Pairing two growth-based, high-throughput selections to fine tune conformational dynamics in oxygenase engineering

Abstract

Cyclohexanone monooxygenases (CHMO) consume molecular oxygen and NADPH to catalyze the valuable oxidation of cyclic ketones. However, CHMO usage is restricted by poor thermostability and stringent specificity for NADPH. Efforts to engineer CHMO have been limited by the sensitivity of the enzyme to perturbations in conformational dynamics and long-range interactions that cannot be predicted. We demonstrate a pair of aerobic, high-throughput growth selection platforms in Escherichia coli for oxygenase evolution, based on NADPH or NADH redox balance. We utilize the NADPH-dependent selection in the directed evolution of thermostable CHMO and discover the variant CHMO GV (A245G-A288V) with a 2.7-fold improvement in residual activity compared to the wild type after 40 °C incubation. Addition of a previously reported mutation resulted in A245G-A288V-T415C which has further improved thermostability at 45 °C. We apply the NADH-dependent selection to alter the cofactor specificity of CHMO to accept NADH, a less expensive cofactor than NADPH. We identified the variant CHMO DTNP (S208D-K326T-K349N-L143P) with a 21-fold cofactor specificity switch from NADPH to NADH compared to the wild type. Molecular modeling indicates that CHMO GV experiences more favorable residue packing and backbone torsions, and CHMO DTNP activity is driven by cooperative fine-tuning of cofactor contacts. Our introduced tools for oxygenase evolution enable the rapid engineering of properties critical to industrial scalability. KEY WORDScyclohexanone monooxygenase, Acinetobacter sp., Baeyer-Villiger monooxygenase, directed evolution, thermostability, NAD(P)H cofactor specificity, redox balance, high-throughput selection Recently, we described the construction and application of an aerobic growth-based, highthroughput selection platform for engineering NADPH-dependent oxygenases 10 . We demonstrated that this selection platform enabled rapid remodeling of the Pseudomonas aeruginosa 4-hydroxybenzoate hydroxylase (PobA) active site to efficiently accept a non-native substrate 3,4-dihydroxybenzoic acid (3,. The selected variants appear to recognize the new substrate with synergistic hydrogen bond networks, which are difficult to discover without high-throughput searching of protein sequence space. However, the key limitation of this method is that it is not compatible with engineering NADH-dependent oxygenases. While NADPH-dependent oxygenases such as most P450s naturally function in anabolism, the NADH-dependent oxygenases function in catabolism and enable the conversion of various recalcitrant substrates such as xylene 11 and plastics 12 . Here, we report an aerobic growth-based selection for NADH activity, which complements the NADPH-dependent selection platform. Together, these two selection systems may support the full range of engineering capabilities to develop catalytically important oxygenases. SUPPORTING INFORMATIONExperimental and computational methods, plasmids and strains used in this study (Table S1), detailed kinetic analysis of CHMO variants...