Establishment And Optimization Of Agrobacterium Tumefaciens-mediated Aspergillus Niger Exogenous Gene Expression System

Aspergillus niger,an important industrial fermentation microbial strain,contains various enzymes with excellent properties.In addition,being an excellent carrier for active proteins in eukaryotic cells,it has advantegous properties that other expression systems such as E.coli cannot fulfill.Therefore,A.niger is valueble in many industry fields,such as feed,medicine and other fields.However,traditional genetic transformation methods have low transformation efficiency and genetic stability for A.niger,which greatly limits the development of filamentous fungal expression systems.In this study,an efficient A.niger genetic transformation system was established via the Agrobacterium tumefaciens-mediated transformation,and A.niger CICC2629 was selected as the most promising host strain after evaluating the growth rate and hygromycin resistance.Subsequently,different promoters which induced the expression level of the enhanced green fluorescent protein(e GFP)were screened on the basis of A.niger CICC2629 expression host,and gpd A was identified as the most promising promoter.The glucanase gene was expressed in A.niger CICC2629 with the selected promoters,and its enzymatic properties were determined and compared.Finally,the effect of the eukaryotic sequence Kozak sequence(GCCACC)on the translation level of A.niger protein was preliminarily explored.The results of the study are as follows:1.Comparing the growth rate and hygromycin sensitivity of 8 strains of A.niger,CICC2629 was selected as the most suitable host strain,and the hygromycin phosphotransferase gene was successfully integrated into the host strain's genome using ATMT technology.The optimized ATMT transformation conditions were:A.tumefaciens AGL-1 was selected,the AS concentration was set as 200?mol/L,the A.niger spore concentration was 5.0×10⁶/m L-1.0×10⁷/m L,cellophane was used as the co-culture medium,and the co-culture medium was placed at 22°C kept away from light.After culture for 48 h,the maximum number of transformants can be 75±5.The highest positive rate is 93.9%,and the average remains above 89.92%.The transformation efficiency increased 2.03-fold after pre-optimization.The hygroscopic mold can still be detected for 5 consecutive passages.The stability phosphotransferase gene remains.2.Gpd A,Gla A,Tox A promoters were applied to drive the expression of e GFP gene in A.niger.Based on the analysis of the pixel values of the fluorescence intensity of the transformants,the expresion level of the e GFP induced by three promoters are:gpd A>gla A>Tox A.Meanwhile,Quantitative Real-time PCR technology was used to detect the relative expression of e GFP gene.It is discovered that the expression induced by gpd A promoter was 3.26-time and 4.82-time higher than that of gla A and Tox A,respectively.3.Glucanase(An-Glu)derived from the rumen fungus(Piromyces sp)induced by gpd A and gla A were tested in A.niger.Activity of the An-Glu is 74.43 U/m L,the optimum temperature is 60?,and the optimum p H is 6.5.63.1%and 49.4%of its initial activity can be retained after 1 h fermatation at 37?and 50?,respectively.The half-life of An-Glu are 15 min and 7 min at 60?and 65?,respectively.Higher than 66.1%of its initial activity can be retained after incubation in a buffer with a p H value of 3.0-11.0 for 1 h.?-mercaptoethanol,DTT,and glycerol have the strongest enhancement effects on An-Glu,which are 1.94-time,1.91-time and 1.79-time higher than the the untreated group.Li⁺,Tween80,Triton X100,acetic acid,ethanol,methanol,PEG4000,ethyl acetate,and urea also improve An-pi Glu activity.However,Ag⁺and Hg²⁺have a strong inhibiting effect on An-Glu.When adding Kozak segment,the expression level of gla A-An-Glu and gpd A-An-Glu were up-regulated by 1.71-fold and 1.50-fold compared with the control group,indicating that the Kozak sequence improve the expression level of genes in the A.niger system.4.Protein expression level was increased by 5.05-time compared with the initial fermentation conditions after optimizing the fermentation conditions of A.niger.Fermentation conditions was adjust to:temperature is 30?,fermentation time is 168h,fresh spore suspension with a concentration of 1.0×10⁷/m L is inoculated at 3%.When the carbon source was selected as Microcrystalline cellulose,Vitamin C,Xylan,Soybean cake powder is selected as the nitrogen source,the protein expression levels were significantly increased.In summary,the above research results are helpful to establish a stable and efficient filamentous fungus expression system,and provide references for the study of filamentous fungus genetic transformation,protein expression,and fermentation technology.