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

Jun Yao, Yang He, Nannan Su, Sakshibeedu R. Bharath, Yong Tao, Jian-Ming Jin (), Wei Chen (), Haiwei Song () and Shuang-Yan Tang ()
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Jun Yao: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences
Yang He: Institute of Molecular and Cell Biology
Nannan Su: Institute of Molecular and Cell Biology
Sakshibeedu R. Bharath: Institute of Molecular and Cell Biology
Yong Tao: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences
Jian-Ming Jin: Beijing Technology and Business University
Wei Chen: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences
Haiwei Song: Institute of Molecular and Cell Biology
Shuang-Yan Tang: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences

Nature Communications, 2020, vol. 11, issue 1, 1-12

Abstract: 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.

Date: 2020
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DOI: 10.1038/s41467-020-14918-5

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