Abstract
ObjectiveTo thoroughly characterize the Pylb promoter and identify the elements that affect the promoter activity.ResultThe sequences flanking the − 35 and − 10 box of the Pylb promoter were divided into six segments, and six random-scanning mutant promoter libraries fused to an enhanced green fluorescent protein EGFP were made and analyzed by flow cytometry. Our results showed that the four nucleotides flanking the − 35 box could mostly influence the promoter activity, and this influence was related to the GC content. The promoters mutated in these regions were successfully used for expressing the gene ophc2 encoding organophosphorus hydrolase (OPHC2) and the gene katA encoding catalase (KatA).ConclusionOur work identified and characterized the sequence signatures of the Pylb promoter that could tune the promoter strength, providing further information for the potential application of this promoter. Meanwhile, the sequence signatures have the potential to be used for tuning gene expression in enzyme production, metabolic engineering, and synthetic biology.
Highlights
Bacillus subtilis has been developed as a convenient host for the production of heterologous proteins and industrial enzymes
The sequence signatures have the potential to be used for tuning gene expression in enzyme production, metabolic engineering, and synthetic biology
By scanning site-directed mutagenesis on the Pylb promoter, we found that the - 35 and - 10 regions, and the regions flanking the - 35 and - 10 regions were directly involved in the transcriptional activity of Pylb (Yu et al 2015)
Summary
Bacillus subtilis has been developed as a convenient host for the production of heterologous proteins and industrial enzymes (van Dijl and Hecker 2013). More endogenous promoters need to be explored for producing heterologous proteins. Efforts have been made to enhance promoter strength. Mutagenesis of the spacer region between the - 35 and - 10 regions in E. coli and Lactobacillus (Lb.) plantarum has successfully created high-coverage synthetic promoter libraries (De Mey et al 2007). Saturation mutagenesis of a Lactococcus lactis promoter drastically modulates expression (Jensen and Hammer 1998). Randomization of the spacer region of PymdA and PserA in B. subtilis has generated mostly strong to medium promoters (Guiziou et al 2016). Mutagenesis of the spacer region is considered a rational methodology to modify prokaryotic promoter strength
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