Molecular Modification Of Xylanase And Its Efficient Expression In Aspergillus Niger

Xylanases collectively represent a group of enzymes capable of breaking down xylan into xylooligosaccharides.Specifically,β-1,4-endoxylanases,referred to as xylanases,play a key role in catalyzing the hydrolysis of internalβ-1,4-glycosidic bonds within xylan molecules.These enzymes have diverse applications in industries such as animal feed,food production,and bioethanol manufacturing.Among the 400-plus identified microbial xylanases,those belonging to the glycoside hydrolase family 11（GH11）stand out due to their robust p H tolerance,high catalytic activity,and strong substrate specificity,making them a focal point in xylanase research.However,GH11 xylanases commonly exhibit limitations in thermal stability and a relatively modest ability to degrade insoluble xylan.Currently,Aspergillus niger serves as a primary industrial strain for the production of food-grade xylanases.Challenges arise from the low efficiency of gene editing,necessitating reliance on traditional mutagenesis breeding for high-yield xylanase production.Addressing these issues,this study screened xylanase genes from the A.niger AG11 genome that could be expressed in both A.niger and Escherichia coli.Employing rational/semi-rational design and domain fusion strategies,the study succeeded in enhancing the thermal stability of xylanases and their capacity to degrade insoluble xylan.Subsequently,the modified xylanase genes were integrated and expressed in A.niger,resulting in the efficient biosynthesis of a novel xylanase.Key research findings include:（1）Xylanase genes were identified and recombined expressed in A.niger AG11.Using corn cob as an inducer,xylanase activity was detected in the fermentation supernatant of A.niger AG11.Subsequently,five xylanase genes（xynI-V）were identified through whole-genome sequencing.These genes were fused with the expression elements ofβ-glucosidase（bgl）to construct recombinant expression cassettes,which were randomly integrated into the genome of A.niger AG11.Among them,the enzyme activity of xynII（xynA）was the highest(15.8U·mL^-1).Enzymatic property revealed favorable stability for xynA within the p H range of2.0-9.0,but residual activity after incubation at 55℃ for 5 min was only 10%.To establish an enzyme property modification platform,xynA was transferred into E.coli BL21（DE3）for heterologous expression,but encountered a high level of inclusion bodies.Therefore,an orthogonal experiment with different fermentation conditions was designed to enhance xynA expression in E.coli.The optimal conditions were determined as 0.1 m M IPTG induction,20℃ induction temperature,and an OD₆₀₀ value of 0.5,resulting in a maximum xynA activity of 15.6 U·mL^-1 at 36 h,an 83.5%improvement compared to the initial conditions.Using birchwood xylan as a substrate,the determined Km and kcat/Km values were 3.45 g·L^-1 and60.30 L·g^-1·s^-1,respectively,with a specific activity of 175.1 U·mg^-1.These properties were similar to those of xynA expressed in A.niger.（2）HFR modification to improve the thermostability of xylanase.The root mean square fluctuation（RMSF）values of xynA were calculated using NAMD software,and HFRs I-IV were delineated.Then,the saturated mutations were performed on 19 amino acids in HFRs I and II.The combinatorial mutant G92F/G97S/G100K in HFR I exhibited a residual activity increase of 44.8%,while the combinatorial mutant T121V/A124P/I126V/T129L/A130N in HFR II showed an 8.4-fold increase in residual activity compared to xynA.Subsequently,segments corresponding to N-and C-termini of xynA were replaced with those from Nesterenkonia xinjiangensi xylanase and Ev Xyn11^TS,respectively,resulting in the mutant EV-xynA-NX with 95.3%residual activity after treatment at 60℃ for 5 min.Introducing N-glycosylation modification in HFR IV yielded the optimal mutant A55N/D57S/S61N,with94.5%residual activity after incubation at 70℃ for 5 min.The combination of the best mutants from the four HFRs resulted in the mutant xynAm1,which exhibited a half-life of2523.5 min and a specific activity of 402.7 U·mg^-1 at 50℃,representing a 137.6-fold and 1.3-fold increase compared to xynA,respectively.Molecular dynamics（MD）analysis indicated significantly lower root mean square deviation（RMSD）values for xynAm1.Additionally,the number of hydrogen bonds,hydrophobic interactions,and electrostatic interactions in xynAm1 increased by 15.8%,17.3%,and 66.7%,respectively,compared to xynA.（3）Enhancement of xylanase activity on insoluble xylan degradation by fusing CBM.The CBM9-2 from Thermotoga maritima was fused to the N-and C-termini of xynAm1 and expressed in E.coli.The specific activity of C-X was 19.0 U·mg^-1,which was 2.3-fold and1.1-fold higher than that of xynAm1 and X-C,respectively.Then,the addition of a flexible linker（GGGGS）₂ between the two domains resulted in the optimal fusion enzyme C-F₂-X.When the substrates were insoluble beechwood xylan and sugarcane xylan,the k_cat/K_m values of C-F₂-X were 4.2-fold and 8.5-fold higher than those of xynAm1,respectively,so that the catalytic efficiency of C-F₂-X for insoluble xylan was significantly enhanced.The structural comparison revealed that the distance between E379,the catalytic active center,and W176,the CBM-binding center,in C-F₂-X was 8（?）shorter than that in C-X,which might be more favorable for the insoluble substrate to bind to the enzyme and be catalyzed.Finally,6.67U/mL of C-F₂-X and cellulase were added to hydrolyze insoluble wheat bran,respectively,and were able to produce 143.23 n M reducing sugars after 24 h.Integration of the gene C-F₂-X into A.niger yielded the recombinant enzyme xynAm2,whose specific activity(34.06U·mg^-1)was 35.2%higher than that of C-F₂-X,and synergistic degradation of wheat bran with cellulase under the same conditions yielded 137.2 n M reducing sugars.（4）Integrated expression strategy to enhance xylanase expression in A.niger.Utilizing the CRISPR/HDR system,xynA was precisely integrated into the bgl locus to generate the recombinant strain Pb Sx,ensuring stable expression of xynA.Then,the optimization of expression elements（PglaA-Samy A）resulted in a 2.08-fold increase in xynA enzyme activity compared to Pb Sx,and the fusion of a 500-amino acid fragment from glaA at the N-terminus further elevated enzyme activity to 1253.8 U·mL^-1.To simultaneously integrate the optimized expression cassette into the bgl,amy A,and amm A loci,a multi-gene integration system combining visual gene editing and CRISPR/HDR was constructed.In the obtained alb A-deficient white transformants,xynA was successfully integrated into all three loci at a rate of23.5%,leading to an enhanced xynA expression of 1460 U·mL^-1.Using this integration system,codon-optimized gene sequences xynAm1’and xynAm2’were individually inserted into the A.niger genome,resulting in the recombinant strains RLM-xynAm1’and RLM-xynAm2’.The fermentation results of 5-L fermenters showed that RLM-xynAm1’and RLM-xynAm2’achieved the highest activities of 1034.6 U·mL^-1 and 125.6 U·mL^-1,respectively,demonstrating promising industrial application potential.