Skip to main content
SpringerLink
Log in

Reconstruction and visualization of carbohydrate, N-glycosylation pathways in Pichia pastoris CBS7435 using computational and system biology approaches

  • Research Article
  • Published:
Systems and Synthetic Biology

Abstract

Pichia pastoris is an efficient expression system for production of recombinant proteins. To understand its physiology for building novel applications it is important to understand and reconstruct its metabolic network. The metabolic reconstruction approach connects genotype with phenotype. Here, we have attempted to reconstruct carbohydrate metabolism pathways responsible for high biomass density and N-glycosylation pathways involved in the post translational modification of proteins of P. pastoris CBS7435. Both these metabolic pathways play a crucial role in heterologous protein production. We report novel, missing and unannotated enzymes involved in the target metabolic pathways. A strong possibility of cellulose and xylose metabolic processes in P. pastoris CBS7435 suggests its use in the area of biofuels. The reconstructed metabolic networks can be used for increased yields and improved product quality, for designing appropriate growth medium, for production of recombinant therapeutics and for making biofuels.

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
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  • Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    PubMed  CAS  Google Scholar 

  • Ates O, Oner ET, Arga KY (2011) Genome-scale reconstruction of metabolic network for a halophilic extremophile, Chromohalobacter salexigens DSM 3043. BMC Syst Biol 5:12

    PubMed  Google Scholar 

  • Bairoch A, Apweiler R, Wu CH, Barker WC, Boeckmann B, Ferro S, Gasteiger E, Huang H, Lopez R, Magrane M, Martin MJ, Natale DA, O’Donovan C, Redaschi N, Yeh LL (2005) The universal protein resource (UniProt). Nucleic Acids Res 33:154–159

    Google Scholar 

  • Balamurugan G, Reddy VR, Suryanarayan VVS (2007) Pichia pastoris: a notable heterologous expression system for the production of foreign proteins—vaccines. IJBT 6:175–186

    CAS  Google Scholar 

  • Bobrowicz P, Davidson RC, Li H, Potgeiter TI, Nett JH, Hamilton SR, Stadheim TA, Meile RG, Bobrowicz B, Mitchell T, Rausch S, Renfer E, Wildt S (2004) Engineering of an artificial glycosylation pathwayblocked in core oligosaccharide assembly in yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose. Glycobiology 14(9):757–766

    PubMed  CAS  Google Scholar 

  • Boyer F, Viari A (2003) Ab initio reconstruction of metabolic pathways. Bioinformatics 19:26–34

    Google Scholar 

  • Cherry JM, Hong EL, Amundsen C, Balakrishnan R, Binkley G, Chan ET, Christie KR, Costanzo MC, Dwight SS, Engel SR, Fisk DG, Hirschman JE, Hitz BC, Karra K, Krieger CJ, Miyasato SR, Nash RS, Park J, Skrzypek MS, Simison M, Weng S, Wong ED (2012) Saccharomyces genome database: the genomics resource of budding yeast. Nucleic Acids Res 40:700–705

    Google Scholar 

  • Chung BKS, Selavarasu S, Camattari A, Ryu J, Lee H, Ahn J, Lee H, Lee DY (2010) Genome scale metabolic reconstruction and in silico analysis of methylotrophic yeast Pichia pastoris for strain improvement. Microb Cell Fact 9:50

    PubMed  Google Scholar 

  • Davidson RC, Bobrowicz P, Zha D. (2012) Metabolic engineering of a galactose assimilation pathway in the glycoengineered yeast Pichia pastoris. U.S. Patent 20120003695, January 5, 2012

  • DeJongh M, Formsma K, Boillot P, Gould J, Rycenga M, Best A (2007) Towards the automated generation of genome-scale metabolic networks in the SEED. BMC Bioinformatics 8:139

    PubMed  Google Scholar 

  • Durot M, Bourguignon PY, Schachter V (2008) Genome-scale models of bacterial metabolism: reconstruction and applications. FEMS Microbiol Rev 33:164–190

    PubMed  Google Scholar 

  • Feist AM, Scholten JCM, Palsson BO, Brockman FJ, Ideker T (2006) Modeling methanogenesis with a genome-scale metabolic reconstruction of Methanosarcina barkeri. Mol Syst Biol. doi:10.1038/msb4100046

    PubMed  Google Scholar 

  • Feist AM, Herrgard MJ, Thiele I, Reed JL, Palsson BO (2009) Reconstruction of biochemical networks in microbial organisms. Nat Rev Microbiol 7(2):129–143

    PubMed  CAS  Google Scholar 

  • Folger O, Jerby L, Frezza C, Gottleib E, Ruppin E, Shlomi T (2011) Predicting selective drug targets in cancer through metabolic networks. Mol Syst Biol 7:501

    PubMed  Google Scholar 

  • Forster J, Famili I, Fu P, Palsson BO, Nielsen J (2003) Genome scale reconstruction of the Saccharomyces cerevisiae metabolic network. Genome Res 13:244–253

    PubMed  CAS  Google Scholar 

  • Forth T, McConkey GA, Westhead DR (2010) MetNetMaker: a free and open source tool for the creation of novel metabolic networks in SBML format. Bioinformatics 26(18):2352–2353

    PubMed  CAS  Google Scholar 

  • Francke C, Seizen RJ, Teusink B (2005) Reconstructing the metabolic network of a bacterium from its genome. Trends Microbiol 13:550–558

    PubMed  CAS  Google Scholar 

  • Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A (2003) ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31:3784–3788

    PubMed  CAS  Google Scholar 

  • Ha S, Wang Y, Rustandi RR (2011) Biochemical and biophysical characterization of humanized IgG1 produced in Pichia pastoris. MAbs 3(5):453–460

    PubMed  Google Scholar 

  • Hu F, Li X, Lu J, Mao PH, Jin X, Rao B, Zheng P, Zhou YL, Liu SY, Ke T, Ma XD, Ma LX (2011) A visual method for direct selection of high-producing Pichia pastoris clones. BMC Biotechnol 11:23

    PubMed  CAS  Google Scholar 

  • Inan M, Meagher MM (2001) Non-repressing carbon sources for Alcohol Oxidase (AOX1) promoter of Pichia pastoris. J Biosci Bioeng 92(6):585–589

    Google Scholar 

  • Jewison T, Knox C, Neveu V, Djoumbou Y, Guo AC, Lee J, Liu P, Mandal R, Krishnamurthy R, Sinelnikov I, Wilson M, Wishart DS (2012) YMDB: the yeast metabolome database. Nucleic Acids Res 40:815–820

    Google Scholar 

  • Kanehisa M, Goto S, Hattori M, Aoki-Kinoshita KF, Itoh M, Kawashima S, Katayama T, Araki M, Hirakawa M (2006) From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res 34:354–357

    Google Scholar 

  • Karp PD, Riley M, Paley SM, Pellegrini-toole A (2002) The metacyc database. Nucleic Acids Res 30:59–61

    PubMed  CAS  Google Scholar 

  • Kharchenko P, Vitkup D, Church GM (2004) Filling gaps in a metabolic network using expression information. Bioinformatics 20:178–185

    Google Scholar 

  • Kuberl A, Schneider J, Thallinger GG, Anderl I, Wibberg D, Hajek T, Jaenicke S, Brinkrolf K, Goesmann A, Szczepanowski R, Puhler A, Schwab H, Gleider A, Pichler H (2011) High-quality genome sequence of Pichia pastoris CBS7435. J Biotechnol 154(4):312–320

    PubMed  Google Scholar 

  • Kumar VS, Dasika MS, Maranas CD (2007) Optimization based automated curation of metabolic reconstructions. BMC Bioinformatics 8:212

    Google Scholar 

  • Kurtzman CP (2005) Description of Komagataella phaffii sp. nov. and the transfer of Pichia pseudopastoris to the methylotrophic yeast genus Komagataella. Int J Syst Evol Microbiol 55:973–976

    PubMed  CAS  Google Scholar 

  • Mazurie A, Bonchev D, Schwikowski B, Buck GA (2010) Evolution of metabolic network organization. BMC Syst Biol 4:59

    PubMed  Google Scholar 

  • Milne CB, Eddy JA, Raju R, Ardekani S, Kim PJ, Senger RS, Jin YS, Blaschek HP, Price ND (2011) Metabolic network reconstruction and genome-scale model of butanol-producing strain Clostridium beijerinckii NCIMB 8052. BMC Syst Biol 5:130

    PubMed  CAS  Google Scholar 

  • Montagud A, Navarro E, de Cordoba PF, Urchueguia JF, Patil KR (2010) Reconstruction and analysis of genome-scale metabolic model of a photosynthetic bacterium. BMC Syst Biol 4:156

    PubMed  Google Scholar 

  • Notebaart RA, van Enckevort FHJ, Francke C, Seizen RJ, Teusink B (2006) Accelerating the reconstruction of genome-scale metabolic networks. BMC Bioinformatics 7:296

    PubMed  Google Scholar 

  • Oberhardt MA, Palsson BO, Papin JA (2009) Applications of genome-scale metabolic reconstructions. Mol Syst Biol 5:320

    PubMed  Google Scholar 

  • Osterman A, Overbeek R (2003) Missing genes in metabolic pathways: a comparative genomics approach. Curr Opin Chem Biol 7:238–251

    PubMed  CAS  Google Scholar 

  • Schellenberger J, Park JO, Conrad TM, Palsson BO (2010) BiGG: a biochemical genetics and genomics knowledgebase of large scale metabolic reconstructions. BMC Bioinformatics 11:213

    PubMed  Google Scholar 

  • Schutter KD, Lin YC, Tiels P, Hecke AV, Glinka S, Lehmann JW, Rouze P, de Peer YV, Callewaert N (2009) Genome sequence of recombinant protein production host Pichia pastoris. Nat Biotechnol 7:561–566

    Google Scholar 

  • Seo S, Lewin HA (2009) Reconstruction of metabolic pathways for cattle genome. BMC Syst Biol 3:33

    PubMed  Google Scholar 

  • Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13:2498–2504

    PubMed  CAS  Google Scholar 

  • Thiele I, Palsson BO (2010) A protocol for generating a high quality genome-scale metabolic reconstruction. Nat Protoc 5(1):93–121

    PubMed  CAS  Google Scholar 

  • Van Rensburg P, Van Zyl WH, Pretorius IS (1998) Engineering yeast for efficient cellulose degradation. Yeast 14(1):67–76

    PubMed  Google Scholar 

  • Weidner M, Taupp M, Hallam SJ (2010). Expression of recombinant proteins in methylotrophic yeast Pichia pastoris. J Vis Exp. 36

  • 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(9):4961–4969

    PubMed  CAS  Google Scholar 

  • Zhang W, Liu C, Wang G, Ma Y, Zhang K, Zou S, Zhang M (2012) Comparison of expression in Saccharomyces cerevisiae of endoglucanase II from Trichoderma reesei and Endoglucanase I from Aspergillus aculeatus. BioResource 7(3):4031–4045

    CAS  Google Scholar 

Download references

Acknowledgments

The authors are thankful to Department of Biotechnology, IET, Lucknow & TERI University, New Delhi for providing the facility and technical support during the preparation of manuscript.

Author information

Authors and Affiliations

  1. Department of Biotechnology, Institute of Engineering and Technology, G.B. Technical University, Sitapur Road, Lucknow, 226021, India

    Akriti Srivastava & Bhartendu Nath Mishra

  2. Department of Biotechnology, TERI University, 10 Institutional Area, Vasant Kunj, New Delhi, 110070, India

    Pallavi Somvanshi

Authors
  1. Akriti Srivastava
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pallavi Somvanshi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bhartendu Nath Mishra
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bhartendu Nath Mishra.

Additional information

Akriti Srivastava and Pallavi Somvanshi share first authorship.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Srivastava, A., Somvanshi, P. & Mishra, B.N. Reconstruction and visualization of carbohydrate, N-glycosylation pathways in Pichia pastoris CBS7435 using computational and system biology approaches. Syst Synth Biol 7, 7–22 (2013). https://doi.org/10.1007/s11693-012-9102-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11693-012-9102-2

Keywords

Search

Navigation