Functional role of a novel zinc finger protein, AoZFA, in growth and kojic acid synthesis in Aspergillus oryzae.

Kojic acid (KA) is a valuable secondary metabolite that is regulated by zinc finger proteins in Aspergillus oryzae. However, only two such proteins have been characterized to function in kojic acid production of A. oryzae to date. In this study, we identified a novel zinc finger protein, AoZFA, required for kojic acid biosynthesis in A. oryzae. Our results showed that disruption of AozfA led to increased expression of kojA and kojR involved in kojic acid synthesis, resulting in enhanced kojic acid production, while overexpression of AozfA had the opposite effect. Furthermore, deletion of kojR in the AozfA disruption strain abolished kojic acid production, whereas overexpression of kojR enhanced it, indicating that AoZFA regulates kojic acid production by affecting kojR. Transcriptional activation assay revealed that AoZFA is a transcriptional activator. Interestingly, when kojR was overexpressed in the AozfA overexpression strain, the production of kojic acid failed to be rescued, suggesting that AozfA plays a distinct role from kojR in kojic acid biosynthesis. Moreover, we found that AozfA was highly induced by zinc during early growth stages, and its overexpression inhibited the growth promoted by zinc, whereas its deletion had no effect, suggesting that AoZFA is non-essential but has a role in the response of A. oryzae to zinc. Overall, these findings provide new insights into the roles of zinc finger proteins in the growth and kojic acid production of A. oryzae.IMPORTANCEKojic acid (KA) is an economically valuable secondary metabolite produced by Aspergillus oryzae due to its vast biological activities. Genetic modification of A. oryzae has emerged as an efficient strategy for enhancing kojic acid production, which is dependent on the mining of genes involved in kojic acid synthesis. In this study, we have characterized a novel zinc-finger protein, AoZFA, as a negative regulator of kojic acid production by affecting kojR. AozfA is an excellent target for improving kojic acid production without any effects on the growth of A. oryzae. Furthermore, the simultaneous modification of AozfA and kojR exerts a more significant promotional effect on kojic acid production than the modification of single genes. This study provides new insights for the regulatory mechanism of zinc finger proteins in the growth and kojic acid production of A. oryzae.

The C2H2-type zinc-finger regulator AoKap5 is required for the growth and kojic acid synthesis in Aspergillus oryzae.

Aspergillus oryzae is an important filamentous fungus widely used for the industrial production of fermented foods and secondary metabolites. The clarifying of the mechanism of the growth and secondary metabolites in A. oryzae is important for its industrial production and utilization. Here, the C2H2-type zinc-finger protein AoKap5 was characterized to be involved in the growth and kojic acid production in A. oryzae. The Aokap5-disrupted mutants were constructed by the CRISPR/Cas9 system, which displayed increased colony growth but decreased conidial formation. Deletion of Aokap5 enhanced the tolerance to cell-wall and oxidative but not osmotic stress. The transcriptional activation assay revealed that AoKap5 itself didn't have transcriptional activation activity. Disruption of Aokap5 resulted in the reduced production of kojic acid, coupled with the reduced expression of the kojic acid synthesis genes kojA and kojT. Meanwhile, overexpression of kojT could rescue the decreased production of kojic acid in Aokap5-deletion strain, indicating that Aokap5 serves upstream of kojT. Furthermore, the yeast one-hybrid assay demonstrated that AoKap5 could directly bind to the kojT promoter. These findings suggest that AoKap5 regulates kojic acid production through binding to the kojT promoter. This study provides an insight into the role of zinc finger protein in the growth and kojic acid biosynthesis of A. oryzae.

Overexpression of a novel gene Aokap2 affects the growth and kojic acid production in Aspergillus oryzae.

BACKGROUND: Aspergillus oryzae is an industrially important filamentous fungus for the production of fermentative food, commercial enzyme and valuable secondary metabolites. Although the whole genome of A. oryzae has been sequenced in 2005, there is currently not enough research on functional genes that affect the growth and secondary metabolites of A. oryzae. This study aimed to identify and characterize functional genes involved in the growth and secondary metabolites of A. oryzae. METHODS AND RESULTS: Our previous work on the developmental transcriptome of A. oryzae found that an uncharacterized gene Aokap2 was repressed during the development of A. oryzae. In this study, the gene expression pattern was verified by qRT-PCR. Phylogenetic analysis revealed that AoKAP2 has the species specificity of Aspergillus. Furthermore, Aokap2 was overexpressed using the A. oryzae amyB promoter and overexpression of Aokap2 caused the inhibition in mycelium growth, conidia formation and biomass. Additionally, overexpression of Aokap2 increased the production of kojic acid. In accordance with the enhanced kojic acid, the overexpression of Aokap2 led to elevated transcription levels of the key kojic acid synthesis gene kojA and the global transcriptional regulator gene of secondary metabolism laeA. Moreover, the expression of Aokap2 was down-regulated significantly in the laeA mutant. Meanwhile, overexpression of Aokap2 in the kojA disrupted strain resulted in a ΔkojA strain-like phenotype with significant inhibition in kojic acid production. CONCLUSION: Taken together, these data suggest that a novel gene Aokap2 is involved in the growth and overexpression of Aokap2 increased kojic acid production through affecting the expression of laeA and kojA. The identification of Aokap2 provides a new target for genetic modification of the growth and the production of kojic acid in A. oryzae.

Construction of single, double, or triple mutants within kojic acid synthesis genes kojA, kojR, and kojT by the CRISPR/Cas9 tool in Aspergillus oryzae.

Kojic acid is an industrially important secondary metabolite produced by Aspergillus oryzae. The construction of genetic materials for kojic acid related genes is important for understanding the mechanism of kojic acid synthesis in A. oryzae. However, multigene simultaneous knockout mutants for kojic acid synthesis genes remain limited because A. oryzae is multinuclear and good selectable markers are scarce. Here, we firstly successfully obtained single mutants of kojA, kojR, and kojT by our previously constructed CRISPR/Cas9 system in A. oryzae, which demonstrated the feasibility of the targeting sgRNAs for kojA, kojR, and kojT. Then, the AMA1-based genome-editing system for multiplex gene editing was developed in A. oryzae. In the multiplex gene-editing system, two guide RNA expression cassettes were ligated in tandem and driven by two U6 promoters in the AMA1-based autonomously replicating plasmid with the Cas9-expression cassette. Moreover, the multiplex gene-editing technique was applied to target the kojic acid synthesis genes kojA, kojR, and kojT, and the double and triple mutants within kojA, kojR, and kojT were obtained successfully. Additionally, the selectable marker pyrG was knocked out in the single and triple mutants of kojA, kojR, and kojT to obtain the auxotrophic strains, which can facilitate to introduce a target gene into the single and triple mutants of kojA, kojR, and kojT for investigating their relationship. The multiplex gene-editing system and release of these materials provide a foundation for further kojic acid research and utilization.

Identification and characterization of a novel gene Aokap1 involved in growth and kojic acid synthesis in Aspergillus oryzae.

The elucidation of the mechanism for the growth and secondary metabolites in Aspergillus oryzae is important for industrial production and utilization. Here, we found that the expression of a novel gene Aokap1 was induced during the growth of A. oryzae. Sequence analysis revealed that AoKap1 has four transmembrane regions and is conserved in Aspergillus species. Disruption of Aokap1 caused the inhibition in mycelium growth and conidia formation, corresponding with reduced expression of brlA and abaA. Furthermore, deletion of Aokap1 resulted in elevated production of kojic acid, and the expression of kojA, kojR and kojT was up-regulated in Aokap1-disrupted strain. Meanwhile, overexpression of kojR resulted in the decreased expression of Aokap1, suggesting that disruption of Aokap1 increased kojic acid production by affecting the expression of kojA, kojR and kojT. The discovery of Aokap1 provides a new target for genetic modification of the growth and kojic acid production in A. oryzae.

Identification and characterization of Nramp transporter AoNramp1 in Aspergillus 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 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02998-z.

A highly efficient identification of mutants generated by CRISPR/Cas9 using the non­functional DsRed assisted selection in Aspergillus oryzae.

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 non­functional 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.

Identification and functional characterization of glycerol dehydrogenase reveal the role in kojic acid synthesis in Aspergillus oryzae.

Glycerol dehydrogenase has been identified and characterized functionally in many species. However, little is known about glycerol dehydrogenase genes and their functions in Aspergillus oryzae. Here, a total of 45 glycerol dehydrogenase genes in Aspergillus oryzae were identified and renamed from AoGld1 to AoGld45 according to their chromosome distribution. They were classified into three groups based on phylogenetic analysis. Synteny analysis revealed that thirteen AoGld genes are conserved among Aspergillus species. Promoter analysis displayed that AoGld3 and AoGld13 harbored multiple binding elements of GATA-type transcription factors and zinc-finger protein msnA that were involved in nitrogen and kojic acid metabolism, respectively. Moreover, the AoGld3 deletion strain Δgld3 was generated by the CRISPR/Cas9 system, which had no visible growth defects compared with the control wild-type strain under the control and osmotic stress treatments. However, disruption of AoGld3 led to the inhibition of kojic acid production, and the expression of kojA, kojR was down-regulated in the Δgld3 strain. Furthermore, when kojA or kojR was overexpressed in the Δgld3 strain, the yield of kojic acid was restored, suggesting that AoGld3 is involved in kojic acid production through affecting the expression of kojR and kojA. Taken together, these findings provide new insights into our understanding of glycerol dehydrogenase and establish foundation for further study of their roles in Aspergillus oryzae.

Identification and characterization of the ZRT, IRT-like protein (ZIP) family genes reveal their involvement in growth and kojic acid production in Aspergillus oryzae.

The ZRT, IRT-like protein (ZIP) family exists in many species and plays an important role in many biological processes, but little is known about ZIP genes in Aspergillus oryzae. Here, 10 ZIP genes in A. oryzae were identified and these were classified into four groups based on phylogenetic analysis. The structures of these AoZip genes were determined, which indicated a great divergence of AoZip members from different groups. Synteny analysis revealed that AoZip7, AoZip8, and AoZip10 are conserved among Aspergillus species. We also found that the promoter regions of AoZip2, AoZip7, AoZip8, and AoZip10 contain multiple conserved response elements. Expression analysis revealed that AoZips exhibited different expression patterns in response to different metal treatments. Moreover, overexpression and RNA-interference (RNAi) of AoZip2 led to a decrease in mycelium growth diameter and inhibited conidia formation. AoZip2 overexpression and RNAi strains showed distinct sensitivity to severely Zn/Mn-depleted stress. In addition, kojic acid production was markedly lower in AoZip2 overexpression and RNAi strains than in the control strains, and the expression of kojA, kojR, and kojT was down-regulated in AoZip2 overexpression and RNAi strains. This study provides new insights into our understanding of ZIP genes and lays a foundation for further investigation of their roles in Aspergillus oryzae.