Advertisement

Uricase production by a recombinant Hansenula polymorpha strain harboring Candida utilis uricase gene

  • Zhiyu Chen
  • Zhaoyue Wang
  • Xiuping He
  • Xuena Guo
  • Weiwei Li
  • Borun Zhang
Biotechnological Products and Process Engineering

Abstract

Uricase is an important medical enzyme which can be used to determine urate in clinical analysis, to therapy gout, hyperuricemia, and tumor lysis syndrome. Uricase of Candida utilis was successfully expressed in Hansenula polymorpha under the control of methanol oxidase promoter using Saccharomyces cerevisiae α-factor signal peptide as the secretory sequence. Recombinant H. polymorpha MU200 with the highest extracellular uricase production was characterized with three copies of expression cassette and selected for process optimization for the production of recombinant enzyme. Among the parameters investigated in shaking flask cultures, the pH value of medium and inoculum size had great influence on the recombinant uricase production. The maximum extracellular uricase yield of 2.6 U/ml was obtained in shaking flask culture. The yield of recombinant uricase was significantly improved by the combined use of a high cell-density cultivation technique and a pH control strategy of switching culture pH from 5.5 to 6.5 in the induction phase. After induction for 58 h, the production of recombinant uricase reached 52.3 U/ml (about 2.1 g/l of protein) extracellularly and 60.3 U/ml (about 2.4 g/l) intracellularly in fed-batch fermentation, which are much higher than those expressed in other expression systems. To our knowledge, this is the first report about the heterologous expression of uricase in H. polymorpha.

Keywords

Candida utilis Recombinant uricase pH control Hansenula polymorpha 

References

  1. Adams A, Gottschling DE, Kaiser CA, Stearns T (1997) Methods in yeast genetics. A Cold Spring Harbor laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  2. Bonnete F, Vivares D, Robert CH, Colloch N (2001) Interactions in solution and crystallization of Aspergillus flavus urate oxidase. J Cryst Growth 232:330–339CrossRefGoogle Scholar
  3. Cereghino GP, Cereghino JL, Ilgen C, Cregg JM (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 13:329–332CrossRefGoogle Scholar
  4. Fitzpatrick DA, McGeeney KF (1975) Antigenic independence of some microbial urate oxidases. Infect Immun 12:1237–1241Google Scholar
  5. Gellissen G, Piontek M, Dahlems U, Jenzelewski V, Gavagan JE, DiCosimo R, Anton DL, Janowicz ZA (1996) Recombinant Hansenula polymorpha as a biocatalyst: coexpression of the spinach glycolate oxidase (GO) and the Saccharomyces cerevisiae catalase T (CTT1) gene. Appl Microbiol Biotechnol 46:46–54CrossRefGoogle Scholar
  6. Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54:741–750CrossRefGoogle Scholar
  7. Gellissen G, Kunze G, Gaillardin C, Cregg JM, Berardi E, Veenhuis M, van der Klei I (2005) New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica—a comparison. FEMS Yeast Res 5:1079–1096CrossRefGoogle Scholar
  8. Gietl C, Faber KN, van der Klei IJ, Veenhuis M (1994) Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using the heterologous host Hansenula polymorpha. Proc Natl Acad Sci USA 91:3151–3155CrossRefGoogle Scholar
  9. Inan M, Meagher MM (2001) Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris. J Biosci Bioeng 92:585–589CrossRefGoogle Scholar
  10. Jahic M, Gustavsson M, Jansen AK, Martinelle M, Enfors SO (2003) Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes. J Biotechnol 102:45–53CrossRefGoogle Scholar
  11. Kang HA, Kang W, Hong WK, Kim MW, Kim JY, Sohn J, Choi ES, Choe KB, Rhee SK (2001) Development of expression systems for the production of recombinant human serum albumin using the MOX promoter in Hansenula polymorpha DL-1. Biotechnol Bioeng 76:175–85CrossRefGoogle Scholar
  12. Klabunde J, Kleebank S, Piontek M, Hollenberg CP, Hellwig S, Degelmann A (2007) Increase of calnexin gene dosage boosts the secretion of heterologous proteins by Hansenula polymorpha. FEMS Yeast Res 7:1168–1180CrossRefGoogle Scholar
  13. Klose S, Stoltz M, Munz E, Portenhauser R (1978) Determination of uric acid on continuous-flow (AutoAnalyzer II and SMA) systems with a uricase/phenol/4-aminophenazone color test. Clin Chem 24:250–255Google Scholar
  14. Koyama Y, Ichikawa T, Nakano E (1996) Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding the Candida utilis urate oxidase (uricase). J Biochem (Tokyo) 120:969–973Google Scholar
  15. Laborde C, Chemardin P, Bigey F, Combarnous Y, Moulin G, Boze H (2004) Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone. Yeast 21:249–263CrossRefGoogle Scholar
  16. Ledeboer AM, Edens L, Maat J, Visser C, Bos JW, Verrips CT, Janowicz Z, Eckart M, Roggenkamp R, Hollenberg CP (1985) Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha. Nucleic Acids Res 13:3063–3082CrossRefGoogle Scholar
  17. Leplatois P, Le Douarin B, Loison G (1992) High-level production of a peroxisomal enzyme: Aspergillus flavus uricase accumulates intracellularly and is active in Saccharomyces cerevisiae. Gene 122:139–145CrossRefGoogle Scholar
  18. Li J, Chen Z, Hou L, Fan H, Weng S, Xu C, Ren J, Li B, Chen W (2006) High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli. Protein Expr Purif 49:55–59CrossRefGoogle Scholar
  19. Linder S, Schliwa M, Kube-Granderath E (1996) Direct PCR screening of Pichia pastoris clones. Biotechniques 20:980–982Google Scholar
  20. Liu J, Li G, Liu H, Zhou X (1994) Purification and properties of uricase from Candida sp. and its application in uric acid analysis in serum. Appl Biochem Biotechnol 47:57–63CrossRefGoogle Scholar
  21. Lotfy WA (2008) Production of a thermostable uricase by a novel Bacillus thermocatenulatus strain. Bioresour Technol 99:699–702CrossRefGoogle Scholar
  22. Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270CrossRefGoogle Scholar
  23. Mayson BE, Kilburn DG, Zamost BL, Raymond CK, Lesnicki GJ (2003) Effects of methanol concentration on expression levels of recombinant protein in fed-batch cultures of Pichia methanolica. Biotechnol Bioeng 81:291–298CrossRefGoogle Scholar
  24. Moon H, Kim H, Rhee SK, Choi ES, Kim IH, Hong SI (2002) Optimal strategy of pH control in the production of recombinant human epidermal growth factor by Hansenula polymorpha. Process Biochem 38:487–495CrossRefGoogle Scholar
  25. Muller F, Tieke A, Waschk D, Muhle C, Muller F, Seigelchifer M, Pesce A, Jenzelewski V, Gellissen G (2002) Production of IFNa-2a in Hansenula polymorpha. Process Biochem 38:15–25CrossRefGoogle Scholar
  26. Nelson DL, Cox MM (2004) Lehninger’s principles of biochemistry, 4th edn. Freeman, New York, pp 866–876Google Scholar
  27. Oda M, Satta Y, Takenaka O, Takahata N (2002) Loss of urate oxidase activity in hominoids and its evolutionary implications. Mol Biol Evol 19:640–653Google Scholar
  28. Oldfield V, Perry CM (2006) Rasburicase: a review of its use in the management of anticancer therapy-induced hyperuricaemia. Drugs 66:529–545CrossRefGoogle Scholar
  29. Sambrook J, Russell DW (2001) Molecular cloning, a laboratory manual, 3rd edn. Cold Spring Harbor, New YorkGoogle Scholar
  30. Shi X, Karkut T, Chamankhah M, Alting-Mees M, Hemmingsen SM, Hegedus D (2003) Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris. Protein Expr Purif 28:321–330CrossRefGoogle Scholar
  31. Sinha J, Plantz BA, Zhang W, Gouthro M, Schlegel V, Liu C-P, Meagher MM (2003) Improved production of recombinant ovine interferon-t by Mutt strain of Pichia pastoris using an optimized methanol feed profile. Biotechnol Prog 19:794–802CrossRefGoogle Scholar
  32. Suzuki H, Verma DP (1991) Soybean nodule-specific uricase (Nodulin-35) is expressed and assembled into a functional tetrameric holoenzyme in Escherichia coli. Plant Physiol 95:384–389CrossRefGoogle Scholar
  33. Suzuki K, Sakasegawa S, Misaki H, Sugiyama M (2004) Molecular cloning and expression of uricase gene from Arthrobacter globiformis in Escherichia coli and characterization of the gene product. J Biosci Bioeng 98:153–158Google Scholar
  34. van der Heide M, Hollenberg CP, van der Klei IJ, Veenhuis M (2002) Overproduction of BiP negatively affects the secretion of Aspergillus niger glucose oxidase by the yeast Hansenula polymorpha. Appl Microbiol Biotechnol 58:487–494CrossRefGoogle Scholar
  35. Vogt B (2005) Urate oxidase (rasburicase) for treatment of severe tophaceous gout. Nephrol Dial Transplant 20:431–433CrossRefGoogle Scholar
  36. Wu XW, Lee CC, Muzny DM, Caskey CT (1989) Urate oxidase: primary structure and evolutionary implications. Proc Natl Acad Sci USA 86:9412–9416CrossRefGoogle Scholar
  37. Yazdi MT, Zarrini G, Mohit E, Faramarzi MA, Setayesh N, Sedighi N, Mohseni FA (2006) Mucor hiemalis: a new source for uricase production. World J Microbiol Biotechnol 22:325–330CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  1. 1.Laboratory of Yeast Molecular Genetics and Breeding, Institute of MicrobiologyChinese Academy of SciencesBeijingPeople’s Republic of China
  2. 2.Graduate University of Chinese Academy of SciencesBeijingPeople’s Republic of China

Personalised recommendations