Zhe Zhang

Kojic acid, a significant secondary metabolite primarily synthesized by Aspergillus oryzae, has extensive applications across various industries. It is imperative to identify effective gene targets that can enhance kojic acid production and to elucidate its biosynthetic regulation for yield optimization in A. oryzae. In this study, we identify the Aokap9 gene, which encodes an uncharacterized protein containing two transmembrane domains, as being closely associated with kojic acid synthesis. Disruption of Aokap9 resulted in increased kojic acid production, whereas mutations in either kojR or laeA within the ΔAokap9 background abolished synthesis, indicating that Aokap9 regulates kojic acid synthesis through kojR and laeA. Conversely, AozfA overexpression suppressed kojic acid accumulation in the ΔAokap9 strain. Furthermore, disruption of AozfA or overexpression of kojR in the Aokap9 mutant resulted in significantly elevated yields, unlike the effects observed with laeA overexpression. Transcriptome profiling further revealed that AoKap9 plays a role in the oxidative stress response through influencing the expression of superoxide dismutase, catalase, and copper amine oxidase in A. oryzae. AoKap9 mediates kojic acid synthesis through a regulatory pathway involving AoZFA, LaeA, and KojR, making it as an important gene target for enhancing kojic acid in A. oryzae. These findings provide essential insights into the regulatory mechanisms governing kojic acid biosynthesis and highlight Aokap9 as a promising target for metabolic engineering in A. oryzae.

Although the kojic acid gene cluster in Aspergillus oryzae was identified in 2010, the functions of neighbouring genes remain poorly understood. Here, we characterise Aokap7, a gene adjacent to this cluster that encodes a novel nucleus-localised zinc finger protein without transcriptional activation activity. Disruption of Aokap7 markedly accelerated spore germination, hyphal growth, and conidial formation, but impaired kojic acid production. Overexpression of kojR or laeA restored kojic acid production in the Aokap7 disruption strain, whereas their disruption abolished it, indicating that Aokap7 regulates kojic acid production by kojR and laeA. Furthermore, disrupting Aokap7 in the AozfA overexpression or disruption strains reversed their kojic aicd production, suggesting that Aokap7 acts downstream of AozfA. The Aokap7 mutant also exhibited reduced expression of reactive oxygen species (ROS)-scavenging genes and heightened oxidative stress sensitivity. Biochemical assays revealed that Aokap7 preferentially binds to the motif 5’-CGGCTCGG-3’, and directly interacts with the AoGPX1 promoter. Disruption of AoGPX1 increased the sensitivity of A. oryzae to oxidative stress. Our findings elucidate the pivotal role of Aokap7 in coordinating growth, oxidative stress response and kojic acid production, advancing understanding of the regulatory network of kojic acid synthesis in A. 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 . IMPORTANCE: Kojic 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 .

CRISPR/Cas technology is a powerful tool for genome engineering in Aspergillus oryzae as an industrially important filamentous fungus. Previous study has reported the application of the CRISPR/Cpf1 system based on the Cpf1 (LbCpf1) from Lachnospiraceae bacterium in A. oryzae. However, multiplex gene editing have not been investigated using this system. Here, we presented a new CRISPR/Cpf1 multiplex gene editing system in A. oryzae, which contains the Cpf1 nuclease (FnCpf1) from Francisella tularensis subsp. novicida U112 and CRISPR-RNA expression cassette. The crRNA cassette consisted of direct repeats and guide sequences driven by the A. oryzae U6 promoter and U6 terminator. Using the constructed FnCpf1 gene editing system, the wA and pyrG genes were mutated successfully. Furthermore, simultaneous editing of wA and pyrG genes in A. oryzae was performed using two guide sequences targeting these gene loci in a single crRNA array. This promising CRISPR/Cpf1 genome-editing system provides a powerful tool for genetically engineering A. 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 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.

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.

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.

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.