Journal Article

Developing a highly efficient hydroxytyrosol whole-cell catalyst by de-bottlenecking rate-limiting steps

Su, Nannan, Chen, Wei, Tang, Shuang-Yan, Yao, Jun, Tao, Yong, Bharath, Sakshibeedu R., Jin, Jian-Ming, Song, Haiwei, He, Yang

2020

Overview

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. Whole-cell catalyst-based hydroxytyrosol production is low. Here, the authors increase the efficiency of its production in E. coli by de-bottlenecking two enzymatic steps catalyzed by monooxygenase and tyramine oxidase using structure-based enzyme redesign or in vivo-directed evolution with the aid of a newly developed biosensor.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 49/31 / 49/47 / 631/1647/1888 / 631/1647/2163 / 631/1647/2258/1266 / 631/61/318 / Amine oxidase (flavin-containing) / Antioxidants / Bacteria / Bacterial Proteins - genetics / Bacterial Proteins - metabolism / Biosensing Techniques / Biosensors / Biosynthesis / Biosynthetic Pathways - genetics / Catalysts / Constraining / Corynebacterium glutamicum - enzymology / Corynebacterium glutamicum - genetics / Directed evolution / Directed Molecular Evolution - methods / E coli / Enzymes / Escherichia coli / Escherichia coli - enzymology / Escherichia coli - genetics / Escherichia coli Proteins - genetics / Escherichia coli Proteins - metabolism / Evolution / Feasibility Studies / Flavin / Free Radical Scavengers - metabolism / Free radicals / Humanities and Social Sciences / Hydroxylase / Metabolic Engineering / Mixed Function Oxygenases - genetics / Mixed Function Oxygenases - metabolism / multidisciplinary / Mutagenesis, Site-Directed / Mutation / Oxidase / Phenylethyl Alcohol - analogs & derivatives / Phenylethyl Alcohol - metabolism / Redesign / Science / Science (multidisciplinary) / Transcription Factors - genetics / Transcription Factors - metabolism / Tyramine / Tyrosine / Tyrosine - metabolism / Tyrosine 3-monooxygenase / Tyrosine 3-Monooxygenase - genetics / Tyrosine 3-Monooxygenase - metabolism / Vanillic acid / Vanillic Acid - metabolism