Effect of Expression of Genes in the Sphingolipid Synthesis Pathway on the Biosynthesis of Ceramide in Saccharomyces cerevisiae

Abstract

Ceramide is important not only for the maintenance of the barrier function of the skin but also for the waterbinding capacity of the stratum corneum. Although the exact role of ceramide in the human skin is not fully understood, ceramide has become a widely used ingredient in cosmetic and pharmaceutical industries. Compared with other microorganisms, yeast is more suitable for the production of ceramide because yeast grows fast and is non-toxic. However, production of ceramide from yeast has not been widely studied and most work in this area has been carried out using Saccharomyces cerevisiae. Regulating the genes that are involved in sphingolipid synthesis is necessary to increase ceramide production. In this study, we investigated the effect of the genes involved in the synthesis of ceramide, lcb1, lcb2, tsc10, lac1, lag1, and sur2, on ceramide production levels. The genes were cloned into pYES2 high copy number vectors. S. cerevisiae was cultivated on YPDG medium at 30 o C. Ceramide was purified from the cell extracts by solvent extraction and the ceramide content was analyzed by HPLC using ELSD. The maximum production of ceramide (9.8 mg ceramide/g cell) was obtained when the tsc10 gene was amplified by the pYES2 vector. Real-time RT-PCR analysis showed that the increase in ceramide content was proportional to the increase in the tsc10 gene expression level, which was 4.56 times higher than that of the control strain.

Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome

“…S10C). As an essential gene, YBR265W encodes 3-ketosphinganine reductase (Tsc10p) that catalyzes the second step in the pathway for sphingolipid synthesis in S. cerevisiae (25). Sphingolipids are reportedly involved in the regulation of the yeast high-osmolarity glycerol (HOG) response pathway (26).…”

An inducible ER–Golgi tether facilitates ceramide transport to alleviate lipotoxicity

“…To test this, we investigated whether Nvj2p allows cells to tolerate elevated ceramide synthesis and prevents ceramide accumulation. It has been found that overexpression of the ceramide synthase Lag1p increases ceramide levels and decreases growth rate (Kim et al, 2010). We introduced into wild-type cells a plasmid expressing Lag1p under the galactose-inducible GAL1 promoter.…”

Seipin negatively regulates sphingolipid production at the ER–LD contact site

“…Among all sphingolipid biosynthetic enzymes, Tsc10 localizes specifically to LDs with no functional implication (Currie et al, 2014). Tsc10 overexpression increases LCB and ceramide levels (Kim et al, 2010), whereas TSC10-DAmP mutant accumulated Figure 7. Blocking sphingolipid synthesis does not affect LD biogenesis, but LD morphology is affected by sphingoid intermediates.…”