Auto-induction Screening Protocol for Ranking Clonal Libraries of Pichia pastoris MutS Strains

• Published in: Biotechnology and bioprocess engineering 2022-08, Vol.27 (4), p.572-585
• Main Authors: Wollborn, David, Müller, Rebecca Luise, Munkler, Lara Pauline, Horstmann, Rebekka, Germer, Andrea, Blank, Lars Mathias, Büchs, Jochen
• Format: Article
• Language: English
• Subjects: Alcohol oxidase; Biotechnology; Chemistry; Chemistry and Materials Science; Complex media; Cultivation; Ethanol; Fluorescence; Glucose; Glycerol; Green fluorescent protein; Industrial and Production Engineering; Libraries; Methanol; Monitoring; Monitoring systems; Oxygen transfer; Pichia pastoris; Proteins; Research Paper; Screening
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/s12257-022-0006-z

Screening Pichia pastoris ( Komagataella phaffii ) strain libraries utilizing the methanol inducible alcohol oxidase 1 ( AOX1 ) promoter requires addition of methanol as an inducer during cultivation. As the AOX1 promoter is repressed in the presence of glucose or glycerol, common screening procedures include long incubation times before methanol induction by successive multiple additions. In this study, a P. pastoris Mut S strain, secreting a green fluorescent protein (GFP), was used to characterize a standard screening protocol in complex medium. Based on these results, an auto-induction screening method for P. pastoris strains was developed using mineral SYN6 medium, where methanol was added from the beginning. To gain more insight into growth and production behavior, the cultures were online-monitored, using the Respiration Activity Monitoring System and the BioLector system. Monitoring the oxygen transfer rate indicated that prolonged cultivation phases before induction can be avoided and that high glucose concentrations hinder inducible protein production. The results show the effects of using 20 g/L glucose, and the resulting ethanol formation on the screening process. The newly developed auto-induction method was then used to screen and rank a strain library of high, medium, and low GFP producers. These results demonstrate that the auto-induction method facilitates the identification of high producers during primary screening. It is significantly less time consuming and labor intensive. Nonetheless, it leads to the same conclusion as the conventional screening with manual methanol additions.