The Improvement Of Bacillus Subtilis Production By Knocking-out Lysis Related Genes

Bacillus subtilis and related species are designated generally recognized as safe microorganisms, and they are widely used in agriculture and industrial biotechnology, such as biocontrol agents, plant growth accelerator, probiotics, producing industrial enzymes and other recombinant proteins. However, cell autolysis is ubiquitous in Bacillus strains and significantly decreases its yield in liquid cultures. Genetically, numerous factors mediate the lysis of B. subtilis, such as cannibalism factors, prophages, and peptidoglycan (PG) hydrolases.In this work we investigated the effect of the different types of lysis genes on cell lysis and biomass through multiple gene silencing, and finally aimed to develop a new strategy to prevent cell lysis and enhance biomass as well as the production of recombinant proteins. Bacillus subtilis168, the type strain which is convenient in genetic manipulation, was used in this work, and five genes or genetic elements representing three different functional categories were studied as follows:lytC encoding PG hydrolases, the prophage genes xpf and yqxG-yqxH-cwlA (yGIA), and skfA and sdpC that encode cannibalism factors. Applying the temperature sensitive integrated vector pNNB194we constructed the knock-out vectors of these five genes, and then a series of single or multiple gene-deletion mutants were achieved.The biomass was highly enhanced by deleting individually skfA, sdpC, and lytC, the inactivation of xpf could also contribute in the biomass production but with less effect, while deletion of yGIA had little effect. When more genes were deleted, the highest biomass was gotten in the triple or double mutant, and mutating more genes did not significantly increase biomass further. Meanwhile, we detected the autolysis activity by adding sodium azide (NaN3). It was indicated that only single deletion of xpf or lytC could inhibit cell lysis effectively, while deletion of skfA or sdpC exerted a addition effect when xpf or lytC were mutated, and the cell lysis of multiple deletion mutants LM2531was inhibited at the greatest extent, in which15%of the cells lysed in4h compared which92%in168. Therefore, we recommend LM2531, a combined deletion of lytC, sdpC, xpf, and skfA, for constructing B. subtilis cell factories, which harboring both higher biomass and the least cell lysis.Moreover, we tried to express several recombinant proteins in LM2531to detect its protein production capacity. Unfortunately the expression of IL32was failed, however we found that the fluorescence intensity of GFP was largely decrease when fusion expressed with IL32. Hereby we presumed that the recombinant protein IL32would be declined by the host strain Bacillus subtilis so that it could not be detected.Then two expression vectors were constructed for producing recombinant proteins (?-galactosidase and nattokinase) under the control of the P43promoter. Cultures of LM2531and wild-type transformants produced13741U/ml and7991U/ml of intracellular ?-galactosidase, respectively (1.72-fold increase). Further, the level of secreted nattokinase produced by strain LM2531increased by2.6-fold compared with wild-type (5226IU/ml vs.2028IU/ml, respectively). It was demonstrated that our systematic multigene deletion approach could also enhance the production of recombinant proteins by B. subtilis as well as inhibiting cell lysis and increasing the biomass.