Analysis Of Exopolysaccharide Biosynthesis Pathway Of Lactobacillus Helveticus LZ-R-5 And Study On The Function Of Key Gene YveK

Tibetan Kefir,also known as"Tibetan Snow Lotus",is a natural yogurt from Nyingchi,Tibet.It is rich in 16 kinds of essential amino acid and trace elements.Through long-term consumption,it was proved that Tibetan Kefir had the magical effect of enhancing body resistance,stabilizing blood pressure,delaying aging,eliminating fatigue and alleviating gastrointestinal ulcer.Lactobacillus helveticus LZ-R-5,isolated from Tibetan Kefir by our research group,is good at fermenting milk and endowing milk with excellent texture.It can produce extropolysaccharide（EPS）,which has immune-activating activity and can be used as a potential immune-regulating agent.Previous studies showed that EPS isolated from L.helveticus LZ-R-5 contained capsular exopolysaccharide（c-EPS）and slime exopolysaccharide（s-EPS）.These two types of EPS were similar in structure and molecular weight,both 5.41×105 Da.The linear repeating unit of EPS was composed of heterozygous monosaccharide,and its structure was characterized as[→6)-β-D-Galp-（1→3）-β-D-Glcp-（1→3）-β-D-Glcp-（1→3）-β-D-Glcp-（1→3）-β-D-Glcp-(1→]_n.In this paper,the biosynthesis pathway of EPS isolated from L.helveticus LZ-R-5 was analyzed by whole genome sequencing,bioinformatics analysis and transcriptional analysis.Then,the key genes in EPS biosynthesis gene cluster were knocked out in order to systematically investigate the relationship between gene,EPS biosynthesis and surface characteristics.Firstly,the characteristics and whole genome sequence of L.helveticus LZ-R-5 were analyzed by growth curve determination,medium optimization,self-coagulation ability determination and surface hydrophobicity determination.The results showed that MRS-GL was the best medium for growth.The morphology and surface characteristics of L.helveticus LZ-R-5 were affected by the logarithmic growth phase.During this period,the length-width ratio of L.helveticus LZ-R-5 increased,the self-agglutinative ability increased by 50%,and the surface hydrophobicity decreased by 35%.Pacific three-generation sequencing was used to obtain the whole genome sequence information of L.helveticus LZ-R-5.The genome of the bacterium consists of a single chromosome without natural plasmids,and the chromosome genome is circular with a size of 2.08 M,a GC content of36.92%,a gene number of 1706,a proportion of coding regions of 70.95%,an average gene length of 866.83 bp,and contains 12 r RNA,64 t RNA,and 3 CRISPR clusters.Software Glimmer was used to predict genomic coding genes,and annotation information of predictive genes was obtained from NCBI databases such as NR,Swiss Prot,COG,KEGG and GO.A genomic circle map was drawn by software Circos,and phylogenetic trees were drawn based on 16S r DNA gene sequence,core gene sequence and amino acid sequence respectively.Based on sequence alignment and gene function analysis,an EPS biosynthesis gene cluster was located in the chromosome of L.helveticus LZ-R-5.The gene cluster contained17 genes related to EPS biosynthesis,5 genes related to cytoplasmic transport of sugar,sugar-phosphate synthesis,5 genes related to the generation of repeating units,6 genes related to aggregation and EPS export.According to the function of the genes involved,the EPS biosynthesis gene cluster of L.helveticus LZ-R-5 was divided into transcriptional regulatory regions（eps1）,chain length determining regionⅠ（eps2-eps4）,repeating unit synthetic regionⅠ（eps5-eps8）,chain length determining regionⅡ（eps9-eps10）,polymerizing and exporting regionⅠ（eps11-eps12）,repeating unit synthetic regionⅡ（eps13）,polymerizing and exporting regionⅡ（eps14-eps17）.The promoter information of the EPS biosynthesis gene cluster was predicted,and the co-transcriptional information of the EPS biosynthesis gene cluster of L.helveticus LZ-R-5 was obtained by reverse transcription PCR.The gene cluster contained eps2-eps4 and eps5-eps8 in total,and the correctly predicted promoters were P₉₂₀,P₈₉₅,P₈₇₅,P₈₆₅,P₈₆₀ and P₈₄₀;RT-PCR was used to obtain the changes of transcription level of key genes in the gene cluster during the biosynthesis of EPS isolated from L.helveticus LZ-R-5.The results showed that the gene transcription level changed with the growth stage,and the variation trend of gene transcription level was related to the transcription unit and the isozyme-coding genes.Finally,the biosynthesis pathway of EPS derived from heteropolysaccharide In vitro experiments on lactobacilli proliferation showed that L.helveticus LZ-R-5 EPS could be rapidly utilized by lactobacilli to achieve the health benefits of rapid enrichment of probiotics.In order to further understand the biosynthesis pathway of L.helveticus LZ-R-5 EPS,gene editing method was used to understand the role of core genes in the biosynthesis gene cluster of L.helveticus LZ-R-5 EPS and its effect on the surface activity of the strain.The protocol of electro conversion of L.helveticus LZ-R-5 was established:mid prophase preparation of competent state in logarithmic growth,the addition of 1μg plasmid,electric shock at pulse intensity of 2.0 k V for 5 ms,incubation for 3 h.Bioinformatic analysis of the native Lactobacillus plasmid p LHs predicted an ORI region corresponding to the rep A gene.Five shuttle vectors,p ELH1,p ELH2,p ELH3,p ELH4 and p ELH5,were constructed by one-step cloning to produce a Cre protein expression plasmid based on fragment LH3.We constructed a knockout vector targeting the gene Yve K encoding EPS phosphotyrosine protein phosphatase in L.helveticus LZ-R-5,replaced the Yve K fragment in genomic DNA by homologous recombination,and used Cre/lox to remove the resistance marker gene.Compared with the wild-type strain,theΔYve K mutant form of L.helveticus LZ-R-5showed a 42.5%decrease in EPS,a thinner of the cell wall surface envelope by 74.1-80.6%,increase of hydrophobicity,a decrease in the ability of agglutinate and biofilm formation.This suggested that the Yve K gene affected strain surface properties by regulating EPS production.In summary,this paper integrated bioinformatics and transcriptional analysis to investigate the EPS biosynthesis gene cluster and EPS biosynthesis process in L.helveticus,which compensated for the anhedonia of using gram-negative bacteria such as Streptococcus pneumonia as a model for the study of EPS biosynthesis metabolism.The gene editing system of L.helveticus LZ-R-5 was established based on the principle of homologous recombination,and the knockout vector T-ery-Yve K was successfully constructed,and this vector was used to obtain theΔYve K type mutant strain of L.helveticus LZ-R-5,to explore the effect of Yve K on the phenotype of L.helveticus.In this study,the biosynthesis pathway of L.helveticus LZ-R-5 EPS was analyzed to provide an important theoretical basis for the synthesis of extracellular polysaccharides in vitro and in vivo by using synthetic biology bacteria.The transformation and gene editing scheme of L.helveticus LZ-R-5 established in this paper provided a powerful tool for the study of the mechanism of L.helveticus LZ-R-5 biological activity.