Predicting high recombinant protein producer strains of Pichia pastoris MutS using the oxygen transfer rate as an indicator of metabolic burden

Abstract

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a widely used host for recombinant protein production. In this study, a clonal library of P. pastoris MutS strains (S indicates slow methanol utilization) was screened for high green fluorescent protein (GFP) production. The expression cassette was under the control of the methanol inducible AOX promoter. The growth behavior was online-monitored in 48-well and 96-well microtiter plates by measuring the oxygen transfer rate (OTR). By comparing the different GFP producing strains, a correlation was established between the slope of the cumulative oxygen transfer during the methanol metabolization phase and the strain’s production performance. The correlation corresponds to metabolic burden during methanol induction. The findings were validated using a pre-selected strain library (7 strains) of high, medium, and low GFP producers. For those strains, the gene copy number was determined via Whole Genome Sequencing. The results were consistent with the described OTR correlation. Additionally, a larger clone library (45 strains) was tested to validate the applicability of the proposed method. The results from this study suggest that the cumulative oxygen transfer can be used as a screening criterion for protein production performance that allows for a simple primary screening process, facilitating the pre-selection of high producing strains.

Assessing the capabilities of 2D fluorescence monitoring in microtiter plates with data-driven modeling for secondary substrate limitation experiments of Hansenula polymorpha

“…This advantage renders the technology an invaluable tool during early-stage bioprocess development [24][25][26]. Successful applications have been reported for clone screening and genetic construct selection [27][28][29][30][31], substrate feeding [32,33] and induction profiling [10,34,35]. Also, a systematic evaluation of media compositions and cultivation conditions for improved microbial growth and product formation has been demonstrated [36][37][38][39].…”

Otimização Da Produção De Activina a Em Komagataella Phaffii: Extensão Do Período De Indução E Uso De Sorbitol

“…A levedura Komagataella phaffi é uma hospedeira ideal para a produção de PR devido a sua capacidade de crescer até altas densidades, secretar proteínas dobradas e funcionais e seu promotor AOX1 (álcool oxidase I), induzido por metanol (Yamada et al, 1995;Wollborn et al, 2022). Apesar disso, desafios como regulação inadequada da expressão gênica, instabilidade da proteína e baixo rendimento de produção permanecem, sendo necessária a otimização das condições de cultivo (Türkanoğlu Özçelik et al, 2019;Bustos et al, 2022;Brabander et al, 2023).…”