Genome Squencing Of An Efficient Flocculant Producing Strain And Study On Its Omics Relating To Extracellular Polysaccharide Synthesis

The microbial flocculants(MBF)are natural polymers produced by microorganisms and secreted into the extracellular.The predominant components of them are polysaccharide,glycoprotein,protein,and nucleic acid.Because of its efficient flocculation effect and non-toxic properties,microbial flocculant has become one of the popular microbial water treatment agents at home and abroad.Howeve,the high production costs and unstable flocculating effect has limit its use to more widely aeras.In this study,a variety of detection techniques were used to detect the biological and genomic characteristics of strain A9,as well as the differential expression of transcriptomes and proteomes in different carbon source culture conditions.The mechanism of the strain A9 producing flocculants was studied from the molecular level.The differentially expressed genes,proteins and regulation law of the strain A9 during the process of MBF producing were investigated from the perspective of transcriptome and proteomics.The regulatory methods wad provided for the industrial production of MBF.The results have a certain theoretical and practical significance to promote the development and utilization of microbial flocculant resources,and reveal the mechanism and regulation of MBF production of A9.In order to ascertain taxonomic position of strain A9,the polyphasic taxonomical identification was performed.The ferment products were separated and purified and then were analyzed by ultraviolet scanning spectrum(UV)and infrared spectrum(IR).Based on the results of the study,it is concluded that strain A9 represents a novel species of the genus Paenibacillus.The UV scanning and IR spectra data showed that bioflocculant produced by strain A9 was mainly composed of polysaccharides.The flocculation mechanism of flocculant produced by strain A9 was analyzed,and the adsorption and bridging was the main mechanism of its flocculation.The genome sequence of A9 was determined using Solexa paired-end sequencing technology.The gene sequence related to exopolysaccharide biosynthesis protein of strain A9 was determined.The draft genomecontains 92 contigs,which can be assembled into 67 scaffolds.There were a total of 4,932 genes was predicted according to Glimmer,of which 4,284(86.9%)similary with NCBI-NR database.A total of 1,689 proteins were assigned to Clusters of Orthologous Groups(COG)families.Functional annotation and metabolic pathway analysis based on KEGG database based on blastp predicted 165 metabolic pathways,including EMP,TCA,PPP.Based on the results of genomic sequencing of strain A9,it is predicted that the extracellular polysaccharide of the strain A9 contains glucose,galactose,glucuronic acid,N-acetylglucosamine and mannose.The extracellular polysaccharide synthesis pathway of strain A9 with glucose as carbon source was also predicted.Related enzymes involved in the synthesis of extracellular polysaccharides include glucose phosphate mutase,UDP-glucose pyrophosphorylase,UDP-glucose dehydrogenase and so on.The differences of transcriptional expression of strain A9 under the two culture conditions were analyzed.The transcriptome of A9 were sequenced by RNA-Seq technology.RNA-Seq datas showed that,there are 70 genes significant differentially expressed under ordinary broth medium and dextrose medium culture conditons.The results of transcriptome analysis showed that:compared to ordinary broth medium,many genes related to carbohydrate synthesis and metabolic were up-regulated in the glucose medium.To study the differential proteome expression of strain A9 cultured in ordinary broth medium,dextrose medium and mannose medium.The proteins were compared and analyzed by two-dimensional gel electrophoresis(2-DE)and identified by mass spectrometry.The results showed significant differences of proteome expression among test groups cultured with different carbon sources.There were 54 differential proteins identified by mass spectrometry,including dihydrolipoyl dehydrogenase,triosephosphate isomerase,fructose-1,6-bisphosphatase,etc,which mainly related with synthesis and metabolism of carbohydrates.Through a number of shake flask tests,the related regulation laws in production of bioflocculant were researched and the relationship between fermentation conditions,functions of differentially expressed genes and proteins,and changes in producing bioflocculant were established.Three dimentional regulating strategies,such as rejuvenation of bioflocculant producing bacteria,substrate and fermentation parameters,were brought up to regulate bioflocculant producing bacteria to produce bioflocculant in industry.