Producing engineering fungal composite materials that sense and capture ß-lactam antibiotics from clinical wastewater to reduce antibiotics burden in sewage treatment plants
Metabolism, Biochemistry and Genetics of Microorganisms
Final Report Abstract
Beta lactam antibiotics are crucial for treating devastating human diseases, yet they are poorly cleared from wastewater using existing technologies. The main aim of this research project was to demonstrate proof of principle that biomass of the basidiomycete Fomes fomentarius can be coated with bifunctional fusion proteins that consist of a cell-wall binding domain and a penicillin-binding protein (PBP), thereby capturing ß-lactam antibiotics from a liquid. Ultimately, this may reduce antimicrobial resistance to beta lactams in an efficient and environmentally sustainable way. A secondary aim of this research project was to improve the transformation efficiency of this non-model species fungus and the heterologous expression of bifunctional fusion proteins. Ultimately, both aims are focused on the goal of generating engineered living materials using a fungal organism. To address the first aim, full-length and truncated variants of the coagulation factor G (CFG) from Tachypleus tridentatus fused to the fluorescent protein mStayGold were expressed in E. coli and binding to the fungal cell wall was confirmed with fluorescence microscopy. Subsequently, the binding of ampicillin to penicillin binding proteins (PBPs) fused to CFG or the enzymatic cleavage of penicillin G by ß-lactamases fused to CFG was investigated by means of HPLC and confirmed that the tested enzymes were active when bound to F. fomentarius. The second aim of this research project, the advancement of genetic engineering of F. fomentarius was realized by increasing the number of protoplasts that can be obtained by changing the cultivation conditions. Further, two promoters were tested successfully for their ability to drive expression of the resistance marker gene natR, conferring resistance against nourseothricine. Overall, this project shows that small (14kDa) glucan binding domains are suitable for immobilizing fusion proteins to the outer glucan layer of F. fomentarius and proteins stay catalytically active when bound to the cell wall.
Publications
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Poster: 18.09.2024 – 20.09.2024 Conference on Engineered Living Materials 2024, Saarbrücken, Germany (poster #47 Modification of Fomes fomentarius cell wall properties for the removal of beta lactam antibiotics from wastewater streams, presented by Dr. Stephan Starke
Stephan Starke
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Uncovering the transcriptional landscape of Fomes fomentarius during fungal-based material production through gene co-expression network analysis. Fungal Biology and Biotechnology, 12(1).
Cairns, Timothy; Freidank-Pohl, Carsten; Birke, Anna Sofia; Regner, Carmen; Jung, Sascha & Meyer, Vera
