Genomic Analysis And Exopolysaccharide Synthesis Of Lactobacillus Delbrueckii Subsp.bulgaricus LDB-C1

Lactobacillus delbrueckii subsp.bulgaricus is one of the most economically valuable homo-fermenting lactic acid bacteria strains.It has good fermentation performances and probiotic functions,and is widely used in the fermentation production industry of dairy products.Exopolysaccharide is an important functional metabolite synthesized by lactic acid bacteria in the fermentation process,and has multiple probiotic effects such as anti-oxidation,anti-tumor,and anti-virus.Therefore,the excavation of the L.bulgaricus strains is very important for the application of the species.In this study,L.bulgaricus LDB-C1 was isolated and screened in the lab,and the complete genome have been determined using Illumina Hiseq combined with Pacbio.This study conducted a comprehensive and in-depth analysis of five aspects of the strain,including the CRISPR-Cas system,antibiotic resistance genes,potential virulence factors,movable elements and bacteriocin biosynthesis genes.The genomic sequence differences between this strain and the other 11 strains of L.bulgaricus that have been published in the NCBI database were compared.The results showed that the type of CRISPR-Cas system in LDB-C1 is CRISPR 2,and the number of spacers in the CRISPR-Cas system of this strain is significantly higher than that of other strains,so it is speculated that this strain has good resistance to phage and plasmid infection.At the same time,the whole genome sequence comparison analysis revealed that the LDB-C1 genome does not contain prophage-related genes,that is,it does not have the threat of becoming a potential phage host.The resistance gene of LDB-C1 does not exist on the movable element,and the movable element is highly homologous to the gene derived from L.bulgaricus.The genes related to potential virulence factors in LDB-C1 are related to basal metabolism and do not directly encode toxic substances.In summary,LDB-C1 has good stability.The eps gene cluster is responsible for the biosynthesis of EPS.In order to clarify the distribution of eps gene cluster in this species,this study analyzed the published sequences of eps gene cluster in L.bulgaricus.It was found that there are usually two eps gene clusters in this species,namely eps1 and eps2.The upstream gene at the 5'end of the former cluster is hflx,which is composed of regulatory genes,chain length control genes,glycosyltransferase-related genes,and EPS output regulatory genes;the latter has a more conservative gene sequence,similar to eps1,but lacks chain length control genes.LDB-C1 also contains two eps gene clusters.Bioinformatics shows that this strain has the ability to synthesize EPS.Carbohydrates are precursors of EPS synthesis,which have a greater impact on EPS biosynthesis and the structure of EPS.In this study,the ability of LDB-C1 to utilize 19 carbon sources was evaluated,and fructose,galactose,glucose,oligofructose,and inulin were selected as the fermentation carbon source for subsequent EPS synthesis.The results showed that under all the tested conditions,the strain EPS produced the highest yield in the skim milk medium supplemented with2% inulin,which could reach 1.71±0.56 g/L.In order to further clarify the effect of inulin on EPS synthesis,follow-up experiments used inulin as an external carbon source to extract a large amount of EPS.The composition and antioxidant properties of EPS monosaccharides were analyzed.The results showed that the EPS produced by the strain in the 2% inulin within skim milk medium had higher antioxidant capacity than the skim milk control group.And after adding inulin,the monosaccharide composition of EPS also changed significantly.In this study,primers were designed based on the EPS synthesis-related gene clusters(eps1 and eps2),the sugar metabolism regulatory gene ccp A,and the two-component system gene hpk/rr in the LDB-C1 genome,and the RT-q PCR method was used for the analysis of gene expressions of this strain under different fermentation conditions.According to the differences in the composition of EPS monosaccharides under different medium conditions,it is speculated that the eps gene cluster of LDB-C1 is mainly responsible for regulating the transport of galactosyl and glucose groups in EPS and regulating the ratio of the two.It is speculated that eps1 plays a leading role in the synthesis of EPS in the two eps gene clusters of LDB-C1,and the function of the gene eps1 N,which is responsible for regulating the output of EPS,is replaced by eps1 K.The two-component system can also indirectly affect the level of EPS synthesis by regulating the activity of strains.