Functional Study Of Cell Surface Cellulase System And Chu_hypz1 In Cytophaga Hutchinsonii

With the oil and other non-renewable resources consumed and exhausted,people have to turn their attention to use the other renewable resources.Therefore,how to convert cellulose which is the most abundant renewable resource on earth into energy that can be used directly is one of the important ways for humans to solve the energy crisis.Cytophaga hutchinsonii is a gram-negative bacterium that is widely distributed in the soil.The cellulose degradation mechanism of C.hutchinsonii is different from the free cellulase mechanism used by known aerobic microorganisms and the cellusome mechanism applied by anaerobic microorganisms,and maybe a novel cellulose degradation mechanism was used by C.hutchinsonii.Recently,the development of C.hutchinsonii genetic manipulation technology established the foundation for the study of the cellulose degradation mechanism.Genome analysis identified nine genes that are predicted to encode endoglucanases,of which five are predicted to be located on the cell surface.In this study,Ce19D was knocked out based on four-knockout strain of cellulase.The role of surface cellulases in the cellulose degradation were studied.Meanwhile,the transposon mutagenesis technology was used to identify a gene that affected cellulose degradation,and the function of the gene was discussed in this study.1.Research on the effects of cell surface cellulases in cellulose degradation of C.hutchinsonii.Genome analyses identified nine genes encode endoglucanases,of which five are predicted to be located on the cell surface:Cel5A,Cel9A,Ce19B,Ce19D and Ce19E.In 2016,Zhu et al.constructed an unmarked deletion mutant called CH581,which was knocked four cellulases,Cel5A,Ce19A,Cel9B,and Ce19E.Only Ce19D could not be knocked out.The CH581 mutant could use Avicel as the carbon and energy source.Based on the CH581 mutant,we successfully knocked out Ce19D,which was predicted to be located on the cell surface.This strain was designated CH681.While CH681 was still able to use cellulose as the carbon and energy source,except that the degrading zone on the filter paper plate was significantly smaller than that of the wild type.Analysis of the growth curve in the liquid medium with different carbon sources,we found that there is no delay with glucose and cellobiose as the sole carbon source.However,there is a significant delay when the mutant grew in RAC or Avicel.It shows that the growth rate of mutant was slow down,which can also be seen from the substrate utilization rate of Avicel.Therefore,we speculated that the lack of cell surface endoglucanases reduces the rate of cellulose utilization.When we examined the endoglucanase activities on the cell surface,we found that even the all five known endoglucanases were missing,the endoglucanases activity still could be determined.How is this residual endoglucanases activity produced?Through the gene analysis,four candidate ?-glycosidase predicted to be located on the cell surface were found,namely bglE,bglF,bglG and bgll,the glycosidase substrate is non-specific,whether their existence led to the residual enzyme activity?Therefore,we first detected the transcription levels of these glycosidase genes in the mutant and wild type by qPCR,and found that their transcription levels were increased to varying degrees.In particular,the difference in bglI was the most significant and increased by about 9 times,this suggests that they may compensate for the lack of cellulases on the surface.Thus,we designed their single knockout and multiple deletions on the basis of the CH681 to see if they played a role in cellulose degradation.There was no significant change in the cellulose degradation when they were knocked out individually.It shows that the deletion of a single glycosidase does not affect the degradation of cellulose,and the multiple deletion process has not yet finished.A preliminary phenotypic verification of the CH685 strain?bglE,bglF,bglH and bgll was knockout based on CH681?revealed that the filter paper degradation ability was reduced compared with the strain CH681,the endoglucanase activity was lower than that of the CH681 strain but not obvious.It shows that the glycosidase on the cell surface has a certain effect on the endoglucanase activity,but not essential.2.Functional study of chu_hypZ1 related to crystalline cellulose degradation.The establishment of transposon-mutagenesis allows us to obtain genes related to cellulose degradation through a large number of screenings.A mutant that lacks the ability to degrade filter paper,was identified by Tn4351 transposon.Reverse PCR showed the transposon was inserted into gene locus chu_hypZ1.After chu_hypZ1 was knocked out,the mutant strain AhypZl could not degrade filter paper and its phenotype could be restored by gene complementation,indicating that the phenotypic defect was caused by chu_hypZ1.RT-PCR showed that the transcription of nearby genes of chu_hypZ1 in AhypZ1 was not affected by deletion of chu_hypZ1.indicating that chu_hypZ1 is a key gene involved in cellulose degradation.The molecular weight of CHU_hypZ1 is 131 kDa,which is a hypothetical protein,and there is no conserved domain.The similar proteins that were found by Blast P were hypothetical proteins,and some of them belonged to the Cytophaga,indicating the evolutionary conservation of this protein.Analysis of the growth of AhypZl in different carbon sources,showed that the mutant could grow with glucose,cellobiose and RAC as the carbon source,but the growth rate and the maximum biomass were slightly lower than the wild type.However,there was a significant growth defect in Avicel.These results suggested that deletion of chu_hypZ1 affected the degradation of crystalline region of cellulose.X-ray diffraction?XRD?was used to detect the crystallinity of Avicel.It was found that the crystallinity of Avicel treated with wild-type decreased,while the crystallinity of Avicel treated with mutant increased,which further demonstrated that chu_hypZ1 is a key gene for crystalline cellulose degradation.In the AhypZl mutant,the endoglucanases activity on the cell surface was significantly reduced.We found that this is caused by the decreased transcriptional level of these endoglucanases.On the other hand,it is also because of the endoglucanase located on the cell surface cannot be normally anchored on the outer membrane.In addition,the outer membrane proteins bound to cellulose of the mutant was missing several bands compared to the wild type,most of them were hypothetical proteins and others have been identified as component proteins of the type ?secretion system.Is the absence of chu_hypZ1 affecting the type ? secretion system?By detecting the substrate protein CHU₀344 of the type IX secretion system,it was found that the band of CHU₀344 was detected in the mutant strain,indicating that the absence of chu_hypZ1 has no effect on the type IX secretion system.Through the identification of extracellular differential proteins,it was found that the proteins originally on the membrane could be detected extracellularly,such as Cel5A and Bg1F.Bg1F is the missed protein on the outer membrane.We speculated that the deletion of chu_hypZ1 may lead to the inability of normal anchoring of some membrane proteins,resulting in the defective degradation of cellulose.In this study,we had firstly taken the unique cellulase profile of C.hutchinsonii as the start point.We found that although there are multiple endoglucanases and glycosidases on the cell surface,they are not essential for the cellulose degradation.We speculated that these cellulases may perform the initial digestion within amorphous regions and produce oligosaccharide chains that are transported into the periplasm for further digestion.And we identified a key gene chu_hypZ1 which was important for degradation of crystalline region of cellulose by transposon mutagenesis.Functional study of chu_hypZ1 found that its deletion caused some membrane proteins can not be anchored on the cell membrane and led to degradation defect.This study about cell surface cellulase system and chu_hypZ1 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.