利用CRISPR-Cas9技术失活黑曲霉中果胶酶基因及突变株性能评价*

我国果胶酶制剂使用广泛但专一性不高,高效、专一的果胶酶制剂在市场上仍然匮乏。利用基因工程技术改造果胶酶生产菌株——黑曲霉来生产单一成分的果胶酶成为解决果胶酶应用需求的一种有效方案。构建一种高效的CRISPR-Cas9基因编辑技术,可为构建高产单一性果胶酶的黑曲霉底盘菌株提供有效的基因编辑工具。首先敲除产果胶酶黑曲霉基因组上的pyrG基因构建尿嘧啶营养缺陷型菌株AnΔpyrG,并在AnΔpyrG菌株的pyrG基因位点定点整合Cas9基因表达盒和pyrG基因表达盒,构建组成型表达Cas9基因的黑曲霉菌株AnCas9,再构建含有gpdA启动子、锤头结构核酶、HDV核酶的稳定性表达sgRNA的pLM2-sgRNA质粒,建立CRISPR-Cas9基因编辑体系。利用该技术失活AnCas9菌株中的2个聚半乳糖醛酸酶基因4978020和4983861来检测构建的CRISPR-Cas9基因编辑效率并检测4978020基因功能缺失菌株的表型变化和产酶变化,结果表明果胶酶基因编辑效率大于50%,AnΔ4978020的表型和果胶酶酶活性与出发菌株均无明显变化。在黑曲霉中成功构建了高效的Cas9基因编辑技术,4978020基因功能缺失也不影响菌株表型,为构建高产单一性果胶酶黑曲霉底盘菌株奠定基础。

CRISPR/Cas9-mediated Inactivation of the Pectinase Gene in Aspergillus niger and Evaluation of the Mutant Strain

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.