Authors: Jun Yao, Yang He, Nannan Su, Sakshibeedu R. Bharath, Yong Tao, Jian-Ming Jin, Wei Chen, Haiwei Song, Shuang-Yan Tang
Published: 2020-03-23
DOI: 10.1038/s41467-020-14918-5
Source: Full article
AbstractHydroxytyrosol is an antioxidant free radical scavenger that is biosynthesized from tyrosine. In metabolic engineering efforts, the use of the mouse tyrosine hydroxylase limits its production. Here, we design an efficient whole-cell catalyst of hydroxytyrosol inEscherichia coliby de-bottlenecking two rate-limiting enzymatic steps. First, we replace the mouse tyrosine hydroxylase by an engineered two-component flavin-dependent monooxygenase HpaBC ofE. colithrough structure-guided modeling and directed evolution. Next, we elucidate the structure of theCorynebacterium glutamicumVanR regulatory protein complexed with its inducer vanillic acid. By switching its induction specificity from vanillic acid to hydroxytyrosol, VanR is engineered into a hydroxytyrosol biosensor. Then, with this biosensor, we use in vivo-directed evolution to optimize the activity of tyramine oxidase (TYO), the second rate-limiting enzyme in hydroxytyrosol biosynthesis. The final strain reaches a 95% conversion rate of tyrosine. This study demonstrates the effectiveness of sequentially de-bottlenecking rate-limiting steps for whole-cell catalyst development.