Transport-controlled growth decoupling for self-induced protein expression with a glycerol-repressible genetic circuit.

Decoupling cell formation from recombinant protein synthesis is a potent strategy to intensify bioprocesses. Escherichia coli strains with mutations in the glucose uptake components lack catabolite repression, display low growth rate, no overflow metabolism, and high recombinant protein yields. Fast growth rates were promoted by the simultaneous consumption of glucose and glycerol, and this was followed by a phase of slow growth, when only glucose remained in the medium. A glycerol-repressible genetic circuit was designed to autonomously induce recombinant protein expression. The engineered strain bearing the genetic circuit was cultured in 3.9 g L-1 glycerol + 18 g L-1 glucose in microbioreactors with online oxygen transfer rate monitoring. The growth was fast during the simultaneous consumption of both carbon sources (C-sources), while expression of the recombinant protein was low. When glycerol was depleted, the growth rate decreased, and the specific fluorescence reached values 17% higher than those obtained with a strong constitutive promoter. Despite the relatively high amount of C-source used, no oxygen limitation was observed. The proposed approach eliminates the need for the substrate feeding or inducers addition and is set as a simple batch culture while mimicking fed-batch performance.

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, and biomedicine) where it tends to make cellular response unpredictable. However, little is known about the diversification profiles that can be adopted by a cell population. Here, we characterize 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). Our results suggest that the diversification dynamics and the fitness cost associated with cell switching are coupled. To quantify the contribution of the switching cost on population dynamics, we design a stochastic model that let us reproduce the dynamics observed experimentally and identify 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 use a cell-machine interface called Segregostat to demonstrate that different levels of control can be applied to these diversification regimes, enabling applications involving more precise cellular responses.

Avoiding the All-or-None Response in Gene Expression During E. coli Continuous Cultivation Based on the On-Line Monitoring of Cell Phenotypic Switching Dynamics.

Different expression vectors are available for the effective production of recombinant proteins by bacterial populations. However, the productivity of such systems is limited by the inherent noise of the gene circuits used for the synthesis of recombinant products. An extreme case of cell-to-cell heterogeneity that has been previously reported for the ara- and lac-based expression systems in E. coli is the all-or-none response. According to this mode of response, two subpopulations of cells are generated, i.e., a "low-" subpopulation exhibiting a shallow expression level and a "high-" subpopulation exhibiting a high-expression level. The "low-" subpopulation can be considered as a cluster of non-producing cells contributing to the loss of productivity. Here we describe the setup, design, and operation of a continuous culture where inducer addition is operated based on microbial population dynamics. The determination of population dynamics is done based on an automated flow cytometry (FC) procedure previously denoted as segregostat. We illustrate how this setup can be used to control the activation of an ara-based expression system and avoid phenotypic diversification leading to an all-or-none response. Upon the determination of the natural frequency of the gene circuit used as an expression system, our current protocol can be set up without the requirement of a feedback controller.