Production of Genistein from Naringenin Using Escherichia coli Containing Isoflavone Synthase-Cytochrome P450 Reductase Fusion Protein

• Published in: Journal of microbiology and biotechnology Vol. 19; no. 12; pp. 1612 - 1616
• Main Authors: Kim, D.H., Konkuk University, Seoul, Republic of Korea, Kim, B.G., Konkuk University, Seoul, Republic of Korea, Jung, N.R., Konkuk University, Seoul, Republic of Korea, Ahn, J.H., Konkuk University, Seoul, Republic of Korea
• Format: Journal Article
• Language: English
• Published: Seoul Korean Society for Applied Microbiology 01.12.2009; 한국미생물·생명공학회
• Subjects: Biological and medical sciences; Biotechnology; Biotransformation; CITOCROMO P450; Culture Media; CYTOCHROME P450; cytochrome P450 reductase; DNA, Plant - genetics; Escherichia coli - metabolism; Flavanones - metabolism; Fundamental and applied biological sciences. Psychology; GENISTEIN; Genistein - metabolism; GENISTEINA; GENISTEINE; Industrial Microbiology; NADPH-Ferrihemoprotein Reductase - chemistry; NADPH-Ferrihemoprotein Reductase - genetics; NADPH-Ferrihemoprotein Reductase - metabolism; naringenin; Oryza - genetics; Oxygenases - chemistry; Oxygenases - genetics; Oxygenases - metabolism; Protein Structure, Tertiary; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Trifolium - genetics; 생물학; Isoflavone; Enzyme; Cytochrome P450; Escherichia coli; Synthase; naringenin; cytochrome P450 reductase; genistein; Production; Bacteria; Oxidoreductases; Fusion protein; Enterobacteriaceae; Reductase
• ISSN: 1017-7825; 1738-8872
• DOI: 10.4014/jmb.0905.05043

Isoflavonoids are a class of phytoestrogens. Isoflavonone synthase (IFS) is responsible for the conversion of naringenin to genistein. IFS is a cytochrome P450 (CYP), and requires cytochrome P450 reductase (CPR) for its activity. Additionally, the majority of cytochrome P450s harbor a membrane binding domain, making them difficult to express in Escherichia coli. In order to resolve these issues, we constructed an in-frame fusion of the IFS from red clover (RCIFS) and CPR from rice (RCPR) after removing the membrane binding domain from RCIFS and RCPR. The resultant fusion gene, RCIFS-RCPR, was expressed in E. coli. The conversion of naringenin into genistein was confirmed using this E. coli transformant. Following the optimization of the medium and cell density for biotransformation, 60 μM of genistein could be generated from 80 μM of naringenin. This fusion protein approach may be applicable to the expression of other P450s in E. coli.