Through genomic mutation and high-throughput screening, we obtained a strain L. lactis WH101 with good acid resistance at pH 5.0 from 35,000 strains. Next, we compared the NZ9000 and WH101 by transcriptomics analysis and mathematical model and obtain key acid resistant components—msmK gene.
The survival rate of L.lactis NZ3900/pNZ8149-msmK and L.lactis NZ3900/pNZ8149 in acid stress result shows that msmK is an effective anti-acid gene, which can make the recombinant strain has 213-flood higher survival rate than parent one after 3 hours at pH 4.0.
Fig 1 Number of colonies at acid stress (pH 4.0).
Fig 2 Survival rate at acid stress (pH 4.0).
As for the composite part msmK-cspD2，The strain Lactococcus lactis NZ3900/pNZ8149-msmK-cspD2 and Lactococcus lactis NZ3900/pNZ8149 were tested for acid resistance and cold resistance.
Deal with the samples under pH 4.0 with nisin as an inducer.
Fig 3 Number of colonies at acid stress (pH 4.0).
Fig 4 Survival rate at acid stress (pH4.0).The result shows that composite part can make the recombinant strain has 243-fold higher survival rate than parent one under acid stress.
Fig 5 Electron microscopy of L.lactis NZ3900/pNZ8149-msmk-cspD2-gfp and L.lactis NZ3900/pNZ8149 before and after acid stress.Before the acid stress, the cell structure of the control strain and the recombinant strain remained intact. After 3 h of pH 4.0 stress, the cell membrane thickness became thinner and the surface became rough, and the cell membrane of some control strains ruptured. In comparison, the cell structure of the recombinant strain remains more intact, thereby effectively reducing the damage caused by acid stress on the cells.
L.lactis NZ3900/pNZ8149-msmk-cspD2-gfp and L.lactis NZ3900/pNZ8149 strains were inoculated with 4% inoculation. When cultured at 30 °C for 2.5 h (OD=0.35), add 0, 0.5 ng/mL of Nisin, and then culture for 8 ∼ 10 h (OD=0.8), centrifuge at 4000 r/min. Resuspend them in the same volume of fresh M17 medium and count the number of colonies. Four freeze-thaw stimulations were performed on all samples by 1 mL of the sample after counting, and placed in a refrigerator at −20 °C to cool rapidly, frozen for 24 h, then slowly frozen at 4 min and 30 °C. Count separately and calculate the survival rate.
Fig 6 The Comparison curve of survival rate under cold stress.After 4 consecutive repeated freeze-thaw tests, the recombinant strain was 47.5 times more viable than the control strain, indicating the antifreeze survival rate of the strain with increased overexpression of cspD2.
Fig 7 Electron microscopy before and after repeated freezing and thawing.Before freezing and thawing, the cell structure of the control strain and the recombinant strain remained intact. After repeated freezing and thawing for 4 times, the cell membrane of the cell became thin and rough, and some intracellular substances overflowed. In comparison, the cell structure of the recombinant strain remains more intact, and the damage of the cell membrane is alleviated, thereby effectively reducing the damage caused by freezing stress on the cells.