Abstract
Hydroxytyrosol 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 in Escherichia coli by de-bottlenecking two rate-limiting enzymatic steps. First, we replace the mouse tyrosine hydroxylase by an engineered two-component flavin-dependent monooxygenase HpaBC of E. coli through structure-guided modeling and directed evolution. Next, we elucidate the structure of the Corynebacterium glutamicum VanR 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.
Highlights
Hydroxytyrosol is an antioxidant free radical scavenger that is biosynthesized from tyrosine
To improve the low efficiency of the tyrosine hydroxylation step catalyzed by mouse tyrosine hydroxylase[13], a microbial tyrosine hydroxylase was redesigned by engineering HpaBC, a twocomponent flavin-dependent monooxygenase from E. coli
To explore the mechanism of the catalytic versatility of HpaBC, we modeled an active HpaBC bound with FAD and 4-HPA based on the reported structures of EcHpaBC (E. coli)[17] and ThHpaBC (Thermus thermophiles)[18]
Summary
Hydroxytyrosol is an antioxidant free radical scavenger that is biosynthesized from tyrosine. By switching its induction specificity from vanillic acid to hydroxytyrosol, VanR is engineered into a hydroxytyrosol biosensor With this biosensor, we use in vivo-directed evolution to optimize the activity of tyramine oxidase (TYO), the second ratelimiting enzyme in hydroxytyrosol biosynthesis. Rate-limiting steps of the biosynthetic pathways in combinatorial whole-cell catalysts might not be the same as those in the original hosts, due to the different protein expression levels, enzymatic activities and substrate or cofactor levels in the engineered host cells. We aim to demonstrate the feasibility of developing a powerful hydroxytyrosol whole-cell catalyst using E. coli, by sequentially solving the activities of enzymes catalyzing the rate-limiting steps of the pathway. Strategies of structure-based enzyme redesign and in vivo-directed evolution are adopted, until all the rate-limiting steps are solved and the conversion rate of end product reaches 95%
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