Expression analysis of Aohps1 in A. oryzae
To explore the expression pattern of Aohps1, samples from three different stages of A. oryzae were taken, which included that stage I mycelia expansion spans about 24 h; stage II early sporulation spans about 48 h and stage III mature sporulation spans about 72 h. RT-qPCR analysis showed that the expression of Aohps1 was induced in three development stages of A. oryzae (Fig. 1). After three days of growth under H2O2 treatment, the transcription level of Aohps1 had no significant changes compared to that without H2O2 treatment (Fig. 1).
Characterization of Aohps1
The Aohps1 gene encodes an uncharacterized protein of 306 amino acid residuals according to Aspergillus Genome Database (Fig. 2A). In silico analysis of Aohps1 protein showed that AoHPS1 had no given domains and contained 54% random coil, 29% α-helix and 10% extended strand (Fig. 2A). The prediction of transmembrane region of AoHPS1 sequence had four distinct hydrophobic regions (Fig. 2B). Multiple alignments showed that AoHPS1 is conserved in Aspergillus species (Fig. 2C).
Generation of Aohps1 overexpression and deletion mutants
To reveal the function of Aohps1, Aohps1 overexpression and deletion mutants were constructed using A. oryzae amyB gene promoter and the CRISPR/Cas9 system, respectively (Fig. 3). In the Aohps1-overexpressing strain (OE-Aohps1), qPCR analysis showed that the transcription level of Aohps1 increased by 5-fold relative to the control WT strain on the starch-containing medium (Fig. 3C). For the Aohps1-disrupted strain (ΔAohpi1), one base-pair deletion in the second exon of Aohps1 led to the termination of translation (Fig. 3A). But disruption of Aohps1 didn’t result in the reduced expression of Aohps1 (Fig. 3C).
Phenotypic characterization of Aohps1 overexpression and disruption strains
After cultivation for three days on CD agar medium, the growth of Aohps1 overexpression and disruption strains slightly decreased compared with the WT strain, while the Aohps1 overexpression strain exhibited a more severe growth defect than the Aohps1 deletion mutant and the WT strain under H2O2 treatment (Fig. 4A, B). Moreover, Aohps1 overexpression and disruption strains exhibited severe conidiation defects (Fig. 4C). Additionally, the biomass of Aohps1 overexpression and disruption strains markedly reduced relative to the WT strain with H2O2 stress, whereas the Aohps1 deletion mutant had no changes in the biomass compared with the WT strain without H2O2 treatment (Fig. 4D).
The effects of overexpression and disruption of Aohps1 on kojic acid synthesis
To study the effects of Aohps1 on secondary metabolism of A. oryzae, the control WT, OE-Aohps1 and ΔAohpi1 strains were cultivated on CD agar medium with ferric ions chelated by kojic acid, showing a red color. The control WT, OE-Aohps1 and ΔAohpi1 strains all displayed a visible red color on the agar medium after cultivation for three days (Fig. 5A). But the control WT strains showed a red color much more intense than OE-Aohps1 and ΔAohpi1 strains (Fig. 5A). Quantitative analysis displayed that the yield of kojic acid in the control WT was about 1.7 and 2.1 times higher than those in the OE-Aohps1 and ΔAohpi1 strains without H2O2 treatment, respectively (Fig. 5A). Under H2O2 treatment, the production of kojic acid in the WT, OE-Aohps1 and ΔAohpi1 strains was down-regulated and kojic acid production was significantly lower in the OE-Aohps1 and ΔAohpi1 strains than in the control strain (Fig. 5). Moreover, the yield of kojic acid in the OE-Aohps1 strain decreased more than that in ΔAohpi1 strains compared with WT (Fig. 5). To clarify whether the declined production of kojic acid is related to transcription levels, the expression levels of LaeA, kojA, kojR and kojT involved in kojic acid synthesis were analyzed. We found that kojA but not kojR and kojT was decreased significantly in the OE-Aohps1 and ΔAohpi1 strains without H2O2 treatment (Fig. 6B, C and D). However, the expression profiles of LaeA, kojR and kojT in the OE-Aohps1 and ΔAohpi1 strains were up-regulated markedly under H2O2 treatment relative to those without H2O2 treatment (Fig. 6A, B and C). Impressively, the transcription level of kojA in the OE-Aohps1 strain had no significant changes with and without H2O2 treatment whereas kojA expression in the ΔAohpi1 strain was increased significantly after H2O2 treatment (Fig. 6B), consistent with the fact that kojic acid production was significantly less in the OE-Aohps1 strain than in the ΔAohpi1 mutant under H2O2 treatment (Fig. 5B).