[2S04m] 18. Biorefinery and Bioproduction - Part 1

10:05 AM - 10:40 AM JST(1:05 AM - 1:40 AM UTC)

[2BRBP-02-KL] Yeast as a Chassis for the Secretory Production of Human-type Sphingoid Bases

○Hyun Ah Kang¹ (1. Chung-Ang University (Korea))

Keywords:

Sphingolipid Pathway,Sphingoid Bases,Yeast,Targeted Engineering,Secretory Production

Ceramide is the simplest basic backbone of sphingolipids, and sphingoid bases are the fundamental building blocks of all sphingolipids. Ceramides and sphingoid bases have various biotechnological applications as antibacterial and antifungal agents, pharmaceutical and cosmeceutical ingredients, or nutraceutical formulation. Here, we report our recent effort on exploration and exploitation of sphingolipid biosynthetic pathways in several yeast species for the secretory production of sphingoid bases, either in acetylated or non-acetylated forms.
To produce human-type sphingoid bases and ceramides in the oleaginous yeast Yarrowia lipolytica, yeast specific-sphingolipid biosynthetic pathways was blocked by deleting SUR2, encoding sphinganine C4-hydroxylase, and SLD1, encoding fungal specific Δ8 sphingolipid desaturase. The resultant Ylsur2Δsld1Δ strain showed remarkably increased secretory production of dihydroshpingosin (DHS) and sphingosine (SO) at the cell surface even without acetylation, along with the elevated production of human-type glucosylceramides. Additional introduction of mouse ceramidase into the Ylsur2Δsld1Δ further improved the production yield of sphingoid bases.
With the traditional yeast Saccharomyces cerevisiae, which lacks the DES1 gene encoding sphingolipid delta 4-desaturase, thus producing PHS-containing sphingolipids without generating SO-containing ceramides. The recombinant S. cerevisiae strains expressing human DES1 and Fusarium graminearum DES1 led to a significant production of SO-containing ceramides in the sur2Δ background. Notably, SO production was observed only in the recombinant S. cerevisiae strain retaining ceramidase activities. Furthermore, the co-expression of W. ciferrii SLI1, encoding N/O-acetyltransferase, together with S. cerevisiae YPC1, encoding a ceramidase, enhanced the secretory production of acetylated sphingoid bases in both WT and sur2Δ strains.
To explore sphingolipid metabolism in the industrial tetraacetylated phytosphingosine (TAPS)-producing yeast Wickerhamomyces ciferrii LCS, we performed de novo whole-genome sequencing and identified the strain as a heterozygous diploid. To facilitate genetic manipulation by improving homologous recombination efficiency, we developed the lig4(A)Δ/lig4(B)Δ strain, lacking a key enzyme in the non-homologous end joining pathway, and subsequently generated sur2(A)Δ/sur2(B)Δ mutants through sequential deletion of two W. ciferrii SUR2 genes, encoding sphingolipid C4-hydroxylase. The W. ciferrii sur2(A)Δ/sur2(B)Δ strain showed significant flux redirection from TAPS to 2-acetylated DHS as a major secretory sphingoid base, demonstrating the genome-informed targeted engineering of sphingolipid pathways as efficient strategy to develop a host platform for production of specific sphingoid bases.