Production of Ortho-hydroxydaidzein Derivatives By a Recombinant Strain of Pichia pastoris Harboring a Cytochrome P450 Fusion Gene

• Main Authors: Yu-Cheng Chen, 陳昱丞
• Other Authors: Te-Sheng Chang
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/97253201702735636421

碩士 === 國立臺南大學 === 生物科技學系碩士班 === 102 === CYP57B3 from Aspergillus oryzae was recently discovered to catalyze the ortho-hydroxylation of the soy-isoflavone genistein. In the present study, the gene encoding CYP57B3 was fused with the reductase domain of the CYP102A1 gene (BM3R) from Bacillus megaterium, and recombinant Pichia pastoris harboring the P450 fusion gene was evaluated for its ability to produce ortho-hydroxydaidzein derivatives from daidzein. The results showed that 8-hydroxydaidzein (8-OHDe), 3’-hydroxydaidzein (3’-OHDe), and 6-hydroxydaidzein (6-OHDe) were produced during fermentation with a maximal conversion of 1.04%, 0.2% and 6.48%, respectively. In advance, CYP57B3 was fused with cytochrome P450 reductase (CPR) from Saccharomyces cerevisiae and the activity of isoflavone biotransformation by the recombinant strain P. pastoris harboring the fusion gene was determined. The results showed that the recombinant strain produced 8-OHDe, 3’-OHDe and 6-OHDe with a maximal conversion of 19.08%, 4.52% and 53.2%, respectively. In contrast, a recombinant strain harboring CYP57B3 only produced little ortho-hydroxydaidzein. The maximal yield of 6-OHDe in the present study was 13.30 µg/ml. To our knowledge, both the maximal yield and the conversion efficiency of 6-OHDe from daidzein in the present study are the highest among those reported in the literatures to date. Taken the results together, our results showed that CYP57B3 selectively produced 6-OHDe from daidzein biotransformation, and CPR from S. cerevisiae was better reductase partner than BMR from B. megaterium for its catalytic activity. In addition, the present study is also the first to demonstrate production of ortho-hydroxydaidzein derivatives using a fusion fungus cytochrome P450 enzyme. Keyword: Aspergillus oryzae；Cytochrome P450；Pichia pastoris；Daidzein；ortho-hydroxydaidzein。