Abstract
Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 is characterized as having wide substrate versatility for the biooxidation of (cyclic) ketones into esters and lactones with high stereospecificity. Despite industrial potential, CHMO usage is restricted by poor thermostability. Limited high-throughput screening tools and challenges in rationally engineering thermostability have impeded CHMO engineering efforts. We demonstrate the application of an aerobic, high-throughput growth selection platform in Escherichia coli (strain MX203) for the discovery of thermostability enhancing mutations for CHMO. The selection employs growth for the easy readout of CHMO activity in vivo, by requiring nicotinamide adenine dinucleotide phosphate (NADPH)-consuming enzymes to restore cellular redox balance. In the presence of the native substrate cyclohexanone, variant CHMO GV (A245G-A288V) was discovered from a random mutagenesis library screened at 42 °C. This variant retained native activity, exhibited ~4.4-fold improvement in residual activity after 30 °C incubation, and demonstrated ~5-fold higher cyclohexanone conversion at 37 °C compared to the wild type. Molecular modeling indicates that CHMO GV experiences more favorable residue packing and supports additional backbone hydrogen bonding. Further rational design resulted in CHMO A245G-A288V-T415C with improved thermostability at 45 °C. Our platform for oxygenase evolution enabled the rapid engineering of protein stability critical for industrial scalability.
Highlights
Baeyer–Villiger monooxygenases (BVMO) are valuable biocatalysts known for catalyzing the oxidation of ketones into esters and lactones through the utilization of reduced nicotinamide adenine dinucleotide (NAD(P)H)
◦ C (Figure 1), we discovered the variant Cyclohexanone monooxygenase (CHMO) GV (A245G-A288V) which retained of selection at single round of selection at 42 °C (Figure 1), we discovered the variant CHMO GV (A245G-A288V)
nicotinamide adenine dinucleotide phosphate (NADPH)-dependent selection2A), strain (MX203) that the activity level of is considerably reduced at elevated temperatures, which is at 37 °C in minimal medium on glucose, but failed to restore the growth at 42 °C
Summary
Baeyer–Villiger monooxygenases (BVMO) are valuable biocatalysts known for catalyzing the oxidation of (cyclic) ketones into esters and lactones through the utilization of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H). These enzymes have been studied for their industrial potential in the production of nylon monomers [1], (Z)-11-(heptanoyloxy)undec-9-enoic acid [2], and prostaglandins [3]. There have been extensive efforts to expand the substrate scope of BVMOs which have good stability but relatively narrow substrate ranges [3,6,7,8]. Protein engineering efforts have been applied to make the broad
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