Ammonium is a source of fermentable inorganic nitrogen essential for the growth and development of filamentous fungi. It is involved in several cellular metabolic pathways underlying nitrogen transport and assimilation. Ammonium can be transferred into the cell by an ammonium transporter. This study explored the role of ammonium transporters in nitrogen metabolism and cell biomass production in Aspergillus oryzae strain BCC 7051. Specific sequences encoding ammonium transporters (Amts) in A. oryzae were identified using genomic analysis. Four of the identified ammonium transporter genes, aoamt1-aoamt4, showed similarity in deduced amino acid sequences to the proteins in the ammonium transporter/methylammonium permease (AMT/MEP) family. Transcriptional analysis showed that the expression of aoamt2 and aoamt3 was ammonium-dependent, and was highly upregulated under ammonium-limited conditions. Their functional roles are characterized by genetic perturbations. The gene disruption and overexpression of aoamt3 indicated that the protein encoded by it was a crucial ammonium transporter associated with nitrogen metabolism and was required for filamentous growth. Compared with the wild type, the aoamt3-overexpressing strain showed superior growth performance, high biomass yield, and low glucose consumption. These results shed light on further improvements in the production of potent bioproducts by A. oryzae by manipulating the ammonium uptake capacity and nitrogen metabolism.

An optimal culture medium and highly stable biometabolites are important in industrial production processes. The response surface methodology with a Box–Behnken design was performed to determine the optimal culture medium of an engineered Aspergillus oryzae strain for cordycepin production by submerged fermentation. The influences of glucose, yeast extract, and adenine concentrations on cordycepin production were explored, and their concentrations were used for experimental design. The results reveal that the optimal culture components involved 30.0 g/L of glucose, 9.8 g/L of yeast extract, and 1.5 g/L of adenine. As predicted, the maximum cordycepin concentration (1724.53 ± 18.30 mg/L) was obtained with a short fermentation time (2 days). A significant increase in cordycepin yield (>50% increase) was observed in the culture grown in the optimized culture medium compared to that grown in the basal medium. A xanthine oxidase inhibitory activity assay demonstrated that the cordycepin product had a pharmacological function. It exhibited strong stability under high thermal and acidic conditions, with over 95% product recovery. The findings of this study are valuable for developing cost-effective processes for producing health-benefiting products.

Enzyme-mediated protein degradation is a major concern in industrial fungal strain improvement, making low-proteolytic strains preferable for enhanced protein production. Here, we improved food-grade Aspergillus oryzae BCC7051 by manipulating the transcriptional regulation of protease-encoding genes. Genome mining of the transcription factor AoprtR and computational analysis confirmed its deduced amino acid sequence sharing evolutionary conservation across Aspergillus and Penicillium spp. The AoPrtR protein, which is classified into the Zn(II)2-Cys6-type transcription factor family, manipulates both intra- and extracellular proteolytic enzymes. Our transcriptional analysis indicated that the regulation of several protease-encoding genes was AoPrtR-dependent, with AoPrtR acting as a potent activator for extracellular acid-protease-encoding genes and a likely repressor for intracellular non-acid-protease-encoding genes. An indirect regulatory mechanism independent of PrtR may enhance proteolysis. Moreover, AoPrtR disruption increased extracellular esterase production by 2.55-fold, emphasizing its role in protein secretion. Our findings highlight the complexity of AoPrtR-mediated regulation by A. oryzae. Manipulation of regulatory processes through AoPrtR prevents secreted protein degradation and enhances the quantity of extracellular proteins, suggesting the low-proteolytic variant as a promising platform for the production of these proteins. This modified strain has biotechnological potential for further refinement and sustainable production of bio-based products in the food, feed, and nutraceutical industries.

Heterologous protein secretion in filamentous fungi is often constrained by limitations in signal peptide recognition and intracellular trafficking. Aspergillus oryzae, a food-grade industrial fungus, has a robust native secretory system. However, its capacity for recombinant protein secretion remains suboptimal. Here, we developed a two-step, carrier-free engineering strategy to enhance protein secretion in A. oryzae. We identified endogenous signal peptides among highly secreted proteins using a green fluorescent protein (GFP) reporter. The oryzin signal peptide SPAoalp1 increased GFP secretion 5.50-fold compared with a no-signal-peptide control. We co-overexpressed Aosly1, a Sec1/Munc18 family protein that regulates soluble N-ethylmaleimide-sensitive factor attachment protein receptor–mediated vesicle trafficking, which, in combination with SPAoalp1, increased secretion approximately two-fold compared with SPAlp1 control and ten-fold with no-SP control. Applying the engineered platform for genetic improvement of heterologous bovine κ-casein increased secretion from 0.11 to 0.24 mg/L. Physiological optimization further increased secretion. The developed system provided initial evidence for secretion of a ~12 kDa band consistent with Aopafb transcription, with MIC₉₀ values of 4.56–8.24% (v/v) against two Candida albicans strains and 4.68% (v/v) against Aspergillus niger. The system offers a modular framework for engineering fungal secretion and expands the utility of A. oryzae for recombinant protein production.