| A.niger is a safer and most widely used filamentous microorganism in industry,it can better secretion of small molecule compounds and proteases when compared with other filamentous microorganisms,such as A.oryzae and A.nidulans.A.niger usually used as a cell factory for the production of citric acid and gluconic acid,and becomes one of the most important hosts for food enzyme production.The A.niger NG1306 isolated and preserved in our laboratory can secrete a variety of glycoside hydrolases and can efficiently transform ginsenosides,but the difficulty of genetic manipulation and insufficiency of expression strategies severely limited the further optimization of this strain.To solve this problem,this study constructed an effective genetic manipulation tool to domesticate A.niger NG1306 and improve its gene editing efficiency.The CRISPR-Cas9 system is simple and efficient to operate,and is widely used for editing of genes.In order to improve homologous recombination ability,this research used the modified CRISPR-Cas9 as a gene editing tool to construct a chassis strain and introduced the recombinant xylosidase gene(Xln D)to verify the efficiency of homologous recombination.The specific methods were as follows:(1)a 5s r RNA sequence was constructed as a g RNA promoter and fused to the Cas9 expression plasmid p FC332 to improve the targeting efficiency,this recombinant plasmid was introduced into A.niger NG1306 by PEG-mediated transformation of protoplasts for knocking out the melanin phenotype.The targeting efficiency of CRISPR-Cas9 was verified through transformant phenotype and molecular identification.(2)Used the modified CRISPR-Cas9 system to improve homologous recombination efficiency of A.niger NG1306,and constructed a chassis strain(?ku70::amdS)that temporarily lacked non-homologous recombination ability;The method was to use seamless cloning in E.coli to recombine amdS and homologous arm sequences that targeted the ku70 editing site to construct a circular gene targeting substrate,and used PEG-mediated protoplast transformation to complete the transformation,then screened the target transformants.(3)In order to verify the homologous recombination efficiency of the A.niger NG1306(?ku70::amdS),Xln D derived from A.niger NG1306 was recombined with the promoter,signal peptide,and kozak sequence and introduced into the alb A site in order to improve target protein expression and exocrine capacity.In this paper,three different recombinant expression cassettes Re Xln D-1,Re Xln D-2,and Re Xln D-3 were constructed in E.coli.The experimental group expressed the Re Xln D-3 in the A.niger chassis strain(?ku70::amdS)and the control group expressed Re Xln D-3 in the original strain by CRISPR-Cas9 system.In this paper,the construction of 5s r RNA sequence as g RNA promoter can improve the targeting efficiency.When alb A was knocked out,the targeting efficiency reached to100%,which proved that the CRISPR-Cas9 tool can be used to domesticate the NG1306 strain;When using the CRISPR-Cas9 tool to construct the A.niger NG1306(?ku70::amdS)chassis strain that temporarily lacked non-homologous recombination ability,the targeting efficiency was 20%;When verified the homologous recombination efficiency of the A.niger NG1306(?ku70::amdS),the experimental group failed to recover the resistance plasmid used for chassis modification,resulting in the same resistance could not be edited for the second time,therefore,no mutants were produced.The control group can homologously recombine Re Xln D-3 at the target site with a knock-in efficiency of 5%.This article successfully used CRISPR-Cas9 as a chassis domestication tool,completed the knock-out and knock-in of target genes,and laid the foundation for the engineering application of NG1306.But it is still necessary to improve the efficiency of homologous recombination of chassis strains. |
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