Screening And Analysis Of Acid-Related Genes In Streptococcus Thermophilus During Fermentation

Streptococcus thermophilus is an important industrial bacteria, which is a gram-positive, facultative anaerobic bacteria. But, the cells will continue to grow and produce lactic acid before drinking. And it appears difficult for customers to accept the taste. This problem has plagued many companies producers and researchers. So it is significant to investigate the acid-related genes of S. thermophilus.According to the pH changes and acid production rate when S.thermophilus was fermented in12%skim milk, four acid-related point was firstly determined:point1, fermentation for20-45min; point2, fermentation for110-130min; point3, fermentation to pH4.6; point4, fermentation for24h. In this paper, transcriptomics method was used to analyze the transcripsome of the four points. Point1was used as a reference, and the other point were compared with point1to analysis acid-related genes of S. thermophilus. Among the1962expressed genes,115genes were found expressed differently in sample2, of which65genes were up-regulated and50genes were down-regulated;104genes were found differently expressed in sample3, of which33genes were up-regulated and81genes were down-regulated;125genes were found expressed differently in sample3, of which43genes were up-regulated and82genes were down-regulated. Among the genes, ptsG plays a role in glucose utilization of S. thermophilus, and it also may be involved in the regulation of certain metabolism. The protein encoded by atpF plays an important role in the acid tolerance. Two-component system, Nudix family hydrolase and XRE regulator family also play important regulatory roles in the acid tolerance. Some of differently expressed genes which related to acid production and aicd tolerance were further analyzed using RT-qPCR. Compared to sample1, genes Y1U_C1378, Y1U_C1466and Y1U_C1888of sample2exhibited1.22,1.51and2.22folds of up regulation, which were consistent with the results of transcriptome. In comparison with sample1, genes Y1U_C0179, Y1U_C0462and Y1UC1378of sample3were up-regulated3.01,6.19and1.48folds, which were also consistent with the results of transcriptome. To further confirm the role of genes in acid production and acid tolerance,2plasmid used for gene knockout were successfully constructed (pMD18-T:XRE:Cl and pMD18-T:gly:Cl), which were used to knockout the corresponding genes. This study provides a theoretical foundation for modification of the engineering strain at genomic level.