Constructing Of High Nisin Producing Strain By Genome Shuffling And Properties Analysis

Nisin, produced by Lactococcus lactis, is one of the antibiotic polypeptide. As itcould be hydrolyzed by protease and would do no harm to our body, nisin is widelyused in food industry for preservation. The objective of this study was to improvenisinZ production of the Lactococcus lactis subsp. Lactis YF11. The DES mutantmethod and the enrichment culture of the UV irradiation mutant stored in our lab weredone for the construction of genome shuffling mutant pool. Four round of genomeshuffling were employed to obtain the high nisin production strain. After shuffling themutant strain named F44was obtained. The response surface methodology wasconducted to optimize the fermentation media and conditions of F44. FESEManalysis was done to study the cell mropholgy.The fluorescence quantitative PCRwas conducted to analyze the expression of the nisin biosynthesis genes. The mainresults were as follows.The wide type strain YF11was mutated by two rounds of DES mutagesis, threestrains with high nisin and glucose tolerance were obtained. The three strains with thethree UV irradiation strains stored in the lab were used for the construction of parentpool for genome shuffling.Six strains were used as the starter strains, the nisin and glucose tolerance wereused as the screening marker. After four rounds of genome shuffling, a strain namedF44was obtained, which could be tolerance of14000IU/mL nisin and of16%glucose.Its nisin production was2994IU/mL, which was2.92times of YF11, and2.12timesof the highest parent strain U2.From the response surface analysis it could be obtained that when the intial pHof the fermentation media was7.15, the cys was0.28g/L, the sucrose was30.9g/L, thenisin titer was the highest,3400.23IU/mL, improved17%when compared with theoriginal conditions. Fed–batch–fermentation with the optimal conditions conductedin3L fermentor showed that F44could produce4023IU/mL nisin, increased18.3%when compared with fermentation with fedding process.FESEM analysis showed that the cell morpholoy was different between theF44and YF11, the size of F44was1.217times of YF11. And the the fluorescencequantitative PCR analysis showed the expression of nisin construction gene nisZ ofF44was about1.48times of the YF11. And the expression of nisin immunity gene nisI of F44was2.3times of YF11. The expression of post-translational modificationgenes nisB, C, T were all higher than that of YF11. And the expression of nisFEGwas somehow lower than that of YF11. These results were very useful for the furtherunderstanding of nisin biosynthesis genes and would be very helpful for improvingnisin production.