Job openings in Program 1

Background:

Biomass and products from biomass are rich in complex molecules. Often, the unambiguous structural elucidation is necessary to identify unwanted byproducts (e. g. chromophores), to increase the understanding of a process (e. g. degradation products, unintended byproducts), and to describe newly discovered bioactive compounds. The availability of the compounds of interest is often limited either because the effort to isolate sizeable amounts is prohibitive, or because the compounds occur only in minute amounts. Current methodology – the combination of mass spectroscopy after chromatography, nuclear magnetic resonance spectroscopy, infrared spectroscopy – quickly reaches its limit when the available amount of substance is small (microgram). Crystallography allows to determine the structure of organic compounds directly and without ambiguity. The main requirement and obstacle – the availability of single crystals – can be mitigated by two recent developments: crystalline sponges and electron diffraction. These make even volatile and oily substances accessible for structural analysis by crystallography.

Crystalline sponges evade the need to grow single crystals and are compatible with widely available instrumentation for X-ray diffraction. They are porous crystalline metal-organic frameworks (MOFs) that act as a crystalline template for the compounds of interest. The target compounds are dissolved and allowed to diffuse into the MOF that soaks them up like a sponge. In the MOF, the target compounds are adsorbed in an orderly, practically crystalline fashion, which allows a structure elucidation by crystallography.

The field of Crystalline Sponges offers the option to elucidate compounds that so far have evaded a comprehensive analysis or were not available as crystals (examples see “Research objectives”). The combination with chromatographic techniques has been demonstrated and is especially powerful if an adsorption chromatographic method such as supercritical fluid chromatography or normal-phase chromatography is used. Normal-phase thin-layer chromatography with direct bioautographic detection of bioactive molecules that are then embedded in a crystalline sponge for crystallography could become an extremely rapid approach for bioactivity studies.

Research Objectives:

• Synthesis of established crystalline sponges (apolar, polar, carbohydrate-based) for first tests with model compounds.

• Application of the crystalline sponges to the identification of the target compounds which so far have evaded a comprehensive analysis as they were not available as crystals: a) di- and oligosaccharides of celluloses and hemicelluloses, b) chromophores and byproducts formed during pulp processing, fiber manufacturing and cellulose aging, c) extractives and bioactive compounds form plants (secondary metabolites).

• Establishing the combination with preceding chromatographic techniques: supercritical fluid chromatography and high-performance thin-layer chromatography with direct bioautographic detection of bioactive molecules.

Methods:

• Synthesis of established crystalline sponges (apolar, polar, carbohydrate based) for first tests with model compounds.

• Tests of suitability of different MOFs for different model compounds. Type of MOF and the properties of the solvent for infusion will be screened. Inclusion rate is determined from the supernatant before crystallography. Robustness of the developed methodology is evaluated. If necessary: definition of requirements of MOFs that need to be developed.

• Combination of established chromatographic methods – gas chromatography, thin-layer chromatography, liquid chromatography, supercritical fluid chromatography – for selected use cases, e. g. identification of bioactive compounds after bioautography, identification of unknown peaks in gas chromatography

Main supervisor: Univ.-Prof. Thomas Roesenau

Co-supervisors: Univ.-Prof. Antje Potthast, Priv.-Doz. Dr. Stefan Böhmdorfer

Location: BOKU University (UFT Tulln)

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

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

Main supervisor: Univ.-Prof. Ruth Birner-Gruenberger

Co-supervisors: Priv.-Doz. Dr. Doris Ribitsch

Duration: 4 years (30 h/week)

Location: TU Wien, Research Group Bioanalytics: https://www.tuwien.at/en/tch/bioanalytics