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Genome-wide identification and evaluation of constitutive promoters in streptomycetes.

Li S, Wang J, Li X, Yin S, Wang W, Yang K - Microb. Cell Fact. (2015)

Bottom Line: Four promoters were used in a plug-and-play platform to control the expression of the cryptic cluster of jadomycin B in S. venezuelae ISP5230 and resulted in different levels of the production of jadomycin B that corresponded to promoter strength.This work identified and evaluated a set of constitutive promoters with different strengths in streptomycetes, and it enriched the presently available promoter toolkit in this genus.These promoters should be valuable in current platforms of metabolic engineering and synthetic biology for the activation of cryptic biosynthetic clusters and the optimization of pathways for the biosynthesis of important natural products in Streptomyces species.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China. lishanshan8427@gmail.com.

ABSTRACT

Background: Streptomycetes attract a lot of attention in metabolic engineering and synthetic biology because of their well-known ability to produce secondary metabolites. However, the available constitutive promoters are rather limited in this genus.

Results: In this work, constitutive promoters were selected from a pool of promoters whose downstream genes maintained constant expression profiles in various conditions. A total of 941 qualified genes were selected based on systematic analysis of five sets of time-series transcriptome microarray data of Streptomyces coelicolor M145 cultivated under different conditions. Then, 166 putative constitutive promoters were selected by following a rational selection workflow containing disturbance analysis, function analysis, genetic loci analysis, and transcript abundance analysis. Further, eight promoters with different strengths were chosen and subjected to experimental validation by green fluorescent protein reporter and real-time reverse-transcription quantitative polymerase chain reaction in S. coelicolor, Streptomyces venezuelae and Streptomyces albus. The eight promoters drove the stable expression of downstream genes in different conditions, implying that the 166 promoters that we identified might be constitutive under the genus Streptomyces. Four promoters were used in a plug-and-play platform to control the expression of the cryptic cluster of jadomycin B in S. venezuelae ISP5230 and resulted in different levels of the production of jadomycin B that corresponded to promoter strength.

Conclusions: This work identified and evaluated a set of constitutive promoters with different strengths in streptomycetes, and it enriched the presently available promoter toolkit in this genus. These promoters should be valuable in current platforms of metabolic engineering and synthetic biology for the activation of cryptic biosynthetic clusters and the optimization of pathways for the biosynthesis of important natural products in Streptomyces species.

No MeSH data available.


Related in: MedlinePlus

Real-time RT-qPCR assays of the selected promoters. a The relative mRNA of gfp controlled by different promoters in S. coelicolor M1146. b The relative mRNA of gfp controlled by different promoters in S. venezuelae WVR2006. c The relative mRNA of gfp controlled by different promoters in S. albus J1074. The values are means  ±  SD from three independent experiments. Data obtained from different growth stages of each strain were not statistically significant
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Fig4: Real-time RT-qPCR assays of the selected promoters. a The relative mRNA of gfp controlled by different promoters in S. coelicolor M1146. b The relative mRNA of gfp controlled by different promoters in S. venezuelae WVR2006. c The relative mRNA of gfp controlled by different promoters in S. albus J1074. The values are means  ±  SD from three independent experiments. Data obtained from different growth stages of each strain were not statistically significant

Mentions: Samples for experiments were harvested at the exponential phase (24 h), transitional phase (48 h), and stationary phase (60 h) (Additional file 6: Figure S1A). For each strain, there was no significant difference among the intensity of GFP fluorescence in different growth stages, meaning all of the tested promoters drove constant gene expression profiles (Fig. 3a), which was consistent with the stable transcript levels of gfp determined by real-time RT-qPCR (Fig. 4a). The orders of promoter strength reflected by the GFP reporter and real-time RT-qPCR were identical to the result generated by RNA-Seq (RPKM value). Moreover, positive linear relationships (R2 > 0.85) existed among the RPKM value, relative mRNA level of gfp and fluorescence intensity of GFP (Fig. 5a). These observations demonstrate the high consistency of the results of systematic analysis and experiments, and they suggest that the 166 constitutive promoters might be very reliable in S. coelicolor.Fig. 3


Genome-wide identification and evaluation of constitutive promoters in streptomycetes.

Li S, Wang J, Li X, Yin S, Wang W, Yang K - Microb. Cell Fact. (2015)

Real-time RT-qPCR assays of the selected promoters. a The relative mRNA of gfp controlled by different promoters in S. coelicolor M1146. b The relative mRNA of gfp controlled by different promoters in S. venezuelae WVR2006. c The relative mRNA of gfp controlled by different promoters in S. albus J1074. The values are means  ±  SD from three independent experiments. Data obtained from different growth stages of each strain were not statistically significant
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4625935&req=5

Fig4: Real-time RT-qPCR assays of the selected promoters. a The relative mRNA of gfp controlled by different promoters in S. coelicolor M1146. b The relative mRNA of gfp controlled by different promoters in S. venezuelae WVR2006. c The relative mRNA of gfp controlled by different promoters in S. albus J1074. The values are means  ±  SD from three independent experiments. Data obtained from different growth stages of each strain were not statistically significant
Mentions: Samples for experiments were harvested at the exponential phase (24 h), transitional phase (48 h), and stationary phase (60 h) (Additional file 6: Figure S1A). For each strain, there was no significant difference among the intensity of GFP fluorescence in different growth stages, meaning all of the tested promoters drove constant gene expression profiles (Fig. 3a), which was consistent with the stable transcript levels of gfp determined by real-time RT-qPCR (Fig. 4a). The orders of promoter strength reflected by the GFP reporter and real-time RT-qPCR were identical to the result generated by RNA-Seq (RPKM value). Moreover, positive linear relationships (R2 > 0.85) existed among the RPKM value, relative mRNA level of gfp and fluorescence intensity of GFP (Fig. 5a). These observations demonstrate the high consistency of the results of systematic analysis and experiments, and they suggest that the 166 constitutive promoters might be very reliable in S. coelicolor.Fig. 3

Bottom Line: Four promoters were used in a plug-and-play platform to control the expression of the cryptic cluster of jadomycin B in S. venezuelae ISP5230 and resulted in different levels of the production of jadomycin B that corresponded to promoter strength.This work identified and evaluated a set of constitutive promoters with different strengths in streptomycetes, and it enriched the presently available promoter toolkit in this genus.These promoters should be valuable in current platforms of metabolic engineering and synthetic biology for the activation of cryptic biosynthetic clusters and the optimization of pathways for the biosynthesis of important natural products in Streptomyces species.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China. lishanshan8427@gmail.com.

ABSTRACT

Background: Streptomycetes attract a lot of attention in metabolic engineering and synthetic biology because of their well-known ability to produce secondary metabolites. However, the available constitutive promoters are rather limited in this genus.

Results: In this work, constitutive promoters were selected from a pool of promoters whose downstream genes maintained constant expression profiles in various conditions. A total of 941 qualified genes were selected based on systematic analysis of five sets of time-series transcriptome microarray data of Streptomyces coelicolor M145 cultivated under different conditions. Then, 166 putative constitutive promoters were selected by following a rational selection workflow containing disturbance analysis, function analysis, genetic loci analysis, and transcript abundance analysis. Further, eight promoters with different strengths were chosen and subjected to experimental validation by green fluorescent protein reporter and real-time reverse-transcription quantitative polymerase chain reaction in S. coelicolor, Streptomyces venezuelae and Streptomyces albus. The eight promoters drove the stable expression of downstream genes in different conditions, implying that the 166 promoters that we identified might be constitutive under the genus Streptomyces. Four promoters were used in a plug-and-play platform to control the expression of the cryptic cluster of jadomycin B in S. venezuelae ISP5230 and resulted in different levels of the production of jadomycin B that corresponded to promoter strength.

Conclusions: This work identified and evaluated a set of constitutive promoters with different strengths in streptomycetes, and it enriched the presently available promoter toolkit in this genus. These promoters should be valuable in current platforms of metabolic engineering and synthetic biology for the activation of cryptic biosynthetic clusters and the optimization of pathways for the biosynthesis of important natural products in Streptomyces species.

No MeSH data available.


Related in: MedlinePlus