The Ultimate Micro-Exon: A Single Nucleotide Exon Is Required to Assemble Cytochrome P450 CYP621A Orthologs from Fusarium Species

Cytochrome P450 monooxygenases (CYPs/P450s) play a key role in organisms' primary and secondary metabolism in species across all domains of life. Accurate annotation of P450 genes is crucial for identifying their functions, evolution, and, consequently, their biotechnological potential. In this study, we report the identification of an unprecedented one-nucleotide exon required for the correct assembly of <i>CYP621A</i> P450 genes from multiple <i>Fusarium</i> species. Through comparative genomic analysis of 20 orthologous <i>CYP621A</i> genes, supported by an intronless <i>CYP621B1</i> gene from <i>Aspergillus clavatus</i>, we demonstrate that omission of this single-nucleotide exon disrupts exon phase compatibility and prevents reconstruction of a full-length, functional P450 protein. The micro-exon encodes the central nucleotide of the glycine codon in the highly conserved PKG motif, which is essential for maintaining the structural integrity between the EXXR and PERF motifs, a characteristic of P450 enzymes. Importantly, transcriptomic evidence from sequence read archive (SRA) data confirms accurate splicing of this one-nucleotide exon in <i>Fusarium solani</i> and <i>F. acuminatum</i> under multiple growth conditions. This work presents the second example of the smallest exon reported to date for a gene, and the first for a P450 gene or a fungal gene. The study's findings have broad implications for genome annotation pipelines, underscoring the need for careful manual curation and improved algorithms to detect ultra-small exons in functionally constrained regions of eukaryotic genes.