SpringerLink
Log in

Codon optimization of the synthetic 3-ketosphinganine reductase (3KSR) protein for enhancing sphingolipid biosynthetic enzyme expression

  • Original Article
  • Published:
Molecular & Cellular Toxicology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Angov E, Legler PM, Mease RM (2011) Adjustment of codon usage frequencies by codon harmonization improves protein expression and folding. In: Heterologous Gene Expression in E. coli, pp 1–13

  • Beeler T et al (1998) The Saccharomyces cerevisiae TSC10/YBR265w gene encoding 3-ketosphinganine reductase is identified in a screen for temperature-sensitive suppressors of the Ca2+-sensitive csg2Δ mutant. J Biol Chem 273:30688–30694

    Article  CAS  Google Scholar 

  • Buede R, Rinker-Schaffer C, Pinto W, Lester R, Dickson R (1991) Cloning and characterization of LCB1, a Saccharomyces gene required for biosynthesis of the long-chain base component of sphingolipids. J Bacteriol 173:4325–4332

    Article  CAS  Google Scholar 

  • Buhr F et al (2016) Synonymous codons direct cotranslational folding toward different protein conformations. Mol Cell 61:341–351

    Article  CAS  Google Scholar 

  • Burenjargal M et al (2007) Endogenous sphingolipid metabolites related to the growth in Sphingomonas chungbukensis. Arch Pharmacal Res 30:317–322

    Article  CAS  Google Scholar 

  • Burgess-Brown NA et al (2008) Codon optimization can improve expression of human genes in Escherichia coli: a multi-gene study. Protein Expr Purif 59:94–102

    Article  CAS  Google Scholar 

  • Castegnaro M et al (1998) Analytical method for the determination of sphinganine and sphingosine in serum as a potential biomarker for fumonisin exposure. J Chromatogr B Biomed Sci Appl 720:15–24

    Article  CAS  Google Scholar 

  • Fornarotto M et al (2006) Sphingolipid biosynthesis in pathogenic fungi: Identification and characterization of the 3-ketosphinganine reductase activity of Candida albicans and Aspergillus fumigatus. Biochem Biophys Acta 1761:52–63

    CAS  PubMed  Google Scholar 

  • Gupta RS, Mok A (2007) Phylogenomics and signature proteins for the alpha proteobacteria and its main groups. BMC Microbiol 7:1–20

    Article  CAS  Google Scholar 

  • Hale RS, Thompson G (1998) Codon Optimization of the gene encoding a domain from human type 1 neurofibromin protein results in a threefold improvement in expression level in Escherichia coli. Protein Expr Purif 12:185–188

    Article  CAS  Google Scholar 

  • Harrison PJ, Dunn TM, Campopiano DJ (2018) Sphingolipid biosynthesis in man and microbes. Nat Prod Rep 35:921–954

    Article  CAS  Google Scholar 

  • He F (2011) Laemmli-sds-page. Bio-protocol, pp e80–e80

  • Imamura T, Kusano H, Kajigaya Y, Ichikawa M, Shimada H (2007) A rice dihydrosphingosine C4 hydroxylase (DSH1) gene, which is abundantly expressed in the stigmas, vascular cells and apical meristem, may be involved in fertility. Plant Cell Physiol 48:1108–1120

    Article  CAS  Google Scholar 

  • Kim SJ, Chun J, Bae KS, Kim YC (2000) Polyphasic assignment of an aromatic-degrading Pseudomonas sp. strain DJ77 in the genus Sphingomonas as Sphingomonas chungbukensis sp. nov. Int J Syst Evol Microbiol 50(4):1641–1647. https://doi.org/10.1099/00207713-50-4-1641

    Article  CAS  PubMed  Google Scholar 

  • Krebs S, Medugorac I, Röther S, Strässer K, Förster M (2007) A missense mutation in the 3-ketodihydrosphingosine reductase FVT1 as candidate causal mutation for bovine spinal muscular atrophy. Proc Natl Acad Sci 104:6746–6751

    Article  CAS  Google Scholar 

  • Kruger NJ (2009) The Bradford method for protein quantitation. In: The protein protocols handbook, pp 17–24

  • Lin M-H et al (2019) Fatty acid transport protein 4 is required for incorporation of saturated ultralong-chain fatty acids into epidermal ceramides and monoacylglycerols. Sci Rep 9:1–18

    Google Scholar 

  • Lowther J et al (2010) Inhibition of the PLP-dependent enzyme serine palmitoyltransferase by cycloserine: evidence for a novel decarboxylative mechanism of inactivation. Mol BioSyst 6:1682–1693

    Article  CAS  Google Scholar 

  • Martin VJ, Pitera DJ, Withers ST, Newman JD, Keasling JD (2003) Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol 21:796–802

    Article  CAS  Google Scholar 

  • McDonald JP, Levine AS and Woodgate R (1997) The Saccharomyces cerevisiae RAD30 gene, a homologue of Escherichia coli dinB and umuC, is DNA damage inducible and functions in a novel error-free postreplication repair mechanism. Genetics 147:1557–1568

    Article  CAS  Google Scholar 

  • Merrill A Jr et al (1986) Inhibition of phorbol ester-dependent differentiation of human promyelocytic leukemic (HL-60) cells by sphinganine and other long-chain bases. J Biol Chem 261:12610–12615

    Article  CAS  Google Scholar 

  • Min J-K, Yoo H-S, Lee E-Y, Lee W-J, Lee Y-M (2002) Simultaneous quantitative analysis of sphingoid base 1-phosphates in biological samples by o-phthalaldehyde precolumn derivatization after dephosphorylation with alkaline phosphatase. Anal Biochem 303:167–175

    Article  CAS  Google Scholar 

  • Nagiec MM, Baltisberger JA, Wells GB, Lester RL, Dickson RC (1994) The LCB2 gene of Saccharomyces and the related LCB1 gene encode subunits of serine palmitoyltransferase, the initial enzyme in sphingolipid synthesis. Proc Natl Acad Sci 91:7899–7902

    Article  CAS  Google Scholar 

  • Patterson SS, Dionisi HM, Gupta RK, Sayler GS (2005) Codon optimization of bacterial luciferase (lux) for expression in mammalian cells. J Ind Microbiol Biotechnol 32:115–123

    Article  CAS  Google Scholar 

  • Pechmann S, Chartron JW, Frydman J (2014) Local slowdown of translation by nonoptimal codons promotes nascent-chain recognition by SRP in vivo. Nat Struct Mol Biol 21:1100–1105

    Article  CAS  Google Scholar 

  • Pinto WJ, Wells GW, Lester RL (1992) Characterization of enzymatic synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae: mutant strains exhibiting long-chain-base auxotrophy are deficient in serine palmitoyltransferase activity. J Bacteriol 174:2575–2581

    Article  CAS  Google Scholar 

  • Richard G, White TW, Smith LE, Bailey RA, Compton JG, Paul DL, Bale SJ (1998) Functional defects of Cx26 resulting from a heterozygous missense mutation in a family with dominant deaf-mutism and palmoplantar keratoderma. Human Genetics 103(4):393–399. https://doi.org/10.1007/s004390050839

    Article  CAS  PubMed  Google Scholar 

  • Rim K-T (2021) Application of the adverse outcome pathway framework to predict the toxicity of chemicals in the semiconductor manufacturing industry. In: Molecular & cellular toxicology, pp 1–21

  • Spencer PS, Siller E, Anderson JF, Barral JM (2012) Silent substitutions predictably alter translation elongation rates and protein folding efficiencies. J Mol Biol 422:328–335

    Article  CAS  Google Scholar 

  • Tessema EN, Gebre-Mariam T, Neubert RH, Wohlrab J (2017) Potential applications of phyto-derived ceramides in improving epidermal barrier function. Skin Pharmacol Physiol 30:115–138

    Article  CAS  Google Scholar 

  • Villalobos A, Ness JE, Gustafsson C, Minshull J, Govindarajan S (2006) Gene designer: a synthetic biology tool for constructing artificial DNA segments. BMC Bioinformatics 7:1–8

    Article  Google Scholar 

  • Williams P, Macdearmid A, Innes G, Gauld S (1984) Ammonia-treated barley straw and rolled barley offered either together, in a mixed ration, or successively to beef steers. Anim Feed Sci Technol 10:247–255

    Article  Google Scholar 

  • Yadava A, Ockenhouse CF (2003) Effect of codon optimization on expression levels of a functionally folded malaria vaccine candidate in prokaryotic and eukaryotic expression systems. Infect Immun 71:4961–4969

    Article  CAS  Google Scholar 

  • Yi H, Bojja RS, Fu J, Du L (2005) Direct evidence for the function of FUM13 in 3-ketoreduction of mycotoxin fumonisins in Fusarium verticillioides. J Agric Food Chem 53:5456–5460

    Article  CAS  Google Scholar 

  • Yu C-H et al (2015) Codon usage influences the local rate of translation elongation to regulate co-translational protein folding. Mol Cell 59:744–754

    Article  CAS  Google Scholar 

  • Zabin I, Mead JF (1953) The biosynthesis of sphingosine: I The utilization of carboxyl-labeled acetate. J Biol Chem 205:271–277

    Article  CAS  Google Scholar 

  • Zhao C (1994) Suppressors (scsl-scs7) of CSG2, a Gene Required by Saccharomyces cerevisiae for Growth in Media Containing 10 mMCa (2+), Identify Genes Required for Sphingolipid Biosynthesis. (Uniformed Services University Of The Health Sciences Bethesda United States

  • Zheng W et al (2006) Ceramides and other bioactive sphingolipid backbones in health and disease: lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy. Biochem Biophys Acta 1758:1864–1884

    Article  CAS  Google Scholar 

  • Zhou M et al (2013) Non-optimal codon usage affects expression, structure and function of clock protein FRQ. Nature 495:111–115

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1A6A1A06046235). This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. NRF-2019R1A2C1010860).

Author information

Author notes

  1. Hyun-Ju Um and Woo-Ri Shin have contributed equally to this work.

Authors and Affiliations

  1. School of Biological Sciences, Chungbuk National University, Chungdae-Ro, Seowon-Gu, Cheongju, 28644, South Korea

    Hyun-Ju Um, Woo-Ri Shin, Simranjeet Singh Sekhon, Young-Chang Kim, Ji-Young Ahn & Yang-Hoon Kim

  2. Department of Biological Sciences and Biotechnology, Chungbuk National University, Cheongju, Chungbuk, 28644, South Korea

    Woo-Ri Shin, Ji-Young Ahn & Yang-Hoon Kim

  3. Department of Food Science and Biotechnology, Shin Ansan University, 135, Sinansandaehak-Ro, Danwon-Gu, Ansan, 15435, South Korea

    Sung Min Woo

Authors
  1. Hyun-Ju Um
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Woo-Ri Shin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Simranjeet Singh Sekhon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sung Min Woo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Young-Chang Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ji-Young Ahn
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yang-Hoon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H-JU, W-RS, SSS, and SMW: Experiments design and results analysis; YCK, J-YA and Y-HK: Investigation and data analysis. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Young-Chang Kim, Ji-Young Ahn or Yang-Hoon Kim.

Ethics declarations

Conflict of interest

Authors H.-J.U, W.-R.S, J.-Y.A, S.S.S, S.M.W, Y.-C.K, and Y.-H.K. declares that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 851 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Um, HJ., Shin, WR., Sekhon, S.S. et al. Codon optimization of the synthetic 3-ketosphinganine reductase (3KSR) protein for enhancing sphingolipid biosynthetic enzyme expression. Mol. Cell. Toxicol. 17, 453–464 (2021). https://doi.org/10.1007/s13273-021-00153-6

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13273-021-00153-6

Keywords

Search

Navigation