Abstract
Bacillus subtilis is an attractive host for production of recombinant proteins. Promoters and expression plasmid backbones have direct impacts on the efficiency of gene expression. To screen and isolate strong promoters, a promoter trap vector pShuttleF was developed in this study. Using the vector, approximately 1000 colonies containing likely promoters from Bacillus licheniformis genomic DNA were obtained. Amongst them, pShuttle-09 exhibited the highest β-Gal activities in both Escherichia coli and B. subtilis. The activity of pShuttle-09 in B. subtilis was eight times of that of the P43 promoter, a commonly used strong promoter for B. subtilis. A sequence analysis showed that pShuttle-09 contained PluxS and truncated luxS in-frame fused with the reporter gene as well as another fragment upstream of PluxS containing a putative promoter. This putative promoter was a hybrid promoter and its β-Gal activity was higher than PluxS. Reconstructing the hybrid promoter from pShuttle-09 to PlapS further improved the β-Gal production by 60%. The usefulness of our promoter trap system is likely due to random shuffling and recombination of DNA fragments and adoption of a rapid and high-throughput screening. Thus, our data provide additional evidence to support the concept of using a promoter trap system to create new promoters.
Highlights
Bacillus subtilis is a non-pathogenic gram-positive bacterium and has been an attractive host for production of recombinant proteins from both prokaryotic and eukaryotic origins [1], [2], [3]
The resultant pShuttle-P43 was transformed into E. coli DH5a and B. subtilis 1A747 and blue colonies on solid LB plate supplemented with X-gal were isolated
The bgaB directed by the P43 in pShuttleF was duly expressed in E. coli and B. subtilis (Figures 1B and 1C)
Summary
Bacillus subtilis is a non-pathogenic gram-positive bacterium and has been an attractive host for production of recombinant proteins from both prokaryotic and eukaryotic origins [1], [2], [3]. The suitability and popularity of B. subtilis as one of main hosts for recombinant protein production is due to several reasons: generally recognized as safe as a result of its lack of pathogenicity and the absence of endotoxins; capable of secreting functional proteins directly into culture media, easy for genetic manipulation and handling, and capable of large-scale fermentation [2], [4], [5], [6]. Strong promoters, a variety of translation/secretion signals, and transcription terminators are continuously being explored. In spite of the progress, it is still beneficial to find even more potent promoters
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