| The microorganisms are highly diverse and functional in mangrove sediments(MGSs).However,some problems still exist in the former studies on the microbial community structures in MGSs,such as the lack of systematicness and representativeness,insufficient sequencing depth,low pratical values of the functional genes isolated from MGSs.The current study aims to detailedly investigate the microbial communities,key driving factors,and the functional genes in MGSs.Agarase genes isolated from MGSs,serve as the reseach target,were expressed in E.coli cells and were furified,and then were modified to obtain novel agarase with valuable industrial potentials.Firstly,the community structures of five microorganisms,including bacteria,archaea,fungi,diazotrophic and denitrifying microorganisms,were investigated from multiple spatiotemperal scales.The results suggested that deph and location could significantly affect the microbial communities in MDSs,but the seasonality had no significant influence on all the investigated microbial communities(ANOSIM and Adonis,p<0.05),indicating that the distribution of the microbial communities in MDSs are regional and stratified,and are stable against time-varying.Besides,linear fitting was conducted to confirm the key drving factors of the community structures of the five microorganism,and the results showed that the particle size of sediments was the key driving factor of the communities of bacteria,archaea,fungi,and denitrifying microorganisms,and salinity was that of the community of diazotrophic microorganisms,suggesting that the particle size of sediments and salinity could indirectly involve in the ecological functions of mangroves by affecting the microbial communities.Secondly,the environmental DNA in MDSs was deeply sequenced,and appropriate 300 Gb of sequences was generated.Agarase genes were selected as the research target in this study,and the abundance,diversity,novelty of agarase genes in the MDSs datasets were analyzed,indicating that the agarase genes in MDSs were of great capacity.In addition,enzymatic properties of the selected agarase genes expressed in E.coli cells showed the agarase resources in MDSs as well harbored great activities for the industrial production in the future.The signal peptides in the potential agarases from MDSs were predicted and analyzed,and the results suggested that the proportion of the agarase harbored signal peptides was obviously low than the reported agarases in public database,indicating the ultilization of agarose in MDSs was probably collaborative.Six draft genomes were recovered and assembled from the MDSs datasets.Thereinto,Cluster 4 possessed the degrading abilities for multipile polysaccharides including agarose,and the degradation pathway of agarose in Cluster 4 was also speculated in this work.All the results in this part showed the great potential of MDS microbiome served as the agarase gene resourceLastly,agarase genes,agaMl and agaM2,isolated from MDS datasets were expressed and purified for the characterization of enzymatic properties of the corresponding proteins.We found that the stability of the recombinant agarase AgaM1(rAgaM1)was stronger than rAgaM2.Therefore,agaMl gene was selected for the further study.Then,180 bp nucleotides were removed from the 3' terminal of agaMl gene,and the truncated gene was re-expressed and purified to obtain the truncated recombinant AgaMl(trAgaMl).By using trAgaMl as a core enzyme tool,we developed a novel method to produce various neoagaro-oligosaccharides with degree polymerizations of 4-12.In addition,the production of trAgaMl was also optimized by using high-density fermentation,and the total agarase production was 842-folds increased.The degree polymerizations of neoagaro-oligosaccharides produced by the reported agarases were always<8 and diverse lowly.The method developed in this study improved those problems,and laid a foundation for the neoagaro-oligosaccharide production in the future. |
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