Biocatalytic Oxyfunctionalization

Tailored Biocatalysis – For Pharmaceutical Active Ingredients, Synthetic Building Blocks, and Fine Chemicals

Selective oxyfunctionalizations are among the key reactions in modern drug synthesis. Using unspecific peroxygenases (UPOs), we perform regio- and stereoselective hydroxylations, epoxidations, as well as N-/O-dealkylations, and develop robust, scalable syntheses for active pharmaceutical ingredients, metabolites, chiral building blocks, and fine chemicals. Our service portfolio covers enzyme selection, reaction and process optimization, scale-up and downstream strategies, as well as comprehensive analytics. We reliably transfer optimized biocatalytic processes from milligram-scale screening to gram-scale production, while ensuring product identity and purity. Beyond product quality, we place particular emphasis on establishing resource-efficient processes.

Characteristics and Benefits of the Technology

  • Broad substrate spectrum, including aliphatic, aromatic, and heterocyclic compounds
  • Direct oxyfunctionalization of non-activated C–H bonds
  • Precise modification through high chemo-, regio-, and stereoselectivity
  • Direct access to the production of drug metabolites
  • Late-stage functionalization of complex natural products (e.g., steroids, terpenes)
  • High-throughput screening for SAR studies leveraging catalytic diversity 

From lab to industry: expertise that drives your innovation.

Our Contribution to Your Project

  • In silico design & cell-free expression of UPOs and variants for functional analysis
  • Selection of suitable UPO candidates for the target synthesis reaction
  • Development and optimization of reaction conditions
  • Scale-up to gram scale and product isolation
  • Analytical support: method development, mass balancing, and structure elucidation 
 

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NGD: Next Generation Drugs

Biotechnologically activated therapeutics – a tailored platform for precision medicine

Fraunhofer IZI-BB optimizes peroxygenases using cell-free systems and develops human liver models to convert drugs such as clopidogrel and tamoxifen into highly potent, well-tolerated metabolites. BTU and Fraunhofer IAP scale up production and develop formulation and cellular testing.

Project (German)

PZ-Syn: Fungal-Based Cell-Free Synthesis Platforms

Cell-free synthesis platforms for the production of innovative biocatalysts

© Alexander Karich
Cystoderma lilacipes in its natural environment.

In this Fraunhofer project group, Fraunhofer IZI-BB, BTU Cottbus-Senftenberg, and TU Dresden are developing novel cell-free synthesis platforms for the rapid production of unspecific peroxygenases from fungi, enabling their use in applications such as the synthesis of new active pharmaceutical ingredients and other specialty chemicals.

Project (German)

Methods

  • In silico structure modeling
  • Cell-free expression of peroxygenases
  • Activity assays in multi-well format for the determination of enzyme parameters
  • LC-MS-based substrate and product mass balancing
  • Design-of-experiments-guided development/optimization of reaction parameters
  • Use of various cosubstrate systems
  • Preparative biocatalytic synthesis in batch or fed-batch mode
  • Isolation of reaction products
  • Mass spectrometry and NMR for structure elucidation 

Equipment

  • Multimode plate reader ClarioStar Plus (BMG Labtech)
  • Spectrophotometer (Shimadzu)
  • 5-L-Bioreaktor (Sartorius)
  • Preparative HPLC (Shimadzu)
  • Mass spectrometer Q-TOF Maxis Impact (Bruker Daltonics)
  • Ultra-sensitive ion trap AmaZon Speed ETD (Bruker Daltonics)
  • Mass spectrometer Q-Trap (Sciex)
  • Gas chromatography (Agilent)
  • UHPLC (Ultimate 3000 nanoRSLC, Dionex)
  • HPLC (Shimadzu)

  • M. Schramm, C. Carrillo Avilés, J. Kalmbach, K.-U. Schmidtke, J. Kiebist, H. Kellner, M. Hofrichter, K. Scheibner (2025): Rapid screening system to identify unspecific peroxygenase activity. Clinical Hemorheologie and Microcirculation, 13860291241306566 (doi: 10.1177/13860291241306566)
  • S. Friedrich, M. Schramm, J. Kiebist, K.-U. Schmidtke, K. Scheibner (2025): Development of translationally active cell lysates from different filamentous fungi for application in cell-free protein synthesis. Enzyme and Microbial Technology, Vol. 185, 110588 (doi: doi.org/10.1016 /j.enzmictec.2025.110588)
  • P. Gomez de Sanztos, A. González-Benjumina, A. Fernandez-Garcia, C. Aranda, Y. Wu, A. But, P. Molina-Espeja, D. Mate Maté, D. Gonzales-Perez, W. Zhang, J. Kiebist, K. Scheibner, M. Hofrichter, K. Swdierek, V. Moliner, J. Sanz-Aparicio, F. Hollmann, A. Gutiérrez, M. Alcalde (2022): Engineering a highly regioselective fungal peroxygenase for the synthesis of hydroxy fatty acids. Angewandte Chemie International Edition, Vol. 62 (9), e202217372 (doi: 10.1002/anie.202217372)
  • R.M. Walter, A. Zemella, M. Schramm, J. Kiebist, S. Kubick (2022): Vesicle-based cell-free synthesis of short and long unspecific peroxygenases. Frontiers in Bioengineering and Biotechnology, 10:964396 (doi: 10.3389/fbioe.2022.964396)
  • R. König, J. Kiebist, J. Kalmbach, R. Herzog, K.-U. Schmidtke, H. Kellner, R. Ullrich, N. Jehmlich, M. Hofrichter, K. Scheibner (2022): Novel unspecific peroxygenases from Truncatella angustata catalyzes the synthesis of bioactive lipid mediators. Microorganisms, Vol. 10, 1267 (doi: 10.3390 /microorganisms10071267)
  • E. Babot, C. Aranda, J. Kiebist, K. Scheibner, R. Ullrich, M. Hofrichter, A.T. Martínez, A. Gutiérrez (2022): Enzymatic epoxidation of long-chain terminal alkenes by fungal peroxygenases. Antioxidants, Vol. 11, 522 (doi:10.3390/antiox11030522).
  • M. Schramm, S. Friedrich, K.-U. Schmidtke, J. Kiebist, P. Panzer, H. Kellner, R. Ullrich, M. Hofrichter, K. Scheibner (2022): Cell-free protein synthesis on basis of fungal lysates offers a novel and rapid platform for the production of unspecific peroxygenases. Antioxidants, Vol. 11 (2) 284 (doi: 10.3390/antiox11020284).
  • M. Hofrichter, H. Kellner, R. Herzog, A. Karich, J. Kiebist, K. Scheibner, R. Ullrich (2022): Peroxide-mediated oxygenation of organic compounds by fungal peroxygenases. Antioxidants, Vol. 11 (1), 163. (doi: 10.3390/antiox11010163)
  • J. Kiebist, K.-U. Schmidtke, M. Schramm, R. König, S. Quint, J. Kohlmann, R. Zuhse, R. Ullrich, M. Hofrichter, K. Scheibner (2021): Biocatalytic syntheses of antiplatelet metabolites of the thienopyridines clopidogrel and prasugrel using fungal peroxygenases. Journal of Fungi, Vol. 7 (9), 752 (doi: 10.3390/jof7090752)
  • P. Gomez de Santos, M. Dat Hoang, J. Kiebist, H. Kellner, R. Ullrich, K. Scheibner, M. Hofrichter, C. Liers, & M. Alcalde (2021): The functional expression in yeast of two unusual acidic peroxygenases from Candolleomyces aberdarensis by adopting evolved secretion mutations. Applied & Environmental Microbiology, AEM-00878 (doi: 10.1128/AEM.00878-21)
  • S. Bello, N. Pérez, J. Kiebist, K. Scheibner, M.I.S. Ruiz, A. Serrano, A.T. Martínez, G. Feijoo, M.T. Moreira (2021): Early-stage sustainability assessment of enzyme production in the framework of lignocellulosic biorefinery. Journal of Cleaner Production, 285, 125461 (doi: 10.1016/j.jclepro.2020.125461)
  • A. González-Benjumea, G. Marques, J. Kiebist, K. Scheibner, J.C. del Río, A.T. Martínez, A. Gutiérrez (2021): High epoxidation yields of vegetable oil hydrolyzates and methyl esters by selected fungal peroxygenases. Frontiers in Bioengineering and Biotechnology, 8, 1470 (doi: 10.3389/fbioe.2020.605854)
  • K.N. Ingenbosch, S. Quint, M. Dyllick-Brenzinger, D.S. Wunschik, J. Kiebist, P. Süss, U. Liebelt, R. Zuhse, U. Menyes, K. Scheibner, C. Mayer, K. Opwis, J.S. Gutmann, K. Hoffmann-Jacobsen (2021): Singlet oxygen generation by peroxidases and peroxygenases for chemo-enzymatic synthesis. ChemBioChem, 22, 398-407 (doi: 10.1002/cbic.202000326)
  • E. Babot, C. Aranda, J. del Río, R. Ullrich, J. Kiebist, K. Scheibner, M. Hofrichter, A. T. Martínez, A. Gutierrez (2020): Selective oxygenation of ionones and damascones by fungal peroxygenases. Journal of Agriculture and Food Chemistry, 68 (19), pp. 5375-5383 (doi: 10.1021/acs.jafc.0c01019)
  • J. Kiebist, S. Steinbrecht, R. König, M. Thiessen, K.-U. Schmidtke, S. Kammerer, J.-H. Küpper, K. Scheibner (2020): Enzymatic synthesis of cyclophosphamide metabolites by a fungal peroxygenase from Marasmius rotula for toxicology studies on human cancer cell lines HepG2 and MCF7. AMB Expr. 10, 128. (doi: 10.1007/s12268-020-1338-x)
  • K. Scheibner, R. Ullrich, J. Kiebist, H. Kellner, M. Hofrichter (2020): Unspezifische Peroxygenasen – Oxyfunktionalisierung außerhalb der Pilzhyphe. BIOspektrum, 26 (1), pp. 103-106 (doi: 10.1007/s12268-020-1338-x)
  • C. Aranda, M. Municoy, V. Guallar, J. Kiebist, K. Scheibner, R. Ullrich, J. C. del Río, M. Hofrichter, A. Gutiérrez (2019): Selective synthesis of 4-hydroxyisophorones and 4-ketoisophorones by fungal peroxygenases. Catalysis Science & Technology, 9, pp. 1398-1405 (doi: 10.1039/c8cy02114g).
  • C. Aranda, A. Olmedo, J. Kiebist, K. Scheibner, J.C. del Río, A.T. Martínez, A. Gutiérrez (2018): Selective epoxidation of fatty acids and fatty acid methyl esters by fungal peroxygenases. ChemCatChem, 10, pp. 3964-3968 (doi: 10.1002/cctc.201800849).
  • C. Aranda, R. Ullrich, J. Kiebist, K. Scheibner, J.C. del Río, M. Hofrichter, A.T. Martínez, A. Gutiérrez (2018): Selective synthesis of the resveratrol analogue 4,4‘-dihydroxy-trans-stilbene and stilbenoids modification by fungal peroxygenases. Catalysis Science & Technology, 8, pp. 2394-2401 (doi: 10.1039/C8CY00272J).
  • A. Olmedo, J.C. del Río, J. Kiebist, R. Ullrich, M. Hofrichter, K. Scheibner, A.T. Martínez, A. Gutiérrez (2017): Fatty Acid Chain Shortening by a Fungal Peroxygenase. Chemistry – A European Journal, 23 (67), pp. 16985-16989 (doi: 10.1002/chem.201701773).
  • J. Kiebist, K.-U. Schmidtke, J. Zimmermann, H. Kellner, R. Ullrich, D. Zänder, M. Hofrichter, K. Scheibner (2017): Novel peroxygenase from Chaetomium globosum enables the selective oxygenation of testosterone. ChemBioChem, 18 (6), pp. 563-569 (doi: 10.1002/cbic.201600677).
  • A. Olmedo, C. Aranda, J.C. del Río, J. Kiebist, K. Scheibner, A.T. Martínez, A. Gutiérrez (2016): From Alkanes to Carboxylic Acids: New Terminal Oxygenation Reactions by Fungal Peroxygenase. Angewandte Chemie Int. Ed., 55, pp. 12248-12251 (doi: 10.1002/anie.201605430R).
  • J. Kiebist, W. Holla, J. Heidrich, M. Poraj-Kobielska, M. Sandvoss, R. Simonis, G. Gröbe, J. Atzrodt, M. Hofrichter, K. Scheibner (2015): One-pot synthesis of human metabolites of SAR548304 by fungal peroxygenases. Bioorganic & Medicinal Chemistry, 23 (15), pp. 4324-4332 (doi: 10.1016/j.bmc.2015.06.035).