Presentation Information
[P01-016]Functional characterization of the Yarrowia lipolytica ceramidase YlYxc1p and its application to enhance sphingoid base production
○Seo Hyeon Shin1, Hye Yun Moon, Eun Joo Jeon, Hyun Ah Kang1 (1. Chung-Ang University (Korea))
Keywords:
Yarrowia lipolytica,Alkaline ceramidase,Sphingolipids,Long Chain bases
Long-chain bases (LCBs; sphingoid bases), building blocks for membrane-localized sphingolipids, are increasingly recognized as functionally and industrially relevant sphingolipid intermediates. In this study, we identified and characterized a putative alkaline ceramidase from the oleaginous yeast Yarrowia lipolytica, designated YlYxc1p, and evaluated its potential as an engineering target for improving LCB production.
YlYxc1p contains the conserved alkaline ceramidase domain (Pfam05875) with seven predicted transmembrane helices, and shares ~42% amino acid identity with the Saccharomyces cerevisiae alkaline ceramidases ScYpc1p and ScYdc1p. Although a canonical C-terminal ER retrieval motif was not apparent, fluorescence microscopy of a YlYXC1–GFP fusion protein showed localization to the ER membrane. Deletion of YlYXC1 (Ylyxc1Δ) did not cause overt growth defects, even under several stress conditions. TLC-based lipid profiling revealed no apparent alteration in major lipid-class patterns between the wild type (WT) and Ylyxc1Δ.
To validate its enzymatic function as a ceramidase, YlYXC1 was heterologously expressed in S. cerevisiae (WT) and a ceramidase-deficient Scypc1Δydc1Δ background. In both contexts, YlYXC1 expression increased intracellular LCB levels, with phytosphingosine (PHS) accumulating more prominently than dihydrosphingosine (DHS), yielding profiles similar to those observed upon ScYPC1 overexpression. In vitro assays using microsomal fractions from Scypc1Δydc1Δ cells expressing YlYXC1 showed preferential hydrolysis of phytoceramide and an alkaline pH optimum, supporting its classification as an alkaline ceramidase.
Finally, we applied the overexpression of YlYXC1 in Y. lipolytica platforms to enhance LCB production. Co-expression of YlYXC1 with the Wickerhamomyces ciferrii acetylation modules WcSLI1 and WcATF2 in the WT background enhanced the secretory production of acetylated LCBs, including tetraacetyl-PHS as the major product, and multi-copy integration of YlYXC1 into a Ylsur2Δsld1Δ chassis increased secretion of non-acetylated DHS as the major species. Collectively, these results demonstrate YlYxc1p as an ER-associated alkaline ceramidase in Y. lipolytica and support its utility as a promising metabolic engineering target for the enhanced production of value-added LCBs for pharmaceutical and cosmeceutical applications.
YlYxc1p contains the conserved alkaline ceramidase domain (Pfam05875) with seven predicted transmembrane helices, and shares ~42% amino acid identity with the Saccharomyces cerevisiae alkaline ceramidases ScYpc1p and ScYdc1p. Although a canonical C-terminal ER retrieval motif was not apparent, fluorescence microscopy of a YlYXC1–GFP fusion protein showed localization to the ER membrane. Deletion of YlYXC1 (Ylyxc1Δ) did not cause overt growth defects, even under several stress conditions. TLC-based lipid profiling revealed no apparent alteration in major lipid-class patterns between the wild type (WT) and Ylyxc1Δ.
To validate its enzymatic function as a ceramidase, YlYXC1 was heterologously expressed in S. cerevisiae (WT) and a ceramidase-deficient Scypc1Δydc1Δ background. In both contexts, YlYXC1 expression increased intracellular LCB levels, with phytosphingosine (PHS) accumulating more prominently than dihydrosphingosine (DHS), yielding profiles similar to those observed upon ScYPC1 overexpression. In vitro assays using microsomal fractions from Scypc1Δydc1Δ cells expressing YlYXC1 showed preferential hydrolysis of phytoceramide and an alkaline pH optimum, supporting its classification as an alkaline ceramidase.
Finally, we applied the overexpression of YlYXC1 in Y. lipolytica platforms to enhance LCB production. Co-expression of YlYXC1 with the Wickerhamomyces ciferrii acetylation modules WcSLI1 and WcATF2 in the WT background enhanced the secretory production of acetylated LCBs, including tetraacetyl-PHS as the major product, and multi-copy integration of YlYXC1 into a Ylsur2Δsld1Δ chassis increased secretion of non-acetylated DHS as the major species. Collectively, these results demonstrate YlYxc1p as an ER-associated alkaline ceramidase in Y. lipolytica and support its utility as a promising metabolic engineering target for the enhanced production of value-added LCBs for pharmaceutical and cosmeceutical applications.
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