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

• Published in: Microbial cell factories 2024-06, Vol.23 (1), p.177-14
• Main Authors: Besleaga, Mihail, Zimmermann, Christian, Ebner, Katharina, Mach, Robert L, Mach-Aigner, Astrid R, Geier, Martina, Glieder, Anton, Spadiut, Oliver, Kopp, Julian
• Format: Article
• Language: English
• Subjects: Analysis; Bi-directionalized promoter; Bioreactors; Catalysis; Derepressed feeding; Electron transport; Glycerin; Glycerol; Heme; Komagataella phaffii; Methanol; Methanol - metabolism; Methanol-free; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Oxidases; Promoter Regions, Genetic; Recombinant protein production; Recombinant proteins; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomycetales - enzymology; Saccharomycetales - genetics; Saccharomycetales - metabolism; Unspecific peroxygenase
• ISSN: 1475-2859; 1475-2859
• DOI: 10.1186/s12934-024-02451-9

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. 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. 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.