Investigation of the robustness of Cupriavidus necator engineered strains during fed-batch cultures

Abstract It is of major interest to ensure stable and performant microbial bioprocesses, therefore maintaining high strain robustness is one of the major future challenges in industrial microbiology. Strain robustness can be defined as the persistence of genotypic and/or phenotypic traits in a syste...

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Autores principales:	Catherine Boy, Julie Lesage, Sandrine Alfenore, Stéphane E. Guillouet, Nathalie Gorret
Formato:	article
Lenguaje:	EN
Publicado:	SpringerOpen 2021
Materias:	Plasmid stabilization systems Single cell analysis Plasmid expression level Cell permeability Kinetics Culture strategies Biotechnology TP248.13-248.65 Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/bd8487889bf64dc085ec22a046d25a6c
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spelling	oai:doaj.org-article:bd8487889bf64dc085ec22a046d25a6c2021-11-21T12:09:07ZInvestigation of the robustness of Cupriavidus necator engineered strains during fed-batch cultures10.1186/s13568-021-01307-42191-0855https://doaj.org/article/bd8487889bf64dc085ec22a046d25a6c2021-11-01T00:00:00Zhttps://doi.org/10.1186/s13568-021-01307-4https://doaj.org/toc/2191-0855Abstract It is of major interest to ensure stable and performant microbial bioprocesses, therefore maintaining high strain robustness is one of the major future challenges in industrial microbiology. Strain robustness can be defined as the persistence of genotypic and/or phenotypic traits in a system. In this work, robustness of an engineered strain is defined as plasmid expression stability, cultivability, membrane integrity and macroscopic cell behavior and was assessed in response to implementations of sugar feeding strategies (pulses and continuous) and two plasmid stabilization systems (kanamycin resistance and Post-Segregational Killing hok/sok). Fed-batch bioreactor cultures, relevant mode to reach high cell densities and higher cell generation number, were implemented to investigate the robustness of C. necator engineered strains. Host cells bore a recombinant plasmid encoding for a plasmid expression level monitoring system, based on eGFP fluorescence quantified by flow cytometry. We first showed that well-controlled continuous feeding in comparison to a pulse-based feeding allowed a better carbon use for protein synthesis (avoiding organic acid excretion), a lower heterogeneity of the plasmid expression and a lower cell permeabilization. Moreover, the plasmid stabilization system Post-Segregational Killing hok/sok, an autonomous system independent on external addition of compounds, showed the best ability to maintain plasmid expression level stability insuring a greater population homogeneity in the culture. Therefore, in the case of engineered C. necator, the PSK system hok/sok appears to be a relevant and an efficient alternative to antibiotic resistance system for selection pressure, especially, in the case of bioprocess development for economic and environmental reasons.Catherine BoyJulie LesageSandrine AlfenoreStéphane E. GuillouetNathalie GorretSpringerOpenarticlePlasmid stabilization systemsSingle cell analysisPlasmid expression levelCell permeabilityKineticsCulture strategiesBiotechnologyTP248.13-248.65MicrobiologyQR1-502ENAMB Express, Vol 11, Iss 1, Pp 1-17 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Plasmid stabilization systems Single cell analysis Plasmid expression level Cell permeability Kinetics Culture strategies Biotechnology TP248.13-248.65 Microbiology QR1-502
spellingShingle	Plasmid stabilization systems Single cell analysis Plasmid expression level Cell permeability Kinetics Culture strategies Biotechnology TP248.13-248.65 Microbiology QR1-502 Catherine Boy Julie Lesage Sandrine Alfenore Stéphane E. Guillouet Nathalie Gorret Investigation of the robustness of Cupriavidus necator engineered strains during fed-batch cultures
description	Abstract It is of major interest to ensure stable and performant microbial bioprocesses, therefore maintaining high strain robustness is one of the major future challenges in industrial microbiology. Strain robustness can be defined as the persistence of genotypic and/or phenotypic traits in a system. In this work, robustness of an engineered strain is defined as plasmid expression stability, cultivability, membrane integrity and macroscopic cell behavior and was assessed in response to implementations of sugar feeding strategies (pulses and continuous) and two plasmid stabilization systems (kanamycin resistance and Post-Segregational Killing hok/sok). Fed-batch bioreactor cultures, relevant mode to reach high cell densities and higher cell generation number, were implemented to investigate the robustness of C. necator engineered strains. Host cells bore a recombinant plasmid encoding for a plasmid expression level monitoring system, based on eGFP fluorescence quantified by flow cytometry. We first showed that well-controlled continuous feeding in comparison to a pulse-based feeding allowed a better carbon use for protein synthesis (avoiding organic acid excretion), a lower heterogeneity of the plasmid expression and a lower cell permeabilization. Moreover, the plasmid stabilization system Post-Segregational Killing hok/sok, an autonomous system independent on external addition of compounds, showed the best ability to maintain plasmid expression level stability insuring a greater population homogeneity in the culture. Therefore, in the case of engineered C. necator, the PSK system hok/sok appears to be a relevant and an efficient alternative to antibiotic resistance system for selection pressure, especially, in the case of bioprocess development for economic and environmental reasons.
format	article
author	Catherine Boy Julie Lesage Sandrine Alfenore Stéphane E. Guillouet Nathalie Gorret
author_facet	Catherine Boy Julie Lesage Sandrine Alfenore Stéphane E. Guillouet Nathalie Gorret
author_sort	Catherine Boy
title	Investigation of the robustness of Cupriavidus necator engineered strains during fed-batch cultures
title_short	Investigation of the robustness of Cupriavidus necator engineered strains during fed-batch cultures
title_full	Investigation of the robustness of Cupriavidus necator engineered strains during fed-batch cultures
title_fullStr	Investigation of the robustness of Cupriavidus necator engineered strains during fed-batch cultures
title_full_unstemmed	Investigation of the robustness of Cupriavidus necator engineered strains during fed-batch cultures
title_sort	investigation of the robustness of cupriavidus necator engineered strains during fed-batch cultures
publisher	SpringerOpen
publishDate	2021
url	https://doaj.org/article/bd8487889bf64dc085ec22a046d25a6c
work_keys_str_mv	AT catherineboy investigationoftherobustnessofcupriavidusnecatorengineeredstrainsduringfedbatchcultures AT julielesage investigationoftherobustnessofcupriavidusnecatorengineeredstrainsduringfedbatchcultures AT sandrinealfenore investigationoftherobustnessofcupriavidusnecatorengineeredstrainsduringfedbatchcultures AT stephaneeguillouet investigationoftherobustnessofcupriavidusnecatorengineeredstrainsduringfedbatchcultures AT nathaliegorret investigationoftherobustnessofcupriavidusnecatorengineeredstrainsduringfedbatchcultures
_version_	1718419210074324992

Investigation of the robustness of Cupriavidus necator engineered strains during fed-batch cultures

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