Optimization of the Expression of Streptomyces venezuelae Cytochrome P450 Hydroxylase (PikC) in Escherichia coli

Bong Gyun Kim; Sang Kil Lee; Kwangkyoung Liou; Jae Kyung Sohng; Yeo Joon Yoon; Hei Chan Lee

Journal of Industrial and Engineering Chemistry, Vol.10, No.5, 759-765, September, 2004

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Optimization of the Expression of Streptomyces venezuelae Cytochrome P450 Hydroxylase (PikC) in Escherichia coli

Bong Gyun Kim, Sang Kil Lee¹, Kwangkyoung Liou, Jae Kyung Sohng, Yeo Joon Yoon², and Hei Chan Lee^†

Institute of Biomolecule Reconstruction, Sun Moon University, Chungnam 336-840, Korea
¹Division of Chemical Engineering, College of Engineering, Seoul National University, Seoul 151-742, Korea
²Division of Nano Science and Department of Chemstry, Ewha Womans University, Seoul 120-750, Korea

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PikC is a cytochrome P450 (CYP) monooxygenase that is involved in the biosynthesis of pikromycin. In this study, conditions for the overexpression of soluble PikC in Escherichia coli (E. coli) were optimized. More than 67.9% of the total PikC expressed was obtained in the soluble form when the cultures were induced at OD₆₀₀ of 3.84 with 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and harvested after culturing at 20℃ for 24 h. Under these conditions, 72 μg/mL of pikC could be obtained in the soluble fraction whereas 34 μg/mL was in the form of inclusion bodies. The factors controlling PikC expression and their interactions were studied according to the factorial design. Within the designed range, an increased IPTG concentration in the medium not only exerted a negative effect but also it interacted negatively with the induction time and induction period after the addition of IPTG. Furthermore, the latter two factors were found to interact negatively with one another.

Keywords:Escherichia coli;Expression;Hydroxylation;Macrolides;PikC

[References]

Katz L, Chem. Rev., 97(7), 2557 (1997)
Xue Y, Wilson D, Zhao H, Liu H, Sherman DH, Chem. Biol., 5, 661 (1998)
Betlach MC, Kealey JT, Betlach MC, Ashley GW, McDaniel R, Biochemistry, 37, 14937 (1998)
Graziani EI, Cane DE, Betlach MC, Kealey JT, McDaniel R, Bioorg. Med. Chem. Lett., 8, 3117 (1998)
Yoon YJ, Beck BJ, Kim BS, Kang HY, Reynolds KA, Sherman DH, Chem. Biol., 9, 203 (2002)
Lee C, Lee SG, Takahashi S, Kim BG, Biotechnol. Bioeng., 8, 89 (2003)
Wang TH, Lee WC, Biotechnol. Bioeng., 8, 263 (2003)
Ryu JC, Ryu TG, Yoo SJ, Hwang JY, Kim YH, Yang HS, J. Ind. Eng. Chem., 9(6), 735 (2003)
Sambrook J, Russell DW, Molecular Cloning, 3rd Edn., Cold Spring Harbor Laboratory Press, New York (2001)
Kharel MK, Lee HC, Sohng JK, Liou K, J. Ind. Eng. Chem., 8(5), 427 (2002)
Plackett RL, Burman JP, Biometrika, 33, 305 (1946)
Hussain HA, Ward JA, Enzyme Microb. Technol., 32(7), 790 (2003)

[Referenced By]

[1] Katz L, Chem. Rev., 97(7), 2557 (1997)

[2] Xue Y, Wilson D, Zhao H, Liu H, Sherman DH, Chem. Biol., 5, 661 (1998)

[3] Betlach MC, Kealey JT, Betlach MC, Ashley GW, McDaniel R, Biochemistry, 37, 14937 (1998)

[4] Graziani EI, Cane DE, Betlach MC, Kealey JT, McDaniel R, Bioorg. Med. Chem. Lett., 8, 3117 (1998)

[5] Yoon YJ, Beck BJ, Kim BS, Kang HY, Reynolds KA, Sherman DH, Chem. Biol., 9, 203 (2002)

[6] Lee C, Lee SG, Takahashi S, Kim BG, Biotechnol. Bioeng., 8, 89 (2003)

[7] Wang TH, Lee WC, Biotechnol. Bioeng., 8, 263 (2003)

[8] Ryu JC, Ryu TG, Yoo SJ, Hwang JY, Kim YH, Yang HS, J. Ind. Eng. Chem., 9(6), 735 (2003)

[9] Sambrook J, Russell DW, Molecular Cloning, 3rd Edn., Cold Spring Harbor Laboratory Press, New York (2001)

[10] Kharel MK, Lee HC, Sohng JK, Liou K, J. Ind. Eng. Chem., 8(5), 427 (2002)

[11] Plackett RL, Burman JP, Biometrika, 33, 305 (1946)

[12] Hussain HA, Ward JA, Enzyme Microb. Technol., 32(7), 790 (2003)