本研究采用黑曲霉Bdel4菌株为宿主菌，敲除glaA，aamA，An05g02100和An12g06930四个主要的胞外分泌蛋白，为核酸酶P1的表达和分泌提供通路。通过PCR扩增得到黑曲霉Bdel4的rDNA序列，构建了以rDNA序列为整合位点的重组质粒pRpT-nucP1-18S和pRpT-2A-nucP1-18S，将构建的重组质粒pRpT-nucP1-18S和pRpT-2A-nucP1-18S转入黑曲霉Bdel4。通过半荧光定量PCR进行转化子初筛，之后测定核酸酶P1的酶活复筛，选出一株高活性菌株用于工业生产。采用SYBR Green实时荧光定量PCR法对含外源目的基因nuc P1的总基因组样本重复检测，取平均值作为目的基因拷贝数，探究其与酶活表达水平之间的联系。结果显示含9个nuc P1基因拷贝数的菌株的酶活水平达到88.5 U/mL，较核酸酶P1单拷贝菌株增加了3.86倍，较之前研究中异源表达核酸酶产量提高1.20倍，再增加nuc P1的拷贝数时，酶活水平随着拷贝数的增加而降低。

In this study, the Aspergillus niger Bdel4 strain was used as the host strain, and the four major extracellular secretion proteins, glaA, aamA, An05g02100 and An12g06930, were knocked out to provide a pathway for the expression and secretion of nuclease P1. The rDNA sequence of A. niger Bdel4 was amplified by PCR, and the recombinant plasmids, pRpT-nucP1-18S and pRpT-2A-nucP1-18S were constructed through using the rDNA sequence as integration sites. The constructed recombinant plasmids pRpT-nucP1-18S and pRpT-2A-nucP1-18S were then transferred into A. niger Bdel4. Prescreening of the transformants was carried out by semi-quantitative fluorescent PCR, and the activity of nuclease P1 was then measured to screen a strain with high activity for industrial production. The total genomic samples containing the exogenous target gene of nuc P1 were repeatedly detected by SYBR Green real-time fluorescent quantitative PCR, and the average value was taken as the copy number of the target gene nuc P1 to explore the relationship between the copy number and the expression level of the enzyme. The result showed that the enzymatic activity of the strain containing 9 nuc P1 gene copies reached 88.52 U/ml, which was 3.86 times higher than that of the nuclease P1 for the single copy strain, and 1.2-fold as high as that of the heterologous nuclease P1 reported in the previous studies. When the copy number of nuc P1 was increased to over 9, the enzyme activity decreased.

国家自然科学基金项目（31871736；31870024）；广东省自然科学基金（2017A030313097）；广东省调味食品生物发酵先进技术企业重点实验室开放基金项目（2017B030302002）