At-line analysis of population heterogeneities in microbial L-cysteine production
At-line analysis of population heterogeneities
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.
Quantitative assessment of metabolic burden in Escherichia coli during (industrial) bioprocesses using real-time fluorescence monitoring
Quantitative assessment of metabolic burden
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.
