초록
<P><B>Background</B></P><P><I>Bacillus subtilis</I> is widely used in agriculture and industrial biotechnology; however, cell autolysis significantly decreases its yield in liquid cultures. Numerous factors mediate the lysis of <I>B. subtilis</I>, such as cannibalism factors, prophages, and peptidoglycan (PG) hydrolases. The aim of this work was to use molecular genetic techniques to develop a new strategy to prevent cell lysis and enhance biomass as well as the production of recombinant proteins.</P><P><B>Results</B></P><P>Five genes or genetic elements representing three different functional categories were studied as follows: <I>lytC</I> encoding PG hydrolases, the prophage genes <I>xpf</I> and <I>yqxG-yqxH-cwlA</I> (<I>yGlA</I>), and <I>skfA</I> and <I>sdpC</I> that encode cannibalism factors. Cell lysis was reduced and biomass was enhanced by deleting individually <I>skfA</I>, <I>sdpC</I>, <I>xpf</I>, and <I>lytC</I>. We constructed the multiple deletion mutant LM2531 (<I>skfA sdpC lytC xpf</I>) and found that after 4 h of culture, its biomass yield was significantly increased compared with that of prototypical <I>B. subtilis</I> 168 (wild-type) strain and that 15% and 92% of the cells were lysed in cultures of LM2531 and wild-type, respectively. Moreover, two expression vectors were constructed for producing recombinant proteins (β-galactosidase and nattokinase) under the control of the P43 promoter. Cultures of LM2531 and wild-type transformants produced 13741 U/ml and 7991 U/ml of intracellular β-galactosidase, respectively (1.72-fold increase). Further, the level of secreted nattokinase produced by strain LM2531 increased by 2.6-fold compared with wild-type (5226 IU/ml vs. 2028 IU/ml, respectively).</P><P><B>Conclusions</B></P><P>Our novel, systematic multigene deletion approach designed to inhibit cell lysis significantly increased the biomass yield and the production of recombinant proteins by <I>B. subtili</I>s. These findings show promise for guiding efforts to manipulate the genomes of other <I>B. subtilis</I> strains that are used for industrial purposes.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (doi:10.1186/s12934-014-0129-9) contains supplementary material, which is available to authorized users.</P>