| Brown algae are important primary producers.A large number of bacteria live on the surface of brown algae.Epiphytic bacteria can produce specific enzymes to degrade brown algae.Alginate is abundant in the cell walls of brown algae,which can reach 30-60% of the dry weight.Therefore,brown algae are important sources for isolation of alginate-degrading strains.So far,many alginate-degrading strains have been isolated from brown algae,which can metabolize alginate as their carbon source.At present,two bacterial alginate metabolic pathways have been reported: one in Flavobacterium of Bacteroidetes and the other in Sphingomonas of Proteobacteria.Till now,although many culturable epiphytes have been isolated from brown algae,but there are still many epiphytes of brown algae,including alginate-degrading bacteria,that have not been studied.Moreover,the alginate metabolic pathways of many alginatedegrading strains remain unclear.In addition,it is expected to find new alginate-degrading enzymes by screening alginate-degrading bacteria from brown algae,which will lay a foundation for the development of tool enzymes for the preparation of alginate oligosaccharides.Sargassum is a large brown alga distributed in the temperate,tropics and subtropics,containing more than 500 species of 24 genera.Sargassum has a high content of alginate,accounting for more than 50% of its dry weight.Therefore,Sargassum is a good material for isolating alginate-degrading bacteria.However,there are only a few studies on Sargassum epiphytes and alginate-degrading bacteria.The study on the epiphytic alginate-degrading bacteria associated with Sargassum contributes to the better understanding of the interaction between Sargassum and epiphytic bacteria and the development and utilization of the alginatedegrading bacteria and their enzymes.In this dissertation,the Sargassum samples collected from the coastal waters of Shandong Province were selected as the research object,the culturable epiphytic bacteria and alginatedegrading strains were screened,the diversity of strains was analyzed,and the ability of alginatedegrading strains to produce extracellular alginate lyases was detected.Then,the alginate metabolic pathways of strains Vibrio sp.C42 and Pseudoalteromonas sp.M9 with high efficiency in alginate degradation were further analyzed basing on the genome,transcriptome,proteome and secretome analyses.The enzymatic properties of the alginate lyase AlyC8 with dual catalytic domains of strain V.sp.C42 were studied,and the application potential of a novel alginate lyase AlyM2 of strain P.sp.M9 was evaluated.(1)Isolation and diversity analysis of alginate lyase-extracting bacteria from Sargassum natans.Culturable epiphytic bacteria and alginate-degrading strains were isolated from Sargassum samples collected from coastal waters of Shandong Province by dilution plate method,and their diversity was then analyzed.A total of 37 strains were isolated.Based on 16 S r RNA gene sequence analysis,these epiphytes belonged to 21 genera of 3 phyla,with Vibrio(6/37)and Pseudoalteromonas(5/37)as the dominant genera.Fifteen genera were reported as Sargassum epiphytic bacteria for the first time.Eight strains were identified to serete alginate lyases,which distributed in 3 genera Alteromonas(1/8),Pseudoalteromonas(4/8)and Vibrio(3/8).The results showed that V.sp.C42 and P.sp.M9 had the highest extracellular alginate lyase activity.These results provide a basis for better understanding of the epiphytic bacteria of Sargassum,and contribute to the further study of the interaction between Sargassum and epiphytic bacteria,as well as the development and utilization of epiphytic alginate-degrading bacteria and their enzymes.(2)Analysis of the alginate metabolic pathway in Vibrio sp.C42Although some genes and gene clusters related to Vibrio alginate metabolism have been reported,the alginate metabolic pathway of Vibrio is still not completely clear.In order to reveal the alginate metabolic pathway of the epiphytic alginate-degrading bacterium V.sp.C42,we sequenced the genome of strain V.sp.C42,and analyzed the alginate metabolism-related genes,including alginate lyase genes and genes located in the alginate utilization locus(AUL).Transcriptome and proteome analysis identified the genes that may play roles in alginate metabolism.Extracellular alginate lyases were further detected by secretome.Combined with the results of multiple omics analyses,the alginate metabolic pathway of strain V.sp.C42 was predicted.Strain V.sp.C42 degraded alginate into oligosaccharides and monosaccharides by using 9 extracellular exotype or endotype alginate lyases.The products enter the periplasmic space through the porin protein on the outer membrane,enter the cell by the sodium solute transporter and TTT transporter on the inner membrane,and are further degraded into monosaccharides by an exotype alginate lyase in the cell.Monosaccharides are metabolized gradually by a series of enzymes(Deh R,Kdg F,Kdg K and Eda),and eventually enter the glycolysis pathway.Gnt R protein regulates the metabolism of alginate of strain V.sp.C42.The key genes of alginate metabolism in strain V.sp.C42 were basically consistent with those reported in the other Vibrio strains,but the difference was that a TTT transporter on the inner membrane were found in an AUL for the first time,which may be involved in the transport of alginate degradation products.In addition,the alginate metabolic pathway of Vibrio is similar to that of Bacteroidetes,but their transport systems are markedly different.The study on the alginate degradation pathway of Vibrio is beneficial to broaden the understanding of the relationship between Vibrio and Sargassum and lay a foundation for better utilization of Vibrio resources.(3)Analysis of the alginate metabolic pathway in Pseudoalteromonas sp.M9.Pseudoalteromonas is an important alginate-degrading group,but its alginate-degrading pathway is not clear.In order to reveal the alginate metabolic pathway of the epiphytic alginatedegrading bacterium P.sp.M9,we sequenced the genome of strain P.sp.M9,and analyzed the alginate metabolism-related genes,including alginate lyase genes and genes located in the AUL.Transcriptome and proteome analysis identified the genes that may play roles in alginate metabolism.Extracellular alginate lyases were further detected by secretome.Combined with the results of multiple omics analyses,the alginate metabolic pathway of strain P.sp.M9 was predicted.All the five alginate lyases of strain P.sp.M9 were secreted into the extracellular space and completely degraded alginate into monosaccharides.Monosaccharides are eventually transported into the cell by the Sus C transporter on the outer membrane and MFS protein on the inner membrane,and then gradually metabolized by Deh R,Kdg F,Kdg K and Eda,and finally enter glycolysis pathway.For the first time,we predicted the alginate metabolic pathway of Pseudoalteromonas,which is similar to those of Bacteroides and Vibrio,but significantly differential from those in the outer membrane transporters.In addition,no obvious regulation genes of alginate metabolism were found in strain P.sp.M9,suggesting a new regulatory mechanism.In addition,no obvious regulation genes of alginate metabolism were found in strain P.sp.M9,suggesting a potential new regulatory mechanism.The results are helpful to broaden the understanding of Pseudoalteromonas,better understand the relationship between Pseudoalteromonas and Sargassum,and lay a foundation for better utilization of Pseudoalteromonas enzyme resources(4)Heterologous expression and characterization of PL7 family alginate lyase AlyC8 with dual catalytic domains.Most of the reported alginate lyases contain only one alginate catalytic domain.At present,only AlyA and Algb have been reported to have two alginate catalytic domains,but the properties of each catalytic domain have not been analyzed and compared.Our analysis of alginate lyases from strain V.sp.C42 showed that the alginate lyase AlyC8 may contain two PL7 catalytic domains,AlyC8-CD1 and AlyC8-CD2.AlyC8,AlyC8-CD1 and AlyC8-CD2 were then heterologously expressed and purified,and their enzymatic properties were further studied.The optimum temperature of AlyC8 is 30℃ and the optimum p H is 9.0.The optimum temperature and p H of AlyC8-CD1 are 30℃ and 8.0,respectively.The optimal temperature and p H of AlyC8-CD2 are 40℃ and 8.0,respectively,indicating that they can adapt to marine environment,and can play high degradation activities in the similar environment.The analyses on the activity,degradation products and kinetic parameters of AlyC8,Alyc8-CD1 and Alyc8-CD2 showed that Alyc8-CD1 plays a major role in the holoenzyme.However,by analyzing the substrate specificities of the enzymes,we found that AlyC8-CD1 had the highest degradation activity for PM,but almost no degradation activity for PG.In contrast,AlyC8-CD2 had the highest activity for PG and the lowest activity for PM.The complementation of AlyC8-CD1 and AlyC8-CD2 in substrate selectivity resulted in the significant degradation activity of AlyC8 holoenzyme to all the four alginate substrates,which reflected the synergism of AlyC8-CD1 and AlyC8-CD2.Compared with the reports of AlyA and Algb,we found for the first time an alginate lyase with dual catalytic domains having alginate-degrading activity,and revealed the synergistic degradation of alginate by the two domains.(5)Heterologous expression,properties and application potential analysis of the PL6 alginate lyase AlyM2.Alginate oligosaccharides(AOs)have a variety of biological activities and have important applications as prebiotics,nutritional supplements and plant growth accelerator.Alginate lyase has unique advantages in the preparation of AOs.However,to date,only a few alginate lyases have been reported to have the potential to produce AOs with the specific polymerization degree(DPs).Five alginate lyases,AlyM1-M5,were found in strain P.sp.M9.We successfully purified AlyM1,AlyM2,AlyM3 and AlyM5,and then analyzed their degradation products towards sodium alginate.AlyM2 mainly produced trisaccharides,accounting for 79.9% of all products.The further sequence alignment and structure prediction of AlyM2 showed that AlyM2 is a novel PL6 alginate lyase,which may have a new catalytic mechanism.Studies on the enzymatic properties of AlyM2 showed that the optimum temperature was 30℃,the optimum p H was 8.0and the optimum Na Cl concentration was 0.5 M.AlyM2 has degradation activity to four alginate substrates(PM,PG,PMG and sodium alginate),and has the highest degradation activity to PMG.All the predominant degradation products of the four substrates were trisaccharides,which accounted for about 80%.When sodium alginate was used as the substrate,the yield of trisaccharides was 588.4 mg/g.These results indicate its promising potential in preparing different trisaccharides.This paper revealed the diversity of epiphytes and epiphytic alginate-degrading bacteria of Sargassum,and improved the understanding of the alginate metabolic pathway of Vibrio by analyzing the alginate metabolic pathway of V.sp.C42.The alginate metabolic pathway of P.sp.M9 was analyzed for the first time.At the same time,the synergistic effect of the two catalytic domains of the PL7 alginate lyase AlyC8 of strain V.sp.C42 was elucidated,and the potential of AlyM2,a PL6 alginate lyase of strain P.sp.M9,in the preparation of alginate trisaccharides was also evaluated.The results are helpful to better understand the interaction between brown algae and its epiphytes,and lay a foundation for the exploitation of strains and enzymes related to the degradation and utilization of alginate. |
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