Functional Characterization and Application of Yarrowia lipolytica YlYxc1 for Enhanced Production of Sphingoid Bases

Long-chain bases (LCBs; sphingoid bases), derived from ceramide metabolism, are valuable sphingolipid intermediates for industrial applications. Here, we functionally characterized a putative ceramidase from Yarrowia lipolytica (YlYxc1) and evaluated its potential to enhance LCB production. YlYxc1 contains a conserved alkaline ceramidase motif with seven transmembrane helices and shares ~42% amino acid identity with Saccharomyces cerevisiae alkaline ceramidases ScYpc1 and ScYdc1. Despite lacking a canonical ER retention motif, YlYxc1 localized to the ER membrane. Deletion of YlYXC1 caused no major growth or lipid profile changes. Heterologous overexpression of YlYXC1 in S. cerevisiae wild-type and Scypc1Δydc1Δ strains increased phytosphingosine (PHS) production over dihydrosphingosine (DHS), mimicking ScYPC1 overexpression. In vitro activity assays using microsomal fractions from Scypc1Δydc1Δ cells expressing YlYXC1 revealed preferential hydrolysis of phytoceramide with an alkaline pH optimum. Furthermore, YlYXC1 expression in an engineered Y. lipolytica strain expressing heterologous acetyltransferases WcSLI1 and WcATF2 remarkably enhanced acetylated LCB production. Multi-copy integration of YlYXC1 into the Ylsur2Δsld1Δ strain also increased the secretory production of non-acetylated DHS. These findings establish YlYxc1 as a promising metabolic engineering target for producing value-added LCBs for pharmaceutical and cosmeceutical applications.