| Summary: | 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 (CHMO Acineto -Mut) that has high stereoselectivity. CHMO Acineto -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 CHMO Acineto -Mut by a methylotrophic yeast, Pichia pastoris, for the first time. The recombinant CHMO Acineto -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 CHMO Acineto -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.
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