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The kojic acid gene cluster of Aspergillus oryzae plays a key role in kojic acid synthesis. Although the kojic acid gene cluster has been found in 2010, there is little information on the function of the genes located near the kojic acid gene cluster of A. oryzae and whether these genes affect the kojic acid gene cluster containing kojA, kojR and kojT. Here, Aokap6 near the kojic acid gene cluster of A. oryzae was identified and characterized. The Aokap6 disrupted mutants were constructed by the CRISPR/Cas9 system, which exhibited increased mycelium growth and conidial formation. Disruption of Aokap6 enhanced the tolerance to cell wall, oxidative and heat stress but not osmotic stress. Deletion of Aokap6 repressed kojic acid production, together with the reduced expression of kojA, kojR and kojT. Meanwhile, knockout of kojA, kojR and kojT led to the declined expression of Aokap6, indicating that Aokap6 is required for kojic acid production in coordination with kojA, kojR and kojT. Furthermore, overexpression of kojA, kojR and kojT had no effects on the transcript level of Aokap6, and overexpression of kojA in Aokap6 deletion strain could rescue the reduced yield of kojic acid, suggesting that Aokap6 is involved in kojic acid synthesis acting upstream of kojA. These findings provide new insight for the further understanding of kojic acid gene cluster and kojic acid production in A. oryzae. [ABSTRACT FROM AUTHOR]

The Nramp (natural resistance-associated macrophage protein) family of genes has been identified and characterized widely in many species. However, the Nramp genes and their characterizations have not been reported for Aspergillus oryzae. Here, only one Nramp gene AoNramp1 in A. oryzae genome was identified. Phylogenetic analysis revealed that AoNramp1 is not clustered with Nramps from yeast genus. Expression analysis showed that the transcript level of AoNramp1 was strongly induced under both Zn/Mn-replete and -deplete conditions. The GUS-staining assay indicated that the expression of AoNramp1 was strongly induced by Zn/Mn. Moreover, the AoNramp1 deletion and overexpression strains were constructed by the CRISPR/Cas9 system and A. oryzae amyB promoter, respectively. Phenotypic analysis showed that overexpression and deletion of AoNramp1 caused growth defects under Zn/Mn-deplete and -replete conditions, including mycelium growth and conidia formation. Together, these findings provide valuable information for further study on the biological roles of AoNramp1 in A. oryzae. [ABSTRACT FROM AUTHOR]

The CRISPR/Cas9 system has become a great tool for target gene knock-out in filamentous fungi. It is laborious and time-consuming that identification mutants from a large number of transformants through PCR or enzyme-cut method. Here, we first developed a CRISPR/Cas9 system in Aspergillus oryzae using AMA1-based autonomously replicating plasmid and Cas9 under the control of the Aspergillus nidulans gpdA promoter. By the genome editing technique, we successfully obtained mutations within each target gene in Aspergillus oryzae. Then, we put the protospacer sequence of a target gene and its protospacer adjacent motif (PAM) behind the start codon "ATG" of DsRed, yielding the non‑functional DsRed (nDsRed) reporter gene, and the nDsRed reporter gene could be rescued after successful targeted editing. Moreover, this method was also applied by targeting the kojic acid synthesis gene kojA, and the transformants with DsRed activity were found to harbor targeted mutations in kojA. These results suggest that the nDsRed can be used as a powerful tool to facilitate the identification of mutants generated by CRISPR/Cas9 in Aspergillus oryzae. [ABSTRACT FROM AUTHOR]