Comprehensive Analysis of GAL4-like Zn₂Cys₆ Proteins from Aspergillus oryzae Using Data Mining

Aspergillus oryzae is an industrially important filamentous fungus regulated by diverse transcription factors, among which GAL4-like Zn₂Cys₆ proteins constitute a major fun-gus-specific family. However, their genome-wide structural diversity remains insuffi-ciently characterized. We performed a comprehensive data mining–based analysis of 233 GAL4-like Zn₂Cys₆ proteins in A. oryzae RIB40 using biosynthetic gene cluster (BGC) analysis, sequence motif analysis, phylogenetic analysis, and principal component analysis (PCA) of amino acid composition descriptors. Of the 233 genes, 48 were located within 20 kb of predicted BGCs, without significant genome-wide enrichment, although BGC proximity was conserved for specific orthologs across Aspergillus species. DNA-binding domains (DBDs) showed strong conservation in length, position, and cysteine-containing motifs. Phylogenetic comparisons and Mantel tests revealed only moderate concordance between DBD-based relationships and those based on non-DBDs or full-length proteins, supporting diversification outside the conserved DBD framework. PCA revealed physicochemical variation in DBDs, non-DBDs, and full-length proteins. In addition, several atypical proteins exhibited domain duplication, rearrangement, or unusual compositional features. These findings indicate that GAL4-like Zn₂Cys₆ proteins combine conservation of the core zinc-cluster framework with extensive structural and physicochemical diversification.