Jun-ichi Maruyama

DNA-binding transcription factors are broadly characterized as proteins that bind to specific sequences within genomic DNA and modulate the expression of downstream genes. This study focused on KojR, a transcription factor involved in the metabolism of kojic acid, which is an organic acid synthesized in Aspergillus oryzae and is known for its tyrosinase-inhibitory properties. However, the regulatory mechanism underlying KojR-mediated kojic acid synthesis remains unclear. Hence, we aimed to obtain a comprehensive identification of KojR-associated genes using genomic systematic evolution of ligands by exponential enrichment with high-throughput DNA sequencing (gSELEX-Seq) and RNA-Seq. During the genome-wide exploration of KojR-binding sites via gSELEX-Seq and identification of KojR-dependent differentially expressed genes (DEGs) using RNA-Seq, we confirmed that KojR preferentially binds to 5′-CGGCTAATGCGG-3′, and KojR directly regulates kojT, as was previously reported. We also observed that kojA expression, which may be controlled by KojR, was significantly reduced in a ΔkojR strain. Notably, no binding of KojR to the kojA promoter region was detected. Furthermore, certain KojR-dependent DEGs identified in the present study were associated with enzymes implicated in the carbon metabolic pathway of A. oryzae. This strongly indicates that KojR plays a central role in carbon metabolism in A. oryzae.

Transglutaminase (TG) is a ubiquitous enzyme that crosslinks substrates. In humans, TG participates in blood clotting and wound healing. However, the functions related to the cellular protection of microbial TG are unknown. In filamentous fungi, we previously identified SppB, which contains the transglutaminase core (TGc) domain and functions in hyphal protection at the septal pore upon wounding. Here, we further analyzed the cytokinesis-related protein Cyk3 and peptide N-glycanase Png1, as both contain the TGc domain. All three proteins exhibited functional importance in wound-related hyphal protection at the septal pore. Upon wounding, SppB and AoPng1 accumulated at the septal pore, whereas AoCyk3 and AoPng1 normally localized around the septal pore. The putative Cys-His-Asp catalytic triad of SppB is conserved with the human TGc domain–containing kyphoscoliosis peptidase. Catalytic triad disruptive mutants of SppB and AoCyk3 exhibited septal pore plugging defects. Similar to other TGs, SppB underwent wound-induced truncation of the N-terminal region. Notably, TG activity was detected in vivo at the septal pore of wounded hyphae using a fluorescent-labeled substrate; however, the activity was inhibited by the TG inhibitor cystamine. Our study suggests a conserved role for TGc domain–containing proteins in wound-related protection in fungi, similar to that in humans.