In this study, we have mainly evaluated the effects of the prtT (transcriptional factor of proteases), mnn9 (subunit of Goli mannoyltransferase complex), and vsm1 (V-SNARE binding protein) on β-glucosidase (A5IL97, an ionic liquid-tolerant thermophilic cellulase) production in Aspergillus niger via gene disruption. When the individual gene was disrupted, the A5IL97 activity was about 2.6 to 9.2 times higher than the transgenic parent strain (A5IL97). When the vms1 was further deleted from the A5IL97/prtTΔ transgenic strain, the A5IL97 activity was increased 14.9 folds as compared with the transgenic parent strain. This strategy is also applicable to the transgene expression of other related proteins in A. niger. Transgene expression of manganese peroxidase (mnP)was further examined and optimized in A. niger. In this study, we evaluated the transgene expression of all four isoforms of mnP (pcmnP1, pcmnP2, pcmnP3, and pcmnP5) from P. chrysosporium, glmnP1 from Ganoderma lucidum, pomnP4 from Pleurotus ostreatus and found that pcmnP2 had the highest transgene expression by the enzyme activities. When the prtT gene was disrupted in the pcmnP2 transgenic strain, the mnP2 activity was increased by 2.2 folds. Various factors such as, nitrogen, oxygen, temperature, pH, hemoglobin, antioxidants, and spore age all exert their effects on mnP production in A. niger. Further improvement of the mnP production is being investigated by disruption of other related genes such as vms1 or mnn9 or over-expression of other pcmnP isoforms in the pcmnP2/prtTΔ transgenic strain background. The overall optimized conditions for the mnP productions will be established in the A. niger.