Kojic acid (KA) is a fungal metabolite and has a variety of applications in the cosmetics and food industries. Aspergillus oryzae is a well-known producer of KA, and its KA biosynthesis gene cluster has been identified. In this study, we showed that nearly all section Flavi aspergilli except for A. avenaceus had complete KA gene clusters, and only one Penicillium species, P. nordicum, contained a partial KA gene cluster. Phylogenetic inference based on KA gene cluster sequences consistently grouped section Flavi aspergilli into clades as prior studies. The Zn(II)2Cys6 zinc cluster regulator KojR transcriptionally activated clustered genes of kojA and kojT in Aspergillus flavus. This was evidenced by the time-course expression of both genes in kojR-overexpressing strains whose kojR expression was driven by a heterologous Aspergillus nidulans gpdA promoter or a homologous A. flavus gpiA promoter. Using sequences from the kojA and kojT promoter regions of section Flavi aspergilli for motif analyses, we identified a consensus KojR-binding motif to be an 11-bp palindromic sequence of 5'-CGRCTWAGYCG-3' (R = A/G, W = A/T, Y = C/T). A CRISPR/Cas9-mediated gene-targeting technique showed that the motif sequence, 5'-CGACTTTGCCG-3', in the kojA promoter was critical for KA biosynthesis in A. flavus. Our findings may facilitate strain improvement and benefit future kojic acid production.
Keywords: Aspergillus; CRISPR; Penicillium; gene cluster; kojic acid; phylogeny; zinc cluster domain.