Functional characterization of AoKat1, a major facilitator superfamily transporter involved in kojic acid production in Aspergillus oryzae.

Kojic acid (KA) is a fungal secondary metabolite with wide-ranging industrial applications; however, the molecular mechanisms underlying its extracellular export remain incompletely understood. While KojT, a major facilitator superfamily (MFS) transporter, has been implicated in KA transport, evidence suggests it is not the sole contributor. Here, we identify and functionally characterize a MFS transporter, AoKat1, in Aspergillus oryzae. Comparative transcriptomic analysis between the wild-type strain and a high-KA-producing strain (T58) revealed AoKat1 as significantly upregulated during peak KA production. Structural modeling and molecular docking suggested that KA binds with high affinity within the central cavity of AoKat1. Functional studies showed that overexpression of AoKat1 (OEAoKat1) increased KA production to approximately 43.9  g/L, representing a 31 % improvement compared to the wild-type strain. AoKat1 expression also impacted colony morphology, sporulation, carbon source utilization, and oxidative stress sensitivity. Notably, deletion of AoKat1 led to transcriptional upregulation of kojT, suggesting a compensatory mechanism between these transporters. Moreover, antioxidant genes (catB and GSH) were upregulated in the OEAoKat1 strain during early fermentation, then declined as KA accumulated. This study provides new insight into KA biosynthetic regulation and offer a genetic target to optimize KA production through metabolic engineering.