Functional Study Of The ?-glucosidases And Flexirubin-pigment In Cytophaga Hutchinsonii

Wth the improvement of industry and the increase of population,the energy crisis and environment pollution problems become the challenges for the human society.For the fossil resources are increasing scarce in these years,developping new renewable resources has becoming great necessary.Lignocellulose,the main component of plant biomass,is the most abundant renewable resource on the earth.More and more attention was paid to the utilization of lignocelluloses.However,due to its compact strcture,insolubility and crystalline regions,cellulose is difficult to utilize.Investigation the cellulose degradation mechanisms in the nature and improvement of the converting efficiency of crystalline cellulose are the key to the lignocellulose utilization.Cytophaga hutchinsonii is a widely distributed aerobic Gram-negative bacterium which belongs to the phylum Bacteroidetes.C.hutchinsonii can digest crystalline cellulose rapidly and thoroughly,but it does not secrete soluble extracellular cellulolytic enzymes and has no cellulosome-like structure.Besides,there was no carbohudrate binding modules?CBM?in all of its cellulases.C.hutchinsonii may use a new cellulose utilization system which is different from the two well-known free cellulase system and complexed cellulosome system Thus,the study of the novel cellulose degradation strategy might be helpful to reveal the efficient cellulose utilization system and realize the bioconversion of cellulose.However,due to the lack of genetic manipulation tools,the studies on mechanisms of'cellulose degradation by C.hutchinsoinii have remained stagnant.Recently,some progress has been made owing to the development of genetic manipulation tools.Cellobiose is the primary unit of cellulose and an important intermediate product of cellulose degradation.So study the degradation and utilization progresses of the cellobiose have important significance on reveal the cellulose degradation of C.hutchinsonii.In this study,the functions of all the four(3-glucosidases and their effects on cellulose degradation were studied.The cello-oligosaccharide and cellulose degradation products both in the medium and in the cell of C.hutchinsonii were detected by ion chromatography?IC?.Hundreds of cellulose adsorption proteins were extracted and identified by a new methord,and the function of a cellulose induced protein CHU₀344 was studied.A pigment defected mutant B-1 was constructed through transposon mutagenesis,the function of ?-hydroxyacyl-?acyl carrier protein??ACP?dehydratase in flexirubin synthesis and the biological role of flexirubin were studied.1.Research on the function of ?-glucosidases and their effects on cellulose degradation of C.hutchinsonii,detection the cellulose degradation products and the dual pathway degradation mechanism of C.hutchinsonii.We studied the function of ?-glycosidases and their effects on cellulose degradation in C.utchinsonii.We found in this study for the first time that C.utchinsonii could degrade cellulose both on the cell surface and in the cell.Based on these results we proposed a new dual-pathway degradation model.?-glucosidases generally worked in the cellobiose hydrolysis stage of cellulose degradation progress.And some ?-glucosidase possessed both hydrolyzation and transglycosylation activity.Analysis of the C.hutchinsonii genome showed that C.hutchinsonii has four candidate ?-glucosidase genes:bglA?chu₂268?,bglB?chu₂273?,bglC?chu₃577?,and bglD?chu₃784?.All of them belong to glycoside hydrolase family GH3.BglA,BglB,and BglC have previously been predicted to be lipoproteins.There is a signal peptide in BglB,but not in BglA,BglC,and BglD as predicted by SignalP 4.1.However,many hydrophobic amino acids are in the first 30 amino acids of BglA,BglC and BglD,suggesting there might be signal peptides in these proteins.In order to detect the function of the all the four ?-glucosidases in cellobiose and cellulose degradation,the expressipn patterns of these ?-glucosidase were chected both in RNA and protein level by a quantitative RT-PCR assay and a renatured SDS-PAGE assay.BglB was the main ?-glucosidase expressed in glucose culture.BglA and BglB were the two main ?-glucosidase genes expressed in cellulose culture.The expression level of BglC was very low both in glucose and cellulose condition.And the expression of BglD was undetectable under our experimental condition.These results indicated that BglA and BglB play a vital role in cellobiose and cellulose degradation.Then all the four ?-glucosidases were singly deleted by the double-crossover recombination system.The two main ?-glucosidases?bglA and bglB?and the three expressed ?-glucosidases?bglA,bglB,and bglC?were multiply deleted in order to construct?bglA/bglB and?bglA/bglB/bglC mutants,respectivelyIn cellobiose and cellulose medium,?bglB could reach the same cell density as the wild type strain but it had a longer lag phase.?bglA/bglB could not grow either in cellobiose or cellulose mediums,but the other deletion mutants grew as well as the wild type strain.In glucose culture,?bglB and ?bglA/bglB comletely lost P-glucosidase activity,while all the other single deletion mutants had similar?-glucosidases activities as the wild type strain.In cellulose culture,?bglB only kept about 20%of the wild type ?-glucosidase activity while ?bglA/bglB lost all the?-glucosidase activity.All the single deletion mutants except ?bglB showed a similar cellobiose degradation rate as the wild type strain.?bglB started to degrade cellobiose after a delay of 2 h and the degradation rate was much slower than that of the wild type strain.But ?bglA/bglB completely lost the ability to hydrolyze cellobiose.All the results indicated that BglB was the major ?-glucosidase of C.huichinsonii.BglA could be induced by cellulose and complement part of the(3-glucosidase activity of?bglB.All the single deletion mutants retained the ability to digest and grow on cellulose.?bglA/bglB could also digest filter paper on Stanier agar plate,but could not utilize cellulose in liquid culture.We found that the micro-nutrients in agar might facilitate cellulose utilization by ?bglA/bglB,and A bglA/bglB could partially degrade cellulose in the liquid cellulose medium with additional glucose.However,?bglA/bglB had no ?-glucosidase activity and could not degrade cellobiose even with additional glucose.These results indicate that C.hutchinsonii could partially utilize cellulose even without BglA and BglB.Quantitative analysis showed that when? bglA/bglB was grown in cellulose medium with little glucose,more than 55%of the degraded cellulose was converted into cellobiose by?bglA/bglB,and the other part of the degraded cellulose might be converted into glucose to supply the growth of the mutant.The extracellular and intracellular cello-oligosaccharide detection methods were set up based on the ion chromatography method.When wild type strain cells were incubated with cellobiose in Stanier medium,almost all of the cellobiose disappeared and a large amount of glucose accumulated in the medium within 4 h incubation,while there was almost no cellobiose accumulated in the cells throughout the process.Meanwhile,according to the ?-glucosidase and activity determination assay and the Western blot assay,?-glucosidases in C.hutchinsonii were located both in the periplasmic space and on the cell surface.We speculated that exogenous cellobiose was degradation into glucose on the cell surface and then be used by the cells.For a long time,no apparent cellulose degradation products were detected in the medium.In this study,obvious glucose was detected in the initial lag phase of the wild type strain when cultured in cellulose medium.When wild type cells were incubated with cellulose,large amounts of glucose and some cellobiose accumulated in the medium.Meanwhile,there were ?-glucosidases and endoglucanases located on the cell surface,we speculated that C.hutchinsonii could degrade cellulose on the cell surface.The intracellular degradation products of the cells cultured in cellulose were also detected for the first time.There were significant amounts of glucose,cellobiose,cellotriose,and cellotetrose present in the cells.Since cello-oligosaccharides could hardly be tansported into the cell,and there were ?-glucosidases and endoglucanases located in the cell,we speculate that C.hutchinsonii could also degrade cellulose in the cell.Combined with the results of this work and other researchs,we proposed a modified mechanism:?i?there were cell surface and intracellular cellulose degradation pathways,?ii?On cell surface endoglucanases with the help of other outer membrane proteins could digest cellulose into cellodextrins and cellobiose,and then the products are digested by?-glucosidases.?iii?The C.hutchinsonii outer membrane protein could remove individual cellulose molecules and transport them into the periplasmic space,where they would be digested by endoglucanases and?-glucosidases.?iiii?C.hutchinsonii could defectly degrade cellulose without the help of ?-glucosidases.We investigated the function of ?-glucosidase and proposed a new dual cellulose degradation model of C.hutchinsonii in this work.This work improved our understanding of cellulose degradation mechanism of C.hutchinsonii.The study of cellulose degradation in C.hutchinsonii would not only focuse on the periplasmic space but also the cell surface,and this work would be helpful for further revealing the mechanism of cellulose degradation in C.hutchinsonii.2.Functional study of chu₂110 and biological role of flexirubin.Transposon mutagenesis is effective to study gene function.In this study,C.hutchinsonii was mutagenized with transposon HimarEm3 and a transposon mutant B-1 which had no obvious pigment was isolated.The site of HimarEm3 insertion of B-1 was analyzed by inverse PCR.The result showed that the transposon was inserted in gene CHU₂110?Gene symbol:fabZ?in the genome of C.hutchinsoinii.The chu₂110 complement strain could restore the pigment synthesis phenoytypic indicating that the defect of B-1 was caused by mutation in chu₂110.The coding region of gene fabZ is 372 nucleotides in length and codes a protein consisting of 122 amino acids.FabZ is annotated as a?3R?-hydroxymyristoyl-ACP dehydratase since there was a putative FabA_FabZ/hot_dog super-family domain in its N-terminal.FabZ contained no signal peptide sequence predicted by SignalP and was predicted to be located in cytoplasm by PSORTb.FabZ is an enzyme generally worked in the elongation cycle of type ? fatty acid biosynthesis.In this study,we found that FabZ is an important dehydratase in the steps of the polyene elongation cycle of flexirubin biosynthesis through a fatty acid-like synthesis pathway.There are two homologues of dehydratase,FabA and FabZ,in C.hutchinsonii.fabZ is located in the putative aryl-polyene synthesis cluster of C.hutchinsonii,and fabA is located in the fatty acid metabolism cluster.Our results indicated that deletion of FabA did not affect the pigment syntheisis in C.hutchinsonii.Pigments are important secondary metabolites of microorganism.Flexirubin-type pigments were used as chemotaxonomic markers for bacteria of the Bacteroidetes phylum,and we studied the biological roles of flexirubin in C.hutchinsonii.We demonstrated that the flexirubin pigment could protect c.hutchinsonii cells against UV radiation and oxidative stress,which is similar to other pigments.The conjugated polyene chain and other unsaturated bonds in flexirubin might provide chemical reactivity against oxidizing agents and free radicals.Moreover,the flexirubin of C.hutchinsonii could protect cells against alkaline stress.This effect has not been reported in other pigments.3.Identification of cellulose adsorption proteins and the functional study of CHU₀344.The unique cellulose degradation mechanism of C.hutchinsonii need the bacteria directly contact with cellulose.Cellulose adsorption proteins,especially surface ion proteins in bacteria play an important role in the process of adsorption aixl degradation of cellulose.In this study,the total cellulose adsorption protein of cells both cultured in glucose and cellulose medium were extracted.All the proteins in the cell were dissolved by Triton X-100,and the proteins absorbed to cellulose were collected.By using proteomics strategy and high-resolution LC-MS/MS,781 proteins were identified in sample extracted from cellulose cultured cells and 744 proteins were identified in sample extracted from glucose cultured cells.And the relative expression of the proteins could be estimated by the results.The expression level of some proteins were obvious different.All the proteins which have been proved to be associated with cellulose degradation were located in the top 100 of the results.The identification of the cellulose adsorption protein would help to identify the proteins involved in cellulose degradation of C.hutchinsonii.CHU₀344 was selected to be studied for its high expression level in both condition and its expression increases obviously in cellulose condition.CHU₀344 was proved to be located both in the cell membrane and could be secreted to the medium.The disruption of chu₀344 caused cellulose adsorption reduced and degradation defect.This result indicated that CHU₀344 was essential for cellulose adsorption.