Abstract
Objectives
To expand the repertoire of strong promoters for high level expression of proteins based on the transcriptome of Bacillus licheniformis.
Results
The transcriptome of B. licheniformis ATCC14580 grown to the early stationary phase was analyzed and the top 10 highly expressed genes/operons out of the 3959 genes and 1249 operons identified were chosen for study promoter activity. Using beta-galactosidase gene as a reporter, the candidate promoter pBL9 exhibited the strongest activity which was comparable to that of the widely used strong promoter p43. Furthermore, the pro-transglutaminase from Streptomyces mobaraensis (pro-MTG) was expressed under the control of promoter pBL9 and the activity of pro-MTG reached 82 U/ml after 36 h, which is 23% higher than that of promoter p43 (66.8 U/ml).
Conclusion
In our analyses of the transcriptome of B. licheniformis, we have identified a strong promoter pBL9, which could be adapted for high level expression of proteins in the host Bacillus subtilis.
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Acknowledgements
We wish to acknowledge the financial support by the Science and Technology Planning Project of Guangdong Province (Grant No. 2013B010404007), the State 863 Project (Grant No. 2014AA021304).
Supporting information
Supplementary Figure 1—Construction flowchart of pBE-pBL1-bgaB. All the 11 expression plasmids were constructed as follows.
Supplementary Figure 2—The homogeniety of β-Gal expression A qualitative plate assay was developed for measuring the enzyme level in isolated colonies. All materials and concentrations were the same for two plates, except for the portion of the plate that was overlaid with 3 ml of 0.067 M potassium phosphate buffer (pH 6.8) containing 3 mg of o-nitrophenyl-β-D-galactoside (ONPGal).
Supplementary Figure 3—β-Gal activities of all the promoters acting throughout the life cycle (72 h).
Supplementary Figure 4—Construction flowchart of pBE-pBL9-proMTG.
Supplementary Table 1—Bacterial strains and plasmids.
Supplementary Table 2—Primers used.
Supplementary Table 3—Genome-wide pathway analysis and GO analysis.
Supplementary Table 4—Identified operons.
Supplementary Table 5—Candidates were selected. Promoter sequence: The sequences of two operon structural genes were screened. When the sequence length was more than 300 bp, the start codon leader sequence (300 bp) was obtained. Predicted σ-factor and Transcriptional factor were obtained from: http://dbtbs.hgc.jp/.
Supplementary Table 6—Identification TF families and functional description of ten Candidates. Predicted TF families and functional description were obtained from: http://dbtbs.hgc.jp/.
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Liu, X., Yang, H., zheng, J. et al. Identification of strong promoters based on the transcriptome of Bacillus licheniformis . Biotechnol Lett 39, 873–881 (2017). https://doi.org/10.1007/s10529-017-2304-7
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DOI: https://doi.org/10.1007/s10529-017-2304-7