| Probiotics have a variety of physiological functions and are widely used in food,medicine,feed industry and other industries,which has important research value and application prospects.Probiotics with excellent characteristics are the premise and basis of industrial application.However,due to the scarcity of excellent strains,it is urgent to breed them by various methods.As a new frontier breeding technology,space mutagenesis has attracted wide attention in recent years.The superposition of mutagenesis factors such as intense radiation,microgravity and super vacuum has improved the efficiency of mutagenesis.However,there are still limited studies on space mutagenesis microorganisms,especially in probiotics.In this study,Lacticaseibacillus rhamnosus Probio-M9(ProbioM9),which has a variety of probiotic properties,was taken as the research object.The strain was carried into space(apogue 7970 km)by Long March-5B carrier rocket.The effects of space environment on physiological and biochemical characteristics of ProbioM9 and ground control strains were studied,and the genetic characteristics differences of the mutant isolates were revealed in combination with the whole genome information,and the changes of the mutant isolates were further elaborated from the differential expression of genes and proteins,so as to explore the internal mechanism of mutant isolates.The main results of this study are as follows:(1)After space mutagenesis,100 space mutant isolates were isolated from the return samples,and evaluated the 10 representative strains mutation phenotype characteristics of isolates,including morphological characteristics,growth activity,tolerance,carbohydrate utilization and capsular polysaccharide(Capsular polysaccharide,CPS)measurement.The results showed that the space environment could induce the changes of physiological and biochemical characteristics of some isolates.Ten space mutant isolates showed two phenotypic characteristics.Compared with the control strains(Probio-M9,SG-R7970-16),the colony diameter of six mutant isolates(HG-R7970-3,HG-R7970-16,HG-R7970-20,HG-R7970-25,HG-R7970-41,HA-R7970-36)with different phenotypic characteristics were increased significantly(P < 0.05);and the number of viable bacteria was higher and CPS(23.36-33.78 g/L)could be produced.However,the survival rate in the artificial gastric fluid was significantly decreased(68.68-87.73%).The other four mutant isolates(HG-R7970-28,HG-R7970-34,HG-R7970-46,HA-R7970-19)had the same phenotypic characteristics as the control strains,but there was no significant difference.(2)Based on the results of genome sequencing,six high-frequency SNP mutation regions were found in 100 mutant isolates.The mutant isolates with significant morphological changes had a same point mutation in ywq D gene,while no such mutation site was found in the control strains.Further comparative analysis of the complete genome information of the 10 mutant isolates also confirmed that the 6 mutant isolates with significant phenotype changes all had mutations in the ywq D gene,and the isolates with the same mutation site of the ywq D gene had similar CPS yield(The CPS yields of HGR7970-16 and HG-R7970-20 were 24.17 g/L and 23.36 g/L,respectively;the CPS yields of HG-R7970-3 and HG-R7970-41 were 33.66 g/L and 33.78 g/L,respectively).Phenotypic results showed that CPS production was related to point mutation of ywq D gene.(3)From the perspective of transcriptome and proteome,the mutation mechanism of mutant isolates was explored and it was found that the expression levels of m RNA and protein of ground control strains and Probio-M9 were similar,with no significant changes.Compared with the control strains,more differential genes/proteins were identified in the 6phenotypic mutant strains.Most of the differential genes/proteins were concentrated in the pathways related to carbohydrate metabolism,membrane transport,membrane components and secondary metabolites synthesis.The phenotypic changes produced by the mutagenic isolates were more related to carbohydrate metabolism.In particular,the production of CPS directly affected the changes in carbohydrate synthesis and metabolism of the isolates.In addition,there were still some differences in the enrichment pathway of differential gene/protein among different mutant isolates,which was thought to be related to the metabolic pattern of mutant isolates,but not significantly related to the phenotypic changes of mutant isolates.(4)From the perspective of application,the metabolic activities of the two mutant isolates with the highest CPS yield(HG-R7970-3,HG-R7970-41)against 190 carbon sources were determined,so as to further clarify the differences in carbohydrate utilization of the mutant isolates.The results showed that the metabolic activity of the two mutant isolates against six carbon sources(D-trehalose,D-glucose,D-mannose-d-sorbitol,Dsalicylate and D-Glc NAc)was significantly lower than that of the control isolates(P <0.05),which was mainly caused by Pts-specific membrane embedding penetrase(EII)encoded in the glycolysis pathway.Gene regulation of carbohydrate kinase and two ratelimiting enzymes(phosphofructokinase(PFK)and pyruvate kinase(PYK)),and changes in carbon source metabolic activity were associated with CPS production in isolates.(5)Multiple omics results showed that the changes in the mutants were closely related to their CPS production.Combined with the whole genome information,a CPS gene cluster consisting of 17 genes was identified,and the ywq D gene,as a regulatory gene,was located at both ends of the gene cluster.Mutations in the ywq D gene of mutant isolates lead to structural changes in the tyrosine kinase encoded by the mutant isolates,which directly regulate the expression of other genes in the CPS gene cluster,thus activating the production of CPS in mutagenic isolates and binding to the cell surface through the capsule.In this study,the influence of space environment on Probio-M9 was investigated,with the purpose of evaluating the influence of space environment on physiological and biochemical characteristics of probiotic strains,and revealing the molecular biological mechanism of phenotypic differences,so as to obtain new microbial resources with more application potential.The results showed that the space environment could induce CPS production of Probio-M9,and the production of CPS directly affected the carbohydrate metabolic activity of the mutagenic isolates,while the mutation of ywq D gene directly regulated the phenotypic changes of the mutagenic isolates and was the main mechanism of mutation of the mutagenic isolates In this thesis,the phenotype of space mutant isolates,especially the induction of CPS production,was characterized by combining biochemistry and multiomics techniques,which further revealed the mutation mechanism of space mutagenesis isolates,and provided data support and theoretical basis for the development and utilization of lactic acid bacteria(LAB). |
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