Expression Of Heterologous Proteins In Aspergillus Niger ATCC 20611 Using The β-fructofuranosidase Gene Promoter PfopA

Aspergillus niger ATCC 20611 is a major industrial strain for fructooligosaccharides synthesis.Its secretes β-fructofuranosidase(FopA)that is the key enzyme responsible for the conversion of sucrose to fructooligosaccharides(1F-fructooligofructoses).The FopA is the major secreted protein of A.niger ATCC 20611 and its native promoter(PfopA)is a strong promoter,which is induced by sucrose.In the process of catalyzation by FopA for the conversion of sucrose to fructooligosaccharides,the proportion of fructooligosaccharides is generally about 55.00%.Increasing the purity of fructooligosaccharides has always been an important direction for the improvement of A.niger ATCC 20611 strains.At the same time,sucrose is a cheap and easily available carbon source,so the development of this strain as a protein expression system also has important application value.Therefore,making full use of the PfopA promoter for strain improvement will help to enhance the yield of fructooligosaccharides and establish a high-value protein expression system.This study takes A.niger ATCC 20611 as the original strain,with the attempt of establishing a heterologous protein expression system using its native promoter PfopA.At first,the 6G-β-fructofuranosidase gene(Xd-INV)from Xanthophyllomyces dendrorhous was expressed in A.niger ATCC 20611 using PfopA for strain improvement.Then,PfopA was further applied to express the glucose oxidase(GOX)and the peroxidase(POD)in A.niger ATCC 20611 to consume the by-product glucose for enhancing the content of fructooligosaccharides.Finally,PfopA was further used to express the aminal-derived antimicrobial peptides in the A.niger ATCC 2061 1.1.The Xd-INV was expressed in A.niger ATCC 20611 using PfopA.Neokestose(6GFOS)is synthesized by 6G-β-fructofuranosidase(Xd-INV)from X.dendrorhous.The probiotic properties and chemical stability of neokestose are better than that of fructooligosaccharides(1F-FOS).Firstly,the gene replacement strategy was used to express the Xd-INV gene with the replacement of the genomic site of the A.niger ATCC 20611 fopA gene by homologous recombination.On the one hand,the transformant IFC was constructed by fusing Xd-INV with the FopA C-terminal domain(cell wall anchoring).On the other hand,the Xd-INV was fused with the A.oryzae cell wall anchoring protein MP1 to construct the transformant IMP.Both of them successfully localized Xd-INV on the cell wall.The detection and analysis of PCR and glucose plate showed that IFC and IMP were successfully constructed,and the highest enzyme activity at the time of fermentation reached 146.53 U/g and 90.93U/g.Neokestose synthesis experiments found that the yield of IFC strains was higher than that of IMP strains,and the content was 19.08%.In order to further enhance the fructooligosaccharides-synthesis efficiency,the Xd-INV gene expression cassette was used to genet transform using a random insertion genomic manner.The transformants retained the original FopA while expressing Xd-INV,which can produce fructooligosaccharides(1F-FOS)mixture with neokestose(6G-FOS).The conversion strains of the FopA C-terminal domain were named IFCF,while the conversion of MP1 were named IMPF.Fructooligosaccharides synthesis experiments have found that the yield of IFCF strains have reached 62.59%,while IMPF strains have also reached 58.30%.Therefore,the expression of Xd-INV in A.niger ATCC 20611 using PfopA was successful,meanwhile optimized fructooligosaccharides ingredients.2.Using PfopA to express glucose oxidase(GOX)and peroxidase(POD)in A.niger ATCC 20611.30%by-product glucose was generated during fructooligosaccharides synthesis.And glucose can be consumed by the transformants that using PfopA to express GOX and POD,which helps to increase fructooligosaccharides purity.Firstly,the GOX expression cassette was constructed by the PfopA promoter,the fopA signal peptide and the gox gene(from A.niger ATCC 1015).The original strain were A.niger ATCC 20611 and A.niger △fopA,named GOD and DGOD,respectively.The GOX viability was mainly in the fermentation liquid,and the transformant DGOD strain enzyme activity(22.97 U/g)was higher than the transformant GOD(6.05 U/g).In order for localized GOX on the cell wall,which was also possible to optimize fuctooligosaccharides synthesis,the C-terminal domain of FopA was fused with the gox gene,named GOF.GOX viability of GOF was mainly present in the cell wall up to 29.30 U/g,and the purity of the synthetic fructooligosaccharides product was increased from 50.10%of the original strain to 60.63%.Then,PfopA was used to express the POD from the horseradish.And to connect the pod gene with the native proteinencoding gene fopA using kex2 to ensure the expression and secretion of peroxidase.The starting strain was A.niger AfopA and the transformed strain was named DPOF.DPOF appeared red halo around the colonies of glucose detection plate,while the departure strain AfopA did not appear halo,and the fermentation of POD was 5.10 U/g,and its FOPA activity had reached 252.60 U/g.This showed that the FopA and POD fusion expression under the PfopA promoter were successful.Further,the POD was expressed by fused of the C-terminal domain of the FopA to make the POD in the cell wall using PfopA.The expression cassette was transformed into A.niger ΔfopA,and the transformant was named DPOC.DPOC mycelial POD activity could reach 7.70 U/g.The DPOC strain and the GOF strain were used to synthesis fructooligosaccharides experiments,and results showed that the purity of fructooligosaccharides reaches 71.26%.In addition,this study also conducted an attempt to express GOX and POD under the promoter PfopA.The GOX expression cassette(containing only fopA signal peptide)and a POD-FopA fusion expression cassette were converted to A.niger ΔfopA,and the conversion strain was named DGP.GOX and POD enzyme activity were detected in the fermentation of transformants,were 49.24 U/g and 3.73 U/g respectively.The extracellular secretion type GOX and POD laid the foundation for the optimization of the enzyme to purify fructooligosaccharides.In summary,this chapter used PfopA to successfully express the GOX and POD of different secretion methods,which achieved the improvement of the A.niger ATCC 20611 and promoted the synthesis of fructooligosaccharides.3.Antimicrobial peptides were expressed in A.niger ATCC 20611 using the PfopA.Firstly,the expression cassette of the three antimicrobial peptides(LFcinB,LFcinB3,Cecropin AD)were constructed by the PfopA promoter and the fopA signal peptide sequence.Then transformed them to the A.niger ATCC 20611,the transformants were named LFB,LFB3 and CAD,respectively.Three types of transformants had been verified successfully.At the same time,three antimicrobial peptides are also fused with fopA in order to facilitate transformants seletting and protein secretion.Then A.niger ΔfopA was directly transformed.The LFcinB transformants were named LKF(fusion protein sequence was LFcinB-FopA)and FKL(fusion protein as FopA-LFcinB).The LFcinB3 transformants were named L3KF(fusion protein was LFcinB3-FopA)and FKL3(fusion protein was FopA-LFcinB3).The Cecropin AD transformants were named CAKF(fusion protein as Cecropin AD-FopA)and FKCA(fusion protein was FopA-Cecropin AD).The transformants were also verified successfully by screening of PT,glucose detection plate and genomic PCR analysis.Finally,4 transformants,4 transformants and 3 transformants of LFcinB,LFcinB3,Cecropin AD correlation were obtained.These transformants expressing antimicrobial peptides using PfopA were initially constructed successfully,which laid the foundation for the subsequent analysis of antimicrobial properties.