ArticleMicrobial cell factories2024
Bi-directionalized promoter systems allow methanol-free production of hard-to-express peroxygenases with Komagataella Phaffii.
Article in Microbial cell factories, 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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4 citing papers in PubMed.
- Empowering Precision C-H Functionalization: Advances in Peroxygenase Engineering.Angewandte Chemie (International ed. in English) · 2026Review
- N-Glycosylation Influences the Heterologous Expression of an Unspecific Peroxygenase From Marasmius rotula in Saccharomyces cerevisiae.Microbial biotechnology · 2026Article
- Current Understanding on the Heterogenous Expression of Plastic Depolymerising Enzymes inBioengineering (Basel, Switzerland) · 2025Review
- Chances and drawbacks of derepressed recombinant enzyme production in continuous cultivations withFrontiers in bioengineering and biotechnology · 2025Article
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9 authors.
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Abstract
backgroundHeme-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.
resultsIn 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.
conclusionsIn 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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