Agar plate-based activity assay for easy and fast screening of recombinant Pichia pastoris expressing unspecific peroxygenases.
Pichia pastoris
UPO
activity screening
agar plate assay
unspecific peroxygenases
Journal
Biotechnology journal
ISSN: 1860-7314
Titre abrégé: Biotechnol J
Pays: Germany
ID NLM: 101265833
Informations de publication
Date de publication:
04 Dec 2023
04 Dec 2023
Historique:
revised:
14
11
2023
received:
18
08
2023
accepted:
30
11
2023
pubmed:
4
12
2023
medline:
4
12
2023
entrez:
4
12
2023
Statut:
aheadofprint
Résumé
Unspecific peroxygenases (UPOs) are promising biocatalysts that catalyze oxyfunctionalization reactions without the need for costly cofactors. Pichia pastoris (reclassified as Komagataella phaffii) is considered an attractive host for heterologous expression of UPOs. However, integration of UPO-expression cassettes into the genome via a single cross-over yields recombinant Pichia transformants with different UPO gene copy numbers resulting in different expression levels. Selection of the most productive Pichia transformants by a commonly used screening in liquid medium in 96-well plates is laborious and lasts up to 5 days. In this work, we developed a simple two-step agar plate-based assay to screen P. pastoris transformants for UPO activity with less effort, within shorter time, and without automated screening devices. After cell growth and protein expression on agar plates supplemented with methanol and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), an additional top agar layer supplemented with ABTS and peroxide is added. UPO activity is visualized within 15 min by formation of green zones around UPO-secreting P. pastoris transformants. The assay was validated with two UPOs, AbrUPO from Aspergillus brasiliensis and evolved PaDa-I from Agrocybe aegerita. The assay results were confirmed in a quantitative 96-deep well plate screening in liquid medium.
Identifiants
pubmed: 38044796
doi: 10.1002/biot.202300421
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e2300421Subventions
Organisme : Ministry of Innovation, Science and Research of the German State of North Rhine-Westphalia
Informations de copyright
© 2023 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.
Références
Schmid, A., Dordick, J. S., Hauer, B., Kiener, A., Wubbolts, M., & Witholt, B. (2001). Industrial biocatalysis today and tomorrow. Nature, 409, 258-268.
Woodley, J. M. (2013). Protein engineering of enzymes for process applications, Current Opinion in Chemical Biology, 17, 310-316.
Bell, E. L., Finnigan, W., France, S. P., Green, A. P., Hayes, M. A., Hepworth, L. J., Lovelock, S. L., Niikura, H., Osuna, S., Romero, E., Ryan, K. S., Turner, N. J., & Flitsch, S. L. (2021). Nature Reviews Methods Primers, Biocatalysis, 1.
Hauer, B. (2020). Embracing nature's catalysts: a viewpoint on the future of Biocatalysis, ACS Catalysis, 10, 8418-8427.
Hofrichter, M., Kellner, H., Herzog, R., Karich, A., Liers, C., Scheibner, K., Kimani, V. W., & Ullrich, R. (2020). in Grand Challenges in Fungal Biotechnology, ed. H. Nevalainen, ch. 14, pp. 369-397.
Ullrich, R., Nuske, J., Scheibner, K., Spantzel, J., & Hofrichter, M. (2004). Novel haloperoxidase from the agaric basidiomycete Agrocybe aegerita oxidizes aryl alcohols and aldehydes, Applied and Environmental Microbiology, 70, 4575-4581.
Rotilio, L., Swoboda, A., Ebner, K., Rinnofner, C., Glieder, A., Kroutil, W., & Mattevi, A. (2021). Structural and biochemical studies enlighten the unspecific peroxygenase from Hypoxylon sp. EC38 as an efficient oxidative biocatalyst, ACS Catalysis, 11, 11511-11525.
Schmitz, F., Koschorreck, K., Hollmann, F., & Urlacher, V. B. (2023). Aromatic hydroxylation of substituted benzenes by an unspecific peroxygenase from Aspergillus brasiliensis. Reaction Chemistry & Engineering, 8, 2177-2186.
Conesa, A., Van De Velde, F., Van Rantwijk, F., Sheldon, R. A., van Den Hondel, C. A., & Punt, P. J. (2001). Expression of the Caldariomyces fumago Chloroperoxidase in Aspergillus niger and characterization of the recombinant enzyme. Journal of Biological Chemistry, 276, 17635-17640.
Babot, E. D., del Rio, J. C., Kalum, L., Martinez, A. T., & Gutierrez, A. (2013). Oxyfunctionalization of aliphatic compounds by a recombinant peroxygenase from Coprinopsis cinerea. Biotechnology and Bioengineering, 110, 2323-2332.
Molina-Espeja, P., Ma, S., Mate, D. M., Ludwig, R., & Alcalde, M. (2015). Tandem-yeast expression system for engineering and producing unspecific peroxygenase. Enzyme and Microbial Technology, 73-74, 29-33.
Püllmann, P., Knorrscheidt, A., Münch, J., Palme, P. R., Hoehenwarter, W., Marillonnet, S., Alcalde, M., Westermann, B., & Weissenborn, M. J. (2021). A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases. Communications Biology, 4, 562.
Bormann, S., Kellner, H., Hermes, J., Herzog, R., Ullrich, R., Liers, C., Ulber, R., Hofrichter, M., & Holtmann, D. (2022). Broadening the biocatalytic toolbox-Screening and expression of new unspecific peroxygenases. Antioxidants, 11, 223.
Kimani, V. W. (2019). PhD Doctoral Thesis, Technical University Dresden.
Kiebist, J., Schmidtke, K. U., Zimmermann, J., Kellner, H., Jehmlich, N., Ullrich, R., Zander, D., Hofrichter, M., & Scheibner, K. (2017). A peroxygenase from Chaetomium globosum catalyzes the selective oxygenation of testosterone. ChemBioChem, 18, 563-569.
Ebner, K., Pfeifenberger, L. J., Rinnofner, C., Schusterbauer, V., Glieder, A., & Winkler, M. (2023). Discovery and heterologous expression of unspecific peroxygenases. Catalysts, 13, 206.
Linde, D., Santillana, E., Fernandez-Fueyo, E., Gonzalez-Benjumea, A., Carro, J., Gutierrez, A., Martinez, A. T., & Romero, A. (2022). Structural characterization of two short unspecific peroxygenases: two different dimeric arrangements. Antioxidants, 11, 891.
Robinson, W. X. Q., Mielke, T., Melling, B., Cuetos, A., Parkin, A., Unsworth, W. P., Cartwright, J., & Grogan, G. (2023). Comparing the catalytic and structural characteristics of a ‘short’ unspecific peroxygenase (UPO) expressed in Pichia pastoris and Escherichia coli. ChemBioChem, 24, 202200558.
Jankowski, N., Koschorreck, K., & Urlacher, V. B. (2020). High-level expression of aryl-alcohol oxidase 2 from Pleurotus eryngii in Pichia pastoris for production of fragrances and bioactive precursors. Applied Microbiology and Biotechnology, 104, 9205-9218.
Bronikowski, A., Hagedoorn, P. L., Koschorreck, K., & Urlacher, V. B. (2017). Expression of a new laccase from Moniliophthora roreri at high levels in Pichia pastoris and its potential application in micropollutant degradation. AMB Express, 7, 73.
Bronikowski, A., Koschorreck, K., & Urlacher, V. B. (2018). Redesign of a new manganese peroxidase highly expressed in Pichia pastoris towards a lignin-degrading versatile peroxidase. ChemBioChem, 19, 2481-2489.
Schwarzhans, J. P., Wibberg, D., Winkler, A., Luttermann, T., Kalinowski, J., & Friehs, K. (2016). Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization. Microbial Cell Factories, 15, 84.
Vogl, T., Gebbie, L., Palfreyman, R. W., & Speight, R. (2018). Effect of plasmid design and type of integration event on recombinant protein expression in Pichia pastoris. Applied and Environmental Microbiology, 84, e02712-e02717.
Schmitz, L. M., Rosenthal, K., & Lutz, S. (2019). Recent advances in heme biocatalysis engineering. Biotechnology and Bioengineering, 116, 3469-3475.
Jankowski, N., & Koschorreck, K. (2022). Agar plate assay for rapid screening of aryl-alcohol oxidase mutant libraries in Pichia pastoris. Journal of Biotechnology, 346, 47-51.
Karnaouri, A., Zerva, A., Christakopoulos, P., & Topakas, E. (2021). Screening of recombinant lignocellulolytic enzymes through rapid plate assays. Methods in Molecular Biology, 2178, 479-503.
Mate, D. M., Gonzalez-Perez, D., Kittl, R., Ludwig, R., & Alcalde, M. (2013). Functional expression of a blood tolerant laccase in Pichia pastoris. BMC Biotechnology [Electronic Resource], 13, 1-12.
Heo, S., Kim, S., & Kang, D. (2020). The role of hydrogen peroxide and peroxiredoxins throughout the cell cycle. Antioxidants, 9, 280.
Francisco, M. (1993). Cumene hydroperoxide explosion. Chemical & Engineering News, 71, 4.
Duh, Y.-S., Kao, C.-S., Hwang, H.-H., & Lee, W. W. L. (1998). Thermal decomposition kinetics of cumene hydroperoxide. Process Safety and Environment Protection, 76, 271-276.
Molina-Espeja, P., Garcia-Ruiz, E., Gonzalez-Perez, D., Ullrich, R., Hofrichter, M., & Alcalde, M. (2014). Directed evolution of unspecific peroxygenase from Agrocybe aegerita. Applied and Environmental Microbiology, 80, 3496-3507.
Adachi, S., Nagano, S., Ishimori, K., Watanabe, Y., Morishima, I., Egawa, T., Kitagawa, T., & Makino, R. (1993). Roles of proximal ligand in heme proteins: Replacement of proximal histidine of human myoglobin with cysteine and tyrosine by site-directed mutagenesis as models for P-450, chloroperoxidase, and catalase. Biochemistry, 32, 241-252.
Matsui, T., Nagano, S., Ishimori, K., Watanabe, Y., & Morishima, I. (1996). Preparation and reactions of myoglobin mutants bearing both proximal cysteine ligand and hydrophobic distal cavity: Protein models for the active site of P-450. Biochemistry, 35, 13118-13124.
Yoshioka, S., Takahashi, S., Ishimori, K., & Morishima, I. (2000). Roles of the axial push effect in cytochrome P450cam studied with the site-directed mutagenesis at the heme proximal site. Journal of Inorganic Biochemistry, 81, 141-151.
Yoshioka, S., Takahashi, S., Hori, H., Ishimori, K., & Morishima, I. (2001). Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450 cam. European Journal of Biochemistry, 268, 252-259.
Ramirez-Escudero, M., Molina-Espeja, P., Gomez de Santos, P., Hofrichter, M., Sanz-Aparicio, J., & Alcalde, M. (2018). Structural insights into the substrate promiscuity of a laboratory-evolved peroxygenase. ACS Chemical Biology, 13, 3259-3268.
Jankowski, N., Urlacher, V. B., & Koschorreck, K. (2021). Two adjacent C-terminal mutations enable expression of aryl-alcohol oxidase from Pleurotus eryngii in Pichia pastoris. Applied Microbiology and Biotechnology, 105, 7743-7755.