该研究探讨了将嗜碱顶孢霉中的GH25溶菌酶基因进行密码子优化后，构建包含糖化酶信号肽的溶菌酶单拷贝和双拷贝表达载体并均由启动子PnaⅡ控制表达，利用双拷贝表达载体和CRISPR/Cas9基因编辑工具，使用PEG介导法转化无孢黑曲霉宿主HL-1，获得高酶活力的溶菌酶表达菌株。重组菌株LyAa-C的溶菌酶活力达到2 291.37 U/mL，是仅使用双拷贝表达载体的LyAa-F菌株酶活力（869.74 U/mL）的2.63倍，是仅使用传统单拷贝表达载体的LyAa-T菌株酶活力（358.41 U/mL）的6.39倍。通过6×His标签使用镍柱亲和层析成功纯化了重组溶菌酶LyAa，并对其酶学性质进行了分析。溶菌酶LyAa蛋白大小约为25.0 ku，最适pH值为4.0，最适温度为30 ℃，并显示出良好的胃蛋白酶稳定性。综上，该研究基于双拷贝载体和CRISPR技术成功优化和提高了溶菌酶在黑曲霉中的表达。

Single- and double-copy expression vectors of lysozyme-containing glucoamylase signal peptide, controlled by promoter Pna II, were constructed following codon optimization of the GH25 lysozyme gene derived from Acremonium alcalophilum. The double-copy expression vector and CRISPR/cas9 techniques were applied. Lysozyme expression strains with high enzyme activity levels were obtained through PEG-mediated transformation of Aspergillus niger host HL-1. The lysozyme activity of the recombinant LyAa-C strain reached 2 291.37 U/mL, which is 2.63 times that of the LyAa-F strain transformed by double-copy expression vector only (869.74 U/mL) and 6.39 times that of the LyAa-T strain transformed by traditional single-copy expression vector only (358.41 U/mL). The recombinant lysozyme LyAa was purified with 6×His-tagging through nickel affinity chromatography, and its enzymatic properties were analyzed. The purified lysozyme LyAa had a size of 25.0 ku, an optimal pH value of 4.0, an optimal temperature of 30 ℃, and exhibited good pepsin stability. In conclusion, this study successfully optimized and improved the lysozyme expression in Aspergillus niger using double-copy vector and CRISPR systems.

国家重点研发计划项目（2021YFC2100200）