为了进一步提高木聚糖酶（Xylanase, XynB）的分泌表达量，该研究以黑曲霉内源高表达的糖化酶（Glucoamylase, GlaA）位点作为基因整合靶点。基于glaA基因六拷贝强启动子，分别利用糖化酶信号肽SglaA和酸性蛋白酶B（Acid Protease B, PepB）信号肽SpepB调控XynB的表达与分泌，并探讨缺失高背景蛋白酸稳定ɑ-淀粉酶（Acid-stable Amylase, AsAA）对XynB分泌的影响。结果显示，重组菌株TgX、TpX、ΔAgX和ΔApX在约22 kDa处均显现出明显的XynB蛋白条带。其中，菌株ΔApX的木聚糖酶活性（21413.74 U/mL）较菌株TgX、TpX及ΔAgX提高了70.67%、14.92%和35.42%。信号肽对xynB基因及UPR标志基因的转录水平无显著影响。缺失高背景蛋白AsAA能够提升xynB基因的转录水平，促进重组蛋白质的折叠与分泌，从而改善内质网应激。综上，该研究基于高效分泌信号肽和敲除高背景蛋白成功优化和提高了XynB在黑曲霉中的表达，为后续持续提升XynB的研究奠定了基础。

To further enhance the secretion and expression of xylanase (Xylanase, XynB), the locus of glucoamylase（Glucoamylase, GlaA）with endogenous high-expression of Aspergillus niger was utilized as the target site for gene integration. Based on the strong promoter of the six copies of the glaA gene, the expression and secretion of xynB were regulated by the signal peptide SglaA from glucoamylase and the signal peptide SpepB from acid protease B (Acid Protease B, PepB), respectively. Additionally, the impact of deleting the high-background protein acid-stable alpha-amylase （Acid-stable alpha-amylase, AsAA）on XynB secretion was also investigated. The results indicated that the recombinant strains TgX, TpX, ΔAgX, and ΔApX all displayed distinct XynB protein bands at approximately 22 kDa. Notably, the xylanase activity of strain ΔApX (21413.74 U/mL) was elevated by 70.67%, 14.92%, and 35.42% compared to strains TgX, TpX, and ΔAgX, respectively. The signal peptide exerts no significant effect on the transcription levels of the xynB gene and UPR marker genes. The absence of high-background protein AsAA can enhance the transcription level of the xynB gene and facilitate the folding and secretion of the recombinant protein, threrby alleviating endoplasmic reticulum stress. In summary, this study successfully optimized and enhanced the expression of XynB in Aspergillus niger through the utilization of efficient secretion signal peptides and the deletion of high-background proteins, thereby establishing a foundation for further continuous improvement of XynB.

中国博士后科学基金