Job openings in Program 2

Background

Single carbon substrates gain increasing interest in biotechnology, as they are independent from agricultural production. Among them, methanol and formate offer the advantage of being liquid, easy to store and transport, and to mix with aqueous culture media. Native C1 assimilation pathways are not necessarily evolved for best carbon efficiency, but rather for enabling fast growth rates. E.g., methanol oxidation to formaldehyde is catalyzed in yeast by an alcohol oxidase, enabling fast turnover rates at the expense of yield. We will therefore apply model-driven synthetic pathway designs to enable more energy and carbon efficient pathways that consequently lead to higher product yields. Characterization of such novel pathways relies on 13C tracer studies which are analyzed with mass spectrometry.

Research Objectives

• Implementation of synthetic methanol/formate assimilation pathways in yeast

• Implementation of Adh for methanol oxidation, to enhance NADH yield

• 13C tracer studies for pathway characterization

• Integration of C1 pathway strain with nitrogen assimilation pathway

Methods

• Synthetic biology based genome editing (Golden Gate cloning, CRISPR-Cas9)

• Small scale and bioreactor cultivation, HPLC analysis, carbon balancing

• Adaptive evolution, whole genome sequencing

• 13C tracer studies, evaluation with metabolic flux modeling

Main supervisor: Univ.-Prof. Diethard Mattanovich

Co-supervisors: Univ.-Prof. Stephan Hann, Associate Prof. Matthias Steiger, Univ.-Prof. Jürgen Zanghellini

Location: BOKU University (Muthgasse, Vienna)

Submission Deadline: 31.03.2025

Description of project

Development of rapid, high purity isolation methods of native and synthetic organelles in yeast for subcellular metabolomics.

Background

In nature, dedicated biochemical conversions are carried out in specific compartments or organelles, which contain a specialized subset of enzymes, confine potentially toxic pathway intermediates and provide a defined chemical environment (e.g. redox potential, cofactors, pH). Cellular metabolomics is at the core of metabolic engineering, driving microbial cell factories towards sustainability. Despite its importance, most metabolomics measurements are conducted in whole cells, primarily due to technical limitations. Thus, there is an urgent demand for subcellularly resolved metabolomics data. The miniaturization of the complete analytical process, to be developed by the research team of Gunda Koellensperger at the University of Vienna, relies on a key prerequisite: the rapid isolation of highly pure organelles.

Research Objectives

The primary goal of this project is to develop rapid, high-purity organelle isolation methods to enable unbiased subcellular metabolomics in yeast. The focus will be on isolating key organelles, including mitochondria, microsomes, vacuoles/lysosomes, peroxisomes, secretory organelles, and newly developed synthetic compartments. The project aims to overcome the limitations of traditional centrifugation-based techniques by introducing novel approaches that ensure high efficiency and precision in organelle isolation.

Methods

• Engineer yeast cells with suitable organelle-specific tags.

• Validate the tagged organelles using fluorescent microscopy and proteomics to ensure accuracy and purity.

• Test novel and faster experimental procedures for subcellular fractionation, such as solid-phase separations and differential filtration.

• Develop and apply strains with tagged organelles for affinity purification methods to ensure rapid and precise organelle isolation.

Main supervisor: Associate Prof. Brigitte Gasser

Co-supervisor: Univ.-Prof. Gunda Köllensperger, Assistant Prof. Matthias Steiger

Location: BOKU University (Muthgasse, Vienna)

Submission Deadline: 31.03.2025

Description of project
Research will focus on innovative approaches to advance understanding of protein folding mechanisms by use of synthetic mini-compartments and their application.

Background:

Organelles are important subcellular chassis for biochemical conversions, because the enrichment of specialized enzymes allows for higher conversion rates. Their chemical environment—including redox potential, cofactors, and pH—can be uniquely tailored compared to the rest of the cell. By re-engineering organelles, a favorable redox or cofactor environment can be created to enhance biochemical pathways.

Oxidative protein folding presents such a compartmentalized pathway, located within the ER of eukaryotic cells. Strikingly, even in single cell eukaryotes such as yeast, several functionally redundant isoforms exist for each step. So far, their distinct functions and substrate specificity has been studied in deletion mutant cells, however, these studies are limited as during the lack of one isoform the cells compensate by the other isoforms.

Artificial organelles, such as synthetic ER-mimetic environments, have the potential to address current challenges in protein folding, enzyme production, and the sustainable synthesis of key proteins. Design and development of artificial organelles in yeast cells will enable novel solutions for production of recombinant proteins of high societal impact and high demand. Such technological innovations are crucial to overcome the current limitations in sustainable and resource efficient bioproduction.

Research Objectives:

This research aims to advance the understanding of protein folding mechanisms using synthetic mini-compartments and to develop novel approaches for engineering and production of proteins and enzymes. The project focuses on:

1. Creation of a synthetic yet contextualized ER mimetic environment in a cell-free setup to study the individual roles of redundant eukaryotic oxidative protein folding catalysts and their requirements during substrate folding kinetics and transport.

2. Design of tailored synthetic ER folding modules in vivo to meet the requirements for the identified best suited folding catalysts.

3. Using the gained knowledge and synthetic organelles for compartmentalized protein synthesis to accelerate the sustainable, cost-efficient production of recombinant proteins and enzymes.

Methods:

Creation of synthetic ER folding modules:

Artificial membrane-surrounded organelles will be created to serve as ER-mimetics, and equipped with a tailored non-redundant subset of chaperones and folding enzymes. High throughput modular cloning tools established for yeast will be employed.

These modules will enable the spatial and time-resolved study of chaperone-substrate interactions, cofactor requirements, and so far unresolved processing steps in translocation and oxidative protein folding.

Cell-Free and Cell-Based Protein Synthesis:

Synthetic ER-mimetic organelles will be employed in cell-free systems to study protein folding kinetics and explore enzyme redundancy under controlled conditions.

In vivo systems in yeast cells (focusing especially on Komagataella phaffii/Pichia pastoris) will be designed and optimized for the production of recombinant proteins with high societal impact, such as food proteins, growth factors, and enzymes.

Metabolic and Kinetic Analyses:

Compartment-specific metabolomics, developed and carried out in collaboration with the groups of Stephan Hann and Gunda Koellensperger, will provide metabolic insights into rewired cells and guide the further design of synthetic compartments.

Protein folding kinetics will be monitored using established methodologies at BOKU Core Facilities.

Main supervisor: Associate Prof. Brigitte Gasser

Location: BOKU University (Muthgasse, Vienna)

Submission Deadline: 31.03.2025

Description of project

Novel pathways are highly likely to require a tailored synthetic cellular environment. In nature, organelles can carry out dedicated biochemical conversions, because the enrichment of a specialized subset of enzymes allows for higher conversion rates. Additionally, pathway intermediates are contained, reducing side reactions in other pathways or potential toxic effects. Other chemical parameters (redox potential, cofactors, pH) can be adjusted in an organelle compared to the rest of the cell, enabling different types of bioconversions in different compartments.

A functional lipid bilayer is required for essential biocatalytic processes like metabolite transport and electrochemical gradient formation. Currently available compartmentalized systems lack lipid membranes (protein nanocages) or are difficult to prepare (proteoliposomes).

Thus, artificial liposomal particles a new class of biocatalysts for biochemical conversions will be developed in this project. These particles are created by modification of enveloped virus like particles (VLPs) that are produced in yeast cells and contain both soluble and membrane bound enzymes in a defined amount and metabolic environment. Synthetic particles can be loaded with metabolic pathways and shall be used as functionalized exosomes outside of the cell.

The new synthetic exosomes can be created in situ in yeast cells by forming virus-like particles that can incorporate membrane proteins in their surrounding lipid bilayer and contain a tailored set of enzymes and cofactors to enable targeted bioconversions.

Research Objectives:

• Which yeast system (S. cerevisiae or K. phaffii) is better suited to produce lipid enveloped VLPs?

• Which method to permeabilize the yeast cell wall is most effective to increase the yield of VLP production and how many enzymes can be simultaneously packed into a VLP? (How can VLPs penetrate the yeast cell wall?)

• Which scaffolding or tagging system is most efficient to enrich proteins within VLPs?

Methods:

• Strains will be constructed by genetic engineering techniques suitable for yeasts (GoldenGate, transformation, CRISPR/Cas9 etc.) and will be used and further developed for library approaches.

• Purification and enrichment  of particles will be carried out via ultracentrifugation and affinity based purification protocols for particles will be developed.

• Catcher/tag techniqus will be used to enrich proteins within particles

• Enzymatic assays, proteomics, isotope labelling will be used to measure the activity of engineered pathways inside particels. (in collaboration with Hann, Köllensperger, Birner-Grünberger)

• Strains will be cultivated in small scale (microtiter, biolector or shake flasks experiments) to carry out screenings.

• Selected strains will be cultivated in controlled bioreactors for a full characterization of physiological parameters (in collaboration with Spadiut).

Main supervisor: Associate Prof. Matthias Steiger

Co-supervisors: Associate Prof. Brigitte Gasser, Univ.-Prof. Oliver Spadiut

Location: TU Wien (Vienna)

Submission Deadline: 31.03.2025

Organelle specific metabolomics

Background:

Top notch (sub)cellular metabolomics at the core of metabolic engineering will support the development of synthetic organelles and new to nature pathways.

Research Objectives:

The major objective, is to develop strategies allowing organelle specific metabolomics.

Methods:

For unbiased subcellular metabolomics, a major emphasis will be the development of rapid, high purity organelle isolation methods targeting mitochondria, microsomes, lysosomes and peroxisomes. Powerful recombinant protein-based affinity purification methods ensuring rapid isolation will be developed. This work will pave the way to subcellular flux tracer studies and multi-omics strategies.

Main supervisor: Univ.-Prof. Gunda Köllensperger

Co-supervisors: Univ.-Prof. Stephan Hann, Associate Prof. Brigitte Gasser, Assistant Prof. Matthias Steiger, Univ.-Prof. Diethard Mattanovich

Location: University of Vienna

Submission Deadline: 31.03.2025

Background

Aerobic hydrogen-oxidizing bacteria (HOBs) use the high energy of recombination of H2 and O2 for CO2 assimilation. This approach presents both technological opportunities and challenges: Green hydrogen has excellent potential as an energy carrier for the conversion of CO2 into chemicals under mild and environmentally friendly reaction conditions. The efficient supply of the cells with H2 and O2 mixtures requires, however, new technologies for the introduction of the gases into the aqueous reaction mixture. Several gas fermentation plants for CO₂ assimilation by HOBs were developed and commissioned in Graz in accordance with ATEX requirements (approval by external safety consultants) (Lambauer and Kratzer, 2022; Lambauer et al., 2023). With the developed gas fermenters and process control systems a cell concentration of the HOB Cupriavidus necator of around 60 g/L dry cell weight and a bioplastic content of ≥75% (carbon storage material) were achieved. Furthermore, a C. necator strain was designed that produces 22 U/L of plasmid-based phytase on CO₂ alone, one of the highest amounts for a GRAS organism (Arhar et al., 2024).

Research Objectives

• New-to-nature metabolic pathways in C. necator strains

• Design of recombinant strains based on a genome-scale metabolic model (GSM) of C. necator.

• Production of mutants (knock-out and cloning of genes) using recombineering tools developed at TU Graz.

• Verification of the targeted properties (heterotrophic and autotrophic cultivations).

Methods:

• Equipment for gas fermentations of hydrogen oxidizing bacteria. ATEX compliant bioreactors for use in the ex-zone (H2, O2 mixtures). Automatic control of bioreactor operation for safe processing, finely tuned gas supply, pH control. Gas fermentation protocols for cultivations of C. necator (high cell density) (Lambauer et al., 2023; Lambauer and Kratzer., 2022).

• Use of genome-scale metabolic model of C. necator (Pearcy et al., 2022).

• Genetic tools for manipulation of C. necator (Arhar et al., 2024).

Main supervisor: Associate Prof. Regina Kratzer

Co-supervisors: Univ.-Prof. Robert Kourist

Location: TU Graz

Submission Deadline: 31.03.2025

Background:

In addition to carbon, oxygen, and hydrogen, nitrogen plays a critical role in sustaining life. Although nitrogen is abundant in the atmosphere, bioavailable forms are limited. Substantial energy is required to produce ammonia and nitrate as agricultural fertilizers, while additional energy is consumed in wastewater treatment plants to remove nitrogen from wastewater, often releasing it back into the atmosphere instead of capturing it in bio-accessible forms.

This project will explore nitrogen recycling by a series of unprecedented yeast strains: re-assimilating ammonia or urea from different technical wastewater sources and facilitating nitrate assimilation to enable biomass production without reliance on traditional, resource-intensive nitrogen compounds. To broaden the spectrum of accessible nitrogen sources, pathways for utilizing urea and nitrate/nitrite will be evaluated and optimized for greater uptake and efficiency where necessary.

Furthermore, a library of novel strains for more energy-efficient and carbon-efficient C1 assimilation generated by model-driven synthetic biology will be characterized and respective, sustainable bioprocess will be developed. One potential strategy for C1 assimilation involves a linear pathway centered on the enzyme formolase, which can be expressed in the peroxisomes of Komagataella phaffii (Pichia pastoris).

Developing these novel pathways often requires a customized synthetic cellular environment. In nature, organelles perform specialized biochemical reactions by concentrating specific enzymes, which enhances conversion rates. These compartments also sequester pathway intermediates, reducing undesirable side reactions or toxic effects, and allow for unique chemical conditions (e.g., redox potential, cofactors, pH) that differ from the rest of the cell. A functional lipid bilayer is essential for critical biocatalytic processes, including metabolite transport and the establishment of electrochemical gradients. Existing compartmentalized systems, such as protein nanocages or proteo-liposomes, either lack lipid membranes or are challenging to produce. To address this, this project will also investigate novel yeast strains which produce artificial liposomal particles as a novel class of biocatalysts for biochemical conversions.

Research Objectives:

• Which technical wastestreams providing N and C1-sources, such as urea, cyanate, nitrate, uric acid, and creatinine as well as methanol and CO2 can be used by native yeast strains (both K. phaffii and S. cerevisiae)?

• How can sustainable bioprocesses for these waste substrates be designed?

• What is a suitable PAT strategy to allow scalable and reproducible bioprocesses?

• How does the environmental footprint compare to state-of-the-art bioprocesses?

• Can these bioprocesses be described in models?

• Can these bioprocesses by intensified to further decrease the environmental footprint – even model-based?

• How do respectively engineered strains perform?

Methods:

• Bioreactor cultivations in batch, fed-batch and continuous mode (scale 0.1-10 L)

• Series of analytical technologies (e.g. HPLC, spectroscopy, protein analytics, analyses for physiology)

• OMICs fingerprints of selected samples in collaboration with Birner-Grünberger

• Data evaluation and interpretation

• Data science in collaboration with Zanghellini

• Bioprocess modeling

Main supervisor: Univ.-Prof. Oliver Spadiut

Co-supervisors: Associate Prof. Matthias Steiger, Univ.-Prof Jürgen Zanghellini, Univ.-Prof. Ruth Birner-Grünberger

Location: TU Wien (Vienna)

Submission Deadline: 31.03.2025

Background

Fed-batch processes are a cornerstone of industrial biotechnology due to their simplicity, scalability, and precise control over critical variables. By gradually adding substrate during the feed phase without removing fermentation broth, these systems maintain optimal substrate concentrations, preventing inhibition. They can be tailored to the metabolic needs of various microorganisms and cell lines, including bacteria, yeast, and mammalian cells. This adaptability supports the biosynthesis of target products while ensuring high product quality, making fed-batch processes indispensable for producing pharmaceuticals, enzymes, biofuels, and fine chemicals.

Designing effective feeding strategies is critical for maximizing product titer, rate, yield, and quality. However, optimizing feeding profiles is challenging due to the many influencing factors. While standard approaches like exponential or constant feeding are widely used for their simplicity and practicality, they often fail to fully exploit the production potential of host systems.

Mathematical modeling provides a cost-effective way to design and refine feeding strategies by simulating profiles, predicting outcomes, and reducing the need for extensive experimental trials. However, existing tools often require advanced coding skills, making them inaccessible to many laboratory scientists.

To address this gap, this project will develop process-specific models and optimization approaches, packaged into a simple, user-friendly software suite with a graphical interface. This tool enables researchers to quickly identify and optimize fed-batch feeding strategies without the need for advanced technical expertise.

Research Objectives

• Develop strain- or cell-line-specific mathematical models for production hosts.

• Create fast and scalable optimization approaches to define optimal environments.

• Investigate the integration of life-cycle aspects into the optimal design.

• Develop a user-friendly interface for the software suite.

• Continuously expand the platform to include new model systems and environments, such as continuous fermentation processes.

Methods

• Constraint-based modeling of metabolism

• Data science analysis of bioprocess data (in collaboration with partners)

• Bioprocess and mathematical modeling

Main supervisor: Univ.-Prof Jürgen Zanghellini

Co-supervisors: Univ.-Prof Diethard Mattanovich, Assistant Prof. Matthias Steiger, Univ.-Prof. Oliver Spadiut

Location: University of Vienna

Submission Deadline: 31.03.202

Background

Compartmentalization is a fundamental feature of cellular organization that enables precise spatial and chemical control over metabolic processes. By sequestering specific reactions into dedicated compartments, cells can optimize enzyme activity, enhance reaction rates, and reduce interference from competing pathways. This isolation of intermediates not only minimizes side reactions but also mitigates potential toxicity, allowing cells to perform complex and highly efficient bioconversions.

Additionally, compartments provide unique microenvironments tailored to specific biochemical needs. These include differences in pH, redox potential, and cofactor availability, which are often critical for driving reactions that would be inefficient or incompatible within the general cytosolic environment. For example, organelles such as peroxisomes, and lysosomes serve specialized metabolic roles, enabling diverse processes like fatty acid degradation, and macromolecule recycling.

In biotechnology, leveraging compartmentalization can significantly enhance the efficiency of engineered pathways. Membrane-bound compartments can isolate engineered pathways, concentrating substrates and enzymes while protecting the rest of the cell from toxic intermediates. By fine-tuning the chemical conditions within these compartments, non-native reactions that mimic natural processes can be enabled.

The goal of this project is to predict the optimal compartmentalization of a production pathway. By strategically allocating specific reactions to distinct cellular compartments, we aim to optimize the thermodynamic properties of the pathway, enhance efficiency, minimize energy losses, and improve metabolic performance.

Research Objectives

• Establish a computational framework to calculate and analyze the thermodynamic profiles of biochemical pathways across various compartments.

• Identify compartmentalization strategies that maximize thermodynamic driving forces, enhance reaction rates, and minimize energy losses within a pathway.

• Assess the computational framework's accuracy, by comparing model predictions with available datasets (e.g., on mitochondria).

• Incorporate practical biological constraints, such as transport efficiencies, and resource limitations, to refine the model's real-world applicability.

Methods

• Constraint-based modeling of metabolism

• Thermodynamic flux analysis

Main supervisor: Univ.-Prof Jürgen Zanghellini

Co-supervisors: Univ.-Prof Diethard Mattanovich, Assistant Prof. Matthias Steiger

Location: University of Vienna

Submission Deadline: 31.03.202