香兰素是世界上最重要的风味和芳香化合物之一,用于食品和化妆品。在植物中,据报道香草醛是通过水合酶/裂解酶型酶VpVAN由阿魏酸生物合成的。然而,在生物技术和生物催化应用中,VpVAN的使用限制了香兰素的生产。虽然微生物酶作为植物酶的替代品很有帮助,使用微生物酶在一个步骤中从阿魏酸合成香兰素仍然是一个挑战。这里,我们开发了一种单一酶,通过合理设计类胡萝卜素裂解加氧酶家族中的微生物双加氧酶,以不依赖辅酶的方式催化阿魏酸生产香草醛。该酶通过将突变引入活性中心以增加其对阿魏酸的亲和力而获得对阿魏酸的催化活性。我们发现,单酶不仅可以催化由阿魏酸生产香草醛,还可以催化由对香豆酸合成其他醛,芥子酸,和松柏醇.这些结果表明,本研究中使用的方法可以极大地扩展双加氧酶家族酶可用的底物范围。该工程酶能够从可再生的木质素衍生化合物有效生产香草醛和其他增值醛。
目的:据报道,植物中香草醛生物合成的最后一步是由酶VpVAN催化的。在我们研究之前,VpVAN是唯一报道的将阿魏酸直接转化为香草醛的酶。然而,由于VpVAN的许多特征仍然未知,这种酶还不适合生物催化应用。我们表明,可以通过修饰微生物双加氧酶型酶来构建一步将阿魏酸转化为香草醛的酶。工程酶作为通过生物催化过程和代谢工程生产香草醛和相关化合物的工具具有生物技术重要性。这项研究的结果也可能为理解植物中香草醛的生物合成提供有用的见解。
Vanillin is one of the world\'s most important flavor and fragrance compounds used in foods and cosmetics. In plants, vanillin is reportedly biosynthesized from ferulic acid via the hydratase/lyase-type enzyme VpVAN. However, in biotechnological and biocatalytic applications, the use of VpVAN limits the production of vanillin. Although microbial enzymes are helpful as substitutes for plant enzymes, synthesizing vanillin from ferulic acid in one step using microbial enzymes remains a challenge. Here, we developed a single enzyme that catalyzes vanillin production from ferulic acid in a coenzyme-independent manner via the rational design of a microbial dioxygenase in the carotenoid cleavage oxygenase family using computational simulations. This enzyme acquired catalytic activity toward ferulic acid by introducing mutations into the active center to increase its affinity for ferulic acid. We found that the single enzyme can catalyze not only the production of vanillin from ferulic acid but also the synthesis of other aldehydes from p-coumaric acid, sinapinic acid, and coniferyl alcohol. These results indicate that the approach used in this study can greatly expand the range of substrates available for the dioxygenase family of enzymes. The engineered enzyme enables efficient production of vanillin and other value-added aldehydes from renewable lignin-derived compounds.
OBJECTIVE: The final step of vanillin biosynthesis in plants is reportedly catalyzed by the enzyme VpVAN. Prior to our study, VpVAN was the only reported enzyme that directly converts ferulic acid to vanillin. However, as many characteristics of VpVAN remain unknown, this enzyme is not yet suitable for biocatalytic applications. We show that an enzyme that converts ferulic acid to vanillin in one step could be constructed by modifying a microbial dioxygenase-type enzyme. The engineered enzyme is of biotechnological importance as a tool for the production of vanillin and related compounds via biocatalytic processes and metabolic engineering. The results of this study may also provide useful insights for understanding vanillin biosynthesis in plants.