The CRISPR/Cas9 system has become a great tool for target gene knock-out in lamentous fungi. It is laborious and time-consuming that identi cation mutants from a large number of transformants through PCR or enzyme-cut method. Here, we rst 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 identi cation of mutants generated by CRISPR/Cas9 in Aspergillus oryzae.
Kojic acid is an important secondary metabolite of industrial importance produced by Aspergillus oryzae. The kojic acid gene cluster plays an essential role in kojic acid production, and harbors kojA, kojR and kojT. The deletion of kojT, encoding a major facilitator superfamily (MFS) transporter, did not completely abolish kojic acid production, implying that other transporters are required for the transport of kojic acid. The aim of this study is to look for the transporters involved in kojic acid production. Here, Aokap4 encoding a novel MFS transporter was identified, which was adjacent to kojT in the kojic acid gene cluster. The deletion of Aokap4 contributed to the hyphal growth, conidial production and biomass of A. oryzae. Moreover, Aokap4 is required for heat- and cell-wall-stress tolerance but not oxidative and osmotic stress. The disruption of Aokap4 impaired kojic acid production with the reduced expression of kojA, kojR and kojT. Furthermore, when kojT was deleted in the Aokap4-disrupted strain, the yield of kojic acid declined to the same level as that of the kojT-deletion mutant, whereas the production of kojic acid was recovered when kojT was overexpressed in the Aokap4 knockout strain, suggesting that kojT acts downstream of Aokap4. AoKap4 was the second identified MSF transporter involved in kojic acid production after kojT was found a decade ago. This study contributes to a better understanding of the biological roles of the MFS transporter and lays a foundation for future studies on kojic acid synthesis in A. oryzae.
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 AoN-ramp1 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.
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