羟基化
阿魏酸
酶动力学
化学
产量(工程)
还原酶
抗氧化剂
多酚
生物合成
突变体
催化作用
酶
立体化学
生物化学
代谢工程
活动站点
材料科学
基因
冶金
作者
Peilong Sun,Pu Zheng,Pengcheng Chen,Dan Wu,Shuping Xu
标识
DOI:10.1016/j.ijbiomac.2024.130545
摘要
Polyphenolic compounds have natural antioxidant properties, and their antioxidant activity is usually related to the number and position of hydroxyls. Here, we successfully applied the engineered 4-hydroxyphenylacetate 3-hydroxylases (4HPA3Hs) derived from Pseudomonas aeruginosa to catalyze ferulic acid (FA) synthesis of ortho-hydroxyferulic acid (5-hydroxyferulic acid, 5-OHFA). Through optimization of co-expression, the oxygenase component (PaHpaB) and the reductase component (PaHpaC) in E. coli, and optimization of whole-cell catalytic conditions, the engineered strain BC catalyzed ortho-hydroxylation of 2 g/L of FA with a yield of 75 % from 39 %. Through tunnel engineering of PaHpaB, the obtained mutants F301A and Q376A almost completely transformed 2 g/L of FA. Further, a multiple mutant L214A/F301A/Q376A converted 4 g/L FA into 5-OHFA within 12 h, and the yield reached 99.9 %, which was approximately 2.39-fold of the wild type. The kcat/Km value of L214A/F301A/Q376A was about 307 times greater than that of the wide type. Analysis of three-dimensional structural models showed that L214, F301, and Q376 mutated into Ala, which greatly shortened the side chain and broadened the tunnel size, thereby significantly improving the catalytic efficiency of L214A/F301A/Q376A. This biosynthesis of 5-OHFA is simple, efficient, and green, suggesting that it is useful for efficient biosynthesis of polyphenolic compounds.
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