Scalable protein production by Komagataella phaffii enabled by ARS plasmids and carbon source-based selection

• Published in: Microbial Cell Factories
• Main Authors: Florian Weiss, Guillermo Requena-Moreno, Carsten Pichler, Francisco Valero, Anton Glieder, Xavier Garcia-Ortega
• Format: Article in Journal/Newspaper
• Language: English
• Published: BMC 2024
• Subjects: Komagataella phaffii; Pichia pastoris; Episomal ARS plasmid; Lipase CalB; Carbon source marker; GUT1; Microbiology; QR1-502; Antarc*; Antarctica
• Online Access: https://doi.org/10.1186/s12934-024-02368-3; https://doaj.org/article/49035643a7b9475a8110594f150942fa

Abstract Background Most recombinant Komagataella phaffii (Pichia pastoris) strains for protein production are generated by genomic integration of expression cassettes. The clonal variability in gene copy numbers, integration loci and consequently product titers limit the aptitude for high throughput applications in drug discovery, enzyme engineering or most comparative analyses of genetic elements such as promoters or secretion signals. Circular episomal plasmids with an autonomously replicating sequence (ARS), an alternative which would alleviate some of these limitations, are inherently unstable in K. phaffii. Permanent selection pressure, mostly enabled by antibiotic resistance or auxotrophy markers, is crucial for plasmid maintenance and hardly scalable for production. The establishment and use of extrachromosomal ARS plasmids with key genes of the glycerol metabolism (glycerol kinase 1, GUT1, and triosephosphate isomerase 1, TPI1) as selection markers was investigated to obtain a system with high transformation rates that can be directly used for scalable production processes in lab scale bioreactors. Results In micro-scale deep-well plate experiments, ARS plasmids employing the Ashbya gossypii TEF1 (transcription elongation factor 1) promoter to regulate transcription of the marker gene were found to deliver high transformation efficiencies and the best performances with the reporter protein (CalB, lipase B of Candida antarctica) for both, the GUT1- and TPI1-based, marker systems. The GUT1 marker-bearing strain surpassed the reference strain with integrated expression cassette by 46% upon re-evaluation in shake flask cultures regarding CalB production, while the TPI1 system was slightly less productive compared to the control. In 5 L bioreactor methanol-free fed-batch cultivations, the episomal production system employing the GUT1 marker led to 100% increased CalB activity in the culture supernatant compared to integration construct. Conclusions For the first time, a scalable and methanol-independent ...