Evaluation of reference genes for transcript analyses in Komagataella phaffii (Pichia pastoris)

Besleaga, Mihail; Vignolle, Gabriel A.; Kopp, Julian; Spadiut, Oliver; Mach, Robert L.; Mach-Aigner, Astrid R.; Zimmermann, Christian

doi:10.1186/s40694-023-00154-1

Cited by 2 publications

(2 citation statements)

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“…In order to monitor the effects of the executed cultivations on a molecular level, RT-qPCR analyses of selected targets, amongst them the recombinant Ano UPO and PDI genes as well as K. pfhaffii endogenous genes, were performed as described recently [ 52 ].…”

Section: Resultsmentioning

confidence: 99%

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Bi-directionalized promoter systems allow methanol-free production of hard-to-express peroxygenases with Komagataella Phaffii

Besleaga,

Zimmermann,

Ebner

et al. 2024

Microb Cell Fact

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Background Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii. Results In initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system. Conclusions In this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.

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Section: Resultsmentioning

confidence: 99%

“…All reactions were performed in technical duplicates on a Rotor-Gene Q system (Qiagen, Hilden, Germany). Calculations of the relative transcript levels were performed according to the Pfaffl method [ 51 ] using the reference genes RSC1 and TAF10 for normalization according to [ 52 ].…”

Section: Methodsmentioning

confidence: 99%