PhD-position | Functional proteomics of underexplored microorganisms for biocatalyst discovery

Description of project:

The identification of novel enzymes for industrial applications has been significantly advanced by omics technologies, bioinformatics, and engineering. The identification of new enzyme scaffolds is the first essential step in the development of new biocatalysts. The aim of this project is to identify novel enzymes including oxidoreductases and hydrolases through functional proteomics approaches. To this end, functional proteomic screening of anaerobic fungi and archaea as well as strain collections isolated from selected environments such as wastewater treatment plants, or microbial communities such as rumen or elephant gut microbiome, will be performed in collaboration with Doris Ribitsch to discover new enzyme activities without prior information about their sequence or structure. Three different proteomics approaches will be pursued: Secretomics will reveal proteins expressed and secreted in response to the substrate of interest (e.g. native and artificial polymers, such as lignocellulose). Thermal proteome profiling will reveal proteins binding to native substrates. Activity-based screens using probes for hydrolases and oxidoreductases will allow to identify new enzyme scaffolds. The latter two approaches offer the advantage to screen also otherwise non-culturable microorganisms, in particular new bacterial taxa or unknown bacteria, since it has a high probability of yielding genes encoding novel enzymes. The identified sequences will be produced in expression hosts like E.coli or P.pastoris, purified, validated and characterized in collaboration with Doris Ribitsch.

Background:

The working group has experience in functional proteomics [1-5].

Research Objective:

- Identification of new biocatalysts for decomposition of natural and synthetic polymers

- Decomposition of natural and synthetic polymer substrates into defined, low-molecular-weight products that will be used to develop novel and sustainable materials

Methods:

- Establishment of functional proteomics screening assays with target substrates (secretomics, thermal proteome profiling)

- Establishment of activity-based proteomics of hydrolases and oxidoreductases

- Establishment of habitat specific metaproteomics workflows

- Screening of strains (anaerobe, aerobe) and microbial communities from underexplored sources for new enzyme activities

- Recombinant production of enzymes and validation of enzymatic activities

References:

1) Krammer L, Darnhofer B, Kljajic M, Liesinger L, Schittmayer M, Neshchadin D, Gescheidt G, Kollau A, Mayer B, Fischer RC, Wallner S, Macheroux P, Birner-Gruenberger R*, Breinbauer R. (2025) A general approach for activity-based protein profiling of oxidoreductases with redox-differentiated diarylhalonium warheads. Chem Sci; doi: 10.1039/d4sc08454c. Epub ahead of print. PMID: 40103729; PMCID: PMC11912224. (#co-first author, *co-corresponding author)

2) Honeder SE, Tomin T, Schinagl M, Pfleger R, Hoehlschen J, Darnhofer B, Schittmayer M, Birner-Gruenberger R. (2023) Research advances through activity-based lipid hydrolase profiling. Israel Journal of Chemistry; doi: 10.1002/ijch.202200078

3) Schittmayer, M., Vujic, N., Darnhofer, B., Korbelius, M., Honeder, S., Kratky, D., Birner-Gruenberger, R. (2020) Spatially Resolved Activity-based Proteomic Profiles of the Murine Small Intestinal Lipases. Molecular & Cellular Proteomics, 19:2104-2115. DOI:10.1074/mcp.RA120.002171

4) Wallace, P. W., Haernvall, K., Ribitsch, D., Zitzenbacher, S., Schittmayer, M., Steinkellner, G., Gruber, K., Guebitz, G. M., Birner-Gruenberger, R. (2017) PpEst is a novel PBAT degrading polyesterase identified by proteomic screening of Pseudomonas pseudoalcaligenes. Applied Microbiology and Biotechnology, 101:2291–2303. DOI:10.1007/s00253-016-7992-8

5) Sturmberger, L., Wallace, P. W., Glieder, A., Birner-Gruenberger, R. (2016) Synergism of proteomics and mRNA sequencing for enzyme discovery. Journal of Biotechnology, 235:132-138. DOI:10.1016/j.jbiotec.2015.12.015

Where to apply:

https://www.circularbioengineering.at/