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Engineering class I cytochrome P450 by gene fusion with NADPH-dependent reductase and S. avermitilis host development for daidzein biotransformation

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Abstract

Daidzein C6 hydroxylase (6-DH, nfa12130), which is a class I type of cytochrome P450 enzyme, catalyzes a hydroxylation reaction at the C6-position of the daidzein A-ring and requires auxiliary electron transfer proteins. Current utilization of cytochrome P450 (CYP) enzymes is limited by low coupling efficiency, which necessitates extramolecular electron transfers, and low driving forces, which derive electron flows from tightly regulated NADPH redox balances into the heterogeneous CYP catalytic cycle. To overcome such limitations, the heme domain of the 6-DH enzyme was genetically fused with the NADPH-reductase domain of self-sufficient CYP102D1 to enhance electron transfer efficiencies through intramolecular electron transfer and switching cofactor preference from NADH into NADPH. 6-DH-reductase fusion enzyme displayed distinct spectral properties of both flavoprotein and heme proteins and catalyzed daidzein hydroxylation more efficiently with a k cat/K m value of 120.3 ± 11.5 [103 M−1 s−1], which was about three times higher than that of the 6-DH-FdxC-FdrA reconstituted system. Moreover, to obtain a higher redox driving force, a Streptomyces avermitilis host system was developed for heterologous expression of fusion 6-DH enzyme and whole cell biotransformation of daidzein. The whole cell reaction using the final recombinant strain, S. avermitilisΔcyp105D7::fusion 6-DH (nfa12130), resulted in 8.3 ± 1.4 % of 6-OHD yield from 25.4 mg/L of daidzein.

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References

  • Agarwal AK, Auchus RJ (2005) Minireview: cellular redox state regulates hydroxysteroid dehydrogenase activity and intracellular hormone potency. Endocrinology 146(6):2531–2538

    Article  PubMed  CAS  Google Scholar 

  • Bartek T, Blombach B, Zonnchen E, Makus P, Lang S, Eikmanns BJ, Oldiges M (2010) Importance of NADPH supply for improved L-valine formation in Corynebacterium glutamicum. Biotechnol Prog 26(2):361–371

    PubMed  CAS  Google Scholar 

  • Bernhardt R (2006) Cytochromes P450 as versatile biocatalysts. J Biotechnol 124(1):128–145

    Article  PubMed  CAS  Google Scholar 

  • Blank LM, Ebert BE, Buehler K, Buhler B (2010) Redox biocatalysis and metabolism: molecular mechanisms and metabolic network analysis. Antioxid Redox Signal 13(3):349–394

    Article  PubMed  CAS  Google Scholar 

  • Budde M, Maurer SC, Schmid RD, Urlacher VB (2004) Cloning, expression and characterisation of CYP102A2, a self-sufficient P450 monooxygenase from Bacillus subtilis. Appl Microbiol Biotechnol 66(2):180–186

    Article  PubMed  CAS  Google Scholar 

  • Buhler B, Park JB, Blank LM, Schmid A (2008) NADH availability limits asymmetric biocatalytic epoxidation in a growing recombinant Escherichia coli strain. Appl Environ Microbiol 74(5):1436–1446

    Article  PubMed  PubMed Central  Google Scholar 

  • Chin JW, Cirino PC (2011) Improved NADPH supply for xylitol production by engineered Escherichia coli with glycolytic mutations. Biotechnol Prog 27(2):333–341

    Article  PubMed  CAS  Google Scholar 

  • Chin JW, Khankal R, Monroe CA, Maranas CD, Cirino PC (2009) Analysis of NADPH supply during xylitol production by engineered Escherichia coli. Biotechnol Bioeng 102(1):209–220

    Article  PubMed  CAS  Google Scholar 

  • Choi KY, Jung E, Jung DH, An BR, Pandey BP, Yun H, Sung C, Park HY, Kim BG (2012a) Engineering of daidzein 3′-hydroxylase P450 enzyme into catalytically self-sufficient cytochrome P450. Microb Cell Factories 11:81

    Article  CAS  Google Scholar 

  • Choi KY, Jung E, Jung DH, Pandey BP, Yun H, Park HY, Kazlauskas RJ, Kim BG (2012b) Cloning, expression and characterization of CYP102D1, a self-sufficient P450 monooxygenase from Streptomyces avermitilis. FEBS J 279(9):1650–1662

    Article  PubMed  CAS  Google Scholar 

  • Choi KY, Kim TJ, Koh SK, Roh CH, Pandey BP, Lee N, Kim BG (2009) A-ring ortho-specific monohydroxylation of daidzein by cytochrome P450s of Nocardia farcinica IFM10152. Biotechnol J 4(11):1586–1595

    Article  PubMed  CAS  Google Scholar 

  • Chowdhary PK, Alemseghed M, Haines DC (2007) Cloning, expression and characterization of a fast self-sufficient P450: CYP102A5 from Bacillus cereus. Arch Biochem Biophys 468(1):32–43

    Article  PubMed  CAS  Google Scholar 

  • De Mot R, Parret AH (2002) A novel class of self-sufficient cytochrome P450 monooxygenases in prokaryotes. Trends Microbiol 10(11):502–508

    Article  PubMed  Google Scholar 

  • Dietrich M, Eiben S, Asta C, Do TA, Pleiss J, Urlacher VB (2008) Cloning, expression and characterisation of CYP102A7, a self-sufficient P450 monooxygenase from Bacillus licheniformis. Appl Microbiol Biotechnol 79(6):931–940

    Article  PubMed  CAS  Google Scholar 

  • Dodhia VR, Fantuzzi A, Gilardi G (2006) Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego. J Biol Inorg Chem 11(7):903–916

    Article  PubMed  CAS  Google Scholar 

  • Fasan R, Chen MM, Crook NC, Arnold FH (2007) Engineered alkane-hydroxylating cytochrome P450(BM3) exhibiting nativelike catalytic properties. Angew Chem Int Ed Engl 46(44):8414–8418

    Article  PubMed  CAS  Google Scholar 

  • Fasan R, Crook NC, Peters MW, Meinhold P, Buelter T, Landwehr M, Cirino PC, Arnold FH (2011) Improved product-per-glucose yields in P450-dependent propane biotransformations using engineered Escherichia coli. Biotechnol Bioeng 108(3):500–510

    Article  PubMed  CAS  Google Scholar 

  • Girvan HM, Seward HE, Toogood HS, Cheesman MR, Leys D, Munro AW (2007) Structural and spectroscopic characterization of P450 BM3 mutants with unprecedented P450 heme iron ligand sets. New heme ligation states influence conformational equilibria in P450 BM3. J Biol Chem 282(1):564–572

    Article  PubMed  CAS  Google Scholar 

  • Gonzalez RJ, Tarloff JB (2001) Evaluation of hepatic subcellular fractions for Alamar blue and MTT reductase activity. Toxicol In Vitro 15(3):257–259

    Article  PubMed  CAS  Google Scholar 

  • Goto H, Terao Y, Akai S (2009) Synthesis of various kinds of isoflavones, isoflavanes, and biphenyl-ketones and their 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activities. Chem Pharm Bull (Tokyo) 57(4):346–360

    Article  CAS  Google Scholar 

  • Grogan G (2011) Cytochromes P450: exploiting diversity and enabling application as biocatalysts. Curr Opin Chem Biol 15(2):241–248

    Article  PubMed  CAS  Google Scholar 

  • Haga T, Hirakawa H, Nagamune T (2013) Fine tuning of spatial arrangement of enzymes in a PCNA-mediated multienzyme complex using a rigid poly-L-proline linker. PLoS ONE 8(9):e75114

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Hannemann F, Bichet A, Ewen KM, Bernhardt R (2007) Cytochrome P450 systems—biological variations of electron transport chains. Biochim Biophys Acta 1770(3):330–344

    Article  PubMed  CAS  Google Scholar 

  • Hung TV, Ishida K, Parajuli N, Liou K, Lee HC, Sohng JK (2006) Enhanced clavulanic acid production in Streptomyces clavuligerus NRRL3585 by overexpression of regulatory genes. Biotechnol Bioproc Eng 11(2):116–120

    Article  CAS  Google Scholar 

  • Ishikawa J, Yamashita A, Mikami Y, Hoshino Y, Kurita H, Hotta K, Shiba T, Hattori M (2004) The complete genomic sequence of Nocardia farcinica IFM 10152. Proc Natl Acad Sci U S A 101(41):14925–14930

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • 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(9):738–743

    Article  PubMed  CAS  Google Scholar 

  • Lamb DC, Ikeda H, Nelson DR, Ishikawa J, Skaug T, Jackson C, Omura S, Waterman MR, Kelly SL (2003) Cytochrome p450 complement (CYPome) of the avermectin-producer Streptomyces avermitilis and comparison to that of Streptomyces coelicolor A3(2). Biochem Biophys Res Commun 307(3):610–619

    Article  PubMed  CAS  Google Scholar 

  • Lee WH, Kim MD, Jin YS, Seo JH (2013) Engineering of NADPH regenerators in Escherichia coli for enhanced biotransformation. Appl Microbiol Biotechnol 97(7):2761–2772

    Article  PubMed  CAS  Google Scholar 

  • McLean KJ, Girvan HM, Munro AW (2007) Cytochrome P450/redox partner fusion enzymes: biotechnological and toxicological prospects. Expert Opin Drug Metab Toxicol 3(6):847–863

    Article  PubMed  CAS  Google Scholar 

  • McLean KJ, Sabri M, Marshall KR, Lawson RJ, Lewis DG, Clift D, Balding PR, Dunford AJ, Warman AJ, McVey JP, Quinn AM, Sutcliffe MJ, Scrutton NS, Munro AW (2005) Biodiversity of cytochrome P450 redox systems. Biochem Soc Trans 33(Pt 4):796–801

    PubMed  CAS  Google Scholar 

  • Munro AW, Girvan HM, McLean KJ (2007) Cytochrome P450—redox partner fusion enzymes. Biochim Biophys Acta 1770(3):345–359

    Article  PubMed  CAS  Google Scholar 

  • O’Reilly E, Kohler V, Flitsch SL, Turner NJ (2011) Cytochromes P450 as useful biocatalysts: addressing the limitations. Chem Commun (Camb) 47(9):2490–2501

    Article  Google Scholar 

  • Omura T, Sato R (1964) The carbon monoxide-binding pigment of liver microsomes. II. Solubilization, purification, and properties. J Biol Chem 239:2379–2385

    PubMed  CAS  Google Scholar 

  • Otey CR, Landwehr M, Endelman JB, Hiraga K, Bloom JD, Arnold FH (2006) Structure-guided recombination creates an artificial family of cytochromes P450. PLoS Biol 4(5):e112

    Article  PubMed  PubMed Central  Google Scholar 

  • Pandey BP, Roh C, Choi KY, Lee N, Kim EJ, Ko S, Kim T, Yun H, Kim BG (2010) Regioselective hydroxylation of daidzein using P450 (CYP105D7) from Streptomyces avermitilis MA4680. Biotechnol Bioeng 105(4):697–704

    PubMed  CAS  Google Scholar 

  • Park JS, Kim DH, Lee JK, Lee JY, Kim HK, Lee HJ, Kim HC (2010) Natural ortho-dihydroxyisoflavone derivatives from aged Korean fermented soybean paste as potent tyrosinase and melanin formation inhibitors. Bioorg Med Chem Lett 20(3):1162–1164

    Article  PubMed  CAS  Google Scholar 

  • Park JS, Park HY, Kim DH, Kim HK (2008) Ortho-dihydroxyisoflavone derivatives from aged Doenjang (Korean fermented soypaste) and its radical scavenging activity. Bioorg Med Chem Lett 18(18):5006–5009

    Article  PubMed  CAS  Google Scholar 

  • Peters MW, Meinhold P, Glieder A, Arnold FH (2003) Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3. J Am Chem Soc 125(44):13442–13450

    Article  PubMed  CAS  Google Scholar 

  • Robin A, Roberts GA, Kisch J, Sabbadin F, Grogan G, Bruce N, Turner NJ, Flitsch SL (2009) Engineering and improvement of the efficiency of a chimeric [P450cam-RhFRed reductase domain] enzyme. Chem Commun (Camb)(18):2478-80

  • Sadeghi SJGG (2013) Chimeric P450 enzymes: activity of artificial redox fusions driven by different reductases for biotechnological applications. Biotechnol Appl Biochem 60(1):102–110

    Article  PubMed  CAS  Google Scholar 

  • Schuckel J, Rylott EL, Grogan G, Bruce NC (2012) A gene-fusion approach to enabling plant cytochromes p450 for biocatalysis. Chembiochem 13(18):2758–2763

    Article  PubMed  Google Scholar 

  • Tee KL, Schwaneberg U (2006) A screening system for the directed evolution of epoxygenases: importance of position 184 in P450 BM3 for stereoselective styrene epoxidation. Angew Chem Int Ed Engl 45(32):5380–5383

    Article  PubMed  CAS  Google Scholar 

  • Urlacher VB, Eiben S (2006) Cytochrome P450 monooxygenases: perspectives for synthetic application. Trends Biotechnol 24(7):324–330

    Article  PubMed  CAS  Google Scholar 

  • Ying W (2008) NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological consequences. Antioxid Redox Signal 10(2):179–206

    Google Scholar 

  • Zhang JD, Li AT, Yu HL, Imanaka T, Xu JH (2011) Synthesis of optically pure S-sulfoxide by Escherichia coli transformant cells coexpressing the P450 monooxygenase and glucose dehydrogenase genes. J Ind Microbiol Biotechnol 38(5):633–641

    Google Scholar 

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Acknowledgments

This work was supported by the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Science, ICT&Future Planning (2013060084). And this work was also partially supported by the Energy Efficiency & Resources of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grant funded by the Korea Government Ministry of Trade, Industry and Energy (20133030000300) and Polar Academic Program (PAP, PD13010) from KOPRI. We thank Prof. Hattori from Kitasato University in Japan for donating Nocardia farcinica IFM10152.

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Authors and Affiliations

  1. Department of Environmental Engineering, College of Engineering, Ajou University, Suwon, Gyeonggi-do, 443-749, South Korea

    Kwon-Young Choi

  2. School of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea

    EunOk Jung & Byung-Gee Kim

  3. Institute of Bioengineering, Seoul National University, Seoul, South Korea

    Byung-Gee Kim

  4. Department of Bioscience and Biotechnology, Konkuk University, Seoul, South Korea

    Hyungdon Yun

  5. Department of Microbial Engineering, College of Engineering, Konkuk University, Seoul, South Korea

    Yung-Hun Yang

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  1. Kwon-Young Choi
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Correspondence to Byung-Gee Kim.

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Choi, KY., Jung, E., Yun, H. et al. Engineering class I cytochrome P450 by gene fusion with NADPH-dependent reductase and S. avermitilis host development for daidzein biotransformation. Appl Microbiol Biotechnol 98, 8191–8200 (2014). https://doi.org/10.1007/s00253-014-5706-7

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  • DOI: https://doi.org/10.1007/s00253-014-5706-7

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