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

Abstract

Inducer-free integrative vectors are often used to create <i>B. subtilis</i> strains for industrial purposes, but employing strong promoters to produce high levels of recombinant proteins in <i>B. subtilis</i> results in high leaky expression that can hamper cloning in <i>Escherichia coli</i>. To overcome the problem, we used strong IPTG-inducible P<i>grac</i> promoters harboring <i>lac</i> operators to construct inducer-free integrative vectors able to integrate into the <i>B. subtilis</i> genome at either the <i>lacA</i> or the <i>amyE</i> locus, or both and examined their ability to repress the β-galactosidase (<i>bgaB</i>) gene in <i>E. coli</i> and to overexpress BgaB in <i>B. subtilis</i>. The P<i>grac</i>01 vectors could repress <i>bgaB</i> expression about 24-fold in <i>E. coli</i> to low background levels. The integrated P<i>grac</i>01-<i>bgaB</i> 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<i>grac</i>100-<i>bgaB</i> and P<i>grac</i>212-<i>bgaB</i> yielded 20.9 % and 42 % of total intracellular proteins after 12 h of incubation, respectively. Incorporation of the P<i>grac</i>212-<i>bgaB</i> into both <i>amyE</i> and <i>lacA</i> loci resulted in BgaB expression up to 53.4 %. In conclusion, integrative vectors containing the P<i>grac</i> promoter family have great potential for inducer-free overproduction of recombinant proteins in <i>B. subtilis</i>.