Reasearch On The Effect Of LspA On The Maturation Of NisI And The Nisin Yield Of Lactococcus Lactis

Nisin,a natural preservative produced by Lactococcus lactis,is widely used in food protection and medical industry.However,the nisin producing L.lactis strains could be affected by the high antimicrobial activity of nisin,and they usually prevent this suicidal behavior through expressing the lipoprotein NisI.However,due to the continuous accumulation of nisin and the limited nisin tolerance of the strains in the fermentation process,the normal growth of Lactococcus lactis is inhibited,which affects the improvement of the nisin yield.In this study,we found that the lipoprotein signal peptidase(LspA)can promote the maturation of NisI and improve the nisin yield of Lactococcus lactis.In this study,the nisin yield of Lactococcus lactis was enhanced from 2884 IU/mL to 3697 IU/mL by over-expressing lspA gene,which was 28% higher than that of the original strain L.lactis F44.Additionally,knocking out the lspA gene leaded to the decrease of nisin yield from 2884 IU/mL to 2388 IU/mL,which was 17% lower than that of F44.These results showed that lspA played an important role in the nisin yield of L.lactis F44.To explore the effect of LspA on the maturation process of NisI and the nisin immunity of L.lactis,the nisI over-expressing strain,nisI and lspA co-expressing strain were constructed.Results showed that the nisin yield of nisI over-expressing strain was improved from 2884 IU/mL to 3762 IU/mL,furthermore the nisin yield of nisI and lspA co-expressing strain could reached 3973 IU/ mL,which was 37% higher than that of L.lactis F44.Meanwhile,nisin tolerance assay showed that the survival rate of nisI and lspA co-expressing strain was 100-fold higher than that of F44 when the nisin concentration of solid plate was 20000 IU/mL.Contrary,the survival rate of deletion strain was 1000-fold lower than that of F44 when the nisin concentration of solid plate was 12000 IU/mL.Finally,through the enzyme activity experiments in vitro and in vivo showed that LspA could excise the signal peptide of pre-NisI and promote the maturation of NisI,which improved the nisin tolerance and nisin yield of L.lactis.Our study informs possible strategies for the improvement of nisin tolerance and provided new insight into the mechanism of NisI in L.lactis.