Forschung

KoSyn is a joint collaboration of researchers of different groups of the Technische Universität Dresden. Within the project, we aim to establish a peptide-mediated cell-to-cell communication system between yeasts (Saccharomyces (S.) cerevisiae, Schizosaccharomyces (S.) pombe) and bacterial (Bacillus (B.) subtilis) cells. In order to proof the applicability of this controllable inter-regnum communication system, we will functionalize these cells to serve as biological sensor or actor cells. As a proof-of-concept, genetically modified bacteria will be capable to sense the presence of an antibiotic, leading to the transfer of this information to functionalized yeasts. In turn, the yeasts will serve as actor cells, secreting degradative enzymes in order to decompose the antibiotic.

The project is divided into four different fields, which are explained in more detail below.

Inhaltsverzeichnis

1. 1. Molecular biological work with yeasts
   2. Molecular biological work with bacteria
   3. Bioprocess engineering work for the cultivation of consortia
   4. Analytical work for the detection of signal peptides

                           

Molecular biological work with yeasts

The generation of specific functionalized yeast cells is carried out in the group “Biological Sensor-Actorsystems” at the Institute of Genetics. This includes the engineering of yeast cells to be able to express and secrete bacterial signaling peptides. For this purpose, S. cerevisiae and S. pombe are used, both of which have been shown to be able to build up an "inter-species" communication system. The heterologous expression of the bacterial signaling peptides is analyzed via methods of protein biochemistry, as well as by the usage of specific bacterial reporter strains, allowing a visualization of the peptide-production by fluorescence or luminescence signals. Vice versa, yeast cells will be modified to act as reporter cells, which react in the presence of yeast-specific pheromones heterologously produced by genetically modified bacteria with the expression of fluorescent proteins. Furthermore, yeasts are engineered in order to modulate their growth depending on the presence of pheromones.

                            

Molecular biological work with bacteria

The chair of General Microbiology performs molecular biological work with bacteria. Within the KoSyn-project, the experiments are conducted with Bacillus subtilis, a Gram-positive, motile, rod-shaped and facultative aerobe prokaryote. It is mostly found in soil and near to vegetation. B. subtilis has the ability to form endospores to survive harsh environmental conditions. Furthermore, the microorganism produce and secrete antimicrobial substances and a variety of small peptides. Those peptides are involved in a complex signaling communication system called quorum sensing.

As already pointed out, the aim of this project is to establish and investigate an interspecies communication between B. subtilis and yeasts, namely S. cerevisiae or S. pombe. The cell-cell communication should occur via signal peptides. Once, we want to express signal peptides from yeast (α- and P-factor) by using protein engineering and secretion pathways in B. subtilis and analyse if yeast reporter cells receive the “signals”. On the other hand, we want to find out if B. subtilis can recognize the species-specific signal peptide CSF that is produced in yeast.

Bioprocess engineering work for the cultivation of consortia

During previous studies, artificial cell-to-cell communication systems between different yeast cells of S. cerevisiae and S. pombe (Hennig et al., 2015; Hoffmann et al., 2019) have been established. Within the current KoSyn project, the pheromone-based cell-to-cell communication shall be extended to co-cultures consisting of the yeasts S. cerevisiae or S. pombe and the bacterium Bacillus subtilis. This co-cultivation experiments are performed at the chair of bioprocess engineering. A prerequisite for maintaining a stable co-culture in one cultivation vessel is an nearly identical growth rate of the respective species. However, the microorganisms differ in regard of their maximum specific growth rates, the preferred cultivation temperature and medium pH.

Ongoing experiments are determining an optimal temperature-pH combination at which S. cerevisiae and B. subtilis exhibit the same growth rate. The investigation started with the development of a synthetic minimal medium that enables growth of both species. Then, a mathematical model was established to predict the behavior of such co-cultures. This model with two species-specific µ(ϑ,pH) functions was at first parameterized by means of data from the literature. Subsequently, the pH and temperature dependencies of the growth rates were studied for both species in pure cultures using the optimized medium and stirred bioreactors. The determined µ(ϑ,pH) functions will be used in further model simulations for predicting stable operating points. Finally, the model-based predictions will be verified in real co-cultures using a temperature and pH-controlled stirred bioreactor.

Analytical work for the detection of signal peptides

The analytical work is conducted at the Institute of Water Chemistry. This includes the development of an analysis method for the detection of the specific signal peptides and the degraded antibiotics based on the tandem mass spectroscopy (LC-MS/MS).

The peptides (α- and P-factor, CSF) secreted from yeast (S. cerevisiae/ S. pombe) or bacteria (B. subtilis) and the enzyme-degraded antibiotics are present at low concentration in complex matrices (culture media).  Therefore, preanalytical steps such as SPE (solid phase extraction), SEC (size exclusion chromatography) or ultrafiltration are required to suppress the influence of the matrix without sample loss. For quantification and qualification of the molecules we will use tandem mass spectroscopy (LC-MS/MS) which provide a rapid analysis of different substances at low concentrations at the same time. Finally, a holistic analysis method from the detection of both signal peptides up to the degraded antibiotics will be developed.