Abstract
Aspergillus aculeatus produces cellulolytic enzymes in the presence of their substrates. We screened a library of 12,000 A. aculeatus T-DNA-inserted mutants to identify a regulatory factor involved in the expression of their enzyme genes in response to inducers. We found one mutant that reduced the expression of FIII-avicelase (chbI) in response to cellulose. T-DNA was inserted into a putative protein kinase gene similar to AN10082 in A. nidulans, serine–arginine protein kinase F, SrpkF. Fold increases in srpkF gene expression in response to various carbon sources were 2.3 (d-xylose), 44 (Avicel®), 59 (Bacto™ Tryptone), and 98 (no carbon) compared with d-glucose. Deletion of srpkF in A. aculeatus resulted in a significant reduction in cellulose-responsive expression of chbI, hydrocellulase (cel7b), and FIb-xylanase (xynIb) genes at an early induction phase. Further, the srpkF-overexpressing strain showed upregulation of the srpkF gene from four- to nine-fold higher than in the control strain. srpkF overexpression upregulated cbhI and cel7b in response to cellobiose and the FI-carboxymethyl cellulase gene (cmc1) and xynIb in response to d-xylose. However, the srpkF deletion did not affect the expression of xynIb in response to d-xylose due to the less expression of srpkF under the d-xylose condition. Our data demonstrate that SrpkF is primarily involved in cellulose-responsive expression, though it has a potential to stimulate gene expression in response to both cellobiose and d-xylose in A. aculeatus.
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Acknowledgements
This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science under Grant 19K05777. Part of this work was also supported by the New Energy and Industrial Technology Development Organization Project under Grant P07015.
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This work was supported by JSPS KAKENHI under Grant 19K05777. Part of this work was also supported by the New Energy and Industrial Technology Development Organization (NEDO) Project under Grant P07015.
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S.T. and T.K. conceived and designed the project. R.K. and N.K. conducted the experiments. S.T wrote the paper. All authors read and approved the final manuscript.
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Supplementary file1 Supplementary Fig. S1. Construction of ΔsrpkF and srpkF+ strains. The upper portion of the figure shows restriction enzyme maps of srpkF loci in MR12, Δ srpkF, and srpkF+ strains. Underlined DNA regions were used as DNA probes for Southern blotting to detect DNA fragments after digestion with BamHI (lower portion of figure). Supplementary Fig. S2. Construction of ΔCsrpkF and complement strains (CsrpkF+). The upper portion of the figure shows restriction enzyme maps of srpkF loci in MR12, ΔCsrpkF, and complement strains. Asterisks indicate positions of stop codons. Underlined DNA regions were used as DNA probes for Southern blotting to detect DNA fragments after digestion with DraI (lower portion of figure). Supplementary Fig. S3. Construction of the OEsrpkF strain. The upper portion of the figure shows restriction enzyme maps of srpkF loci in MR12 and OEsrpkF strains. Underlined DNA regions were used as DNA probes for Southern blotting to detect DNA fragments after digestion with PstI (lower portion of figure). Supplementary Fig. S4. Effect of srpkF deletion on expression of cbhI and xynIb. qRT-PCR results for each gene in MR12 (M), ΔsrpkF (Δ), srpkF+ (+), ΔCsprkF (ΔC), and the complement strain of ΔCsprkF (C+) incubated for 9 h under noninducing and the 1% Avicel®-inducing conditions. Expression of genes was normalized to gpdA expression. Fold induction of test genes (gene expression under inducing conditions divided by that under the noninducing condition) is presented as means of three independent experiments, and error bars indicate standard deviations. Letters indicate significant differences between groups (p < 0.05, one-way ANOVA). Supplementary Fig. S5. Expression of srpkF in the overexpression strain. qRT-PCR analysis of srpkF in MR12 (M) and the srpkF-overexpressing strain (OEsrpkF, OE). RNA was prepared from strains grown for 24 h in the presence of 0.1% cellobiose with 50 μg/L DNJ (a) or 1% d-xylose (b). Relative expression is the ratio of mean expression in srpkF divided by gpdA expression. Relative expression is presented as means of three independent experiments, and error bars indicate standard deviations. M: MR12, filled bars; OE: OEsrpkF, striped bars. (PDF 1593 kb)
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Katayama, R., Kobayashi, N., Kawaguchi, T. et al. Serine–arginine protein kinase-like protein, SrpkF, stimulates both cellobiose-responsive and d-xylose-responsive signaling pathways in Aspergillus aculeatus. Curr Genet 68, 143–152 (2022). https://doi.org/10.1007/s00294-021-01207-x
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DOI: https://doi.org/10.1007/s00294-021-01207-x