ABSTRACTWe report an aerobic, growth-based selection platform founded on NADP(H) redox balance restoration in Escherichia coli, and demonstrate its application in high-throughput evolution of oxygenase. A single round of selection enabled Pseudomonas aeruginoasa 4-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.
Absorption spectra of radical forms of 2,4-dihydroxybenzoic acid, a substrate for p-hydroxybenzoate hydroxylase