Efficient Expression Of Fungal-derived Lysozyme In Aspergillus Niger

Lysozyme(EC3.2.1.17)hydrolyzes the β-1,4 glycosidic bond between the peptidoglycan N-acetylmuramic acid and N-acetylglucosamine resulting in the bacteria die.Lysozyme is not only a natural but also an important non-specific immune factor,which is widely used in food,biomedicine,feed,and other fields.Fungi lysozymes may have the advantages of acid resistance and high-temperature resistance that commercially available lysozymes do not have.The research on lysozyme mainly focuses on egg-white lysozyme and human lysozyme.There are few reports on fungal lysozyme,and the yield is relatively low.The low-background Aspergillus niger HL-1 constructed by our research group died prematurely in the fermentation medium,resulting in a significant decrease in the yield of the target protein.After comparison,it was found that the knockout of the Glucoamylase Gene made the host unable to use the carbon source material in the fermentation medium and die.After supplementing gla A,the host can grow normally in the fermentation medium,which meets the conditions of industrial stable fermentation.Then three fungal lysozyme genes with the potential application were selected to express and screen in Aspergillus niger,and the highly active lysozyme gene Ly Aa was obtained.The high expression strategy of the double copy expression vector with the CRISPR/cas9 gene-editing tool was used to improve the yield.After the fermentation broth was purified by nickel column affinity chromatography,the enzymatic properties of lysozyme were characterized.The main research contents are as follows:(1)The glucoamylase promoter Tgla A,glucoamylase gla A,and linearized universal vector Gp were connected into an expression vector,transformed into Aspergillus niger HL-1,and successfully expressed glucoamylase.In the fermentation medium,the original host could not effectively use the starch carbon source,and the bacteria began to break and die on the third day of fermentation;When the carbon source is replaced by glucose,the growth can return to normal,and the expression of foreign protein will decrease significantly.After transformation and optimization,the host still has a high bacterial volume from the 6th day of fermentation,and the mycelial growth state is normal,which meets the conditions of industrial stable fermentation.(2)The hybrid promoter Pna II and different lysozyme genes were respectively connected with the linearized universal vector UEV to construct three expression vectors of different lysozymes.After transformation into Aspergillus niger HL-1,the GH25 enzyme system with high enzyme activity lysozyme Ly Aa from Acremonium alcalophilum.The highest enzyme activity of the fermentation supernatant of the corresponding transformant Ly Aa-T was 358.41U/m L.(3)Another expression frame composed of hybrid promoter Pna II,lysozyme Ly Aa,and glucoamylase Pgla A terminator was inserted into the lysozyme single copy expression vector and inversely connected with the original copy to obtain double copy lysozyme Ly Aa expression vector.Two copy transformant Ly Aa-F and four copy transformant Ly Aa-C were obtained by transformation alone and using the CRISPR/cas9 tool.The highest enzyme activity of Ly Aa-C fermentation supernatant was 2291.37 U/m L,2.63 times that of Ly Aa-F(869.74U/m L),and 6.39 times that of Ly Aa-T(358.41 U/m L).SDS-PAGE showed that the target band(25.0 k Da)of Ly Aa-C fermentation supernatant was the most concentrated.Lysozyme Ly Aa was purified by nickel column affinity chromatography.The enzymatic properties showed that the optimum temperature was 30℃,and it had good thermal stability below 70℃;When the p H value is 3.5～5.0,the property is stable;Compared with egg lysozyme,lysozyme has good pepsin stability and has good application potential in the field of feed.The efficient expression of lysozyme was successfully realized by using multi-copy and CRISPR/Cas9 technology,and the related enzymatic properties were characterized.