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bioRxiv · 10.1101/2020.05.11.088898

A growth-based, high-throughput selection platform enables remodeling of 4-hydroxybenzoate hydroxylase active site

Maxel, S.·Aspacio, D.·King, E.·Zhang, L.·Acosta, A. P.·Li, H.
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Abstract

We 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.

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Maxel, S., Aspacio, D., King, E., Zhang, L., Acosta, A. P., Li, H.. 2020-05-13. A growth-based, high-throughput selection platform enables remodeling of 4-hydroxybenzoate hydroxylase active site. https://doi.org/10.1101/2020.05.11.088898

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