Acid-resistance Mechanism And Gene Function Analysis Of Recombinant Lactococcus Lactis

Nisin is a non-toxic side-effect green food preservative that has been widely used around the world for preservation of meat,dairy products,etc.However,the lactic acid secreted by lactic acid bacteria in the fermentation process will acidify the fermentation broth and affect the normal growth and the productionof nisin.In order to investigate the acid tolerance mechanism of Lactococcus lactis,we used genome-wide sequencing and comparative genomics to analyze the genome differences between genome shuffling recombinant strain G423 and the original F44.Transcription differential genes were identified by using RNA-seq and qRT-PCR.The expression sRNA strains were analyzed for their phenotypes,and the sRNAs with better acid resistance were subjected to target prediction and possible function analysis.At the same time,the CRISPR/dCas9 system of Lactococcus lactis was preliminarily constructed and validated in order to speed up gene screening and gene function analysis.In order to further excavate the acid resistance mechanism of the recombinant strains,DNA of acid-resistant strain G423 was fragmented,library constructed,sequenced,assembled,annotated,and the whole genome sequence map of recombinant strain G423 was obtained.In the genome-wide comparison,it was found that there were six large fragment insertions in the G423 genome and one large fragment loss that only changed the gene copy number without losing the gene function.A comparison of COG classifications revealed that there were 18 COG cluster extensions in G423 and these COG clusters were associated with the process of cell membrane/wall biosynthesis and H~+transport.According to the COG classification of differentially expressed genes,it is found that cell walls and cell membranes play an important role in the adaptation of lactic acid bacteria to the acidic environment.Transcriptome analysis also revealed that four novel non-coding transcripts existed in G423 but not presenting in F44.The results of RT-PCR validation showed that the transcription of NC-1 and NC-4 was increased by 2.2-fold and 1.8-fold respectively,after 1 h of stress treatment at pH 3.0.After constructing sRNA overexpression or expression engineered strains,acid stress test and fermentation experiments showed that NC-1 not only could increase the acid tolerance of strains F44 and G423,but also had certain effect on the improvement of nisin production.The bioinformatics software was used to analyze the secondary structure and target mRNA of NC-1.Results showed that it may be related to the energy metabolism of the strain.At the same time,the CRISPR/dCas9 system of L.lactis was successfully constructed and verified to be able to regulate gene transcription.In this study,it was found that sRNA NC-1 plays an important role in the acid tolerance of the recombinant strain G423,however,the whole reason for the high acid resistance of the recombinant strain was not be completely clarified.Due to the complexity of the tolerance mechanism of lactic acid bacterial and large number of inserted genes,it is necessary to further analyze the acid resistance mechanism of recombinant strains using a well-established CRISPRa/i system.