Secretory expression of cyclohexanone monooxygenase by methylotrophic yeast for efficient omeprazole sulfide bio-oxidation

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.

Keywords

Cyclohexanone monooxygenase / Omeprazole sulfoxide / Pichia pastoris / Secretory expression / Asymmetric oxidation

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Funding

National Natural Science Foundation of China(21922804)

National Key Research and Development Program of China(2019YFA09005000)

Fundamental Research Funds for the Central Universities(22221818014)

Program of Shanghai Academic Research Leader(21XD1400800)