Construction Of Non-resistant Aspergillus Niger Expression System And The Expression Of Lipase

Aspergillus niger has been used for high-level production of a wide range of protein products.In this study,firstly we selected a strain of A.niger?CICC2169?with a lower protein background and Hygromycin B-sensitivity as the expression host.The methods of Agrobacterium-mediated transformation and PEG-protoplast transformation were optimized.Then,the homologous and heterologous lipases were expressed in A.niger.Improvement of A.niger lipase?ANL?expression was conducted in the level of transcription and post-transcription.ANL was purified and characterized.Finally,the CRISPR/cas9 technology was applied in the A.niger expression system,of which a uracil-deficient strain was constructed.The main studies and innovative results are listed as follows:?1?The methods of Agrobacterium-mediated transformation and PEG-protoplast transformation were optimized.The optimum conditions for Agrobacterium-mediated transformation of A.niger are10⁷?mL^-11 non-germinated fresh spores and OD₆₀₀ of Agrobacterium cultured to 0.9-1.0 in a ratio of1:1,and the IM plate with acetosyringone concentration of 200?mol·L^-1.The mixed cells were then transfected at 23 ^oC for 48 h,and the number of transformants reached 60±5 transformants in 10⁶spores,and the positive rate reached over 90%.The preparation conditions of PEG-protoplasts are10⁷?mL^-1 A.niger spores were cultured with PD at 30 ^oC and 200 r·min^-1 for 24 h,collected the mycelium and the lyase(0.8 mol·L^-1 MgSO₄,1%cellulase,0.5%snail enzyme,0.25%lysozyme)was added in a ratio of 1:10?m/v?.Protoplasts were collected after enzymolysis at 37 ^oC and 120r·min^-1 for 2.5 h,the number of protoplasts was about 6.8×10⁵ cells?mL^-1.The regeneration rate was about 63±2.5%.?2?The homologous and heterologous proteins were expressed in A.niger.The homologous Aspergillus niger lipase?ANL?was successfully expressed while the heterologous Rhizopus chinensis lipase RCL was not expressed in CICC2169.However,the expression level of ANL was low.Promoter transcription level analysis showed that the inducible promoter glaA transcription level was increased by 50.35-fold while the constitutive promoter gpdA transcription level was increased by994.86-fold compared to wild type.Three ways were used for the improvement of the expression of ANL,including supplement with introns?improve the stability of the mRNA?,add the kozak sequence?enhance the translation efficiency?,signal peptides optimization?enhance the efficiency of extracellular secretion of the protein?.The transformants enzyme activity of glaS-ANL1000 were about 75.80 U?mL^-1 and the transformants enzyme activity of koglaS-ANL were about 61.33 U?mL^-1while the initial transformant?glaS-ANL?could not detect the enzyme activity.Different signal peptides have different effects on expression,which is cbh?signal>ANL signal>glaA signal.Integration of three strategies,the best transformant was kocbhS-ANL1000 with a maximum enzyme activity of 314.67 U?mL^-1.Under the premise of containing the kozak sequence,compared to the ANL transformants,the expression of ANL1000 transformants with glaA,ANL,and cbh?signal peptides were increased by 1.64-fold,1.94-fold,and 3.61-fold.Enzymatic Characterization studies showed that the optimum pH of ANL was 3.0 and the optimum temperature was 45 ^oC.?3?The CRISPR/cas9 gene editing technology was applied in A.niger,and the CRISPR/cas9plasmid was transformed into A.niger by the protoplast transformation.The sgRNA targets the pyrG gene,and the transformant was screened to obtain?pyrG strain.The mutation site was determined by sequencing and located in the sgRNA region.The targeting efficiency is about 25%.The sgRNA targets the hyg gene attempts to edit the hyg gene by CRISPR,but no positive transformants were obtained.