The Mechanism Of Alginate Degradation In Vibrio Alginolyticus ATCC 17749

Alginate is the main component in the cell wall of brown algae,and its degradation product--alginate oligosaccharides have important biological activities,such as immunomodulatory,anti-tumor,antibacterial,and anti-oxidation.In recent years,with the continuous progress of synthetic biology technology,the biorefining platform using alginate as raw material has made significant development,which has successfully realized the bioconversion of a series of high value-added products from alginate to 2,3-butanediol,lycopene and bioethanol.However,these bacteria constructed by synthetic biology technology are currently suffering from low efficiency of alginate degradation and low yield of products.Therefore,it is of significance to elucidate the mechanism of alginate degradation in order to promote the high-value utilization of alginate and the development of alginate-related biorefinery technology platform.In this thesis,Vibrio alginolyticus ATCC 17749 was used to investigate the action mechanism and regulation mechanism of alginate degradation process.Firstly,the alginate degradation pathway of V.alginolyticus ATCC 17749 was reconstructed at the genomic level through bioinformatic analysis.Then biochemistry and molecular genetic technologies were employed to analyze the biological function of different alginate lyases and determine their roles in alginate degradation.Finally,the regulatory network of alginate degradation process was mapped,and two transcriptional regulators which played a role in alginate degradation process were identified,and the transcriptional regulatory mechanism involved in alginate degradation was understood.The contents are as follows:1.Reconstruction of alginate degradation pathway of V.alginolyticus ATCC 17749 on the genomic levelThe genome of V.ATCC 17749 contains an alginate degradation related gene cluster,including extracellular alginate lyase genes aly1,aly2 and aly3;intracellular alginate oligosaccharide lyase genes aly4 and aly5;polysaccharide transport genes kdgM and toaA;ED(Entner-Doudoroff)metabolic pathway-related genes kdgF,dehR,kdgK and kdgA;and potential regulators fadRl and fadR2.Bioinformatics analysis revealed that Aly1,Aly2 and Aly3 belong to alginate lyase PL7 family.All of them have signal peptides,suggestiong a role in extracellular alginate degradation.Among them,Aly1 contains two catalytic domains;Aly2 contains an Nterminal CBM32 carbohydrate-binding domain and a C-terminal catalytic domain;Aly3 has only one catalytic domain.Structural modeling analysis showed that the catalytic domains of three enzymes are typical β jelly roll structure.In addition,unlike the catalytic cavities of Aly1 and Aly2,more loop structures occur above the active center of Aly3 and together form a pore-like catalytic cavity,suggesting that it might act as an exonuclease.The surface potential analysis displayed that the non-reducing end of Aly1 has more basic amino acids,while Aly2 and Aly3 contain more acidic amino acids.The distribution patterns of amino acids may be related to substrate specificity and catalytic mechanism of the enzyme.Aly4 and Aly5 belong to PL17 family,and neither of them contain signal peptides.Therefore,they probably degrade alginate oligomers intracellular.Domain composition of Aly4 and Aly5 are relatively similar,including an N-terminal conserved catalytic domain on and a C-terminal heparin degradation related domain.The structural modeling exhibited that the active centers of Aly4 and Aly5 are pocket-like structures.2.Biological function analysis of different alginate lyasesThe analysis of enzymatic properties of alginate lyases revealed that the optimum temperature of all five enzymes(Aly1,Aly2,Aly3,Aly4 and Aly5)is 20-30℃,the optimum pH is 7.5-8.0,and the optimum NaCl concentration of Aly1,Aly2 and Aly3 is 0.3-0.4 M.The results of substrate specificity analysis showed that Aly1 prefers to degrade polyM;Aly2 has the highest enzymatic activity for three substrates(sodium alginate,polyM and polyG)among five enzymes;Aly3 prefers to degrade polyG.Oligosaccharide lyases Aly4 and Aly5 displayed lower activities for polysaccharide degradation,implying that they are mainly used for intracellular oligosaccharide degradation.Analysis of the enzymatic products revealed that Aly1 and Aly2 exhibited an action of endonuclease mode.The products of Aly1 are unsaturated disaccharides,trisaccharides and tetrasaccharides.Aly2 mainly produce unsaturated trisaccharides as well as partially unsaturated disaccharides.Aly3 is an exonuclease,and the main products is consistent with Aly4,which are unsaturated trisaccharides and DEH(4deoxy-L-erythro-5-hexoseulose uronic acid).The minimum sugar unit recognized by Aly3 and Aly4 may be disaccharide.Aly5 may degrade substrates from both the reducing end and the non-reducing end simultaneously.The main products of Aly5 are unsaturated dicosaccharides,trisaccharides and unsaturated polysaccharides,and the minimum recognition sugar unit may be four sugars.To clarify the biological function of five alginate lyases in bacterial alginate degradation process,five alginate lyases mutant strains were constructed by homologous recombination.The mutant strains could not grow in the medium with alginate as the sole carbon source when aly2 or aly4 was deleted,indicating that aly2 and aly4 were essential for alginate degradation in V.alginolyticus ATCC 17749.Combining the bioinformatics analysis and the results of enzymatic products,as the most important extracellular alginate lyase in V.alginolyticus ATCC 17749,Aly2 can degrade insoluble alginate rapidly,and thus,is the main energy provider for bacterial growth.Meanwhile,Aly4 controls the degradation of oligosaccharide to monosaccharide,which is a prerequisite for downstream metabolic activities.Therefore,aly2 and aly4 are essential genes that play key roles in extracellular and intracellular alginate degradation of V.alginolyticus ATCC 17749,respectively.3.Investigation of the transcriptional regulation mechanism of alginate degradation in V.alginolyticus ATCC 17749Analysis of the transcriptional levels of V.alginolyticus ATCC 17749 under two different carbon sources revealed that the expression of all alginate degradation-related genes were regulated by alginate,except for alginate lyases aly1,aly4,aly5 and the endosomal transporter protein toaA.Regarding its constitutive expression pattern,Aly1 likely acts as a "sentinel enzyme" and is responsible for the activation of alginate degradation gene cluster.Once alginate is present,Alyl is capable of producing oligomers,which serve as signaling molecules and trigger the whole alginate degradation pathway.In addition,analysis of the expression levels at different growth periods indicated that the transcription of extracellular alginate lyase genes,oligosaccharide transport-related genes,and intracellular ED pathway related genes had temporal sequence.Firstly,the extracellular glycan degradation and transport genes were activated in the early stage of growth,and then,ED pathways were further activated during the stationary phase.Moreover,two transcriptional regulators FadR1 and FadR2 are identified to modulate alginate degradation process.FadR2 as a global regulator suppressed expression of almost all genes in the alginate metabolic pathways.FadR1 was possibly responsible for the regulation of alginate extracellular degradation,activating the expression of extracellular glycan degradation and oligosaccharide transport genes.A potential DNA binding motif for FadR1 and FadR2 was further obtained.And EMSA confirmed that FadR1 and FadR2 were capable of binding to the DNA motif and formed protein-DNA complexes.During the regulation process,FadR1 and FadR2 appear to share the same DNA motif to regulate target genes.