Identification and characterization of genes involved in kojic acid biosynthesis in Aspergillus flavus

Abstract

Although Aspergillus flavus is a strong producer strain for kojic acid (KA), up till now there is no report on genes encoding KA. Conversely, KA genes from A. oryzae have been reported, with kojA, kojR and kojT genes, an enzyme gene, a transcription factor and a transporter, respectively, having been cloned and characterized. Thus, in the present study, we aimed to identify and characterize KA genes from A. flavus. To achieve this goal, we first produced by gamma irradiation A. flavus and A. oryzae mutants with improved levels of KA with respect to wild type (WT) strains. Then, we cloned orthologs of A. oryzae kojA, kojR and kojT from A. flavus. Finally, we analyzed the expression levels of KA genes in mutant (MT) and WT strains of both species. The mutants A. flavus HAk1-M2 and A. oryzae HAk2-M26 were found to produce KA at levels 2.5- and 3.02-fold higher than their parent strains after 7–10 days under shaking conditions. The cloned A. flavus genes AFLA_096040, AFLA_096050 and AFLA_096060, exhibited 99–100% similarity to kojA, kojR and kojT genes from A. oryzae, respectively. Gene expression analyses revealed that the expression levels of KA genes nicely overlap with KA levels both in A. flavus and A. oryzae, MT and WT strains. In addition, we cloned the laeA gene, which controls the biosynthesis of secondary metabolites from A. flavus. The results showed that, unlike A. oryzae, the expression of laeA in A. flavus does not correlate with KA biosynthesis. Our findings are of importance and reveal that the gene cluster comprising AFLA_096040, AFLA_096050 and AFLA_096060 is potentially involved in KA biosynthesis.