Presentation Information
[P02-284]Regulatory roles of SrpkF phosphorylation in enzyme production and morphogenesis in Aspergillus aculeatus
○Shuji Tani1, Ririka Nakai1, Kentaro Minamoto1 (1. Osaka Metropolitan University (Japan))
Keywords:
Gene regulation,Filamentous fungi,Cellulase
[Purpose] The cellulolytic filamentous fungus Aspergillus aculeatus strain no. F-50 produces β-glucosidase with strong saccharification activity, capable of hydrolyzing cellooligosaccharides, but a relatively low amount of endoglucanase and cellobiohydrolase. Our goal is to elucidate the regulatory mechanisms underlying cellulase production in this fungus and to establish a platform for high-level enzyme production based on these insights.
[Method] We identified a 416-amino-acid serine–arginine protein kinase F-like protein (SrpkF1–416) as a regulatory factor involved in the induction of cellulase gene expression in A. aculeatus.1 Functional analyses using a gene deletion strain (ΔsrpkF) and a C-terminal truncation strain expressing SrpkF1–327 (ΔCsrpkF) revealed that cellulase gene expression was significantly reduced in both strains. Under 1 M NaCl conditions, however, only ΔCsrpkF exhibited decreased conidiation and increased conidial germination rates.2 These findings suggest that the C-terminal region (residues SrpkF328–416) regulates SrpkF function. To investigate this possibility, we analyzed the roles of five predicted phosphorylation sites (S355, Y362, S369, S377, and S404) within the SrpkF328–416 region predicted by Group-based Prediction System 5.0.
[Results] Yeast two-hybrid analysis demonstrated that phosphomimetic variants SrpkF328–416-Y362E, -S369E, and -S377E interacted with SrpkF1–327, suggesting intramolecular interactions. We then generated mutant strains in which each of these residues was substituted with glutamic acid (phosphomimetic mutation) or alanine (phosphor-deficient mutation). Under 1 M NaCl conditions, no significant differences in conidiation were observed among these strains. However, the conidial germination rate was significantly increased only in the SrpkF-S377A mutant. To further assess the effects of mutated SrpkFs on CAZyme gene expression, we focused on S355 based on preliminary analyses. Strains overexpressing SrpkF-S355E or SrpkF-S355A were constructed in a creA deletion background, which alleviates carbon catabolite repression. When cultured with cellobiose or β-1,4-mannobiose as the carbon source, expression of CAZyme genes was approximately threefold higher in the SrpkF-S355A overexpression strain, whereas SrpkF-S355E had no effect. In contrast, under wheat bran conditions, cellulase production was unaffected by S355 substitution, while β-1,4-mannanase production was significantly increased in the early cultivation stage in the SrpkF-S355E overexpression strain. Consistently, expression of CAZyme genes was also significantly elevated in this strain.
[Consideration] Taken together, these results indicate that dephosphorylation of SrpkF-S355 promotes gene expression in the presence of pure inducers such as cellobiose or β-1,4-mannobiose, whereas phosphorylation of SrpkF-S355 enhances gene expression in the presence of various inducers such as wheat bran.
[Conclusion] These findings reveal a phosphorylation-dependent regulatory mechanism that fine-tunes enzyme production as well as conidiation and germination in A. aculeatus.
1. Katayama et al. Curr Genet. 2022;68(2):143-152; 2. Kobayashi et al. Int Microbiol. 2024; 27(1):91-100
[Method] We identified a 416-amino-acid serine–arginine protein kinase F-like protein (SrpkF1–416) as a regulatory factor involved in the induction of cellulase gene expression in A. aculeatus.1 Functional analyses using a gene deletion strain (ΔsrpkF) and a C-terminal truncation strain expressing SrpkF1–327 (ΔCsrpkF) revealed that cellulase gene expression was significantly reduced in both strains. Under 1 M NaCl conditions, however, only ΔCsrpkF exhibited decreased conidiation and increased conidial germination rates.2 These findings suggest that the C-terminal region (residues SrpkF328–416) regulates SrpkF function. To investigate this possibility, we analyzed the roles of five predicted phosphorylation sites (S355, Y362, S369, S377, and S404) within the SrpkF328–416 region predicted by Group-based Prediction System 5.0.
[Results] Yeast two-hybrid analysis demonstrated that phosphomimetic variants SrpkF328–416-Y362E, -S369E, and -S377E interacted with SrpkF1–327, suggesting intramolecular interactions. We then generated mutant strains in which each of these residues was substituted with glutamic acid (phosphomimetic mutation) or alanine (phosphor-deficient mutation). Under 1 M NaCl conditions, no significant differences in conidiation were observed among these strains. However, the conidial germination rate was significantly increased only in the SrpkF-S377A mutant. To further assess the effects of mutated SrpkFs on CAZyme gene expression, we focused on S355 based on preliminary analyses. Strains overexpressing SrpkF-S355E or SrpkF-S355A were constructed in a creA deletion background, which alleviates carbon catabolite repression. When cultured with cellobiose or β-1,4-mannobiose as the carbon source, expression of CAZyme genes was approximately threefold higher in the SrpkF-S355A overexpression strain, whereas SrpkF-S355E had no effect. In contrast, under wheat bran conditions, cellulase production was unaffected by S355 substitution, while β-1,4-mannanase production was significantly increased in the early cultivation stage in the SrpkF-S355E overexpression strain. Consistently, expression of CAZyme genes was also significantly elevated in this strain.
[Consideration] Taken together, these results indicate that dephosphorylation of SrpkF-S355 promotes gene expression in the presence of pure inducers such as cellobiose or β-1,4-mannobiose, whereas phosphorylation of SrpkF-S355 enhances gene expression in the presence of various inducers such as wheat bran.
[Conclusion] These findings reveal a phosphorylation-dependent regulatory mechanism that fine-tunes enzyme production as well as conidiation and germination in A. aculeatus.
1. Katayama et al. Curr Genet. 2022;68(2):143-152; 2. Kobayashi et al. Int Microbiol. 2024; 27(1):91-100
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