Biology subjects

Telek, S.

Publications and source records attributed to Telek, S..

9 recordsLinked to original sources

bioRxiv2025

Synthetic niches enable co-culture bioprocessing but are prone to mutational escape

Stabilizing microbial co-cultures is a central challenge for bioproduction. While division of labor between strains can enhance efficiency, it often results in population instability over time. Classical strategies, including cross-feeding, quorum sensing, and toxin-antitoxin modules, often rely on complex ecological interactions that are difficult to predict or maintain under bioprocess conditions. Here, we introduce synthetic niches as an alternative framework, using genetic toggle switches that couple growth to defined phenotypic states. We engineered two auxotrophic strains, TOGGLE_green and TOGGLE_yellow, in which growth is linked to either GFP- or YFP-expressing states and assessed their behavior in continuous bioreactor cultures using automated flow cytometry. Unexpectedly, the introduction of auxotrophic pressure reshaped circuit function i.e., instead of maintaining bistability, toggle strains behaved as unidirectional inducible systems that reverted upon inducer withdrawal. This feature enabled simplified control with a single input but also revealed a vulnerability to mutational escape under intensified cultivation. A simple repression-based ODE model recapitulated the reversible dynamics, but deviations under prolonged operation highlighted the rapid evolutionary erosion of control. Our findings demonstrate both the potential and the limitations of synthetic niches for co-culture engineering and emphasize the need to integrate evolutionary robustness into the design of next-generation bioprocess control strategies.

synthetic biology↗

bioRxiv2025

A Fitness Entropy Compensation effect set the trade-off between growth and gene expression in cell populations

We present findings on a Fitness-Entropy Compensation (FEC) mechanism which offsets the activation of gene circuits that compromise survival. It counteracts the resulting fitness reduction by increasing the diversity in gene expression among individual cells within the population. This diversity, quantified by the Shannon entropy, enables cells with lower expression levels to support the survival of the entire population. We investigated the presence of FEC in a range of synthetic and stress-related genetic circuits in continuous culture. Our results reveal that it effectively stabilizes cell populations by mitigating the detrimental trade-offs between growth and gene expression. This stabilization is due to the reduced growth rate of the induced phenotype that leads to environmental changes, decreases induction strength, and promotes escape from unfit states. These findings suggest that the FEC mechanism may be a universal strategy for stabilization in various cellular systems and set the basis for a quantitative description of the trade-off between growth and gene expression and its consequences at the population level.

systems biology↗

bioRxiv2024

Lowering the switching cost related to the activation of burdensome gene circuits promotes cell population homogeneity and productivity

The activation of gene circuits can impose a significant burden on cells, leading to heterogeneous expression and reduced productivity. In this work, we focused on the T7 production system in E. coli BL21, a prime example of a burdensome gene circuit, to investigate the main cause for this gene expression heterogeneity and methods to mitigate it. Based on continuous cultivation analyzed and control by automated flow cytometry, we quantified the trade-off between cellular growth and gene expression and tracked the cell-to-cell heterogeneity in gene expression (measured as entropy). We concluded that the growth reduction associated to the activation of the burdensome gene circuit, i.e., the switching cost, is at the origin of the population heterogeneity. The loss of growth rate imposed by the burdensome activation of the gene is compensated at the population level by the overgrowth of less induced cells that safeguard the population by generating entropy. We tried to homogenize the population by pulsing the inducer with increasing frequency but found that the population escapes control through promoter mutation, leading to a genotype exhibiting reduced gene expression, but also, reduced entropy. To engineer a more homogeneous population without sacrificing gene expression, we decreased the switching cost associated to the induction by lowering the quality of the main carbon source. This strategy successfully led to a more homogeneous and productive population. Our approach allows for a precise quantification of the trade-off between growth and gene expression in cell population cultivated under dynamic conditions and highlights the importance of the switching cost for designing efficient approaches of cell population control.

systems biology↗

bioRxiv2024

Automated adjustment of metabolic niches enables the control of natural and engineered microbial co-cultures

A lot of attention has been given to the understanding of microbial interactions leading to stable co-cultures, but the resulting technologies have been rarely challenged in dynamic cultivation conditions. In this work, substrate pulsing was performed to promote better control of the metabolic niches corresponding to each species, leading to the continuous co-cultivation of diverse microbial organisms. For this purpose, we used a cell-machine interface relying on automated flow cytometry, allowing to adjust the temporal profile of two metabolic niches according to a rhythm ensuring the successive growth of two species i.e., in our case a yeast and a bacterium. The resulting approach, called Automated Adjustment of Metabolic Niches (AAMN), was successfully employed for stabilizing both cooperative and competitive co-cultures. Additionally, AAMN can be considered as an enabling technology for the deployment of co-cultures in bioprocesses, demonstrated here based on the continuous bioproduction of p-coumaric acid. Taken altogether, the data accumulated suggest that AAMN could be used for a wider range of biological systems, but also to gain fundamental insights about microbial interaction mechanisms.

microbiology↗

bioRxiv2023

Release of extracellular DNA by Pseudomonas species as a major determinant for biofilm switching and an early indicator for cell population control

Withdrawal StatementThe authors have withdrawn this manuscript due to a duplicate posting of manuscript number BIORXIV/2021/430776. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The correct preprint can be found at doi: 10.1101/2021.02.11.430776

microbiology↗

bioRxiv2023

Fitness cost associated with cell phenotypic switching drives population diversification dynamics and controllability

Isogenic cell populations can cope with stress conditions by switching to alternative phenotypes. Even if it can lead to increased fitness in a natural context, this feature is typically unwanted for a range of applications (e.g., bioproduction, synthetic biology, biomedicine...) where it tends to decrease the controllability of the cellular response. However, little is known about the diversification profiles that can be adopted by a cell population. We characterized the diversification dynamics for various systems (bacteria and yeast) and for different phenotypes (utilization of alternative carbon sources, general stress response and more complex development patterns). Interestingly, our results suggest that the diversification dynamics and the fitness cost associated with cell switching are coupled. For quantifying the contribution of the switching cost on population dynamics, we built a stochastic model that allowed us to reproduce the dynamics observed experimentally and identified three diversification regimes, i.e., constrained (at low switching cost), dispersed (at medium and high switching cost), and bursty (for very high switching cost). Furthermore, we used a cell-machine interface that we call the Segregostat to demonstrate that different levels of control can be applied to these diversification regimes, enabling applications involving more precise cellular responses.

microbiology↗

bioRxiv2022

Controlling microbial co-culture based on substrate pulsing can lead to stability through differential fitness advantages

Microbial consortia are an exciting alternative for increasing the performances of bioprocesses for the production of complex metabolic products. However, the functional properties of microbial communities remain challenging to control, considering the complex interaction mechanisms occurring between co-cultured microbial species. Indeed, microbial communities are highly dynamic and can adapt to changing environmental conditions through complex mechanisms, such as phenotypic diversification. We focused on stabilizing a co-culture of Saccharomyces cerevisiae and Escherichia coli in continuous cultures. Our preliminary data pointed out that transient diauxic shifts could lead to stable co-culture by providing periodic fitness advantages to the yeast. Based on a computational toolbox called MONCKS (for MONod-type Co-culture Kinetic Simulation), we were able to predict the dynamics of diauxic shift for both species based on a cybernetic approach. This toolbox was further used to predict the frequency of diauxic shift to be applied to reach co-culture stability. These simulations were successfully reproduced experimentally in continuous bioreactors with glucose pulsing. Finally, based on a bet-hedging reporter, we observed that the yeast population exhibited an increased phenotypic diversification process in co-culture compared with mono-culture, suggesting that this mechanism could be the basis of the metabolic fitness of the yeast.

systems biology↗

bioRxiv2021

Reducing phenotypic and genotypic instabilities of microbial population during continuous cultivation based on stochastic switching dynamics

Predicting the fate of a microbial population (i.e., growth, gene expression...) remains a challenge, especially when this population is exposed to very dynamic environmental conditions, such as those encountered during continuous cultivation. Indeed, the dynamic nature of continuous cultivation process implies the potential deviation of the microbial population involving genotypic and phenotypic diversification. This work has been focused on the induction of the arabinose operon in Escherichia coli as a model system. As a preliminary step, the GFP level triggered by an arabinose-inducible ParaBAD promoter has been tracked by flow cytometry in chemostat with glucose-arabinose co-feeding. For a large range of glucose-arabinose co-feeding, the simultaneous occurrence of GFP positive and negative subpopulation was observed. In a second set of experiments, continuous cultivation was performed by adding either glucose or arabinose, based on the ability of individual cells for switching from low GFP to high GFP states, according to a technology called segregostat. In segregostat mode of cultivation, on-line flow cytometry analysis was used for adjusting the arabinose/glucose transitions based on the phenotypic switching capabilities of the microbial population. This strategy allowed finding an appropriate arabinose pulsing frequency, leading to a prolonged maintenance of the induction level with limited impact on phenotypic diversity for more than 60 generations. This result suggests that constraining individual cells into a given phenotypic trajectory is maybe not the best strategy for directing cell population. Instead, allowing individual cells switching around a predefined threshold seems to be a robust strategy leading to oscillating, but predictable, cell population behavior.

microbiology↗

bioRxiv2019

Segregostat: A novel concept to control phenotypic diversification dynamics on the example of Gram-negative bacteria

Controlling and managing the degree of phenotypic diversification of microbial populations is a challenging task. This task not only requires detailed knowledge regarding diversification mechanisms but also advances technical setups for the real-time analyses and control of population behavior on single-cell level. In this work, setup, design and operation of the so called segregostat is described which, in contrast to a traditional chemostats, allows the control of phenotypic diversification of microbial populations over time. Two exemplary case studies will be discussed, emphasizing the applicability and versatility of the proposed approach. In detail the phenotypic diversification of Eschericia coli or Pseudomonas putida based on monitoring membrane permeability will be controlled. We show that upon nutrient limitation, cell population tends to diversify into several subpopulations exhibiting distinct phenotypic (non-permeablized and permeablized cells). On-line analysis leads to the determination of the ratio between cells in these two states, which in turn trigger the addition of glucose pulses in order to maintain a pre-defined diversification ratio. These results prove that phenotypic diversification can be controlled by means of defined pulse-frequency modulation within continuously running bioreactor setups. This lays the foundation for systematic studies, not only of phenotypic diversification but also for all processes where dynamics single cell approaches are required, such as synthetic co-culture processes.

microbiology↗