Nisin is a green, safe and natural food preservative. With the expansion of nisin application, the demand for nisin has gradually increased, which equates to increased requirements for nisin production. In this study, Lactococcus lactis subsp. lactis lxl was used as the original strain, and the compound mutation method was applied to induce mutations. A high-yielding and genetically stable strain (Lactobacillus lactis A32) was identified, with the nisin titre raised by 332.2% up to 5089.29 IU/mL. Genome and transcriptome sequencing was used to analyse A32 and compare it with the original lxl strain. The comparative genomics results show that 107 genes in the A32 genome had mutations and most base mutations were not located in the four well-researched nisin-related operons, nisABTCIPRK, nisI, nisRK and nisFEG: 39 single-nucleotide polymorphisms (SNPs), 34 insertion mutations and 34 deletion mutations. The transcription results show that the expression of 92 genes changed significantly, with 27 of these differentially expressed genes upregulated, while 65 were downregulated. Our findings suggest that the output of nisin increased in L. lactis strain A32, which was accompanied by changes in the DNA replication-related gene dnaG, the ABC-ATPase transport-related genes patM and tcyC, the cysteine thiometabolism-related gene cysS, and the purine metabolism-related gene purL. Our study provides new insights into the traditional genetic mechanisms involved nisin production in L. lactis, which could provide clues for a more efficient metabolic engineering process.

Plasmids are commonly used tools in microbiology and molecular biology and have important and wide-ranging applications in the study of gene function, protein expression, and compound synthesis. The complex relationship between necessary antibiotic addition, compatibility between multiple plasmids, and the growth burden of host bacteria has plagued the wider use of compatibility plasmids. In this study, we constructed the terminal polymerization pathway of PSA by exogenously expressing the neuA, neuD, and neuS genes after the knockdown of Eschesrichia coli BL21 (DE3). Duet series vectors were utilized to regulate the expression level of neuA, neuD, and neuS genes to study the gene expression level, plasmid copy number growth burden, pressure of antibiotic addition, stability of compatible plasmids, and the level of expression stability of exogenous genes, as well as the effect on the biological reaction process. The results showed that the three genes, neuA, neuD, and neuS, were enhanced in the recombinant strain E. coli NA-05, with low copy, medium copy, and high copy, respectively. The effect of PSA synthesis under standard antibiotic pressure was remarkable. The results of this thesis suggest the use of a Duet series of compatible expression vectors to achieve the stable existence and co-expression of multiple genes in recombinant bacteria, which is a good reason for further research.