Affiliation:
1. State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Centre for Biomanufacturing East China University of Science and Technology Shanghai China
Abstract
AbstractThe methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a highly distinguished expression platform for the excellent synthesis of various heterologous proteins in recent years. With the advantages of high‐density fermentation, P. pastoris can produce gram amounts of recombinant proteins. While not every protein of interest can be expressed to such high titers, such as Baeyer–Villiger monooxygenase (BVMO) (AcPSMO) which is responsible for pyrazole sulfide asymmetric oxidation. In this work, an excellent yeast expression system was established to facilitate efficient AcPSMO expression, which exhibited 9.5‐fold enhanced secretion. Subsequently, an ultrahigh throughput screening method based on fluorescence‐activated cell sorting by fusing super folder green fluorescent protein (sfGFP) in the C‐terminal of AcPSMO was developed, and directed evolution was performed. The protein expression level of the superior mutant AcPSMOP1 (S58T/T252P/E336N/H456D) reached 84.6 mg/L at 100 mL shaking flask, which was 4.7 times higher than the levels obtained with the wild‐type. Finally, the optimized chassis cells were used for high‐density fermentation on a 5‐L scale, and AcPSMOP1 protein yield of 3.4 g/L was achieved, representing approximately 85% of the total protein secreted. By directly employing the pH‐adjusted supernatant as a biocatalyst, 20 g/L pyrmetazole sulfide was completely transformed into the corresponding (S)‐sulfoxide, with a 78.8% isolated yield. This work confers dramatic benefits for efficient secretion of other BVMOs in P. pastoris.
Funder
Fundamental Research Funds for the Central Universities
Program of Shanghai Academic Research Leader
National Natural Science Foundation of China
Subject
Applied Microbiology and Biotechnology,Bioengineering,Biotechnology
Cited by
5 articles.
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