A Growth-Based, High-Throughput Selection Platform Enables Remodeling of 4-Hydroxybenzoate Hydroxylase Active Site

• DOI: 10.1021/acscatal.0c01892
• Publication Date: 2020-06-05T18:28:52Z
• Authors: Sarah Maxel; Derek Aspacio; Edward King; Linyue Zhang; Ana Paula Acosta; Han Li
• Keywords: redox balance; Inorganic Chemistry; 0303 health sciences; 03 medical and health sciences; 4-hydroxybenzoate hydroxylase; 3; Organic Chemistry; 4-dihydroxybenzoic acid; directed evolution; Chemical Engineering; NADPH-dependent monooxygenase

ABSTRACTWe report an aerobic, growth-based selection platform founded on NADP(H) redox balance restoration inEscherichia coli, and demonstrate its application in high-throughput evolution of oxygenase. A single round of selection enabledPseudomonas aeruginoasa4-hydroxybenzoate hydroxylase (PobA) to accept 3,4-dihydroxybenzoic acid efficiently, an essential step toward gallic acid biosynthesis. The best variant DA015 exhibited more than 5-fold higher catalytic efficiency compared to previously engineered enzymes. Structural modeling suggests precise re-organization of active site hydrogen bond network, which is difficult to obtain without deep navigation of combinatorial sequence space. We envision universal application of this selection platform in engineering NADPH-dependent oxidoreductases.