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Construction and analysis of novel controllable expression vectors for Bacillus subtilis
(2007)
- Summary The gram-positive bacterium Bacillus subtilis is well-known for its contributions to agricultural, medical, and food biotechnology and for the production of recombinant proteins. At present, about 60% of the commercially available technical enzymes are produced by Bacillus species. Furthermore, a large body of information concerning transcription, translation, protein folding and secretion mechanisms, genetic manipulation, and large-scale fermentation has been acquired. But so far, efficient and inexpensive expression vectors for B. subtilis are still missing. To fill this gap, a glycine-inducible expression system and a lysine-autoinducible one were explored and IPTG-inducible expression plasmids that allow overexpression and purification of proteins were constructed and analyzed. Furthermore, a technique with a useful promoter-probe plasmid to analyze strong promoters in B. subtilis was established, which allowed to study promoter and mRNA stabilizing elements to enhance the transcript level and mRNA stability leading to higher production of recombinant protein. During the development of the glycine-inducible and lysine-autoinducible expression plasmids, the presence of a small transcript termed riboswitch corresponding to the 5´ UTR in the absence of L-glycine or presence of L-lysine and its conversion into the full-length transcript after addition of the L-glycine or removal of L-lysine was confirmed by Northern blot. Next, the promoter and downstream riboswitch was fused to the lacZ reporter gene to measure glycine or lysine-dependent induction. The production potential for the glycine system was analyzed in detail, and the promoter strength improved by using HtpG, Pbp4 and alpha-amylase as model proteins. In summary, the glycine riboswitch can be used successfully for regulatable production of both intra- and extracellular proteins, and the lysine riboswitch can be applied as an auto-inducible expression system allowing production of recombinant proteins when the L-lysine concentration within the growth medium falls below a threshold value. Six commercially available novel plasmid-based IPTG-inducible expression plasmids for B. subtilis were constructed, too. While the first vector allows intracellular production of recombinant proteins (pHT01), the second provides a strong secretion signal (pHT43). The third vector allows addition of the c-Myc epitope tag (pHT10), and the remaining three vectors provide the purification tags His (pHT08) and Strep (pHT09 and pHT24). The versatility of all six vectors was verified by insertion of appropriate reporter genes and by demonstrating high level production of their proteins. A total of 85 different synthetic and groE-modified sigmaA-dependent promoters were introduced into pHT06 and analyzed. Sequences around the transcriptional start site, the -10 region, the -15 region, the -35 region and the upstream region turned out to influence the promoter strength. BgaB activities and Northern blot analyses were used to measure the activity of the different promoters. The measurements of some new combinations of core promoters and UP elements on gene expression revealed that the beta-galactosidase activity expression levels could be increased up to 13-fold and the mRNA levels up to 43-fold as compared to the strong Pgrac promoter. If both elements were combined, an activity roughly 690 times higher than that obtained with the Pspac promoter were obtained, and synthesis of BgaB, under control of these promoters, could reach up to 30% of the total cellular protein. The mRNA stabilizing elements were also analyzed by using a similar experimental approach. First, seven-teen different 3´-mRNA terminal stem-loops have been investigated, which did not significantly influence neither the amount of protein produced nor the mRNA stability. Second, the 5´-mRNA stabilizing elements including a strong RBS, the lacO Controllable Stabilizing Element (CoSE) and the spacer between the RBS and CoSE were examined. The results demonstrated that CoSE together with an appropriate spacer and a strong RBS could increase gene expression 9-fold as compared to the Pgrac promoter, reaching up to 26% of total cellular protein and a half-life of the mRNA of more than 60 min. A combination of strong promoters and stabilizing elements showed that recombinant protein synthesis levels of up to 42% of the total cellular protein could be obtained.
