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 (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.

Colorimetric High‐Throughput Screening Method for Directed Evolution of Prazole Sulfide Monooxygenase

“…[14] Consecutive rounds of error-prone polymerase chain reactions (epPCRs) were conducted to improve the activity of CHMO Acineto in the bioconversion of omeprazole sulfide 2 a. [8,15] Although positive variants with improved activities were identified, screening of the two libraries was realized in a low-throughput manner using headspace gas chromatography-mass spectrometry or high-performance liquid chromatography.…”