Transcriptomic and fluxomic changes in Streptomyces lividans producing heterologous protein

Abstract Background The Gram-positive Streptomyces lividans TK24 is an attractive host for heterologous protein production because of its high capability to secrete proteins—which favors correct folding and facilitates downstream processing—as well as its acceptance of methylated DNA and its low end...

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Main Authors: Wouter Daniels, Jeroen Bouvin, Tobias Busche, Christian Rückert, Kenneth Simoens, Spyridoula Karamanou, Lieve Van Mellaert, Ólafur H. Friðjónsson, Bart Nicolai, Anastassios Economou, Jörn Kalinowski, Jozef Anné, Kristel Bernaerts
Format: Article
Language:English
Published: BMC 2018-12-01
Series:Microbial Cell Factories
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Online Access:http://link.springer.com/article/10.1186/s12934-018-1040-6
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author Wouter Daniels
Jeroen Bouvin
Tobias Busche
Christian Rückert
Kenneth Simoens
Spyridoula Karamanou
Lieve Van Mellaert
Ólafur H. Friðjónsson
Bart Nicolai
Anastassios Economou
Jörn Kalinowski
Jozef Anné
Kristel Bernaerts
author_facet Wouter Daniels
Jeroen Bouvin
Tobias Busche
Christian Rückert
Kenneth Simoens
Spyridoula Karamanou
Lieve Van Mellaert
Ólafur H. Friðjónsson
Bart Nicolai
Anastassios Economou
Jörn Kalinowski
Jozef Anné
Kristel Bernaerts
author_sort Wouter Daniels
collection DOAJ
description Abstract Background The Gram-positive Streptomyces lividans TK24 is an attractive host for heterologous protein production because of its high capability to secrete proteins—which favors correct folding and facilitates downstream processing—as well as its acceptance of methylated DNA and its low endogeneous protease activity. However, current inconsistencies in protein yields urge for a deeper understanding of the burden of heterologous protein production on the cell. In the current study, transcriptomics and $$^{13}\hbox {C}$$ 13C -based fluxomics were exploited to uncover gene expression and metabolic flux changes associated with heterologous protein production. The Rhodothermus marinus thermostable cellulase A (CelA)—previously shown to be successfully overexpressed in S. lividans—was taken as an example protein. Results RNA-seq and $$^{13}\hbox {C}$$ 13C -based metabolic flux analysis were performed on a CelA-producing and an empty-plasmid strain under the same conditions. Differential gene expression, followed by cluster analysis based on co-expression and co-localization, identified transcriptomic responses related to secretion-induced stress and DNA damage. Furthermore, the OsdR regulon (previously associated with hypoxia, oxidative stress, intercellular signaling, and morphological development) was consistently upregulated in the CelA-producing strain and exhibited co-expression with isoenzymes from the pentose phosphate pathway linked to secondary metabolism. Increased expression of these isoenzymes matches to increased fluxes in the pentose phosphate pathway. Additionally, flux maps of the central carbon metabolism show increased flux through the tricarboxylic acid cycle in the CelA-producing strain. Redirection of fluxes in the CelA-producing strain leads to higher production of NADPH, which can only partly be attributed to increased secretion. Conclusions Transcriptomic and fluxomic changes uncover potential new leads for targeted strain improvement strategies which may ease the secretion stress and metabolic burden associated with heterologous protein synthesis and secretion, and may help create a more consistently performing S. lividans strain. Yet, links to secondary metabolism and redox balancing should be further investigated to fully understand the S. lividans metabolome under heterologous protein production.
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spelling doaj.art-812e20fba96a4f5bbe7f7b859a5fa04b2022-12-22T00:41:58ZengBMCMicrobial Cell Factories1475-28592018-12-0117111910.1186/s12934-018-1040-6Transcriptomic and fluxomic changes in Streptomyces lividans producing heterologous proteinWouter Daniels0Jeroen Bouvin1Tobias Busche2Christian Rückert3Kenneth Simoens4Spyridoula Karamanou5Lieve Van Mellaert6Ólafur H. Friðjónsson7Bart Nicolai8Anastassios Economou9Jörn Kalinowski10Jozef Anné11Kristel Bernaerts12Department of Chemical Engineering, Bio- and Chemical Systems Technology, Reactor Engineering and Safety Section, KU LeuvenDepartment of Chemical Engineering, Bio- and Chemical Systems Technology, Reactor Engineering and Safety Section, KU LeuvenCenter for Biotechnology (CeBiTec), Bielefeld UniversityCenter for Biotechnology (CeBiTec), Bielefeld UniversityDepartment of Chemical Engineering, Bio- and Chemical Systems Technology, Reactor Engineering and Safety Section, KU LeuvenDepartment of Microbiology and Immunology, Laboratory of Molecular Bacteriology, KU LeuvenDepartment of Microbiology and Immunology, Laboratory of Molecular Bacteriology, KU LeuvenMatísDivision of Mechatronics, Biostatistics and Sensors (MeBioS), Department of Biosystems (BIOSYST), KU LeuvenDepartment of Microbiology and Immunology, Laboratory of Molecular Bacteriology, KU LeuvenCenter for Biotechnology (CeBiTec), Bielefeld UniversityDepartment of Microbiology and Immunology, Laboratory of Molecular Bacteriology, KU LeuvenDepartment of Chemical Engineering, Bio- and Chemical Systems Technology, Reactor Engineering and Safety Section, KU LeuvenAbstract Background The Gram-positive Streptomyces lividans TK24 is an attractive host for heterologous protein production because of its high capability to secrete proteins—which favors correct folding and facilitates downstream processing—as well as its acceptance of methylated DNA and its low endogeneous protease activity. However, current inconsistencies in protein yields urge for a deeper understanding of the burden of heterologous protein production on the cell. In the current study, transcriptomics and $$^{13}\hbox {C}$$ 13C -based fluxomics were exploited to uncover gene expression and metabolic flux changes associated with heterologous protein production. The Rhodothermus marinus thermostable cellulase A (CelA)—previously shown to be successfully overexpressed in S. lividans—was taken as an example protein. Results RNA-seq and $$^{13}\hbox {C}$$ 13C -based metabolic flux analysis were performed on a CelA-producing and an empty-plasmid strain under the same conditions. Differential gene expression, followed by cluster analysis based on co-expression and co-localization, identified transcriptomic responses related to secretion-induced stress and DNA damage. Furthermore, the OsdR regulon (previously associated with hypoxia, oxidative stress, intercellular signaling, and morphological development) was consistently upregulated in the CelA-producing strain and exhibited co-expression with isoenzymes from the pentose phosphate pathway linked to secondary metabolism. Increased expression of these isoenzymes matches to increased fluxes in the pentose phosphate pathway. Additionally, flux maps of the central carbon metabolism show increased flux through the tricarboxylic acid cycle in the CelA-producing strain. Redirection of fluxes in the CelA-producing strain leads to higher production of NADPH, which can only partly be attributed to increased secretion. Conclusions Transcriptomic and fluxomic changes uncover potential new leads for targeted strain improvement strategies which may ease the secretion stress and metabolic burden associated with heterologous protein synthesis and secretion, and may help create a more consistently performing S. lividans strain. Yet, links to secondary metabolism and redox balancing should be further investigated to fully understand the S. lividans metabolome under heterologous protein production.http://link.springer.com/article/10.1186/s12934-018-1040-6Streptomyces lividansHeterologous protein production and secretion$$^{13}\hbox {C}$$ 13 C -based metabolic fluxRNA-seq analysisGene clustering analysis
spellingShingle Wouter Daniels
Jeroen Bouvin
Tobias Busche
Christian Rückert
Kenneth Simoens
Spyridoula Karamanou
Lieve Van Mellaert
Ólafur H. Friðjónsson
Bart Nicolai
Anastassios Economou
Jörn Kalinowski
Jozef Anné
Kristel Bernaerts
Transcriptomic and fluxomic changes in Streptomyces lividans producing heterologous protein
Microbial Cell Factories
Streptomyces lividans
Heterologous protein production and secretion
$$^{13}\hbox {C}$$ 13 C -based metabolic flux
RNA-seq analysis
Gene clustering analysis
title Transcriptomic and fluxomic changes in Streptomyces lividans producing heterologous protein
title_full Transcriptomic and fluxomic changes in Streptomyces lividans producing heterologous protein
title_fullStr Transcriptomic and fluxomic changes in Streptomyces lividans producing heterologous protein
title_full_unstemmed Transcriptomic and fluxomic changes in Streptomyces lividans producing heterologous protein
title_short Transcriptomic and fluxomic changes in Streptomyces lividans producing heterologous protein
title_sort transcriptomic and fluxomic changes in streptomyces lividans producing heterologous protein
topic Streptomyces lividans
Heterologous protein production and secretion
$$^{13}\hbox {C}$$ 13 C -based metabolic flux
RNA-seq analysis
Gene clustering analysis
url http://link.springer.com/article/10.1186/s12934-018-1040-6
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