| As a new environmental-friendly water treatment agent,the development and application of microbial flocculants have been paid more and more attention by scientists all over the world,which have been the research focus of environmental biotechnology for many years.However,the biosynthetic pathways,metabolic pathways and related genes of microbial flocculants are not clear,so the root causes of instability and low yield of wild strains can not be determined,and the problems of microbial flocculants can not be solved fundamentally.Based on the analysis of genome/transcriptome,this paper was carried out the research on the analysis of biosynthetic pathway and quorum sensing system regulatory mechanism polysaccharide-type microbial flocculants,which provided theoretical support to solve the problem of microbial flocculants.This research was carried out according to the whole genome sequence of A.tumefaciens F2,using bioinformatic and comparative genomic methods to analyze function genes related to the main metabolic pathways and regulatory mechanisms.Meanwhile,the main active component of microbial flocculants produced by F2 was polysaccharide,therefore genes of polysaccharide-type microbial flocculant biosynthesis were located on polysaccharide biosynthesis related genes.84 polysaccharide-type microbial flocculant biosynthesis related genes were found in genome sequence of F2,and succinoglycan biosynthetic exo genes were speculated to be the most possible gene that directed polysaccharide-type microbial flocculant biosynthesis.In addition,the gene cluster of strain F2 was found to be highly similar to the tra R/tra I quorum sensing system in Rhizobium and Agrobacterium.By bioinformatic and comparative genomic analysis,the biosynthetic genes and regulatory genes of polysaccharide-type microbial flocculants were analyzed,which provided enormous genome information for transcriptome sequence analysis.In order to accomplish identification and function analysis of the key genes related to polysaccharide-type microbial flocculant biosynthesis,high-throughput transcriptome and gene expression differences were analyzed,in accordance with the differential phenotypes of polysaccharide-type microbial flocculants producing bacteria when cultured with different nutrient components(FX24/FP24)and different fermentation time(FX9/FX24).65 differential genes related to polysaccharide-type microbial flocculant biosynthesis were identified out of 84 putative genes.Polysaccharide-type microbial flocculants exo gene cluster was accomplished by aligning the monosaccharides components of polysaccharides produced by A.tumefaciens C58 and F2,and the analysis of biosynthetic pathway and the prediction of regulatory genes were also accomplished.The types of biosynthetic genes that cause the difference of yield and potency of polysaccharide-type microbial flocculants were revealed,which provided the theoretical basis for the strategy on the biology-oriented synthesis of polysaccharide-type microbial flocculants.The result of genomics/transcriptomics analysis showed that there were quorum sensing genes in F2.UPLC-MS/MS was used to detect the existence of AHLs in the fermentation broth.C6-HSL and 3-oxo-C8-HSL were added and their effects on polysaccharide-type microbial flocculant biosynthesis were studied.The result showed that C6-HSL and 3-oxo-C8-HSL could enhance the polysaccharide concentration and flocculation rate of microbial flocculants.The optimum quantity of C6-HSL and 3-oxo-C8-HSL addition were 0.4 ?M and 0.2 ?M separately,which increased the polysaccharide concentration by 1.6 times and 1.4 times respectively.Influences of temperature and initial p H on polysaccharide-type microbial flocculant biosynthesis were also studied,and the optimum conditions were calculated through response surface optimization,which significantly raised the polysaccharide concentration and flocculation rate of microbial flocculants,providing new methods and thoughts for enhancement of industrial microbial flocculant produce.To further explore the regulation of quorum sensing system on polysaccharide-type microbial flocculant biosynthesis,the transcriptional-level differences of F2 between quorum sensing system genes and polysaccharide-type microbial flocculant biosynthesis related genes were analyzed through transcriptome sequencing.Trar R-Tra I quorum sensing system was confirmed to exist in the genome of F2,whose regulatory mechanism was also detected: when the concentration of C6-HSL reached threshold,it acted as a self inducer and was able to combine with transcription-regulation protein and induce the expression of target genes of Agau_C100492,Agau_P200324 and Agau_L100081,regulating polysaccharide-type microbial flocculant biosynthesis by controlling the transfer of glucosyl and polysaccharide.In order to understand the regulatory mechanism of microbial flocculant biosynthesis,quorum sensing system regulatory mechanism was confirmed,which provide a theoretical basis for improving the expression of regulatory genes to achieve the directional regulation of polysaccharide-type microbial flocculants. |
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