At-line analysis of population heterogeneities in microbial L-cysteine production

L-cysteine is a sulphur-containing amino acid used in the manufacture of many products such as food supplements, cosmetics, and medicines. An environmentally friendly industrial bio-based production of this amino acid applies genetically engineered microorganisms such as Escherichia coli.

However, on an industrial scale, environmental gradients occur in the bioreactor, which in turn can lead to the formation of phenotypic population heterogeneities of the cell population in the production process. This complicates scale-up, as production output can change unpredictably.

In-vivo-measurements of phenotypic population heterogeneities are possible with fluorescent reporter strains that genetically engineering to express fluorescent proteins together with genes of interest for different cellular characteristics such as growth rate, general stress response and oxygen limitation. The different fluorescence signals of the individual microorganisms can be measured on-line using automated real-time flow cytometry. Population dynamics can thus be identified in real-time, and the formation of phenotypic population heterogeneity can be better understood through correlation with the specific bioprocess conditions during its development.

The aim of this research project is to run fed-batch processes for L-cysteine production with a triple reporter strains Escherichia coli in a multi-compartment bioreactor on a laboratory scale and analyze the effects of environmental gradients caused by long mixing times on an industrial scale on cellular physiology. For this purpose, specific properties of single cells are measured automatically in real-time using a flow cytometer to be able to directly detect the emergence of phenotypic population heterogeneities.

Contact person: José Enrique Oropeza Vargas

Quantitative assessment of metabolic burden in Escherichia coli during (industrial) bioprocesses using real-time fluorescence monitoring

Metabolic burden is a central challenge in industrial biotechnology. In nature, microorganisms have evolved finely tuned regulatory networks that ensure an optimal allocation of cellular resources. When microbial hosts are engineered for biotechnological applications, e.g., by introducing production plasmids to overexpress heterologous proteins or by performing genomic knock-ins or knock-outs, this balance is perturbed. The expression of foreign genes competes with housekeeping processes for limited intracellular resources, including the transcriptional and translational machinery, amino acids, and energy carriers. This competition can ultimately impair cellular fitness and reduce overall bioprocess performance.

To enable the monitoring and quantification of metabolic burden of Escherichia coli during bioprocesses, this project aims to develop fluorescent reporter strains. This strain should allow tracking of intracellular bottlenecks and physiological states of microbial production strains linking the expression of suitable genes of interest to with expression of fluorescent proteins. Subsequently, these fluorescence-based E. coli reporter strains are cultivated in laboratory-scale bioreactors under varying environmental conditions like they are present in industrial scale bioprocesses. Using at-line flow cytometry, fluorescence signals can be monitored throughout the bioprocess. Dynamics in reporter signal intensity can then provide insights into bioprocess-related bottlenecks and stress responses of cellular populations, enabling the discrimination of most robust and productive subpopulations within microbial cultures.

Contact Person: Julia von Irmer 

Robustness and stability of plasmid-based antibiotic-free Escherichia coli cultivation in industrial scale batch and fed-batch cultures

Over the past decades, antibiotic usage in industrial bioprocesses have prompted significant drawbacks due to the rapid emergence of antibiotic-resistant strains. To overcome this problem, the transition away from antibiotic-based selection is being vigorously pursued. The company Gen-H GmbH developed a novel antibiotic-free plasmid-based Escherichia coli expression system, wherein an essential gene on the chromosome is placed under the control of an inducible promoter and is instead expressed under normal growth conditions solely through an IPTG-inducible production plasmid*. In order to monitor product formation, the model product EmeraldGFP, a green fluorescent protein, is expressed from the production plasmid. Additionally, different versions of plasmids are generated by using different origins of replication (ori) as well as proteins that regulate plasmid DNA replication to enable expression with different plasmid copy numbers.

To date, the suitability and long-term stability of antibiotic-free (ABF) production especially in industrial scale bioprocesses remain unknown. To address this gap, the goal of this project is to assess the robustness of these ABF expression systems by determining potential metabolic burden in response to environmental fluctuations and monitor their stability via plasmid copy number determination using qPCR measurement.

Antibiotic-free Escherichia coli strains carrying plasmids with different copy numbers are cultivated in batch and fed-batch mode in laboratory scale stirred-tank bioreactors as well as in a scale-down bioreactor to experimentally mimic industrial scale bioprocess conditions with mixing insufficiencies. Due to the nature of the product being a fluorescent protein, also phenotypic population heterogeneity at the single-cell level can be monitored using flow cytometry analysis.

Contact person: Donald De Leon

Reference

* K. E. Brechun, M. Förschle, M. Schmidt, and H. Kranz, “Inducible complementation for antibiotic-free plasmid-based biomanufacturing in industrially relevant strains,” Frontiers in Industrial Microbiology, vol. 3, Jan. 2026.