| Zinc finger proteins play a crucial role in fungal transcription regulation.Kojic acid,as one of the major secondary metabolites in Aspergillus oryzae,is regulated by zinc finger transcription factors.In A.oryzae,zinc finger proteins are primarily classified into three types:C6,C2H2,and C4.However,the regulatory mechanism of C2H2-type zinc finger proteins in kojic acid biosynthesis of A.oryzae remains unclear.In previous study,we identified the Aozfp1 gene encoding a novel C2H2-type zinc finger protein by transcriptome analysis and strain screening.In this study,we firstly performed sequence analysis of Aozfp1.Subsequently,Aozfp1 knockout strains were generated using the CRISPR/Cas9 technology,and Aozfp1 overexpression strains were constructed by A.oryzae amy B promoter.Based on these strains,we investigated the effects of Aozfp1 on the growth and kojic acid synthesis in A.oryzae.The results elucidated the physiological function of Aozfp1 in A.oryzae and revealed the molecular mechanism of Aozfp1 in regulating kojic acid synthesis.These findings provide a theoretical basis for expanding the application of A.oryzae and improving the efficiency of kojic acid synthesis.The findings are as follows:1.Aozfp1（Ao090012000498）,encoding a novel C2H2-type zinc finger protein,was identified by transcriptomic analysis of A.oryzae.Domain analysis revealed that Ao ZFP1contains three C2H2 zinc finger domains.Multiple sequence alignment showed the zinc finger protein Ao ZFP1 is conserved among Aspergillus species.Phylogenetic analysis indicated that Ao ZFP1 and its orthologous proteins are divided into two groups.Ao ZFP1belongs to the group I,along with members of other filamentous fungi in the genus Aspergillus.The group II includes members from yeasts such as Candida albicans and Saccharomyces cerevisiae,suggesting Ao ZFP1 has potential functional differences with its homologs in yeasts.2.To investigate the impact of Aozfp1 on the growth of A.oryzae,we constructed the engineered strains,including Aozfp1 knockout and overexpression strains.Deletion of Aozfp1 did not affect the growth of A.oryzae,while overexpression of Aozfp1 led to an increase in hyphal diameter but a decrease in the number of conidia and biomass.Additionally,the presence of Zn2+enhanced conidia formation and biomass in the Aozfp1overexpression strains.These findings indicate that overexpression of Aozfp1 exerts an impact on the growth of A.oryzae.3.Quantification of kojic acid showed that knocking out Aozfp1 increased the production of kojic acid in A.oryzae,while overexpression of Aozfp1 inhibited kojic acid synthesis,suggesting that Aozfp1 serves as a negative regulator of kojic acid biosynthesis in A.oryzae.4.We constructed a BD fusion vector containing the Aozfp1 gene and transformed it into a yeast strain with auxotroph to validate its transcriptional activation activity.The self-activation assay in yeast revealed that the BD-Ao ZFP1 transformants were able to grow on SD/-Ade-His-Trp medium,indicating that Ao ZFP1 exhibits self-activation activity.5.RT-PCR analysis revealed that the transcript levels of koj A and koj R involved in kojic acid synthesis,were significantly increased in the Aozfp1 disruption strains,while their expression levels were significantly decreased in Aozfp1 overexpression strains,which was consistent with kojic acid production in the Aozfp1 disruption and overexpression strains,indicating that the involvement of Aozfp1 in the kojic acid production of A.oryzae is related to the expression of koj A and koj R.6.To analyze the relationship between Aozfp1 and the kojic-acid-related genes koj A and koj R.we constructed the double knock out strains:（35）Aozfp1（35）koj A and（35）Aozfp1（35）koj R,and overexpression of koj A or koj R in the Aozfp1 deletion strain:（35）Aozfp1-OE-koj A and（35）Aozfp1-OE-koj R.The determination of kojic acid contents display that the double deletion strain（35）Aozfp1（35）koj A and（35）Aozfp1（35）koj R exhibited undetectable kojic acid production,whereas（35）Aozfp1-OE-koj A and（35）Aozfp1-OE-koj R strains had enhanced yield of kojic acid.These findings indicated that Aozfp1 genetically acts upstream of koj A and koj R to regulate the kojic acid synthesis of A.oryzae. |
