Influence of global gene regulatory networks on single cell heterogeneity of green fluorescent protein production in Bacillus subtilis
Abstract Background Gram-positive bacterium Bacillus subtilis has been extensively studied as a microbial cell factory for high-level producing a wide range of interesting products. Green fluorescent protein (GFP) is commonly used as a marker for determining the strength of a given promoter or for t...
Main Authors: | , |
---|---|
Format: | Article |
Language: | English |
Published: |
BMC
2018-08-01
|
Series: | Microbial Cell Factories |
Subjects: | |
Online Access: | http://link.springer.com/article/10.1186/s12934-018-0985-9 |
id |
doaj-7ea453d9933d4b7b8d2b335f52e26b3c |
---|---|
record_format |
Article |
spelling |
doaj-7ea453d9933d4b7b8d2b335f52e26b3c2020-11-25T02:28:28ZengBMCMicrobial Cell Factories1475-28592018-08-0117111010.1186/s12934-018-0985-9Influence of global gene regulatory networks on single cell heterogeneity of green fluorescent protein production in Bacillus subtilisHaojie Cao0Oscar P. Kuipers1Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of GroningenDepartment of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of GroningenAbstract Background Gram-positive bacterium Bacillus subtilis has been extensively studied as a microbial cell factory for high-level producing a wide range of interesting products. Green fluorescent protein (GFP) is commonly used as a marker for determining the strength of a given promoter or for the subcellular localization of a fusion protein. However, the inherent heterogeneity of GFP expression among individual cells that can arise from global regulation differences in the expression host, has not yet been systematically assessed. B. subtilis strains with single mutation(s) in the two major transcriptional regulators CcpA and/or CodY were earlier found to improve overall heterologous protein production levels. Here, we investigate the dynamic production performance of GFP in the reporter strains with chromosomally integrated P hyspank -sfGFP(Sp). Results The mutation R214C in the DNA-binding domain of CodY effectively enhances GFP production at the population level relative to two other strains, i.e. wildtype (WT) and CcpAT19S. During the late stationary phase, the high- and low-level GFP-producing cells coexist in the WT population, while the CodYR214C population at the single-cell level shows higher phenotypic homogeneity of fluorescence signals. Conclusion Expression of GFP is prominently heterogeneous in the WT B. subtilis cells, and this phenotypic heterogeneity can be significantly reduced by CodYR214C mutation. The rates of production heterogeneity show a high correlation to the overall GFP yields. Moreover, the toolkit of flow cytometry and fluorescence microscopy that can achieve real-time profiles of GFP production performance in various strains may facilitate the further use of B. subtilis as a cell factory.http://link.springer.com/article/10.1186/s12934-018-0985-9Bacillus subtilisSuperfolder green fluorescent protein (sfGFP)Heterogeneous expressionGlobal transcriptional regulationProduction levelSingle cell analysis |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Haojie Cao Oscar P. Kuipers |
spellingShingle |
Haojie Cao Oscar P. Kuipers Influence of global gene regulatory networks on single cell heterogeneity of green fluorescent protein production in Bacillus subtilis Microbial Cell Factories Bacillus subtilis Superfolder green fluorescent protein (sfGFP) Heterogeneous expression Global transcriptional regulation Production level Single cell analysis |
author_facet |
Haojie Cao Oscar P. Kuipers |
author_sort |
Haojie Cao |
title |
Influence of global gene regulatory networks on single cell heterogeneity of green fluorescent protein production in Bacillus subtilis |
title_short |
Influence of global gene regulatory networks on single cell heterogeneity of green fluorescent protein production in Bacillus subtilis |
title_full |
Influence of global gene regulatory networks on single cell heterogeneity of green fluorescent protein production in Bacillus subtilis |
title_fullStr |
Influence of global gene regulatory networks on single cell heterogeneity of green fluorescent protein production in Bacillus subtilis |
title_full_unstemmed |
Influence of global gene regulatory networks on single cell heterogeneity of green fluorescent protein production in Bacillus subtilis |
title_sort |
influence of global gene regulatory networks on single cell heterogeneity of green fluorescent protein production in bacillus subtilis |
publisher |
BMC |
series |
Microbial Cell Factories |
issn |
1475-2859 |
publishDate |
2018-08-01 |
description |
Abstract Background Gram-positive bacterium Bacillus subtilis has been extensively studied as a microbial cell factory for high-level producing a wide range of interesting products. Green fluorescent protein (GFP) is commonly used as a marker for determining the strength of a given promoter or for the subcellular localization of a fusion protein. However, the inherent heterogeneity of GFP expression among individual cells that can arise from global regulation differences in the expression host, has not yet been systematically assessed. B. subtilis strains with single mutation(s) in the two major transcriptional regulators CcpA and/or CodY were earlier found to improve overall heterologous protein production levels. Here, we investigate the dynamic production performance of GFP in the reporter strains with chromosomally integrated P hyspank -sfGFP(Sp). Results The mutation R214C in the DNA-binding domain of CodY effectively enhances GFP production at the population level relative to two other strains, i.e. wildtype (WT) and CcpAT19S. During the late stationary phase, the high- and low-level GFP-producing cells coexist in the WT population, while the CodYR214C population at the single-cell level shows higher phenotypic homogeneity of fluorescence signals. Conclusion Expression of GFP is prominently heterogeneous in the WT B. subtilis cells, and this phenotypic heterogeneity can be significantly reduced by CodYR214C mutation. The rates of production heterogeneity show a high correlation to the overall GFP yields. Moreover, the toolkit of flow cytometry and fluorescence microscopy that can achieve real-time profiles of GFP production performance in various strains may facilitate the further use of B. subtilis as a cell factory. |
topic |
Bacillus subtilis Superfolder green fluorescent protein (sfGFP) Heterogeneous expression Global transcriptional regulation Production level Single cell analysis |
url |
http://link.springer.com/article/10.1186/s12934-018-0985-9 |
work_keys_str_mv |
AT haojiecao influenceofglobalgeneregulatorynetworksonsinglecellheterogeneityofgreenfluorescentproteinproductioninbacillussubtilis AT oscarpkuipers influenceofglobalgeneregulatorynetworksonsinglecellheterogeneityofgreenfluorescentproteinproductioninbacillussubtilis |
_version_ |
1724837810293178368 |