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

Abstract

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

Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform

“…This task is especially challenging when engineering enzymes for biotechnology applications, as most of the industrially important reactions neither exist in natural metabolism nor do they contribute to cell fitness. However, employing the redox balance principle can bypass this bottleneck in engineering redox cofactor-dependent enzyme 15 , 21 – 23 , 25 , 59 . In this work, we established the engineered Gor as a universal reporter for intracellular availability of NMNH, which successfully distinguished the high and low NMN + -reducing variants of two distinct enzymes, PTDH and GDH.…”

“…Growth selection is a powerful tool in enzyme engineering due to its easy readout and unparallel throughput (>10 6 per iteration) 15 , 21 – 28 , compared to 96-well plate-based or agar plate-based colorimetric methods (10 2 –10 4 per iteration) 29 – 32 . Multiple growth-based selection platforms have been designed to engineer NAD(P)H-dependent enzymes 21 – 28 , where the unifying principle behind is that cells can only grow when the life-essential redox reactions have their cofactors recycled continuously in vivo. We extend this principle here for the noncanonical redox cofactor NMN + .…”

Logistic Regression-Guided Identification of Cofactor Specificity-Contributing Residues in Enzyme with Sequence Datasets Partitioned by Catalytic Properties

“…Studies utilizing enzymes with different redox cofactor selectivity from other organisms were reported; however, protein engineering is often needed because heterologous enzymes may not be expressed or have good catalytic activities. There are reports of narrowing down candidate mutant residues from crystal structures of enzymes with bound cofactors − and proliferative screening that alters the cellular metabolic state . However, a high-throughput assay that can screen many metabolic enzymes has not been constructed.…”

“…There are reports of narrowing down candidate mutant residues from crystal structures of enzymes with bound cofactors 28 − 31 and proliferative screening that alters the cellular metabolic state. 32 However, a high-throughput assay that can screen many metabolic enzymes has not been constructed. In this study, we redesigned the cofactor specificity of MaeB from NADP + to NAD + without structure information and screening steps.…”

“…Protein engineering to switch cofactor specificity was primarily developed in metabolic engineering research. − However, many redesigned enzymes are only promiscuous modifications, and a basic understanding of cofactor specificity switching is inadequate. In brief, this study used a logistic regression model to identify the amino acids behind NAD + - and NADP + -dependence for each position.…”