Skip to main content
SpringerLink
Log in

Characterization of the superoxide dismutase SOD1 gene of Kluyveromyces marxianus L3 and improved production of SOD activity

  • Biotechnologically Relevant Enzymes and Proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Superoxide dismutase (SOD) activity is one major defense line against oxidative stress for all of the aerobic organisms, and industrial production of this enzyme is highly demanded. The Cu/Zn superoxide dismutase gene (KmSOD1) of Kluyveromyces marxianus L3 was cloned and characterized. The deduced KmSod1p protein shares 86% and 71% of identity with Kluyveromyces lactis and Saccharomyces cerevisiae Sod1p, respectively. The characteristic motifs and the amino acid residues involved in coordinating copper and zinc and in enzymatic function were conserved. To the aim of developing a microbial production of Cu/Zn superoxide dismutase, we engineered the K. marxianus L3 strain with the multicopy plasmid YG-KmSOD1 harboring the KmSOD1 gene. The production of KmSOD1p in K. marxianus L3 and K. marxianus L3 (pYG-KmSOD1) in response to different compositions of the culture medium was evaluated. The highest specific activity (472 USOD mgprot −1) and the highest volumetric yield (8.8 × 105 USOD l−1) were obtained by the recombinant strain overexpressing KmSOD1 in the presence of Cu2+ and Zn2+ supplements to the culture media. The best performing culture conditions were positively applied to a laboratory scale fed-batch process reaching a volumetric yield of 1.4 × 106 USOD l−1.

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

Similar content being viewed by others

References

  • Bender JP, Mazutti MA, de Oliveira D, Di Luccio M, Treichel H (2006) Inulinase production by Kluyveromyces marxianus NRRL Y-7571 using solid state fermentation. Appl Biochem Biotechnol 129–132:951–958

    Article  Google Scholar 

  • Butt TR, Sternberg EJ, Gorman JA, Clark P, Hamer D, Rosenberg M, Crooke ST (1984) Copper metallothionein of yeast, structure of the gene, and regulation of expression. Proc Natl Acad Sci U S A 81:3332–3336

    Article  CAS  Google Scholar 

  • Campanella JJ, Bitincka L, Smalley J (2003) MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. BMC Bioinformatics 4:29

    Article  Google Scholar 

  • Carroll MC, Girouard JB, Ulloa JL, Subramaniam JR, Wong PC, Valentine JS, Culotta VC (2004) Mechanisms for activating Cu- and Zn-containing superoxide dismutase in the absence of the CCS Cu chaperone. Proc Natl Acad Sci U S A 101:5964–5969

    Article  CAS  Google Scholar 

  • Chen XJ, Saliola M, Falcone C, Bianchi MM, Fukuhara H (1986) Sequence organization of the circular plasmid pKD1 from the yeast Kluyveromyces drosophilarum. Nucleic Acids Res 14:4471–4481

    Article  CAS  Google Scholar 

  • Corvo ML, Jorge JC, van't HR, Cruz ME, Crommelin DJ, Storm G (2002) Superoxide dismutase entrapped in long-circulating liposomes: formulation design and therapeutic activity in rat adjuvant arthritis. Biochim Biophys Acta 1564:227–236

    Article  Google Scholar 

  • Culotta VC, Klomp LW, Strain J, Casareno RL, Krems B, Gitlin JD (1997) The copper chaperone for superoxide dismutase. J Biol Chem 272:23469–23472

    Article  CAS  Google Scholar 

  • Dancis A, Haile D, Yuan DS, Klausner RD (1994) The Saccharomyces cerevisiae copper transport protein (Ctr1p). Biochemical characterization, regulation by copper, and physiologic role in copper uptake. J Biol Chem 269:25660–25667

    CAS  PubMed  Google Scholar 

  • Dellomonaco C, Amaretti A, Zanoni S, Pompei A, Matteuzzi D, Rossi M (2007) Fermentative production of superoxide dismutase with Kluyveromyces marxianus. J Ind Microbiol Biotechnol 34:27–34

    Article  CAS  Google Scholar 

  • Emerit J, Samuel D, Pavio N (2006) Cu-Zn super oxide dismutase as a potential antifibrotic drug for hepatitis C related fibrosis. Biomed Pharmacother 60:1–4

    Article  CAS  Google Scholar 

  • Field LS, Luk E, Culotta VC (2002) Copper chaperones: personal escorts for metal ions. J Bioenerg Biomembr 34:373–379

    Article  CAS  Google Scholar 

  • Fonseca GG, Gombert AK, Heinzle E, Wittmann C (2007) Physiology of the yeast Kluyveromyces marxianus during batch and chemostat cultures with glucose as the sole carbon source. FEMS Yeast Res 7:422–435

    Article  CAS  Google Scholar 

  • Fridovich I (1978) The biology of oxygen radicals. Science 201:875–880

    Article  CAS  Google Scholar 

  • Fridovich I (1998) Oxygen toxicity: a radical explanation. J Exp Biol 201:1203–1209

    CAS  PubMed  Google Scholar 

  • Glerum DM, Shtanko A, Tzagoloff A (1996) Characterization of COX17, a yeast gene involved in copper metabolism and assembly of cytochrome oxidase. J Biol Chem 271:14504–14509

    Article  CAS  Google Scholar 

  • Goulielmos GN, Arhontaki K, Eliopoulos E, Tserpistali K, Tsakas S, Loukas M (2003) Drosophila Cu,Zn superoxide dismutase gene confers resistance to paraquat in Escherichia coli. Biochem Biophys Res Commun 308:433–438

    Article  CAS  Google Scholar 

  • Harris N, Bachler M, Costa V, Mollapour M, Moradas-Ferreira P, Piper PW (2005) Overexpressed Sod1p acts either to reduce or to increase the lifespans and stress resistance of yeast, depending on whether it is Cu(2+)-deficient or an active Cu,Zn-superoxide dismutase. Aging Cell 4:41–52

    Article  CAS  Google Scholar 

  • Hart PJ, Balbirnie MM, Ogihara NL, Nersissian AM, Weiss MS, Valentine JS, Eisenberg D (1999) A structure-based mechanism for copper-zinc superoxide dismutase. Biochemistry 38:2167–2178

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  • Limtong S, Sringiew C, Yongmanitchai W (2007) Production of fuel ethanol at high temperature from sugar cane juice by a newly isolated Kluyveromyces marxianus. Bioresour Technol 98:3367–3374

    Article  CAS  Google Scholar 

  • Lin SJ, Culotta VC (1995) The ATX1 gene of Saccharomyces cerevisiae encodes a small metal homeostasis factor that protects cells against reactive oxygen toxicity. Proc Natl Acad Sci U S A 92:3784–3788

    Article  CAS  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    CAS  Google Scholar 

  • Luk E, Yang M, Jensen LT, Bourbonnais Y, Culotta VC (2005) Manganese activation of superoxide dismutase 2 in the mitochondria of Saccharomyces cerevisiae. J Biol Chem 280:22715–22720

    Article  CAS  Google Scholar 

  • Masoud W, Jespersen L (2006) Pectin degrading enzymes in yeasts involved in fermentation of Coffea arabica in East Africa. Int J Food Microbiol 110:291–296

    Article  CAS  Google Scholar 

  • McCord JM, Fridovich I (1969) Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244:6049–6055

    CAS  Google Scholar 

  • Moradas-Ferreira P, Costa V, Piper P, Mager W (1996) The molecular defences against reactive oxygen species in yeast. Mol Microbiol 19:651–658

    Article  CAS  Google Scholar 

  • Nishikawa M, Nagatomi H, Nishijima M, Ohira G, Chang BJ, Sato E, Inoue M (2001) Targeting superoxide dismutase to renal proximal tubule cells inhibits nephrotoxicity of cisplatin and increases the survival of cancer-bearing mice. Cancer Lett 171:133–138

    Article  CAS  Google Scholar 

  • Rae TD, Schmidt PJ, Pufahl RA, Culotta VC, O’Halloran TV (1999) Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutase. Science 284:805–808

    Article  CAS  Google Scholar 

  • Saliola M, Mazzoni C, Solimando N, Crisa A, Falcone C, Jung G, Fleer R (1999) Use of the KlADH4 promoter for ethanol-dependent production of recombinant human serum albumin in Kluyveromyces lactis. Appl Environ Microbiol 65:53–60

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (2001) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Sherman F, Fink GR, Hicks JB (1986) Methods in Yeast Genetics: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Sturtz LA, Diekert K, Jensen LT, Lill R, Culotta VC (2001) A fraction of yeast Cu,Zn-superoxide dismutase and its metallochaperone, CCS, localize to the intermembrane space of mitochondria. A physiological role for SOD1 in guarding against mitochondrial oxidative damage. J Biol Chem 276:38084–38089

    CAS  PubMed  Google Scholar 

  • Taylor WR (1986) The classification of amino acid conservation. J Theor Biol 119:205–218

    Article  CAS  Google Scholar 

  • Vorauer-Uhl K, Furnschlief E, Wagner A, Ferko B, Katinger H (2001) Topically applied liposome encapsulated superoxide dismutase reduces postburn wound size and edema formation. Eur J Pharm Sci 14:63–67

    Article  CAS  Google Scholar 

  • Wesolowsky-Louvel M, Breunig KD, Fukuhara H (1996) Kluyveromyces lactis. In: Wolf K (ed) Nonconventional Yeasts in Biotechnology. Springer, Berlin, pp 139–201

    Chapter  Google Scholar 

  • Wink DA, Mitchell JB (1998) Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med 25:434–456

    Article  CAS  Google Scholar 

  • Yabe Y, Kobayashi N, Nishihashi T, Takahashi R, Nishikawa M, Takakura Y, Hashida M (2001) Prevention of neutrophil-mediated hepatic ischemia/reperfusion injury by superoxide dismutase and catalase derivatives. J Pharmacol Exp Ther 298:894–899

    CAS  PubMed  Google Scholar 

  • Yoo HY, Kim SS, Rho HM (1999) Overexpression and simple purification of human superoxide dismutase (SOD1) in yeast and its resistance to oxidative stress. J Biotechnol 68:29–35

    Article  CAS  Google Scholar 

  • Yu P (2007) A new approach to the production of the recombinant SOD protein by methylotrophic Pichia pastoris. Appl Microbiol Biotech 74:93–98

    Article  CAS  Google Scholar 

  • Yunoki M, Kawauchi M, Ukita N, Sugiura T, Ohmoto T (2003) Effects of lecithinized superoxide dismutase on neuronal cell loss in CA3 hippocampus after traumatic brain injury in rats. Surg Neurol 59:156–160

    Article  Google Scholar 

  • Zhang Y, Wang JZ, Wu YJ, Li WG (2002) Anti-inflammatory effect of recombinant human superoxide dismutase in rats and mice and its mechanism. Acta Pharmacol Sin 23:439–444

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This study was partially supported by PRIN 2006 research program: “Yeast as sources of biodiversity for the production of molecules of agro-alimentary and pharmaceutical interest” and by Ateneo research funding La Sapienza 2007.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Modena and Reggio Emilia, Modena, Italy

    S. Raimondi, U. M. Pagnoni & M. Rossi

  2. Department of Developmental and Cell Biology, University of Rome “La Sapienza”, Piazza Aldo Moro 5, 00185, Rome, Italy

    D. Uccelletti & C. Palleschi

  3. Department of Pharmaceutical Science, University of Bologna, Bologna, Italy

    D. Matteuzzi

Authors
  1. S. Raimondi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D. Uccelletti
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D. Matteuzzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. U. M. Pagnoni
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M. Rossi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. C. Palleschi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to C. Palleschi.

Electronic supplementary material

Supplementary Fig. 1

Coomassie-stained electrophoresis gel of the protein extracts utilized in the Western blotting against Sod1p reported in Fig. 2. The molecular mass of Sod1p is reported (GIF 1.62 mb)

Supplementary Fig. 1

Coomassie-stained electrophoresis gel of the protein extracts utilized in the Western blotting against Sod1p reported in Fig. 2. The molecular mass of Sod1p is reported (GIF 1.62 mb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Raimondi, S., Uccelletti, D., Matteuzzi, D. et al. Characterization of the superoxide dismutase SOD1 gene of Kluyveromyces marxianus L3 and improved production of SOD activity. Appl Microbiol Biotechnol 77, 1269–1277 (2008). https://doi.org/10.1007/s00253-007-1270-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-007-1270-8

Keywords

Search

Navigation