Skip to main content
SpringerLink
Log in

Evaluation of MutS and Mut+ Pichia pastoris Strains for Membrane-Bound Catechol-O-Methyltransferase Biosynthesis

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) is an enzyme that catalyzes the methylation of catechol substrates, and while structural and functional studies of its membrane-bound isoform (MBCOMT) are still hampered by low recombinant production, Pichia pastoris has been described as an attractive host for the production of correctly folded and inserted membrane proteins. Hence, in this work, MBCOMT biosynthesis was developed using P. pastoris X33 and KM71H cells in shake flasks containing a semidefined medium with different methanol concentrations. Moreover, after P. pastoris glass beads lysis, biologically and immunologically active hMBCOMT was found mainly in the solubilized membrane fraction whose kinetic parameters were identical to its correspondent native enzyme. In addition, mixed feeds of methanol and glycerol or sorbitol were also employed, and its levels quantified using liquid chromatography coupled to refractive index detection. Overall, for the first time, two P. pastoris strains with opposite phenotypes were applied for MBCOMT biosynthesis under the control of the strongly methanol-inducible alcohol oxidase (AOX) promoter. Moreover, this eukaryotic system seems to be a promising approach to deliver MBCOMT in high quantities from fermentor cultures with a lower cost-benefit due to the cheaper cultivation media coupled with the higher titers tipically achieved in biorreactors, when compared with previously reported mammallian cell cultures.

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.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

AOX:

Alcohol oxidase

COMT:

Catechol-O-methyltransferase

E. coli :

Escherichia coli

DNA:

Deoxyribonucleic acid

gDNA:

Genomic DNA

HPLC:

High-peformance liquid chromatography

LLOQ:

Lower limit of quantification

MBCOMT:

Membrane-bound catechol-O-methyltransferase

MP:

Membrane protein

Mut:

Methanol utilization

MutS :

Methanol utilization slow

Mut+ :

Methanol utilization plus

OD600 :

Optical density 600 nm

PCR:

Polymerase chain reaction

P. pastoris :

Pichia pastoris

RID:

Refractive index detection

SAM:

S-adenosyl-L-methionine

SCOMT:

Soluble catechol-O-methyltransferase

SDS-PAGE:

Reducing sodium dodecyl sulfate polyacrylamide

References

  1. Bonifacio, M. J., Palma, P. N., Almeida, L., & Soares-da-Silva, P. (2007). Catechol-O-methyltransferase and its inhibitors in Parkinson’s disease. CNS Drug Reviews, 13(3), 352–379.

    Article  CAS  Google Scholar 

  2. Reenila, I., & Mannisto, P. T. (2001). Catecholamine metabolism in the brain by membrane-bound and soluble catechol-O-methyltransferase (COMT) estimated by enzyme kinetic values. Medical Hypotheses, 57(5), 628–632.

    Article  CAS  Google Scholar 

  3. Kanasaki, K., Palmsten, K., Sugimoto, H., Ahmad, S., Hamano, Y., Xie, L., Parry, S., Augustin, H. G., Gattone, V. H., Folkman, J., Strauss, J. F., & Kalluri, R. (2008). Deficiency in catechol-O-methyltransferase and 2-methoxyoestradiol is associated with pre-eclampsia. Nature, 453(7198), 1117–1121.

    Article  CAS  Google Scholar 

  4. Papaleo, F. J., Crawley, N., Song, J., Lipska, B. K., Pickel, J., Weinberger, D. R., & Chen, J. (2008). Genetic dissection of the role of catechol-O-methyltransferase in cognition and stress reactivity in mice. Journal of Neuroscience, 28(35), 8709–8723.

    Article  CAS  Google Scholar 

  5. Zhu, B. T., & Liehr, J. G. (1993). Inhibition of the catechol-O-methyltransferase-catalyzed O-methylation of 2- and 4-hydroxyestradiol by catecholamine: implications for the mechanism of estrogen-induced carcinogenesis. Archives of Biochemistry and Biophysics, 304(1), 248–256.

    Article  CAS  Google Scholar 

  6. Zhu, B. T. (2002). On the mechanism of homocysteine pathophysiology and pathogenesis: a unifying hypothesis. Histology and Histopathology, 17(4), 1283–1291.

    CAS  Google Scholar 

  7. Lachman, H. M., Papolos, D. F., Saito, T., Yu, Y. M., Szumlanski, C. L., & Weinshilboum, R. M. (1997). Human catechol-O-methyltransferase pharmacogenetics: description of a functional polymorphism and its potential application to neuropsychiatric disorders. Pharmacogenetics, 6(3), 243–250.

    Article  Google Scholar 

  8. Malherbe, P., Bertocci, B., Caspers, P., Zurcher, G., & Da Prada, M. (1992). Expression of functional membrane-bound and soluble catechol-O-methyltransferase in Escherichia coli and a mammalian cell line. Journal of Neurochemistry, 58(5), 1782–1789.

    Article  CAS  Google Scholar 

  9. Vilbois, F., Caspers, P., da Prada, M., Lang, G., Karrer, C., Lahm, H. W., & Cesura, A. M. (1994). Mass spectrometric analysis of human soluble catechol-O-methyltransferase expressed in Escherichia coli. Identification of a product of ribossomal frameshifting and of reactive cysteines involved in S-adenosyl-L-methionine binding. European Journal of Biochemistry, 222(2), 377–386.

    Article  CAS  Google Scholar 

  10. Bai, H. W., Shim, J. Y., Yu, J., & Zhu, B. T. (2007). Biochemical and molecular modeling studies of the O-methylation of various endogenous and exogenous catechol substrates catalyzed by recombinant human soluble and membrane-bound catehcol-O-methyltransferase. Chemical Research in Toxicology, 20(10), 1409–1425.

    Article  CAS  Google Scholar 

  11. Pedro, A. Q., Bonifacio, M. J., Queiroz, J. A., Maia, C. J., & Passarinha, L. A. (2011). A novel prokaryotic expression system for biosynthesis of recombinant human membrane-bound catechol-O-methyltranferase. Journal of Biotechnology, 156(2), 141–146.

    Article  CAS  Google Scholar 

  12. Santos, F. M., Pedro, A. Q., Soares, R. F., Martins, R., Bonifacio, M. J., Queiroz, J. A., & Passarinha, L. A. (2013). Performance of hydrophobic interaction ligands for human membrane-bound catechol-O-methyltransferase purification. Journal of Separation Science, 36(11), 1693–1702.

    Article  CAS  Google Scholar 

  13. Correia, F. F., Santos, F. M., Pedro, A. Q., Bonifácio, M. J., Queiroz, J. A., & Passarinha, L. A. (2013). Recovery of biological active catechol-O-methyltransferase isoforms from Q-sepharose. Journal of Separation Science, 37(1–2), 20–29.

    Google Scholar 

  14. Tilgmann, C., Melen, K., Lundstrom, K., Jalanko, A., Julkunen, I., Kalkkinen, N., & Ulmanen, I. (1992). Expression of recombinant soluble and membrane-bound catechol-O-methyltransferase in eukaryotic cells and identification of the respective enzymes in rat brain. European Journal of Biochemistry, 207(2), 813–821.

    Article  CAS  Google Scholar 

  15. Bertocci, B., Miggiano, V., Da Prada, M., Dembic, Z., Lahm, H. W., & Malherbe, P. (1991). Human catechol-O-methyltransferase: cloning and expression of the membrane-associated form. Proceedings of the National Academy of Sciences of the United States of America, 88(4), 1416–1420.

    Article  CAS  Google Scholar 

  16. Ulmanen, I., Peranen, J., Tenhunen, J., Tilgmann, C., Karhunen, T., Panula, P., Bernasconi, L., Aubry, J. P., & Lundstrom, K. (1997). Expression and intracellular localization of catechol-O-methyltransferase in transfected mammalian cells. European Journal of Biochemistry, 243(1–2), 452–459.

    Article  CAS  Google Scholar 

  17. Junge, F. B., Schneider, B., Reckel, S., Schwarz, V., Dotsch, V., & Bernhard, F. (2008). Large-scale production of functional membrane proteins. Cellular and Molecular Life Sciences, 65(11), 1729–1755.

    Article  CAS  Google Scholar 

  18. Cregg, J. M., Cereghino, J. L., Shi, J., & Higgins, D. R. (2000). Recombinant protein expression in Pichia pastoris. Molecular Biotechnology, 16(1), 23–52.

    Article  CAS  Google Scholar 

  19. Goncalves, A. M., Pedro, A. Q., Maia, C., Sousa, F., Queiroz, J. A., & Passarinha, L. A. (2013). Pichia pastoris, a recombinant microfactory for antibodies and human membrane proteins. Journal of Microbiology and Biotechnology, 23(5), 587–601.

    Article  CAS  Google Scholar 

  20. Ramon, A., & Marin, M. (2011). Advances in the production of human membrane proteins in Pichia pastoris. Biotechnology Journal, 6(6), 700–706.

    Article  CAS  Google Scholar 

  21. Vogl, T., & Glieder, A. (2013). Regulation of Pichia pastoris promoters and its consequences for protein production. New Biotechnology, 30(4), 385–404.

    Article  CAS  Google Scholar 

  22. Burrowes, O. J., Diamond, G., & Lee, T. C. (2005). Recombinant expression of pleurocidin cDNA using the Pichia pastoris expression system. Journal of Biomedicine & Biotechnology, 4, 374–384.

    Article  Google Scholar 

  23. Villatte, F., Hussein, A. S., Bachmann, T. T., & Schmid, R. D. (2001). Expression level of heterologous proteins in Pichia pastoris is influenced by flask design. Applied Microbiology and Biotechnology, 55(4), 463–465.

    Article  CAS  Google Scholar 

  24. Jahic, M., Veide, A., Charoenrat, T., Teeri, T., & Enfors, S. O. (2006). Process technology for production and recovery of heterologous proteins with Pichia pastoris. Biotechnology Progress, 22(6), 1465–1473.

    Article  CAS  Google Scholar 

  25. Cos, O., Ramon, R., Montesinos, J. L., & Valero, F. (2006). Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: a review. Microbial Cell Factories, 5, 17–37.

    Article  Google Scholar 

  26. Chiruvolu, V., Cregg, J. M., & Meagher, M. M. (1997). Recombinant protein production in an alcohol oxidase-defective strain of Pichia pastoris in fed-batch fermentations. Enzyme Microbiology Technology, 21, 277–283.

    Article  CAS  Google Scholar 

  27. Pontes, H., Pinho, P., Casal, S., Carmo, H., Santos, A., Magalhaes, T., Remião, F., Carvalho, F., & Lourdes Bastos, M. (2009). Determination of ethanol, metanol, acetone and acetaldehyde by a simple capillary direct injection gas chromatographic method in human whole blood, vitreous humour and urine and in cell culture medium. Journal of Chromatographic Science, 47(4), 272–278.

    Article  CAS  Google Scholar 

  28. Parpinello, G., & Versari, A. (2000). A simple high-performance liquid chromatography method for the analysis of glucose, glycerol and methanol in a bioprocess. Journal of Chromatographic Science, 38(6), 259–261.

    Article  CAS  Google Scholar 

  29. Almuzara, C., Cos, O., Baeza, M., Gabriel, D., & Valero, F. (2002). Methanol determination in Pichia pastoris cultures by flow injection analysis. Biotechnology Letters, 24, 413–417.

    Article  CAS  Google Scholar 

  30. Zhang, W., Hywood Potter, K. J., Plantz, B. A., Schlegel, V. L., Smith, L. A., & Meagher, M. M. (2003). Pichia pastoris fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement. Journal of Industrial Microbiology & Biotechnology, 30(4), 210–215.

    Article  CAS  Google Scholar 

  31. Katakura, Y., Zhang, W., Zhuang, G., Omasa, T., Kishimoto, M., Goto, Y., & Suga, K.-I. (1998). Effect of the methanol concentration on the production of human beta(2)-glycoprotein I domain V by a recombinant Pichia pastoris: a simple system for the control of methanol concentration using a semiconductor gas sensor. Journal of Fermentation and Bioengineering, 86, 482–487.

    Article  CAS  Google Scholar 

  32. Hellwig, S., Emde, F., Raven, N. P., Henke, M., Van Der Logt, P., & Fischer, R. (2001). Analysis of single-chain antibody production in Pichia pastoris using on-line methanol control in fed-batch and mixed-feed fermentations. Biotechnology and Bioengineering, 74, 344–352.

    Article  CAS  Google Scholar 

  33. Celik, E., Calik, P., & Oliver, S. G. (2009). fed-batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co-substrate sorbitol. Yeast, 26(9), 473–484.

    Article  CAS  Google Scholar 

  34. Tenhunen, J., Salminen, M., Lundstrom, K., Kiviluoto, T., Savolainen, R., & Ulmanen, I. (1994). Genomic organization of the human catechol-O-methyltransferase gene and its expression from two distinct promoters. European Journal of Biochemistry, 223(3), 1049–1059.

    Article  CAS  Google Scholar 

  35. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685.

    Article  CAS  Google Scholar 

  36. Passarinha, L. A., Bonifacio, M. J., & Queiroz, J. A. (2007). Comparative study on the interaction of recombinant human soluble catechol-O-methyltransferase on some hydrophobic adsorbents. Biomedical Chromatography, 21(4), 430–438.

    Article  CAS  Google Scholar 

  37. Passarinha, L. A., Bonifacio, M. J., & Queiroz, J. A. (2006). The effect of temperature on the analysis of metanephrine for catechol-O-methyltransferase activity assay with electrochemical detection. Biomedical Chromatography, 20(9), 937–944.

    Article  CAS  Google Scholar 

  38. Goodrick, J. C., Xu, M., Finnegan, R., Schilling, B. M., Schiavi, S., Hoppe, H., & Wan, N. C. (2001). High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutive Pichia pastoris expression system. Biotechnology and Bioengineering, 74(6), 492–497.

    Article  CAS  Google Scholar 

  39. Oberg, F., Ekvall, M., Nyblom, M., Backmark, A., Neutze, R., & Hedfalk, K. (2009). Insight into factors directing high production of eukaryotic membrane proteins; production of 13 human AQPs in Pichia pastoris. Molecular Membrane Biology, 26, 215–227.

    Article  Google Scholar 

  40. Long, S. B., Tao, X., Campbell, E. B., & Mackinnon, R. (2007). Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. Nature, 450, 376–382.

    Article  CAS  Google Scholar 

  41. Orman, M. A., Çalik, P., & Ozdamar, H. O. (2009). The influence of carbon sources on recombinant-human-growth hormone production by Pichia pastoris is dependent on phenotype: a comparison of MutS and Mut+ strains. Biotechnology and Applied Biochemistry, 52, 245–255.

    Article  CAS  Google Scholar 

  42. Horstkotte, B., Arnau, C., Valero, F., Elsholz, O., & Cerdà, V. (2008). Monitoring of sorbitol in Pichia pastoris cultivation applying sequential injection analysis. Biochemical Engineering Journal, 42, 77–83.

    Article  CAS  Google Scholar 

  43. Food and Drug administration - Guidance for Industry – bioanalytical method validation., U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM) (2001), Available from: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm070107.pdf. Assessed 20/03/2014.

  44. Zhang, P., Zhang, W., Zhou, X., Bai, P., Cregg, J. M., & Zhang, Y. (2010). Catabolite repression of Aox in Pichia pastoris is dependent on hexose transporter PpHxt1 and pexophagy. Applied and Environmental Microbiology, 76(18), 6108–6118.

    Article  CAS  Google Scholar 

  45. Sreekrishna, K., Brankamp, R. G., Kropp, K. E., Blankenship, D. T., Tsay, J. T., Smith, P. L., Wierschke, J. D., Subramaniam, A., & Birkenberger, L. A. (1997). Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene, 190, 55–62.

    Article  CAS  Google Scholar 

  46. Kim, S. I., Wu, Y., Kim, K. L., Kim, G. J., & Shin, H. J. (2013). Improved cell disruption of Pichia pastoris utilizing aminopropyl magnesium phyllosilicate (AMP) clay. World Journal of Microbiology and Biotechnology, 29(6), 1129–1132.

    Article  CAS  Google Scholar 

  47. Giersing, B., Miur, a. K., Shimp, R., Wang, J., Zhou, H., Orcutt, A., Stowers, A., Saul, A., Miller, L. H., Long, C., & Singh, S. (2005). Posttranslational modification of recombinant Plasmodium falciparum apical membrane antigen 1: impact on functional immune responses to malaria vaccine candidate. Infection and Immunity, 73(7), 3963–3970.

    Article  CAS  Google Scholar 

  48. Abad, S., Nahalka, J., Bergler, G., Arnold, S. A., Speight, R., Fotheringam, I., Nidetzsky, B., & Glieder, A. (2010). Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst. Microbial Cell Factories, 9, 24–36.

    Article  Google Scholar 

  49. Daniels, M. J., Wood, M. R., & Yeager, M. (2006). In vivo functional assay of a recombinant aquaporin in Pichia pastoris. Applied Environmental Microbiology, 72(2), 1507–1514.

    Article  CAS  Google Scholar 

  50. Pla, I. A., Damasceno, L. M., Vannelli, T., Ritter, G., Batt, C. A., & Shuler, M. L. (2006). Evaluation of MutS and Mut+ Pichia pastoris phenotypes for high level extracellular scFv expression under feedback control of the methanol concentration. Biotechnology Progress, 22, 881–888.

    Article  CAS  Google Scholar 

  51. Bonifacio, M. J., & Soares-da-Silva, P. (2003). Purification of membrane-bound catechol-O-methyltransferase. Methods in Molecular Biology, 228, 231–238.

    CAS  Google Scholar 

  52. Prive, G. C. (2007). Detergents for the stabilization and crystallization of membrane proteins. Methods, 41(4), 388–397.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

A.Q. Pedro acknowledges a doctoral fellowship (SFRH/BD/81222/2011) from Fundação para a Ciência e Tecnologia and D. Oppolzer acknowledges a fellowship (CENTRO-07-ST24_FEDER-002014-TPCR-2-004) from Programa “Mais Centro” within the scope of QREN–POPH–Advanced Formation programs cofunded by Fundo Social Europeu and MEC. This work was partially funded by Fundação para a Ciência e Tecnologia I.P. (PIDDAC) and Fundo Europeu de Desenvolvimento Regional-FEDER funds through Programa Operacional Factores de Competitividade (POFC)–COMPETE: FCOMP-01-0124-FEDER-027563 and by National Funds through FCT–Fundação para a Ciência e Tecnologia within the scope of Project “EXPL/BBB478/BQB/0960/2012.”

Conflict of Interest

All authors declare they do not have any conflict of interest.

Compliance with Ethical Standards

In this work, no studies involving human participants or animals were carried out.

Author information

Authors and Affiliations

  1. CICS-UBI–Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6201-001, Covilhã, Portugal

    A. Q. Pedro, D. Oppolzer, C. J. Maia, J. A. Queiroz & L. A. Passarinha

  2. Bial–Departamento de Investigação e Desenvolvimento, 4745-457, São Mamede do Coronado, Portugal

    M. J. Bonifácio

Authors
  1. A. Q. Pedro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D. Oppolzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. J. Bonifácio
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. C. J. Maia
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J. A. Queiroz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. L. A. Passarinha
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to L. A. Passarinha.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pedro, A.Q., Oppolzer, D., Bonifácio, M.J. et al. Evaluation of MutS and Mut+ Pichia pastoris Strains for Membrane-Bound Catechol-O-Methyltransferase Biosynthesis. Appl Biochem Biotechnol 175, 3840–3855 (2015). https://doi.org/10.1007/s12010-015-1551-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-015-1551-0

Keywords

Search

Navigation