

Abstract

Bacterial cytochrome P450 (CYP) steroid hydroxylases are effectively useful in the
pharmaceutical industry for introducing hydroxyl groups to a wide range of steroids. We
found a putative CYP steroid hydroxylase (BaCYP106A2) from the bacterium Bacillus sp.
PAMC 23377 isolated from Kara Sea of the Arctic Ocean, showing 94% sequence similarity
with BmCYP106A2 (Bacillus megaterium ATCC 13368). In this study, soluble BaCYP106A2 was
overexpressed to evaluate its substrate-binding activity. The substrate affinity (Kd value) to
4-androstenedione was 387 ± 37 μM. Moreover, the crystal structure of BaCYP106A2 was
determined at 2.7 Å resolution. Structural analysis suggested that the α8–α9 loop region of
BaCYP106A2 is intrinsically mobile and might be important for initial ligand binding. The
hydroxyl activity of BaCYP106A2 was identified using in vitro enzyme assays. Its activity was
confirmed with two kinds of steroid substrates, 4-androstenedione and nandrolone, using
chromatography and mass spectrometry methods. The main products were monohydroxylated
compounds with high conversion yields. This is the second study on the
structure of CYP106A steroid hydroxylases, and should contribute new insight into the
interactions of bacterial CYP106A with steroid substrates, providing baseline data for
studying the CYP106A steroid hydroxylase from the structural and enzymatic perspectives.

Keywords: Bacillus sp., crystal structure, cytochrome P450, steroid hydroxylase, X-ray crystallography

References

  1. Bernhardt R. 1996. Cytochrome P450: structure, function, and generation of reactive oxygen species. Rev. Physiol. Biochem. Pharmacol. 127: 137-221.
    CrossRef
  2. Nelson DR, Koymans L, Kamataki T, Stegeman JJ, Feyereisen R, Waxman DJ, et al. 1996. P450 superfamily:update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6: 1-42.
    Pubmed CrossRef
  3. Urlacher VB, Eiben S. 2006. Cytochrome P450 monooxygenases:perspectives for synthetic application. Trends Biotechnol. 24:324-330.
    Pubmed CrossRef
  4. Milhim M, Gerber A, Neunzig J, Hannemann F, Bernhardt R. 2016. A novel NADPH-dependent flavoprotein reductase from Bacillus megaterium acts as an efficient cytochrome P450 reductase. J. Biotechnol. 231: 83-94.
    Pubmed CrossRef
  5. Ortiz de Montellano PR. 2005. Cytochrome P450 Structure, Mechanism, and Biochemistry, 3rd Ed. Kluwer Academic/Plenum Publishers, New York, USA.
  6. Zhang A, Zhang T, Hall EA, Hutchinson S, Cryle MJ, Wong LL, et al. 2015. The crystal structure of the versatile cytochrome P450 enzyme CYP109B1 Bacillus subtilis. Mol. Biosyst. 11:869-881.
    Pubmed CrossRef
  7. Holland HL. 1999. Recent advances in applied and mechanistic aspects of the enzymatic hydroxylation of steroids by whole-cell biocatalysts. Steroids 64: 178-186.
    CrossRef
  8. Bureik M, Lisurek M, Bernhardt R. 2002. The human steroid hydroxlases CYP1B1 and CYP11B2. Biol. Chem. 383: 1537-1551.
    Pubmed CrossRef
  9. Lee GY, Kim DH, Kim D, Ahn T, Yun CH. 2015. Functional characterization of steroid hydroxylase CYP106A1 derived from Bacillus megaterium. Arch. Pharm. Res. 38: 98-107.
    Pubmed CrossRef
  10. 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: 2831-2838.
    CrossRef
  11. Jozwik IK, Kiss FM, Gricman L, Abdulmughni A, Brill E, Zapp J, et al. 2016. Structural basis of steroid binding and oxidation by the cytochrome P450 CYP109E1 from Bacillus megaterium. FEBS J. 283: 4128-4148.
    Pubmed KoreaMed CrossRef
  12. Makino T, Katsuyama Y, Otomatsu T, Misawa N, Ohnishi Y. 2014. Regio- and stereospecific hydroxylation of various steroids at the 16α position of the D ring by the Streptomyces griseus cytochrome P450 CYP154C3. Appl. Environ. Microbiol.80: 1371-1379.
    Pubmed KoreaMed CrossRef
  13. Bracco P, Janssen DB, Schallmey A. 2013. Selective steroid oxyfunctionalisation by CYP154C5, a bacterial cytochrome P450. Microb. Cell Fact. 12: 95.
    Pubmed KoreaMed CrossRef
  14. Khatri Y, Ringle M, Lisurek M, von Kries JP, Zapp J, Bernhardt R. 2016. Substrate hunting for the myxobacterial CYP260A1 revealed new 1α-hydroxylated products from C-19 steroids. Chembiochem 17: 90-101.
    Pubmed CrossRef
  15. Salamanca-Pinzon SG, Khatri Y, Carius Y, Keller L, Muller R, Lancaster CR, et al. 2016. Structure-function analysis for the hydroxylation of Δ4 C21-steroids by the myxobacterial CYP260B1. FEBS Lett. 590: 1838-1851.
    Pubmed CrossRef
  16. Kiss FM, Schmitz D, Zapp J, Dier TK, Volmer DA, Bernhardt R. 2015. Comparison of CYP106A1 and CYP106A2 from Bacillus megaterium - identification of a novel 11-oxidase activity. Appl. Microbiol. Biotechnol. 99: 8495-8514.
    Pubmed CrossRef
  17. Nguyen KT, Virus C, Gunnewich N, Hannemann F, Bernhardt R. 2012. Changing the regioselectivity of a P450 from C15 to C11 hydroxylation of progesterone. Chembiochem 13: 1161-1166.
    Pubmed CrossRef
  18. Nikolaus J, Nguyen KT, Virus C, Riehm JL, Hutter M, Bernhardt R. 2017. Engineering of CYP106A2 for steroid 9α-and 6β-hydroxylation. Steroids 120: 41-48.
    Pubmed CrossRef
  19. Janocha S, Carius Y, Hutter M, Lancaster CR, Bernhardt R. 2016. Crystal structure of CYP106A2 in substrate-free and substrate-bound form. Chembiochem 17: 852-860.
    Pubmed CrossRef
  20. Omura T, Sato R. 1964. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239: 2370-2378.
  21. Johnston JB, Ouellet H, Ortiz de Montellano PR. 2010. Functional redundancy of steroid C26-monooxygenase activity in Mycobacterium tuberculosis revealed by biochemical and genetic analyses. J. Biol. Chem. 285: 36352-36360.
    Pubmed KoreaMed CrossRef
  22. Minor W, Otwinowski Z. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276:307-326.
    CrossRef
  23. Vagin A, Teplyakov A. 1997. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30:1022-1025.
    CrossRef
  24. Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, et al. 2011. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67: 235-242.
    Pubmed KoreaMed CrossRef
  25. Murshudov GN, Skubák P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, et al. 2011. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67:355-367.
    Pubmed KoreaMed CrossRef
  26. Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, et al. 2010. PHENIX: a comprehensive pythonbased system for macromolecular structure solution. Acta Crystallogr. D 66: 213-221.
    Pubmed KoreaMed CrossRef
  27. Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, et al. 2010. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66: 12-21.
    Pubmed KoreaMed CrossRef
  28. DeLano WL. 2002. The PyMOL molecular graphics system. CCP4 Newsletter on Protein Crystallography 40: 82-92.
  29. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, et al. 2006. The Protein Data Bank, 1999, pp. 675-684. International Tables for Crystallography, Vol. F. John Wiley & Sons, IN.
    CrossRef
  30. Rauschenbach R, Isernhagen M, Noeske-Jungblut C, Boidol W, Siewert G. 1993. Cloning sequencing and expression of the gene for cytochrome P450meg, the steroid-15 betamonooxygenase from Bacillus megaterium ATCC 13368. Mol. Gen. Genet. 241: 170-176.
    CrossRef
  31. Lee CW, L ee J H, R imal H , Park H , Lee J H, Oh TJ . 2016. Crystal structure of cytochrome P450 (CYP105P2) from Streptomyces peucetius and its conformational changes in response to substrate binding. Int. J. Mol. Sci. 17: 813.
    Pubmed KoreaMed CrossRef
  32. Lee CW, Yu SC, Lee JH, Park SH, Park H, Oh TJ, et al. 2016. Crystal structure of a putative cytochrome P450 alkane hydroxylase (CYP153D17) from Sphingomonas sp. PAMC 26605 and its conformational substrate binding. Int. J. Mol. Sci. 17: 2067.
    Pubmed KoreaMed CrossRef
  33. Schenkman JB, Sligar SG, Cinti DL. 1981. Substrate interaction with cytochrome P-450. Pharmacol. Ther. 12: 43-71.
    CrossRef
  34. Brill E. 2013. Identifizierung und charakterisierung neuer cytochrom P450 systeme aus Bacillus megaterium DSM319 (Doctoral dissertation).
  35. Schmitz D, Zapp J, Bernhardt R. 2014. Steroid conversion with CYP106A2 – production of pharmaceutically interesting DHEA metabolites. Microb. Cell Fact. 13: 81.
    Pubmed KoreaMed CrossRef
  36. Holm L. 2010. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38: W545-W549.
    Pubmed KoreaMed CrossRef
  37. Savino C, Montemiglio LC, Sciara G, Miele AE, Kendrew SG, Jemth P, et al. 2009. Investigating the structural plasticity of a cytochrome P450: three-dimensional structures of P450 EryK and binding to its physiological substrate. J. Biol. Chem. 284: 29170-29179.
    Pubmed KoreaMed CrossRef
  38. Zhang A, Zhang T, Hall EA, Hutchinson S, Cryle MJ, Wong LL, et al. 2015. The crystal structure of the versatile cytochrome P450 enzyme CYP109B1 from Bacillus subtilis. Mol. Biosyst. 11: 869-881.
    Pubmed CrossRef
  39. Montemiglio LC, Parisi G, Scaglione A, Sciara G, Savino C, Vallone B. 2016. Functional analysis and crystallographic structure of clotrimazole bound OleP, a cytochrome P450 epoxidase from Streptomyces antibioticus involved in oleandomycin biosynthesis. Biochim. Biophys. Acta 1860: 465-475.
    Pubmed CrossRef
  40. Li S, Tietz DR, Rutaganira FU, Kells PM, Anzai Y, Kato F, et al. 2012. Substrate recognition by the multifunctional cytochrome P450 MycG in mycinamicin hydroxylation and epoxidation reactions. J. Biol. Chem. 287: 37880-37890.
    Pubmed KoreaMed CrossRef
  41. Yasutake Y, Fujii Y, Nishioka T, Cheon WK, Arisawa A, Tamura T. 2010. Structural evidence for enhancement of sequential vitamin D3 hydroxylation activities by directed evolution of cytochrome P450 vitamin D3 hydroxylase. J. Biol. Chem. 285: 31193-31201.
    Pubmed KoreaMed CrossRef
  42. Brill E, Hannemann F, Zapp J, Bruning G, Jauch J, Bernhardt R. 2014. A new cytochrome P450 system from Bacillus megaterium DSM319 for the hydroxylation of 11-keto-β-boswellic acid (KBA). Appl. Microbiol. Biotechnol. 98:1701-1717.
    Pubmed CrossRef
  43. Donova MV, Egorova OV. 2012. Microbial steroid transformations: current state and prospects. Appl. Microbiol. Biotechnol. 94: 1423-1447.
    Pubmed CrossRef
  44. Wang R, S ui P , Hou X, C ao T , J ia L, Lu F , et al. 2017. Cloning and identification of a novel steroid 1α-hydroxylase gene from Absidia coerulea. J. Steroid Biochem. Mol. Biol. DOI:10.1016/j.jsbmb.2017.04.006.
    Pubmed CrossRef