Comparison of plasmid stabilization systems during heterologous isopropanol production in fed-batch bioreactor.

Autor: Boy C; TBI, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France., Lesage J; TBI, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France., Alfenore S; TBI, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France., Gorret N; TBI, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France., Guillouet SE; TBI, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France. Electronic address: guillouet@insa-toulouse.fr.
Jazyk: angličtina
Zdroj: Journal of biotechnology [J Biotechnol] 2023 Mar 20; Vol. 366, pp. 25-34. Date of Electronic Publication: 2023 Mar 03.
DOI: 10.1016/j.jbiotec.2023.02.011
Abstrakt: Strain robustness during production of recombinant molecules is of major interest to ensure bioprocess profitability. The heterogeneity of populations has been shown in the literature as a source of instability in bioprocesses. Thus, the heterogeneity of the population was studied by evaluating the robustness of the strains (stability of plasmid expression, cultivability, membrane integrity and macroscopic cell behavior) during well-controlled fedbatch cultures. On the context of microbial production of chemical molecules, isopropanol (IPA) has been produced by recombinant strains of Cupriavidus necator. Plasmid stability was monitored by the plate count method to assess the impact of isopropanol production on plasmid stability, depending on implanted plasmid stabilization systems for strain engineering designs. With the reference strain Re2133/pEG7c, an isopropanol titer of 15.1 g·L -1 could be achieved. When the isopropanol concentration has reached about 8 g. L -1 , cell permeability increased (up to 25 %) and plasmid stability decreased significantly (up to 1.5 decimal reduction rate) resulting in decreased isopropanol production rates. Bioprocess robustness under isopropanol producing conditions was then investigated with two plasmid construction strategies (1) Post Segregational Killing hok/sok (in Re2133/pEG20) and (2) expression of GroESL chaperon proteins (in Re2133/pEG23). Plasmid stability for strain Re2133/pEG20 (PSK hok/sok) appears to be improved up to 11 g. L -1 of IPA compared to the reference strain (8 g. L -1 IPA). Nevertheless, cell permeability followed the same dynamic as the reference strain with a drastic increase around 8 g. L -1 IPA. On the contrary, the Re2133/pEG23 strain made it possible to minimize the cell permeability (with a constant value at 5 % IP permeability) and to increase the growth capacities in response to increased isopropanol concentrations but plasmid stability was the weakest. The metabolic burden, linked to either the overexpression of GroESL chaperones or the PSK hok/sok system, seems to be deleterious for the overall isopropanol production compared to the reference strain (RE2133/pEG7c) even if we have shown that the overexpression chaperones GroESL improve membrane integrity and PSK system hok/sok improve plasmid stability as long as isopropanol concentration does not exceed 11 g L - 1 .
Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Guillouet reports financial support was provided by French Ministry of Research.
(Copyright © 2023 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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