Abstract
The oryzapsin genes opsA and opsB in Aspergillus oryzae encoding glycosylphosphatidylinositol (GPI)-anchored aspartic endopeptidase are homologs of Saccharomyces cerevisiae yapsins. We recently found another homolog, opsC, in the A. oryzae genome database, which was suggested to be a pseudogene. However, the profiles and roles of the proteins encoded by these genes have not yet been clarified. Toward this end, we first produced opsA- and opsB-overexpression strains and performed enzymatic analyses, revealing that OpsA and OpsB can attack sites other than the carboxyl-terminal peptide bonds of basic amino acids. Moreover, OpsA and OpsB were confirmed to bind to the cell membrane with a GPI anchor. Second, opsA and opsB single-deletion and double-deletion strains (ΔopsA, ΔopsB, and ΔopsAΔopsB) were constructed to explore the expected roles of oryzapsins in cell wall synthesis, similar to the role of yapsins. The transcription level of mpkA in the cell wall integrity pathway was increased in ΔopsB and ΔopsAΔopsB strains, suggesting that OpsB might be involved in processing cell wall synthesis-related proteins. Treatment with an ergosterol biosynthesis inhibitor reduced the growth of the ΔopsAΔopsB strain. Moreover, the mRNA levels of Aoerg1, Aoerg3-1, Aoerg3-2, Aoerg7b, Aoerg11, and Aohmg1,2 showed a decreasing tendency in the ΔopsAΔopsB strain, and the ergosterol content in the membrane was reduced in the ΔopsAΔopsB strain. These results suggest that oryzapsins exist in the cell membrane and play roles in the formation of cell membranes. This is the first report of the involvement of GPI-anchored aspartic endopeptidases in ergosterol biosynthesis.
Key points
• The oryzapsins have wider substrate specificity than yaspins in S. cerevisiae.
• Unlike the yapsins, the oryzapsins might not be involved in the main structure synthesis of the cell wall.
• The oryzapsins would be involved in ergosterol biosynthesis.
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Data Availability
RNA-Seq data was available from DDBJ (Accession number: DRA011494).
Code availability
Not applicable.
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Acknowledgements
This work was partially supported by the Program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN). The authors thank Dr. Junichiro Marui (Applied Microbiology Division, National Food Research Organization) for kindly providing A. oryzae ΔpyrG strain.
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This study was partially supported by the Program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN; H18).
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NS, TK, and YM constructed strains and performed experiments. AM performed experiments using ergosterol synthesis inhibitors. NS, HA, and NS performed RNA-Seq and data processing. KA, TK, HI, K-IK, MT, and YY designed and planned the study. NS TK, YM, and YY drafted the manuscript, all authors revised the manuscript, NS and YY finalized the manuscript. All authors agreed to the final version of the manuscript.
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Shimizu, N., Katagiri, T., Matsumoto, A. et al. Oryzapsins, the orthologs of yeast yapsin in Aspergillus oryzae, affect ergosterol synthesis. Appl Microbiol Biotechnol 105, 8481–8494 (2021). https://doi.org/10.1007/s00253-021-11639-7
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DOI: https://doi.org/10.1007/s00253-021-11639-7