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KMID : 1034820210170040453
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Molecular & Cellular Toxicology
2021 Volume.17 No. 4 p.453 ~ p.464
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Codon optimization of the synthetic 3-ketosphinganine reductase (3KSR) protein for enhancing sphingolipid biosynthetic enzyme expression
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Um Hyun-Ju
Shin Woo-Ri
Sekhon Simranjeet Singh
Woo Sung-Min
Kim Young-Chang
Ahn Ji-Young
Kim Yang-Hoon
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Abstract
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Background: Sphinganine is a key precursor for the synthesis of ceramide during sphingolipid biosynthesis in eukaryotes. In the sphinganine biosynthetic pathway, serine palmitoyltransferase (SPT) and 3-ketosphinganine reductase (3KSR) catalyze the condensation of serine and palmitoyl-CoA to form 3-ketosphinganine, and the conversation of 3-ketosphinganine to dehydrosphingosine (DHS), respectively.
Objective: Phytosphingosine among cosmeceutical ingredients exists primarily on the surface of the skin and serves as a potent anti-microbial and anti-inflammatory agent. To overexpressing produce phytosphingosine in bacteria, we designed and synthesized the oligonucleotide of two major genes (3KSR and SPT) involved in the biosynthesis of sphingolipid for industrial applications.
Results: The genes encoding SPT from the Gram-negative bacterium Sphingobium chungbukense DJ77 and 3KSR from the eukaryotic Saccharomyces cerevisiae were expressed in Escherichia coli BL21 (DE3) to produce DHS efficiently. Codon optimization was utilized for 3KSR expression in E. coli to facilitate the expression of eukaryotic proteins in the bacterial host. The synthetic 3KSR (3sKSR) protein activity was improved approximately 1.8 times compared to the wild-type 3KSR. Additionally, we successfully overexpressed the spt gene obtained from S. chungbukense DJ77, which can be used in efficient production of the sphenoid bases, with codon-optimized 3sKSR. The enhanced accumulation of DHS by SPT-3sKSR suggests that the codon-optimized enzyme (3sKSR) is more efficient for heterogeneous expression in E. coli.
Conclusion: Our results demonstrate that sphingolipid metabolism can be configured in engineered E. coli by the simultaneous overexpression of two key enzymes, SPT and 3sKSR, thereby producing sphingosine.
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KEYWORD
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Sphingolipids, Dihydrosphingosine, 3-Ketosphinganine reductase, Serine palmitoyltransferase, Codon optimization, Sphingobium chungbukense DJ77
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