Abstract
Aspergillus niger is a primary cell factory for food-grade protein (enzyme) production due to its strong protein secretion capacity and unique safety characteristics. The bottleneck issue for the current A. niger expression system is the difference in expression yield of heterologous proteins of non-fungal origin compared to those of fungal origin, which is about three orders of magnitude. The sweet protein monellin, derived from West African plants, has the potential to become a food-grade sweetener due to its high sweetness and the benefit of not containing sugar itself, but it is extremely difficult to establish a research model for heterologous expression in A. niger, owing to extremely low expression, a small molecular weight, and being undetectable with conventional protein electrophoresis. HiBiT-Tag was fused with low-expressing monellin in this work to create a research model for heterologous protein expression in A. niger at ultra-low levels. We increased monellin expression by increasing the monellin copy number, fusing monellin with the endogenous highly expressed glycosylase glaA, and eliminating extracellular protease degradation, among other strategies. In addition, we investigated the effects of overexpression of molecular chaperones, inhibiting the ERAD pathway, and enhancing the synthesis of phosphatidylinositol, phosphatidylcholine, and diglycerides in the biomembrane system. Using medium optimization, we finally obtained 0.284 mg/L of monellin in the supernatant of the shake flask. This is the first time recombinant monellin has been expressed in A. niger, with the goal of investigating ways to improve the secretory expression of heterologous proteins at ultra-low levels, which can serve as a model for the expression of other heterologous proteins in A. niger.