水飞蓟来源黄酮3-羟化酶鉴定及黄杉素发酵优化

Identification of flavonoids 3-hydroxylase from [Silybum marianum (L.) Gaertn] and its application in enhanced production of taxifolin

摘要:

黄杉素是一种重要的黄酮类化合物，具有抗炎症、抗氧化等功效，在医药及保健品行业有着广泛的应用。黄酮3-羟化酶 (Flavanone 3-hydroxylase，F3H) 可以催化圣草酚合成黄杉素等3位羟基化的黄酮类化合物。一直以来，由于黄酮3-羟化酶催化效率低，使得利用微生物法合成黄杉素等多种3位羟基化黄酮的产量较低。文中从水飞蓟转录组中鉴定得到一个黄酮3-羟化酶SmF3H。发酵验证结果表明，SmF3H具有黄酮3位羟基化功能，且催化效率显著高于常用的来源于番茄 (Solanum lycopersicum) 的SlF3H。选取6个转录强度不同的启动子优化从黄酮前体柚皮素到黄杉素的合成途径，发现PGAL7启动子起始转录SmF3H时黄杉素合成产量最高，在摇瓶中产量达到695.90 mg/L；在5-L发酵罐上进行发酵优化，以柚皮素为底物发酵得到3.54 g/L黄杉素，是目前可见报道的最高产量。

Abstract:

(2S)-taxifolin is an important flavonoid that has anti-inflammatory and anti-oxidation effects. It is widely used in pharmaceutical and nutraceutical industries. Flavone 3-hydroxylase (F3H) can catalyze the synthesis of (2S)-taxifolin and other 3-hydroxylated flavonoids from (2S)-eriodictyol. Due to the low catalytic efficiency of F3H, the titer of many 3-hydroxyflavones, such as taxifolin, synthesized by microbial method is relatively low. In this study, a SmF3H was identified from the transcriptome of Silybum marianum (L.) Gaertn. The results of fermentation showed that SmF3H can catalyze the flavone 3-hydroxylation reaction, and its catalytic efficiency was significantly higher than that of commonly used SlF3H from Solanum lycopersicum. Six promoters with different transcription strength were selected to optimize the synthesis pathway from the flavonoid precursor (2S)-naringenin to (2S)-taxifolin. The results showed that the highest titer of (2S)-taxifolin (695.90 mg/L in shake flask) could be obtained when the PGAL7 promoter was used to control the expression of SmF3H. The titer of (2S)-taxifolin was further improved to 3.54 g/L in a 5-L fermenter, which is the highest titer according to current available literatures.