| In previous studies,Aspergillus oryzae,a filamentous fungus,has been found to be an excellent strain with high transcriptional activity,strong protein secretion ability and high safety,and has great potential as a host for heterologous protein expression.However,so far,Aspergillus oryzae has not been used in heterologous protein expression.One of the reasons is that Aspergillus oryzae has four nuclei and it is difficult to make genetic modification.Meanwhile,Aspergillus oryzae has strong protein degradation ability,which is not conducive to the accumulation of heterologous protein products.In this paper,we will establish a gene knockout method for Aspergillus oryzae,and modify the degradation system of Aspergillus oryzae heterologous protein,so as to explore the potential of Aspergillus oryzae as a host for heterologous protein expression.The growth characteristics of Aspergillus oryzae of different strains were different.Therefore,13 strains of Aspergillus oryzae of different strains were screened for the purpose of finding a strain with low protease activity and good mycelium growth and development level.Strain 2035 was obtained by phenotypic comparison,protease activity deter mination and amylase activity deter mination.2035 was further modified and analyzed with strain 3.042 commonly used in our laboratory.In order to evaluate the heterologous protein expression performance of 2035 and 3.042 in the natural state,the heterologous protein FSH was expressed by 2035 and 3.042,respectively.By comparing the amount of FSH expression products and transcription level between the two,it was found that the transcription level of3.042 was much higher than 2035,but the amount of protein expression products had no obvious advantage.These results suggest that high transcriptional activity is not the only factor that enhances the production of alloprotein,but the degradation of alloprotein products may also be an important factor affecting the production of alloprotein.The non-protease degradation pathway of alloprotein is common in different strains of Aspergillus oryzae,in which vacuolar sorter protein receptor Vps10 can recognize alloprotein as misfolded protein and transport its translated product from Golgi apparatus to vacuole for degradation.Knockout of Ao Vps10 gene of 2035 and3.042 can prevent the translation products from being consumed in large quantities,thus increasing the yield.Therefore,in this paper,a CRISPR/Cas9 knockout system was first established to knockout Ao Vps10 gene of 2035 and 3.042.2035-ΔAo Vps10 and 3.042-ΔAo Vps10 were obtained.Subsequently,the protease genes alp A,pep A and tpp A in 3.042-ΔAo Vps10 were continuously knocked out using CRISPR/Cas9 knockout system by the way of labeling recovery,and 3.042-ΔAo Vps10Δalp AΔpep AΔtpp A strains were obtained.The protease activity was compared with that of wild 3.042,wild 2035 and engineering 2035-ΔAo Vps10.It was found that the protease secretion level of strain3.042-ΔAo Vps10Δalp AΔpep AΔtpp A decreased significantly,but its growth level was not affected.Finally,two strains with low protein degradation level were used to express heterologous protein PETaes to evaluate the performance of the strains,further confir ming that the destruction of Aspergillus oryzae protein degradation system can effectively improve the production of heterologous protein.In this study,a CRISPR/ Cas9-based gene knockout system for Aspergillus oryzae was established to address the two shortco mings of Aspergillus oryzae,namely the difficulty of genetic modification and the high level of heterologous protein degradation.Five genes were continuously knocked out in strain 3042 and two genes were continuously knocked out in strain 2035.It provides an efficient tool for Aspergillus oryzae gene modification in the subsequent application of heterologous expression of Aspergillus oryzae,and also provides a direction for the subsequent improvement of heterologous protein production. |
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