| Lactobacillus kefiranofaciens widely exists in fermented dairy products and has important application value.In 2020,this strain has been added as a new resource that can be added to food in China.However,the research on the genomics of this strain is scarce.Analyzing its genomic characteristics and evolution process is of great significance to the development and utilization of the strain.In this study,the whole genes of 73 strains of Lactobacillus kefiranofaciens isolated from koumiss and sour milk were sequenced by Illumina novaseq high-throughput sequencing platform.Combined with the whole gene sequences of only 9 strains in the NCBI database,comparative genomics analysis was carried out to analyze their genome characteristics and analyze the differences between strains from different sources,to provide data support for the screening of excellent strains of Lactobacillus kefiranofaciens.The main results are as follows:(1)A total of 284 strains of lactic acid bacteria were isolated from 18 mare’s milk and koumiss.Lactobacillus helveticus and Lactobacillus kefiranofaciens were the dominant strains koumiss,accounting for 64.8% and 21.9% respectively.Lactobacillus helveticus,Lactococcus lactis,and Leuconostoc mesenteroides are relatively high in mare’s milk,accounting for 29.8%,22.8% and 31.6% respectively.There are some differences in flora composition between mare’s milk and koumiss.(2)The whole-genome sequences of 73 strains of Lactobacillus kefiranofaciens were obtained.The taxonomic status of all strains was determined based on the housekeeping genes(rpo A and phe S)and ANI value,and the taxonomic status and evolutionary relationship of all strains were determined in combination with the phylogenetic tree constructed by the core gene.The ANI value shows that the strain has high homology.The sensitivity and resolution based on the 16 S r RNA gene sequence and housekeeping gene sequence analysis are low.The resolution is improved based on core gene analysis,but most strains fail to determine the attribution of subspecies.It is speculated that there may be problems in the previous subspecies taxonomic status of the strain.(3)Lactobacillus kefiranofaciens is an open genome with 1045 core genes and 7189 pan genes.The diversity of this strain is high.The average genome size is 2.05±0.10 Mb,the average GC content is 37.49±0.16%,and the average number of predicted genes is2153±105.The prediction of functional genes shows that genes related to carbohydrate metabolism,life activities,and information storage account for a large proportion.(4)The genetic evolution of Lactobacillus kefiranofaciens has a segregation source correlation.There were significant differences between kefir grains isolates and sour milk and koumiss isolates,but there was no significant difference between sour milk and koumiss isolates.The main differences are reflected in the number of predicted genes,functional annotation of core genes and specific genes,utilization of carbohydrate-active enzymes,and bacteriocin prediction.The prediction of strain genome function showed that the number of genes of kefir grains isolate was significantly higher than that of other isolates in amino acid metabolism,defense mechanism,cell replication,recombination,and repair.There were differences in the functional distribution of core genes of different isolates.The differences of specific genes of different isolates were reflected in amino acid metabolism,specific transport of various monosaccharides,and CRISPR-Cas defense mechanism.There were significant differences in the number of carbohydrate-active enzymes GH42,GH31,GH36,and AA4.At the same time,kefir grains isolate showed better ability to use plant substrate for fermentation.Kefir grains isolates contained fewer kinds of bacteriocin than sour milk and koumiss isolates.Helveticin J and Lanthipeptide class I were not predicted in kefir grains isolates. |
