## 2 _Aspergillus_ Is an Expression Host for Heterologous Protein Production  
### A. niger  
_A. niger_, as an indispensable industrial strain, has been extensively exploited for homologous and heterologous expressions of many protein products used in food, washing, textile, paper, and other industries [23] due to its high protein secretion efficiency, unique safety characteristics, and low-medium costs [24]. On the one hand, the genome of _A. niger_ contains an extensive repertoire of genes encoding carbohydrate active enzymes (CAZymes) [25], making it an ideal host for CAZyme production and homologous expression. For example, [PERSON] et al. used the tunable Tet-on system to control the _glaA_ gene encoding glucoamylase (GlaA) and generated the _\(\Delta\)racA_ strain via _racA_ gene deletion, leading to a 4-fold increase in GlaA secretion compared to the parental strain [26]. Moreover, [PERSON] et al. constructed gpdA promoters with one to four copies of the gpd box (PgpdA,PgpdA2B, PgpdA3B, PgpdA4B) and used them to regulate \(xynB\) expression in \(A\). _niger_. The strain containing three copies of gpd box (PgpdA3B) demonstrated the highest xylanase activity (3588.38 U/mL), protein expression, and transcription efficiency [27]. Furthermore, [PERSON] et al. cloned two \(\beta\)-glucosidase (BGL) genes from marine \(A\). _niger ZJIBE-1_ and then inserted them into the host genome for constitutive homologous expression. By leveraging the gpdA promoter to drive stable gene expression independent of inducers, they achieved directed expression of BGLs [28]. Similarly, [PERSON] et al. used the strong constitutive gpdA promoter to overexpress the transcription factor gaaR in \(A\). _niger_, enabling constitutive transcription of pectinase-encoding genes, D-galacturonic acid (GA) transporter genes, and catabolic pathway enzymes even under non-inducing conditions [29]. [PERSON] et al. cloned the tannase gene \(tan7\) from \(A\). _niger SH-2_ and overexpressed it in the low-background secretory strain \(A\). _niger Bdel4_ using the glaA promoter, achieving a peak tannase activity of 111.5 U/mL at 168 h with over 70% purity in the supernatant [30]. In addition, \(A\). _niger_ has successfully enabled the homologous expression of amylases, cellulases, and other CAZymes, facilitating their large-scale industrial production [31; 32]. On the other hand, \(A\). _niger_ has also been used as a cell factory to express some heterologous proteins, including lysozyme [33], human lactoferrin [34], chymosin [35], thaumatin [36], lipase [37], and nuclease P1 [38]. However, achieving the efficient expression of most heterologous proteins is still difficult [39]. In recent years, driven by the rapid advancement and widespread application of gene editing technology, the CRISPR/Cas9 system has been effectively employed for genetic manipulation in \(A\). _niger_[40]. For example, this technology enabled a significant enhancement in trehalose MthT production from _Thermothelomyces thermophilus_, with activity reaching up to 1698.83 U/mL [41]. Moreover, a CRISPR/Cas9-mediated multi-copy expression system for an alkaline serine protease from \(A\). _oryzae_ was successfully established in \(A\). _niger_, yielding a protease activity of 11,023.2 U/mL and a protein concentration of 10.8 mg/mL [42].