Growth-Based, High-Throughput Selection for NADH Preference in an Oxygen-Dependent Biocatalyst

• DOI: 10.1021/acssynbio.1c00258
• Publication Date: 2021-09-01T17:59:05Z
• Authors: Sarah Maxel; Samer Saleh; Edward King; Derek Aspacio; Linyue Zhang; Ray Luo; Han Li
• Keywords: Protein Engineering; Saturated mutagenesis; Rational design; Directed Molecular Evolution; Metabolic Engineering; Synthetic Biology

Cyclohexanone monooxygenases (CHMO) consume molecular oxygen and NADPH to catalyze the valuable oxidation of cyclic ketones. However, CHMO usage is restricted by poor stability stringent specificity for NADPH. Efforts engineer have been limited sensitivity enzyme perturbations in conformational dynamics long-range interactions that cannot be predicted. We demonstrate an aerobic, high-throughput growth selection platform Escherichia coli oxygenase evolution based on NADH redox balance. applied this NADH-dependent alter cofactor accept NADH, a less expensive than first identified variant DTNP (S208D-K326T-K349N-L143P) with ∼1200-fold relative switch from compared wild type through semirational design. Molecular modeling suggests activity driven cooperative fine-tuning contacts. Additional random mutagenesis yielded DTNPY ∼2900-fold afforded additional distal mutation, H163Y. These results highlight difficulty engineering functionally innovative variants static models rational designs, need high throughput methods. Our introduced tools accelerate advancements characteristics essential industrial feasibility.