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Temporal distribution of physiological states within pure bacterial cultures producing isopropanol : understanding of microbial subpopulation to process optimization

Abstract : Monoclonal cultures of microorganisms in bioreactors have long been considered homogeneous (i.e. all cells are identical). However, population heterogeneities may occur, due to stochastic changes in gene expression, plasmid instability or concentration gradients. This heterogeneity can negatively impact the performance of the processes by decreasing their robustness, especially in the case of recombinant molecule production. The objective of this work is to identify the sub-populations coming from a pure culture bioreactor, to understand their effectors and to control their distribution. The chosen study model is the production of isopropanol by a recombinant strain of Cupriavidus necator. Tools need to be developed to monitor the behavior of these sub-populations during culture. However, such tools are not yet adapted to the dynamic monitoring of bacterial cultures. Thus, to answer the scientific questions, an eGFP biosensor (coded on a recombinant plasmid in C. necator) has been built to dynamically monitor sub-populations by flow cytometry during bioreactor cultures. Different plasmid stabilization strategies, under different controlled environment conditions, were studied in order to characterize their response and efficacy. Two different but complementary methods for monitoring the stability of plasmid expression have been implemented: plate count and flow cytometry. In a first step, isopropanol production by C. necator in fed batch culture was studied with different plasmid constructs. Isopropanol yields and production rates decreased drastically as the instability of plasmid expression increased. This threshold can be modulated according to the plasmid design (plasmid stabilization system or not). In a second step, a technique for plasmid expression monitoring at the single-cell level has been developed. The gene coding for eGFP was inserted into the recombinant plasmid. The impact of the strength of different constitutive promoters was quantified in order to determine the construct with the lowest metabolic load on host cells while ensuring satisfactory sensitivity of the fluorescence measurements. The tool defined was then confronted with culture conditions allowing the generation of population heterogeneity, in order to understand its responses according to the operating conditions. Thus, the impact of growth rate on plasmid expression level was studied in continuous culture, under stringent conditions of plasmid loss. In addition, the robustness of non-isopropanol-producing strains was evaluated under different conditions of substrate feeding and plasmid stabilization systems. Finally, the plasmid expression monitoring system was inserted into a plasmid coding for isopropanol production. Its insertion led to a decrease in isopropanol productivity, linked to instability of plasmid expression compared to the non-fluorescent strain. Different levels of plasmid expression could be identified and quantified during culture. This work has allowed, through the development of a biosensor, to better understand the link between the operating conditions imposed during bacterial cultures and the appearance of plasmid expression heterogeneity on the robustness of a process.
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Submitted on : Monday, February 28, 2022 - 6:09:08 PM
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  • HAL Id : tel-03591750, version 1

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Catherine Boy. Temporal distribution of physiological states within pure bacterial cultures producing isopropanol : understanding of microbial subpopulation to process optimization. Biochemistry [q-bio.BM]. INSA de Toulouse, 2020. English. ⟨NNT : 2020ISAT0031⟩. ⟨tel-03591750⟩

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