Abstract: In order to improve the ability of a wild-type strain (BY-1) of Pichia pastoris to produce glutathione (GSH), a mutant strain designated BY-1-26 was obtained by atmospheric and room temperature plasma (ARTP) mutagenesis. To further improve the production of glutathione by the mutant during shake flask culture, 1,2,4-triazole was added to the culture medium. Finally, adaptive evolution of the mutant strain was carried out on a microbial microdroplet culture system (MMC) using 1,2,4-triazole as a screening factor. As a result, a high-yield glutathione-producing mutant strain BY-2-24 was obtained, and its genetic stability was investigated. The results showed that a high-yield glutathione-producing mutant strain could be obtained using the wild-type strain BY-1 by ARTP mutagenesis, primary resistance screening on gradient plates, adaptive evolution on MMC and shake-flask secondary screening. The yield of glutathione produced by BY-2-24 in shake flask culture was (312.13 ± 2.62) mg/L, which was 134.26% higher than that produced by the original strain, and this mutant still had good genetic stability after seven passages. Meanwhile, the biomass was increased by 118.33%, indicating that the growth ability of the mutant strain was improved compared to the original strain. The result of this study show that the combination of ARTP and MMC can be used as a simple and effective screening method for excellent mutant strains, which provides a reference for high throughput selection of target strains.

Key words: glutathione; Pichia pastoris; atmospheric and room temperature plasma; adaptive evolution; strain selection