| Microbial surface display systems have a wide range of applications in biotechnology and industrial production.Corynebacterium glutamicum is capable of producing a variety of chemicals,and is a promising host for surface display due to its robustness,high-density fermentation,and availability of a wide range of natural substrates.C.glutamicum-displayed enzymes show good performance in cheap biomass conversion to produce chemical products,and are one of the research hotspots in the field of renewable biomanufacturing.However,the number of anchoring proteins in C.glutamicum is low,and studies demonstrating the technology are relatively few.In order to expand the types of anchor proteins in the surface display system of C.glutamicum,in this study,we took the industrial model bacterium C.glutamicum ATCC 13032 as the research object and combined bioinformatics technology prediction and engineering display technology.The characteristics of the anchor protein in the genome of C.glutamicum ATCC 13032 were screened and confirmed.On this basis,a variety of α-Amylase and β-glucosidase display systems were constructed,and the transformation effects of different display systems and the enzymatic properties of the display systems were explored.Finally,the function of the anchor protein NCgl2775 was initially explored.The specific research contents and results are as follows:(1)Screening and confirmation of novel anchor proteins at the genome-wide levelA globally novel high-throughput screening method for anchored proteins based on the genome of C.glutamicum was developed.C.glutamicum ATCC 13032 was analyzed by subcellular localization(SCL)and parameter prediction of Locate P,and transmembrane structure prediction and visualization of TMHMM(tied-mixture hidden Markov models).Of all the proteins encoded by the genome,25 potential anchor proteins were predicted.Next,14 novel anchoring proteins were screened by displaying reporter proteins e GFP and m Cherry,combined with immunofluorescence reaction,flow cytometry analysis and laser confocal imaging technology.In addition,when using m Cherry as a passenger protein to screen anchor proteins,confocal microscopy imaging and flow cytometry analysis of some strains are better than e GFP screening.(2)Construction and transformation of α-Amylase surface-displayed system of C.glutamicumThe 14 novel anchor proteins excavated were used to display the α-Amylase Amy E(derived from Bacillus subtilis 168),and the α-Amylase activity was detected on the surface of the recombinant strains.The surface display activities of the 10 novel anchor proteins were higher than those of the anchor proteins NCgl1221 and NCgl1337 reported in the literature,proving the high efficiency of these novel anchor proteins.The NCgl0661-Amy E strain had the highest surface hydrolysis activity,reaching 0.36 U/m L(43.85 U/g cell dry mass).At the same time,strains displaying Amy E using NCgl1307,NCgl2775 or NCgl0717 was able to grow in medium with soluble starch as the sole carbon source.In order to improve the stability and catalytic activity of the display strain,the methods of removing the signal peptide and propeptide of Amy E,fermenting with different media,modifying the display system and displaying Amy A were explored.The results showed that the existence of the signal peptide cleavage site in the Amy E sequence may only be one of the reasons for the leakage of Amy E display strains,and there are other unknown reasons that lead to the instability of the display system;The α-amylase activity in the bacterial suspension and fermentation broth supernatant in BHISG medium was significantly higher than that in BHI medium,which may be the reason for the higher biomass of the former;When the display system was modified,the NCgl2775-DI-Amy strain had the highest activity,and the effects of the modification systems of different anchor proteins were affected by the types of anchor proteins;The α-amylase activity was detected in the cell suspension and fermentation broth supernatant of the Amy A display strain,and the surface catalytic activity of the strain was significantly lower than that of the Amy E and Amy display strains..(3)Construction and transformation of β-glucosidase surface-displayed system of C.glutamicumThree β-glucosidases were selected as passenger proteins,and the enzymatic properties of the recombinant strains of C.glutamicum were studied.In order to further improve the catalytic activity of the recombinant strain,based on NCgl1307,NCgl2775,and NCgl0717 as anchor proteins,the Tfu0937 display system was modified by direct fusion,addition of flexible/rigid linker,and anchor protein truncation.Results showed that the activity of NCgl2775s-Tfu0937 was the highest,reaching 23.34 m U/m L(4.02 U/g cell dry mass),which was 0.6 times higher than that of NCgl2775-DI-Tfu0937.At the same time,the influence of the modification method on the display system is not only affected by the type of anchor protein,but also related to the type of passenger protein.It is worth noting that the Tfu1629 and Tfu0937 display systems are relatively stable,and no enzyme activity was detected in the fermentation broth supernatant of the display strains.This also indicates that the stability of the C.glutamicum display system is related to the type of passenger protein.(4)Preliminary study on the function of the anchor protein NCgl2775The basic physical and chemical properties of NCgl2775 were analyzed by bioinformatics techniques,results showed that NCgl2775 is an N-terminally anchored membrane protein,belonging to the cutinase family.Its genetic and evolutionary characteristics were analyzed by genetic mapping,phylogenetic tree construction and sequence alignment.Results showed that NCgl2775 is located in a gene cluster related to cell wall synthesis in C.glutamicum,which is highly conserved in genetic location and evolution.At the same time,the basic enzymatic properties of NCgl2775 were characterized by in vitro purification,indicating that NCgl2775 protein is an alkaline thermophilic esterase and is sensitive to p H conditions.In addition,the importance of NCgl2775 on strain growth was studied.Results showed that although NCgl2775 is not a necessary gene for C.glutamicum,the absence of NCgl2775 affects the growth of the strain. |
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