Functional Study Of Chu₃220 And Chu₁557 In Degradation Of The Crystalline Region Of Cellulose By Cytophaga Hutchinsonii

Cellulose is the most abundant renewable biological resource on earth,and the utilization of cellulose may be one of the important ways to solve the energy crisis.Cellulose consists of line polymers of ?-1,4-linked glucose units that are organized into a higher-order structure,and it contains the crystalline and amorphous structure.Because of the hydrophobic interactions between cellulose sheets,adjacent cellulose chains,cellulose and water molecules,the crystalline structure of cellulose is stable and recalcitrant to degradation.Therefore,loosening and disruption of the crystalline region of cellulose are the first and the key steps in the utilization of crystalline cellulose.Microbial degradation of cellulose plays a key role in nature carbon cycle.Cellulolytic microorganisms have evolved different mechanisms to efficiently degrade cellulose.There are two well-known strategies of cellulose degradation.Most aerobic cellulolytic microorganisms secrete a series of free cellulases,and many of which contain a carbohydrate binding molecule?CBM?.Most anaerobic microbes produce cellulosomes,and all of which contain a scaffold protein that have a CBM domain.CBMs could disrupt the crystalline structure of cellulose by binding and disrupting the hydrogen bonding networks of cellulose.Besides CBMs,expansins,expansin-like proteins,swollenins,and lytic polysaccharide monooxygenases?LPMOs?are thought to disrupt the crystalline structure of cellulose.Cytophaga hutchinsonii is a Gram-negative bacterium and wide-spread in soil,and it could degrade crystalline cellulose,such as filter paper and cotton wool,rapidly and efficiently.Analysis of its genomic sequence reveals that C.hutchinsonii lacks exoglucanases and its possible endoglucanases lacks recognizable CBMs.Direct contact with insoluble cellulose is necessary for C.hutchinsonii to degrade crystalline cellulose,and most of the cellulase acticities are cell associated.C.hutchinsonii also does not encode any proteins containing dockerin or cohesion domains which are characteristics of cellulosomes.It may degrade the crystalline cellulose with a unique and unknown mechanism different from the free cellulases or cellulosomes strategies.For C.hutchinsonii,besides cellulose,glucose and cellooligosaccharides can also be rapidly assimilated,and no reducing sugars could be detected in the culture supernatant when C.hutchinsonii is cultivated with cellulose as the sole carbon source.A possible mechanism for cellulose degradation by C.hutchinsonii is the presence on the outer surface of its outer membrane of a protein complex which could remove the cellulose molecules from cellulose fibers and transport them across the outer membrane into periplasmic space,where they would be degraded by endoglucanases.C.hutchinsonii could degrade the crystalline cellulose efficiently,however,besides CBMs,it also dose not encode proteins similar to expansins,expansin-like proteins,swollenins,or LPMOs to disrupt the crystalline structure of cellulose.The mechanism of crystalline region of cellulose degradation is still a mystery.Recently,some progress has been made in the mechanism of cellulose degradation owing to the development of many genetic manipulation tools in C.hutchinsonii.In this study,a method based on the FLP/FRT recombination system was developed for C.hutchinsonii to express external genes.Two genes involved in cellulose utilization of C.hutchinsonii were identified by transposon mutagenesis,and the factors which are associated with utilization of cellulose of C.hutchinsonii were also discussed.1.Development of exogenous DNA insertion method based on the FLP/FRT recombination system.Presently,plasmid is the common methods to express the exogenous genes in C.hutchinsonii,however,it is instability and required antibiotics to maintain the existence.Recently,our laboratory had successfully inserted the exogenous DNA into the genome of C.hutchinsonii by linear DNA double crossover method.However,two longer homologous fragments which are required limit the size of exogenous DNA fragments.The FLP/FRT recombination system from Saccharomyces cerevisiae is one of the most useful tools for efficient genetic engineering,and has been widely used in gene knockout,gene recombination etc.The unmarked deletion method based on FLP/FRT recombination had successfully been used in C.hutchinsonii.FLP recombinase could identify the FRT sites on genome and the ring exogenous fragments,and then integrate the fragments into genome at the FRT site.In this study,we had developed the exogenous DNA insertion method based on the FLP/FRT recombination system.Firstly,a parent strain was constructed with a single FRT site was introduced into the pseudogene?chu₃328?locus.Secondly,a suicide plasmid was also constructed with a single FRT site,and then lacZ was successfully inserted into the genome of C.hutchinsonii with this plasmid.The exogenous DNA could be inserted into the specific sites of genome with a single FRT site,and no homologous fragments and antibiotics are required.The DNA insertion method could also use the FRT site which is produced by the unmarked deletion method based on FLP/FRT recombination,and accomplish the gene complement in situ.The exogenous DNA insertion method based on the FLP/FRT recombination system is simpler and more stable than plasmid or DNA double crossover method.2.Functional study of chu₃220.C.hutchinsonii contains 3790 open reading frames?ORF?,of which only 1986 were predicted by functional annotation.Our laboratory had developed a method based on transposon-mutagenesis to screen the genes associated with cellulose degradation.A mutant that lacks the ability to completely degrade filter paper,was identified by-HimarEm3 transposon.Inverse PCR showed the transposon was inserted into gene locus chu₃220.chu₃220 deletion mutant was constructed by line DNA double-crossover deletion method and named ?3220.Similar to the transposon-mutagenized mutant.?3220 failed to degrade the filter paper completely.The result of RT-PCR showed that deletion of chu₃220 did not affect the transcription of the surrounding genes,and the phenotype of mutant was caused by deletion of chu₃220.chu₃220 is an essential for C.hutchinsonii to utilize crystalline cellulose.Analysis of the growth of ?3220 with different carbon source,and the results showed that the growth rate and the maximum biomass of mutant were not lower than that of wild type with glucose,cellobiose or amorphous cellulose as the sole carbon source.However,the growth rate and maximum of the mutant were significantly lower than that of wild type with Avicel as the sole carbon source.These results suggested that deletion of chu₃220 affected the degradation of crystalline region of cellulose.X-ray diffraction?XRD?was used to test the crystallinity of Avicel,and the results showed that the crystallinity of Avicel was increased after incubation of wild type cells,while it was reduced after incubation of the mutant cells.The surface morphology change in Avicel was also observed by scanning electron microscopy?SEM?.The surface of untreated Avicel was rough and irregular but after incubation with wild type,Avicel had a relatively smooth and uniform surface that was different from that of untreated Avicel.It seemed that C.hutchinsonii had a peeling effect on the cellulose surface no matter whether it was amorphous or crystalline regions.However,the surface of Avicel treated with the mutant was rough and irregular.All of the results we previous finding that ? 3220 was defective in the utilization of crystalline region of cellulose,suggesting that chu₃220 is an essential for C.hutchinsonii to degrade the crystalline region of cellulose.CHU₃220 is a hypothetical protein with a molecular weight of 205 kDa,and contains a CHU_C domain and 15 PbH1 domains.The CHU_C domain was found to play important role in localization of CHU₃220,and the PbHl domain was predicted to be related to protein interactions.CHU₃220 is a cellulose binding protein and located on the surface of cells.The expression level of chu₃220 was induced by cellulose.Endoglucanase activity on the cell surface was decreased with deletion of chu₃220,while the transcription level of these endoglucanase genes were also reduced.There are several features of CHU₃220 were similar to those of scaffold proteins in cellulosomes,however,no evidence of interaction between CHU₃220 and cellulases has ever been found.Combined with above results,we speculated that CHU₃220 might be a component of the protein complex on the cell surface of C.hutchinsonii to disrupt the crystalline region of cellulose.The products of cellulose hydrolyzed by C.hutchinsonii were analyzed by ion chromatography with integrated pulsed amperometric detection?IC-IPAD?.After the wild type incubated with cellulose for 5 hours,certain amounts of reducing sugar,including glucose,cellobiose,and cellotriose,could be detected in the supernatant culture,indicating that the cellulose could be digest by the cellulases which are located on cell surface.However,the hydrolytic products could not be detected any more when incubation time was prolonged to 24 hours.At the same time,the intracellualr reducing sugar was also determined,and hydrolytic products including glucose,cellobiose,cellotriose,and cellotetraose were also detected in the cells,suggesting that oligosaccharides could be transported into periplasm for further digestion.The same hydrolytic products were also detected in the cells of mutant,suggesting that deletion of chu₃220 had little efect on oligosaccharide transport.In addition to CHU₃220,the energy providing and intact cell structure are required for C.hutchinsonii to disrupt the hydrogen-bonding networks of cellulose.When ATP production was inhibited by NaN₃,cells could not utilize crystalline region but could still degrade the amorphous region of cellulose,suggesting that energy providing is required for C.hutchinsonii to disrupt the crystalline region of cellulose.Triton X-100 can destroy the cell membrane structure,but does not affect the interaction between proteins.The crystallinity of Avicel was increased after incubation of the cells that was treated by Triton X-100,suggesting that the amorphous region of cellulose was digested.The energy providing and intact cell structure are required for C.hutchinsonii to utilize the crystalline cellulose.This is obviously different from other cellulolytic systems,including free cellulase systems and cellulosome systems,in which crystalline cellulose could be disrupted without cells or any external energy supply.3.Functional study of chu₁557.chu₁557 is involved in the degradation of crystalline cellulose that was identified by HimarEm3 tansposon mutagenesis.chu₁557 deletion mutant??1557?was constructed by linear DNA double crossover deletion.And RT-PCR proved that deletion of chu₁557 did not affect the transcription of the surrounding genes.CHU₁557 is a hypothetical protein with a molecular mass of 391.4 kDa,and contains a N-terminal signal peptide and a C-terminal domain?CTD?.CTD was the conserved C-terminal signal of protein substrates which could be secreted across the outer membrane by type IX secretion system?T9SS?,so CHU₁557 might be secreted across the outer membrane by T9SS.CHU₁557 was a cellulose binding protein.The subcellular localization of CHU₁557 was determined by western blot,and the results showed that CHU₁557 was located on the outer surface of outer membrane.The expression level of chu₁557 in the cell cycle was detected with glucose as the carbon source.The results showed that expression level of chu₁557 was obviously increased at lag phase and decreased dramatically at the log phase,suggesting that chu₁557 might take effect in the startup of growth.The expression level of chu₁557 was increased at lag phase with Avicel as the carbon source,and it kept increasing at log phase,indicating that expression of chu₁557 was induced by crystalline cellulose and chu₁557 might take effect in cellulose degradation.The lag phase of ?1557 was associated with the concentration of glucose.It was similar to that of wild type when the cells were cultured with high concentration of glucose?0.4%,w/v?,but it was 60 h longer than that of wild type when the cells were grown on low concentration of glucose?0.1%,w/v?.Different concentrations of glucose were used to test the growth of wild type and ?1557,and a glucose concentration-dependent lag phases of ?1557 were observes with the shortest lag phase occurring at the 0.4%?w/v?glucose.When the cells pre-cultured on low concentration of glucose were inoculated into low concentration of glucose,the lag phase of ?1557 was similar to that of wild type.The lag phase of ?1557 was about 72 h longer than that of wild type with amorphous cellulose and crystalline cellulose,and the shortened lag phase of ?1557 on amorphous cellulose and crystalline cellulose was also observed when the cells pre-grown on low concentration on glucose.The maximum biomass of ?1557 after grown on low and high concentration of glucose was 33%and 62%of that of wild type,respectively,suggesting that the mutant could utilize partial crystalline region of cellulose.chu₁557 is involved in uptake of glucose by C.hutchinsonii,and deletion of chu₁557 resulted in a significantly slower uptake rate of glucose at a low concentration compared with that of wild type,while the mutant displayed a similar uptake rate of glucose at a high concentration to that of wild type.After pre-cultured on low concentration of glucose,the uptake rate of glucose at a low concentration of?1557 was similar to that of wild type.Energy providing is required for C.hutchinsonii to disrupt the crystalline cellulose,and the decreased glucose uptake rate of ?1557 might affect the energy supplying.The intracellular ATP level was significantly decreased and the ATP transcripts was increased with deletion of chu₁557.So we deduced that ?1557 might consume more energy than wild type in cell growth.Based on our results,chu₁557 may have two main functions for cellulose utilization by C.hutchinsonii.Firstly,it is involved in the uptake of low concentration of glucose.Absence of chu₁557 resulted in a compromised assimilation of glucose at a low concentration,which may affect the carbon and energy source supplying for C.hutchinsonii.Secondly,it was a membrane of the protein complex on the cell surface and consumed energy to disrupt the crystalline region of cellulose.More energy was required for utilization of crystalline cellulose when chu₁557 was deleted,so the mutant could degrade crystalline cellulose completely when glucose was added in the medium or increasing the uptake rate of glucose.In this study,we had developed the exogenous DNA insertion method based on the FLP/FRT recombination system.The method eliminates the characteristic of genetic instability of plasmid,and be able to use the FRT site which was produced by the method of non-trace knock out based on FLP/FRT recombination system to achieve the gene complement in situ and gene expression.C.hutchinsonii could degrade the crystalline cellulose efficiently by an unknown strategy,and two cellulose degradation ways had been identified by analyzing the cellulose hydrolysis product in or out the cells.The crystalline region of cellulose degradation is the key step to utilize crystalline cellulose,and in this study we had firstly identified two genes chu₃220 and chu₁557 which were important for degradation of crystalline region of cellulose by by transposon mutagenesis.Functional study of CHU₃220 and CHU₁557 found that they might be the important components of protein complex on the cell surface which could remove the individual cellulose molecular from cellulose fiber.Besides the proteins associated with crystalline region of cellulose degradation,the intact cell structure and energy supplied by live cells were also essential for C.hutchinsonii to digest crystalline cellulose.The mechanism of crystalline cellulose degradation of C.hutchinsonii is obviously different from free cellulase system and cellulosome system,and the study on degradation of crystalline region of cellulose had shed light on the novel strategy of cellulose degradation by C.hutchinsonii and would be also helpful to improve the utilization efficiency of crystalline cellulose.