Skip to main content
SpringerLink
Log in

Cloning and characterization of squalene synthase gene from Fusarium fujikuroi (Saw.) Wr.

  • Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

The gene encoding squalene synthase (GfSQS) was cloned from Fusarium fujikuroi (Gibberella fujikuroi MP-C) and characterized. The cloned genomic DNA is 3,267 bp in length, including the 5′-untranslated region (UTR), 3′-UTR, four exons, and three introns. A noncanonical splice-site (CA-GG, or GC-AG) was found at the first intron. The open reading frame of the gene is 1,389 bp in length, corresponding to a predicted polypeptide of 462 amino acid residues with a MW 53.4 kDa. The predicted GfSQS shares at least four conserved regions involved in the enzymatic activity with the SQSs of varied species. The recombinant protein was expressed in E. coli and detected by SDS–PAGE and western blot. GC–MS analysis showed that the wild-type GfSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene, while the mutant mGfSQS (D82G) lost total activity, supporting the prediction that the aspartate-rich motif (DTXED) in the region I of SQS is essential for binding of the diphosphate substrate.

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

Access this article

Log in via an institution

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
Fig. 7

Similar content being viewed by others

References

  1. Ashby MN, Edwards PA (1990) Elucidation of the deficiency in two yeast coenzyme Q mutants. Characterization of the structural gene encoding hexaprenyl pyrophosphate synthetase. J Biol Chem 265:13157–13164

    CAS  PubMed  Google Scholar 

  2. Bömke C, Rojas MC, Gong F, Hedden P, Tudzynski B (2008) Isolation and characterization of the gibberellin biosynthetic gene cluster in Sphaceloma manihoticola. Appl Environ Microbiol 74:5325–5339

    Article  PubMed  Google Scholar 

  3. Burset M, Seledtsov A, Solovyev VV (2000) Analysis of canonical and noncanonical splice sites in mammalian genomes. Nucleic Acids Res 28:4364–4375

    Article  CAS  PubMed  Google Scholar 

  4. Chong A, Zhang G, Bajic VB (2004) Information for the coordinates of exons (ICE): a human splice sites database. Genomics 84:762–766

    Article  CAS  PubMed  Google Scholar 

  5. Domenech CE, Giordano W, Avalos J, Cerdá -Olmedo E (1996) Separate compartments for the production of sterols, carotenoids and gibberellins in Gibberella fujikuroi. Eur J Biochem 239:720–725

    Article  CAS  PubMed  Google Scholar 

  6. Fernández-Martín R, Domenech C, Cerdá-Olmedo E, Avalos J (2000) Ent-kaurene and squalene synthesis in Fusarium fujikuroi cell-free extracts. Phytochemistry 54:723–728

    Article  PubMed  Google Scholar 

  7. Farrer T, Roller AB, Kent WJ, Zahler AM (2002) Analysis of the role of Caenorhabditis elegans GC-AG introns in regulated splicing. Nucleic Acids Res 30:3360–3367

    Article  CAS  PubMed  Google Scholar 

  8. Goldstein JL, Brown MS (1990) Regulation of the mevalonate pathway. Nature 343:425–430

    Article  CAS  PubMed  Google Scholar 

  9. Gu P, Ishii Y, Spencer TA, Shechter I (1998) Function-structure studies and identification of three enzyme domains involved in the catalytic activity in rat hepatic squalene synthase. J Biol Chem 273:12515–12525

    Article  CAS  PubMed  Google Scholar 

  10. Guan G, Dai PH, Osborne TF, Kim JB, Shechter I (1997) Multiple sequence elements are involved in the transcriptional regulation of the human squalene synthase gene. J Biol Chem 272:10295–10302

    Article  CAS  PubMed  Google Scholar 

  11. Hanley KM, Nicolas O, Donaldson TB, Smith-Monroy C, Robinson GW, Hellmann GM (1996) Molecular cloning, in vitro expression and characterization of a plant squalene synthetase cDNA. Plant Mol Biol 30:1139–1151

    Article  CAS  PubMed  Google Scholar 

  12. Huang Z, Jiang K, Pi Y, Hou R, Liao Z, Cao Y, Han X, Wang Q, Sun X, Tang K (2007) Molecular cloning and characterization of the yew gene encoding squalene synthase from Taxus cuspidata. J Biochem Mol Biol 40:625–635

    CAS  PubMed  Google Scholar 

  13. Inoue T, Osumi T, Hata S (1995) Molecular cloning and functional expression of a cDNA for mouse squalene synthase. Biochim Biophys Acta 1260:49–54

    PubMed  Google Scholar 

  14. Jennings SM, Tsay YH, Fisch TM, Robinson GW (1991) Molecular cloning and characterization of the yeast gene for squalene synthetase. Proc Natl Acad Sci U S A 88:6038–6042

    Article  CAS  PubMed  Google Scholar 

  15. Kennedy MA, Barbuch R, Bard M (1999) Transcriptional regulation of the squalene synthase gene (ERG9) in the yeast Saccharomyces cerevisiae. Biochim Biophys Acta 1445:110–122

    CAS  PubMed  Google Scholar 

  16. Kim OT, Seong NS, Kim MY, Hwang B (2005) Isolation and characterization of squalene synthase cDNA from Centella asiatica (L.) urban. J Plant Biol 48:263–269

    Article  CAS  Google Scholar 

  17. Lange BM, Rujan T, Martin W, Croteau R (2000) Isoprenoid biosynthesis: the evolution of two ancient and distinct pathways across genomes. Proc Natl Acad Sci U S A 97:13172–13177

    Article  CAS  PubMed  Google Scholar 

  18. Lee JH, Yoon YH, Kim HY, Shin DH, Kim DU, Lee IJ, Kim KU (2002) Cloning and expression of squalene synthase cDNA from hot pepper (Capsicum annuum L.). Mol Cells 13:436–443

    CAS  PubMed  Google Scholar 

  19. Lee S, Poulter CD (2008) Cloning, solubilization, and characterization of squalene synthase from Thermosynechococcus elongatus BP-1. J Bacteriol 190:3808–3816

    Article  CAS  PubMed  Google Scholar 

  20. Lewin A, Mayer M, Chusainow J, Jacob D, Appel B (2005) Viral promoters can initiate expression of toxin genes introduced into Escherichia coli. BMC Biotechnol 5:19

    Article  PubMed  Google Scholar 

  21. Liu YG, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8:457–463

    Article  CAS  PubMed  Google Scholar 

  22. LoGrasso PV, Soltis DA, Boettcher BR (1993) Overexpression, purification, and kinetic characterization of a carboxyl-terminal-truncated yeast squalene synthetase. Arch Biochem Biophys 307:193–199

    Article  CAS  PubMed  Google Scholar 

  23. Lu JP, Duan ZB, Liu TB, Lin FC (2007) Cloning, sequencing and expression analysis of the NAR promoter activated during hyphal stage of Magnaporthe grisea. J Zhejiang Univ Sci B 8:661–665

    Article  CAS  PubMed  Google Scholar 

  24. McKenzie TL, Jiang G, Straubhaar JR, Conrad DG, Shechter I (1992) Molecular cloning, expression and characterization of the cDNA for the rat hepatic squalene synthase. J Biol Chem 267:21368–21374

    CAS  PubMed  Google Scholar 

  25. Nakashima T, Inoue T, Oka A, Nishino T, Osumi T, Hata S (1995) Cloning, expression, and characterization of cDNAs encoding Arabidopsis thaliana squalene synthase. Proc Natl Acad Sci U S A 92:2328–2332

    Article  CAS  PubMed  Google Scholar 

  26. Pandit J, Danley DE, Schulte GK, Mazzalupo S, Pauly TA, Hayward CM, Hamanaka ES, Thompson JF, Harwood HJ Jr (2000) Crystal structure of human squalene synthase. A key enzyme in cholesterol biosynthesis. J Biol Chem 275:30610–30617

    Article  CAS  PubMed  Google Scholar 

  27. Paradise EM, Kirby J, Chan R, Keasling JD (2008) Redirection of flux through the FPP branch-point in Saccharomyces cerevisiae by down-regulation squalene synthase. Biotechnol Bioeng 100:371–378

    Article  CAS  PubMed  Google Scholar 

  28. Raeder U, Broda P (1985) Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1:17–20

    Article  CAS  Google Scholar 

  29. Reese MG, Eeckman FH (1995) Novel neural network algorithms for improved eukaryotic promoter site recognition. The Seventh International Genome Sequencing and Analysis Conference, Hilton Head Island

    Google Scholar 

  30. Rep M, Duyvesteijn RG, Gale L, Usgaard T, Cornelissen BJ, Ma LJ, Ward TJ (2006) The presence of GC-AG introns in Neurospora crassa and other euascomycetes determined from analyses of complete genomes: implications for automated gene prediction. Genomics 87:338–347

    Article  CAS  PubMed  Google Scholar 

  31. Robinson GW, Tsay YH, Kienzle BK, Smith-Monroy CA, Bishop RW (1993) Conservation between human and fungal squalene synthetases: similarities in structure, function, and regulation. Mol Cell Biol 13:2706–2717

    CAS  PubMed  Google Scholar 

  32. Rodríguez-Ortiz R, Limón MC, Avalos J (2009) Regulation of carotenogenesis and secondary metabolism by nitrogen in wild-type Fusarium fujikuroi and carotenoid-overproducing mutants. Appl Environ Microbiol 75:405–413

    Article  PubMed  Google Scholar 

  33. Sealey-Cardona M, Cammerer S, Jones S, Ruiz-Pérez LM, Brun R, Gilbert IH, Urbina JA, González-Pacanowska D (2007) Kinetic characterization of squalene synthase from Trypanosoma cruzi: selective inhibition by quinuclidine derivatives. Antimicrob Agents Chemother 51:2123–2129

    Article  CAS  PubMed  Google Scholar 

  34. Shahmuradov KA, Kolchanov NA, Solov’ev VV, Ratner VA (1986) Enhancer-like structures in middle repetitive sequences of the eukaryotic genomes. Genetika 22:357–367

    Google Scholar 

  35. Shimada H, Kondo K, Fraser PD, Miura Y, Saito T, Misawa N (1998) Increased carotenoid production by the food yeast Candida utilis through metabolic engineering of the isoprenoid pathway. Appl Environ Microbiol 64:2676–2680

    CAS  PubMed  Google Scholar 

  36. Skoblov M, Shakhbazov K, Oshchepkov D, Ivanov D, Guskova A, Ivanov D, Rubtsov P, Prasolov V, Yankovsky N, Baranova A (2006) Human RFP2 gene promoter: unique structure and unusual strength. Biochem Biophys Res Commun 342:859–866

    Article  CAS  PubMed  Google Scholar 

  37. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85

    Article  CAS  PubMed  Google Scholar 

  38. Summers C, Karst F, Charles AD (1993) Cloning, expression and characterisation of the cDNA encoding human hepatic squalene synthase, and its relationship to phytoene synthase. Gene 136:185–192

    Article  CAS  PubMed  Google Scholar 

  39. Swofford DL (2004) PAUP*. Phylogenetic analysis using parsimony (and other methods) version 4.0b 10. Sinauer, Sunderland

    Google Scholar 

  40. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    Article  CAS  PubMed  Google Scholar 

  41. Thanaraj TA, Clark F (2001) Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions. Nucleic Acids Res 29:2581–2593

    Article  CAS  PubMed  Google Scholar 

  42. Uchida H, Yamashita H, Kajikawa M, Ohyama K, Nakayachi O, Sugiyama R, Yamato KT, Muranaka T, Fukuzawa H, Takemura M, Ohyama K (2009) Cloning and characterization of a squalene synthase gene from a petroleum plant, Euphorbia tirucalli L. Planta 229:1243–1252

    Article  CAS  PubMed  Google Scholar 

  43. Voss T, Schulte J, Tudzynski B (2001) A new MFS-transporter gene next to the gibberellin biosynthesis gene cluster of Gibberella fujikuroi is not involved in gibberellin secretion. Curr Genet 39:377–383

    Article  CAS  PubMed  Google Scholar 

  44. Withers ST, Keasling JD (2007) Biosynthesis and engineering of isoprenoid small molecules. Appl Microbiol Biotechnol 73:980–990

    Article  CAS  PubMed  Google Scholar 

  45. Zhang D, Jennings SM, Robinson GW, Poulter CD (1993) Yeast squalene synthase: expression, purification, and characterization of soluble recombinant enzyme. Arch Biochem Biophys 304:133–143

    Article  CAS  PubMed  Google Scholar 

  46. Zhao MW, Liang WQ, Zhang DB, Wang N, Wang CG, Pan YJ (2007) Cloning and characterization of squalene synthase (SQS) gene from Ganoderma lucidum. J Microbiol Biotechnol 17:1106–1112

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Professor Chen Ma and Mr. Tong Li at the National Research Center for Analysis of Drugs and Metabolites for the GC–MS analysis. This work was supported by National High Technology Research and Development Program (“863” Program) of China (No.2001AA234021) and Beijing Municipal Natural Science Foundation (No.5992012).

Author information

Authors and Affiliations

  1. Department of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Xian Nong Tan Street, Beijing, 100050, China

    Rui-Yu Zhao, Wei Xiao, Hai-Li Cheng, Ping Zhu & Ke-Di Cheng

  2. Key Laboratory of Biosynthesis of Natural Products, Ministry of Health of PRC, 1 Xian Nong Tan Street, Beijing, 100050, China

    Rui-Yu Zhao, Wei Xiao, Hai-Li Cheng, Ping Zhu & Ke-Di Cheng

  3. Key Laboratory of Bioactive Substances and Resources Utilization, Ministry of Education of PRC, 1 Xian Nong Tan Street, Beijing, 100050, China

    Rui-Yu Zhao, Wei Xiao, Hai-Li Cheng, Ping Zhu & Ke-Di Cheng

Authors
  1. Rui-Yu Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wei Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hai-Li Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ping Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ke-Di Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ping Zhu.

Additional information

R.-Y. Zhao and W. Xiao contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

(DOC 199 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhao, RY., Xiao, W., Cheng, HL. et al. Cloning and characterization of squalene synthase gene from Fusarium fujikuroi (Saw.) Wr.. J Ind Microbiol Biotechnol 37, 1171–1182 (2010). https://doi.org/10.1007/s10295-010-0764-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-010-0764-z

Keywords

Search

Navigation