Journal
JOURNAL OF FUNGI
Volume 9, Issue 2, Pages -Publisher
MDPI
DOI: 10.3390/jof9020259
Keywords
kojic acid; gene cluster; phylogeny; zinc cluster domain; CRISPR; Aspergillus; Penicillium
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In this study, it was found that almost all section Flavi aspergilli, except for A. avenaceus, had complete KA gene clusters. Additionally, the partial KA gene cluster was found in the Penicillium species, P. nordicum. Phylogenetic analysis based on KA gene cluster sequences revealed different evolutionary branches within section Flavi aspergilli. The transcriptional regulator KojR was shown to activate the kojA and kojT genes in Aspergillus flavus, and a critical binding motif for KojR in the kojA promoter region was identified.
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)(2)Cys(6) 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.
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