Integrative expression vectors with P grac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis

Highlights

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  The new inducer-free integrative expression vectors could repress the reporter gene expression in the E. coli cloning strain, thereby facilitating the cloning step.

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  The expression vectors carrying IPTG-inducible P grac promoters allow the production of the recombinant protein at high levels in B. subtilis in the absence of the inducer.

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  The single-copy expression levels of integrative constructs, P grac01- bgaB, P grac100- bgaB, P grac212- bgaB could reach to % and 8%, 20.9 % and 42 % of total cellular proteins after 12 h incubation, respectively.

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  The double integration of P grac212- bgaB into both amyE and lacA loci resulted in BgaB expression up to 53.4 %.

Abstract

Inducer-free integrative vectors are often used to create B. subtilis strains for industrial purposes, but employing strong promoters to produce high levels of recombinant proteins in B. subtilis results in high leaky expression that can hamper cloning in Escherichia coli. To overcome the problem, we used strong IPTG-inducible P grac promoters harboring lac operators to construct inducer-free integrative vectors able to integrate into the B. subtilis genome at either the lacA or the amyE locus, or both and examined their ability to repress the β-galactosidase ( bgaB) gene in E. coli and to overexpress BgaB in B. subtilis. The P grac01 vectors could repress bgaB expression about 24-fold in E. coli to low background levels. The integrated P grac01- bgaB constructs exhibited inducer-free expression and produced 8% of total cellular proteins, only 1.25 or 1.75 times less compared with their cognates as plasmids. The stronger promoters, P grac100- bgaB and P grac212- bgaB yielded 20.9 % and 42 % of total intracellular proteins after 12 h of incubation, respectively. Incorporation of the P grac212- bgaB into both amyE and lacA loci resulted in BgaB expression up to 53.4 %. In conclusion, integrative vectors containing the P grac promoter family have great potential for inducer-free overproduction of recombinant proteins in B. subtilis.