α-Amylase AmyA and Aspartic ProteasePepA Cooperatively Drive Efficient Starch Hydrolysis in Aspergillus niger

Biological synthesis of bulk chemicals offers a green alternative to conventional processes but remains limited by costly carbon sources. Starch, an inexpensive and renewable substrate hydrolyzed into glucose, is a promising carbon source, yet its cooperative hydrolysis mechanisms remain unclear. This study revealed that Aspergillus niger ΔcexA, a high l-malic acid producer, exhibited enhanced starch-hydrolyzing capacity. Under starch induction, ΔcexA secreted higher levels of extracellular proteins, including key starch-hydrolyzing enzymes such as AmyA (α-Amylase). Aspartic protease PepA, the second most abundant extracellular protein, also promoted starch hydrolysis. Deletion of pepA reduced hydrolysis efficiency, whereas overexpression of amyA and pepA in A. niger ATCC 1015 enhanced starch-hydrolyzing activity. These findings reveal a cooperative mechanism involving AmyA and PepA, highlight an unconventional functional role of PepA in starch utilization, and provide molecular targets for engineering fungal strains to efficiently utilize low-cost carbon sources, thereby supporting greener and more economical biomanufacturing.