Genome And Functional Genomic Analysis Of Streptococcus Thermophilus KLDS SM Applied In Dairy Production

Streptococcus thermophilus was “Generally Recognized as Safe,GRAS” strain,and has been widely used in the industrialized production of several important fermented dairy products.It is the second most important industrialized lactic acid bacteria species after Lactococcus lactis,and worth about $40 billion market value.Because of the limitation of molecular biology research methods,most of the traditional microbiological studies focus on,properties and formation mechanisms based on the physiological,biochemical tests and molecular biology experiments.Generally,only function or interaction of a few genes were studied in the genome.At the same time,it also requires a lot of manpower and resources.Microbial genome sequencing is becoming more common,with the gradual maturity of sequencing technology and lower cost.It is much more possible to design more effective and targeted protocols to study physiological metabolisms,such as the metabolism of substances and synthesis,gene function,the interaction of multiple genes and gene expression regulation,at the genetic level.Streptococcus thermophilus KLDS SM is a fast acid producing and high viscosity strain with independent intellectual property rights.In order to further analyze and evaluate the properties of strain KLDS SM when applied to dairy fermentation,and excavate the potential function of it at the genetic level,we sequenced the whole genome and drew the genome map in this study.The COG and KEGG databases were used to annotate the encoded proteins.By using bioinformatics software and methods,three aspects of strain KLDS SM were analyzed in detail,including environmental adaptability(sugar metabolism,proteolysis system,stress response,two component system,synergy,genomic islands and pseudogenes),biosynthesis(exopolysaccharides,amino acid and vitamin B)and defense system(CRISPR/Cas system,restriction modification system and bacteriocin).Comparative genome analysis was performed between the strain and fourteen Streptococcus thermophilus strains which were published on NCBI,focusing on the analysis of exopolysaccharide biosynthesis,amino acids biosynthesis,vitamin B biosynthesis and CRISPR/Cas system.At last,the outstanding functions in genetic analysis of the strain was verified by experiments.The EPS production was determined for single fermentation and combined fermentation with Lactobacillus delbrueckii subsp.bulgaricus.The following results were obtained after analysis:(1)Basic characteristics of strain KLDS SM.The genome of Streptococcus thermophilus KLDS SM consists of a 1,856,787 bp circular chromosome with G+C content of 39.08%.A total of 1,950 genes were predicted,including 1,732 protein-coding genes,129 pseudogenes,6 r RNA operons,67 t RNA genes and 4 nc RNA genes.Furthermore,1,400 and 1,046 protein-coding genes were annotated in COG database and KEGG database.(2)Analysis of environmental adaptability of strain KLDS SM.Strain KLDS SM was able to transport and utilize glucose,lactose,sucrose,mannose and beta-glucosidase and produce L-lactic acid through glycolysis pathway.However,it did not have a complete citrate pathway and pentose phosphate pathway.It had a complete proteolytic system.Some stress-related proteins were identified,which were mostly related to acid,oxygen and heat stress.There are 4 sets of complete and functional two component systems which involved in the processes of mediating the synthesis of bacteriocin and the formation of biofilm.Nine genome islands were predicted and most of them were associated with biosynthesis,metabolism,defense,transport and transcriptional regulation.Pseudogenes accounted for 6.9% protein coding genes,relating to carbohydrates transport and metabolism and amino acids biosynthesis and transport.(3)Analysis of the biosynthesis of strain KLDS SM,we obtained these results.The EPS gene cluster of KLDS SM had 99% nucleotide identity to that in Streptococcus thermophilus ASCC 1275 which was reported to give a high EPS yield.The yield of EPS was about 331 mg/m L in the experiments.This strain couls synthesize eight amino acids,including His,Trp,Ser,Cys,Gly,Val,Leu and Ala,among which Val,Leu and Trp are human essential amino acids.Comparative analysis showed the amino acid biosynthetic ability of 15 strains was relatively conservative.The strain could only synthesize folic acid in B vitamins.There was little difference in the ability of B vitamins biosynthesis of 15 strains.(4)Defense system analysis of strain KLDS SM,we obtained these results.There are four CRISPR/Cas systems in strain KLDS SM genome.The activity of CRISPR loci were predicted to be CRISPR4>CRISPR1>CRISPR3>CRISPR2 in the order from strong to weak,and the CRISPR/Cas system of site 2 was degraded.The R-M systems were not abundant and only 2 complete I type R-M systems existed in strain KLDS SM genome.Two bacteriocin biosynthesis gene clusters were predicted,however,the lantibiotic biosynthesis gene cluster was degraded.(5)Comparative genomic analysis of 15 Streptococcus thermophilus strains,we obtained these results.It was highly conservative collinearity among 15 genomes.Pan-genome was an open type.Pan-genome and core genome was consisted of 4,094 and 1,240 genes of 15 strains.It was showed that strain KLDS SM had the closest phylogenetic relationship with strain MN-BM-A02,strain ASCC 1275 and strain ND07,and was less close to strain LMG 18311 and strain CNRZ1066.(6)EPS yield comparison of single and combined fermentation showed that combined fermentation produced more EPS than single fermentation.With fermentation for 4 to 8 h,the rate of producing EPS for KLDS SM and KLDS 08007 combination fermentation reached the maximum.The rate was about 18.9 mg/L·h,and ultimately most EPS was produced with 158 mg/L.The conclusions of this study are as follows:(1)Genomic analysis confirmed that strain KLDS SM had a high yield EPS gene cluster,and thus it would have the potential of high yield EPS,which provided a reference for screening high-yield EPS of Streptococcus thermophilus.(2)Genomic analysis confirmed strain KLDS SM had comprehensive and rich CRISPR/Cas systems,with the ability to effectively resist phage infection.(3)It was preliminarily verified that EPS got the highest yield,when KLDS SM and KLDS 08007 were combined-fermented for 24 hours.Combinated-fermentation rate of EPS reached maximum at the time of fermentation for 4 h and 8 h.The results of this study provided a theoretical basis for better understanding of the mechanism that combinated-fermentation of Streptococcus thermophilus and Lactobacillus delbrueckii subsp.bulgaricus yielded more EPS.