Department of Chemical Science and Engineering, Kathmandu University, P.O.Box: 6250, Dhulikhel, Kavre, Nepal.
School of Chemical and Biological Engineering, Institute of Molecular Biology and Genetics, Institute of Bioengineering, Seoul National University, Seoul, South Korea.
Appl Biochem Biotechnol. 2018 Mar;184(3):1036-1046. doi: 10.1007/s12010-017-2606-1. Epub 2017 Sep 22.
We have reported that the expression of CYP105D7 in Streptomyces avermitilis produces 112.5 mg L of 7,3',4'-trihydroxyisoflavone (3'ODI) in 15 h of the reaction time, when 7,4'-dihydroxyisoflavone (daidzein) is used as a substrate. Although production is significant, rapid degradation of 3'ODI after 15 h was observed in a whole-cell biotransformation system, suggesting the further modification of 3'ODI by endogenous enzymes. In this present study, the effect of deletion of extracellular tyrosinase (melC2) in S. avermitilis for 3'ODI production as well as the expressions of CYP105D7, ferredoxin (Fdx), and ferredoxin reductase (Fpr) were investigated. The result revealed that daidzein hydroxylation activity in the ∆melC2 mutant decreased by 40% compared with wild-type S. avermitilis. Further, melC2 deletion significantly affects the messenger RNA (mRNA) expression profile of CYP105D7 and its electron transfer counterparts. Real-time PCR analysis of 9 Fdx, 6 Fpr, and CYP105D7 revealed a significant decrease in mRNA expression level compared to wild-type S. avermitilis. The result clearly shows that the decrease in daidzein hydroxylation activity is due to the lower expression level of CYP105D7 and its electron transfer counterpart in the ∆melC2 mutant. Furthermore, melC2 deletion prevents the degradation of 3'ODI.
我们曾报道过，在阿维链霉菌中表达 CYP105D7 时，以 7,4'-二羟基异黄酮（大豆黄素）为底物，反应 15 小时可产生 112.5mg/L 的 7,3',4'-三羟基异黄酮（3'ODI）。尽管产量可观，但在全细胞生物转化体系中，15 小时后 3'ODI 迅速降解，表明内源性酶进一步修饰了 3'ODI。在本研究中，我们研究了缺失阿维链霉菌胞外酪氨酸酶（melC2）对 3'ODI 生产的影响，以及 CYP105D7、铁氧还蛋白（Fdx）和铁氧还蛋白还原酶（Fpr）的表达情况。结果表明，与野生型阿维链霉菌相比，突变株中大豆黄素羟化活性下降了 40%。此外，melC2 缺失显著影响 CYP105D7 及其电子传递对应物的信使 RNA（mRNA）表达谱。与野生型阿维链霉菌相比，9 个 Fdx、6 个 Fpr 和 CYP105D7 的实时 PCR 分析显示其 mRNA 表达水平显著下降。结果清楚地表明，daidzein 羟化活性的降低是由于 ∆melC2 突变体中 CYP105D7 及其电子传递对应物的表达水平较低所致。此外，melC2 缺失可防止 3'ODI 的降解。
