Abstract
Prochiral pyrmetazole can be asymmetrically oxidized into (S)-omeprazole, a proton pump inhibitor that is used to treat gastroesophageal reflux, by an engineered cyclohexanone monooxygenase (CHMOAcineto-Mut) that has high stereoselectivity. CHMOAcineto-Mut is produced by heterologous expression in Escherichia coli, where it is expressed intracellularly. Thus, isolating this useful biocatalyst requires tedious cell disruption and subsequent purification, which hinders its use for industrial purposes. Here, we report the extracellular production of CHMOAcineto-Mut by a methylotrophic yeast, Pichia pastoris, for the first time. The recombinant CHMOAcineto-Mut expressed by P. pastoris showed a higher flavin occupation rate than that produced by E. coli, and this was accompanied by a 3.2-fold increase in catalytic efficiency. At a cell density of 150 g/L cell dry weight, we achieved a recombinant CHMOAcineto-Mut production rate of 1,700 U/L, representing approximately 85% of the total protein secreted into the fermentation broth. By directly employing the pH adjusted supernatant as a biocatalyst, we were able to almost completely transform 10 g/L of pyrmetazole into the corresponding (S)-sulfoxide, with > 99% enantiomeric excess.
Highlights
Baeyer–Villiger monooxygenase (BVMO) is a flavindependent enzyme that catalyzes the regioselective Baeyer–Villiger oxidation of ketones to the corresponding esters or lactones (Fürst et al 2019; Romero et al 2018; de Gonzalo et al 2010)
An approximately 65-kDa protein band increased in intensity in parallel with increasing pyrmetazole oxidation activity when CHMOAcineto-Mut was expressed under the control of the Alcohol oxidase 1 promoter (AOX1) promoter (Fig. 1B)
To identify strains with multiple integration events, X33-pPICZαA-CHMOAcineto-Mut transformants were grown on YPDZ plates with different zeocin concentrations
Summary
Baeyer–Villiger monooxygenase (BVMO) is a flavindependent enzyme that catalyzes the regioselective Baeyer–Villiger oxidation of ketones to the corresponding esters or lactones (Fürst et al 2019; Romero et al 2018; de Gonzalo et al 2010). An approximately 65-kDa protein band increased in intensity in parallel with increasing pyrmetazole oxidation activity (from 12 to 41 U/L) when CHMOAcineto-Mut was expressed under the control of the AOX1 promoter (Fig. 1B).
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