中国生物工程杂志

Pectinase preparations have been widely used in China. However, efficient and specific pectinases are still lacking in the market. Producing a single-component pectinase using modified Aspergillus niger by genetic engineering technology has become an effective solution to meet the growing industrial demand of pectinase. Constructing an efficient CRISPR-Cas9 technology can provide an efficient genome editing tool for the construction of Aspergillus niger chassis strain with high yield single pectinase. First, the pyrG gene on the genome of pectinase-producing Aspergillus niger was knocked out to construct the uracil auxotrophic strain AnΔpyrG, and the Cas9 expression cassette and pyrG expression cassette were integrated precisely at the pyrG site of the AnΔpyrG strain to construct AnCas9 strain constitutively expressing the Cas9 gene. Then, the pLM2-sgRNA plasmid which contained the gpdA promoter, hammerhead ribozyme, and HDV ribozymes for the efficient expression and maturation of sgRNA was constructed. And finally, the CRISPR-Cas9 gene editing system is successfully established. The 4978020 and the 4983861 gene were used to detect the gene editing efficiency of the constructed CRISPR-Cas9 system, and the phenotypic changes and enzyme production changes of the 4978020 gene function deletion strain were detected. The results showed that the pectinase gene editing efficiency is more than 50%, the phenotype and pectinase activity of the strain AnΔ4978020 had no significant changes compared with the original strain. An efficiency CRISPR-Cas9 system was successfully constructed in Aspergillus niger producing pectinase, and losing of the 4978020 gene function did not affect the phenotype of the strain, which laid the foundation for the construction of high yield single pectinase Aspergillus niger chassis strain.