Construction of Chromosomally Located T7 Expression System for Production of Heterologous Secreted Proteins in Bacillus subtilis

Abstract

Bacillus subtilis is most commonly employed for secretion of recombinant proteins. To circumvent the problems caused by using plasmids, the T7 expression system known for its high efficiency was rebuilt in B. subtilis. Accordingly, a markerless and replicon-free method was developed for genomic insertion of DNAs. By the act of homologous recombination via the guide DNA, a suicidal vector carrying the gene of interest was integrated into genomic loci of bacteria. Removal of the inserted selection marker and replicon flanked by FRT sites was mediated by the FLP recombinase. By using the mentioned system, B. subtilis strain PT5 was constructed to harbor a genomic copy of the spac promoter-regulated T7 gene 1 located at wprA (encoding the cell wall-associated protease). Similarly, the T7 promoter-driven nattokinase or endoglucanase E1 of Thermomonospora fusca genes were also integrated into mpr (encoding an extracellular protease) of strain PT5. Consequently, the integrant PT5/Mmp-T7N or PT5/MT1-E1 resulted in a "clean" producer strain deprived of six proteases. After 24 h, the strain receiving induction was able to secret nattokinase and endoglucanase E1 with the volumetric activity reaching 10860 CU/mL and 8.4 U/mL, respectively. This result clearly indicates the great promise of the proposed approach for high secretion of recombinant proteins in B. subtilis.

Characterization and overexpression of a novel keratinase from Bacillus polyfermenticus B4 in recombinant Bacillus subtilis

“…Gene cloning was performed in E. coli DH5α and B. subtilis strain PT5 containing the T7 RNA polymerase for gene expression driven by the T7 promoter (Chen et al 2010). For DNA manipulation, E. coli were grown in 10 mL of Luria-Bertani (LB) medium in a 125-mL shake flask, and cell densities were determined through the optical density at 600 nm (OD 600 ).…”

Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis

“…Moreover, multiplexing is not practical, as the number of available selection and counterselection markers is limited, exposure to multiple antibiotics is not preferable (i.e., may compromise cell physiology), and counterselection will become increasingly difficult (i.e., less efficient or more time-consuming) as the number of simultaneous targets increases. On the other hand, site-specific recombination via the Cre/loxP (49) and FLP/FLP recombination target (FRT) (50) systems has also been applied to markerless recombineering in B. subtilis, with a generally higher efficiency than counterselection methods. Additionally, single-stranded DNA (ssDNA) recombineering mediated by the Red phage ␤-recombinase provides a high editing efficiency (51).…”

Transient expression of optimized and synthesized nattokinase gene in melon (<i>Cucumis melo</i> L.) fruit by agroinfiltration

“…NK was overexpressed in Escherichia coli as an insoluble recombinant protein linked to the C terminus of oleosin by an intein fragment to form a artificial oil (Chiang et al 2005). Chen et al (2010) constructed a chromosomally located T7 expression system in Bacillus subtilis and the secretion of NK up to 10860 CU ml −1 . Li et al (2007) expressed the active NK authentically in Spodoptera frugiperda cells with the modified Bac-to-Bac system and the recombinant NK retained fibrinolytic activity of 60 U ml −1 .…”