Abstract
Kojic acid (KA) is a valuable secondary metabolite that is regulated by zinc finger proteins in Aspergillus oryzae. However, only two such proteins have been characterized to function in kojic acid production of A. oryzae to date. In this study, we identified a novel zinc finger protein, AoZFA, required for kojic acid biosynthesis in A. oryzae. Our results showed that disruption of AozfA led to increased expression of kojA and kojR involved in kojic acid synthesis, resulting in enhanced kojic acid production, while overexpression of AozfA had the opposite effect. Furthermore, deletion of kojR in the AozfA disruption strain abolished kojic acid production, whereas overexpression of kojR enhanced it, indicating that AoZFA regulates kojic acid production by affecting kojR. Transcriptional activation assay revealed that AoZFA is a transcriptional activator. Interestingly, when kojR was overexpressed in the AozfA overexpression strain, the production of kojic acid failed to be rescued, suggesting that AozfA plays a distinct role from kojR in kojic acid biosynthesis. Moreover, we found that AozfA was highly induced by zinc during early growth stages, and its overexpression inhibited the growth promoted by zinc, whereas its deletion had no effect, suggesting that AoZFA is non-essential but has a role in the response of A. oryzae to zinc. Overall, these findings provide new insights into the roles of zinc finger proteins in the growth and kojic acid production of A. oryzae.IMPORTANCEKojic acid (KA) is an economically valuable secondary metabolite produced by Aspergillus oryzae due to its vast biological activities. Genetic modification of A. oryzae has emerged as an efficient strategy for enhancing kojic acid production, which is dependent on the mining of genes involved in kojic acid synthesis. In this study, we have characterized a novel zinc-finger protein, AoZFA, as a negative regulator of kojic acid production by affecting kojR. AozfA is an excellent target for improving kojic acid production without any effects on the growth of A. oryzae. Furthermore, the simultaneous modification of AozfA and kojR exerts a more significant promotional effect on kojic acid production than the modification of single genes. This study provides new insights for the regulatory mechanism of zinc finger proteins in the growth and kojic acid production of A. oryzae.