A Novel Major Facilitator Superfamily Transporter Gene Aokap4 near the Kojic Acid Gene Cluster Is Involved in Growth and Kojic Acid Production in Aspergillus oryzae

Abstract

Kojic acid is an important secondary metabolite of industrial importance produced by Aspergillus oryzae. The kojic acid gene cluster plays an essential role in kojic acid production, and harbors kojA, kojR and kojT. The deletion of kojT, encoding a major facilitator superfamily (MFS) transporter, did not completely abolish kojic acid production, implying that other transporters are required for the transport of kojic acid. The aim of this study is to look for the transporters involved in kojic acid production. Here, Aokap4 encoding a novel MFS transporter was identified, which was adjacent to kojT in the kojic acid gene cluster. The deletion of Aokap4 contributed to the hyphal growth, conidial production and biomass of A. oryzae. Moreover, Aokap4 is required for heat- and cell-wall-stress tolerance but not oxidative and osmotic stress. The disruption of Aokap4 impaired kojic acid production with the reduced expression of kojA, kojR and kojT. Furthermore, when kojT was deleted in the Aokap4-disrupted strain, the yield of kojic acid declined to the same level as that of the kojT-deletion mutant, whereas the production of kojic acid was recovered when kojT was overexpressed in the Aokap4 knockout strain, suggesting that kojT acts downstream of Aokap4. AoKap4 was the second identified MSF transporter involved in kojic acid production after kojT was found a decade ago. This study contributes to a better understanding of the biological roles of the MFS transporter and lays a foundation for future studies on kojic acid synthesis in A. oryzae.