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Bioprocess and Biosystems Engineering

, Volume 27, Issue 6, pp 399–406 | Cite as

Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes

  • Theppanya Charoenrat
  • Mariena Ketudat-Cairns
  • Helle Stendahl-Andersen
  • Mehmedalija Jahic
  • Sven-Olof EnforsEmail author
Original paper

Abstract

An oxygen-limited fed-batch technique (OLFB) was compared to traditional methanol-limited fed-batch technique (MLFB) for the production of recombinant Thai Rosewood β-glucosidase with Pichia pastoris. The degree of energy limitation, expressed as the relative rate of respiration (q O/q O,max), was kept similar in both the types of processes. Due to the higher driving force for oxygen transfer in the OLFB, the oxygen and methanol consumption rates were about 40% higher in the OLFB. The obligate aerobe P. pastoris responded to the severe oxygen limitation mainly by increased maintenance demand, measured as increased carbon dioxide production per methanol, but still somewhat higher cell density (5%) and higher product concentrations (16%) were obtained. The viability was similar, about 90–95%, in both process types, but the amount of total proteins released in the medium was much less in the OLFB processes resulting in substantially higher (64%) specific enzyme purity for input to the downstream processing.

Keywords

Oxygen-limited fed batch (OLFB) Methanol-limited fed batch (MLFB) Pichia pastoris β-glucosidase 

List of symbols

AOX

Enzyme alcohol oxidase

AOX1

Alcohol oxidase gene 1

CPR

Carbon dioxide production rate (mol h−1)

DOT

Dissolved oxygen tension (%)

MLFB

Methanol limited fed-batch

OLFB

Oxygen limited fed-batch

OUR

Oxygen uptake rate (mol h−1)

PI

Propidium iodide

qo

Specific oxygen uptake rate (mol g cell −1 h−1)

qo,max

Maximum specific oxygen uptake rate (mol g cell −1 h−1)

qp

Specific β-glucosidase productivity (U g cell −1 h−1)

Qi

Inlet air flow rate (L h−1)

Qo

Outlet air flow rate (L h−1)

RRR

Relative rate of respiration

V

Medium volume (L)

Vm

Molar volume of gas (L mol−1)

X

Biomass concentration from dry weight (g L−1)

YCCO2/S

Carbon yield coefficient of carbon dioxide from methanol (mol mol−1)

YCX/S

Carbon yield coefficient of biomass from methanol (mol mol−1)

Notes

Acknowledgements

TC is supported by the university lecturer development program from the Ministry of Education and Suranaree University, Thailand. This work is part of the BiMaC Enzyme Factory programme financed by the Södra Skogsägarnas Stiftelse för Forskning, Utveckling och Utbildning.

References

  1. 1.
    Higgins DR, Cregg JM (1998) Methods in molecular biology: Pichia protocols. In: Lin Cereghino GP, Lin Cereghino J, Ilgen C, Cregg JM (eds) Production of recombinant proteins in fermentor cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 13:329–332Google Scholar
  2. 2.
    Cregg JM, Lin Cereghino J, Shi J, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16:23–52Google Scholar
  3. 3.
    Wegner G (1990) Emerging application of methelotrophic yeast. FEMS Microbiol Rev 87:279–284Google Scholar
  4. 4.
    Jahic M, Rotticci-Mulder JC, Martinelle M, Hult K, Enfors S-O (2002) Modelling of growth and energy metabolism of Pichia pastoris producing a fusion protein. Bioprocess Biosyst Eng 24:385–393Google Scholar
  5. 5.
    Couderc R, Baratti J (1998) Oxidation of methanol by the yeast Pichia pastoris: purification and properties of alcohol oxidase. Agric Biol Chem 44:2279–2289Google Scholar
  6. 6.
    Hasslacher M, Schall M, Hayn M, Bona R, Rumbold K, Lückl J, Griengl H, Kohlwein SD, Schwab H (1997) High-level intracellular expression of hydroxynitril lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts. Protein Expression Purif 11:61–71Google Scholar
  7. 7.
    Werten MWT, van den Bosch TJ, Wind RD, Mooibroek H, de Wolf FA (1999) High-yield secretion of recombinant gelatines by Pichia pastoris. Yeast 15:1087–1096Google Scholar
  8. 8.
    Chiruvolu V, Eskridge K, Cregg J, Meagher M (1998) Effect of glycerol concentration and pH on growth of recombinant Pichia pastoris yeast. Appl Biochem Biotechnol 75:63–173Google Scholar
  9. 9.
    Jahic M, Wallberg F, Bollok M, Garcia P, Enfors S-O (2003) Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures. Microbial cell Factories 2:1–6Google Scholar
  10. 10.
    Kobayashi K, Kuwae S, Ohya T, Ohda T, Ohyama M, Ohi H, Tomomitsu K, Ohmura T (2000) High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation. J Biosci Bioeng 89:55–61Google Scholar
  11. 11.
    Zhang W, Bevisn MA, Plantz BA, Smith LA, Meagher MM (2000) Modelling Pichia pastoris growth on methanol and optimizing the production of a recombinant protein, the heavy-chain fragment C of botulinum, serotype A. Biotechnol Bioeng 70:1–8Google Scholar
  12. 12.
    Katakura Y, Zhang W, Zhuang G, Omasa T, Kishimoto M, Goto Y, Suga K-I (1998) Effect of methanol concentration on the production of human β2-glycoprotein I domain V by a recombinant Pichia pastoris: a simple system for the control of methanol concentration using a semiconductor gas sensor. J Ferm Bioeng 86(5):482–487Google Scholar
  13. 13.
    Enfors S-O, Jahic M, Rozkov A, Xu B, Hecker M, Jürgen B, Krüger E, Schweder T, Hamer G, O’Beirne D, Noisommit-Rizzi N, Reuss M, Boone L, Hewitt C, McFarlane C, Nienow A, Kovacs T, Trägårdh C, Fuchs L, Revstedt J, Friberg PC, Hjertager B, Blomsten G, Skogman H, Hjort S, Hoeks F, Lin H-Y, Neubauer P, van der Lans R, Luyben K, Vrabel P, Manelius Å (2001) Physiological responses to mixing in large scale bioreactors. J Biotechnol 85:175–185Google Scholar
  14. 14.
    Trentmann O, Khatri NK, Hoffmann F (2004) Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris. Biotechnol Prog 20:1766–1775Google Scholar
  15. 15.
    Trinh LB, Phue JN, Shiloach J (2003) Effect of methanol feeding strategies on production and yield of recombinant mouse endostatin from Pichia pastoris. Biotechnol Bioeng 82:438–444Google Scholar
  16. 16.
    Lin Cereghino J, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microb Rev 24:45–66Google Scholar
  17. 17.
    Sibirny AA, Ubiyvovk VM, Gonchar MV, Titorenko VI, Voronovsky AY, Kapultsevich YG, Bliznik KM (1990) Reaction of direct formaldehyde oxidation to CO2 are not-essential for energy supply of yeast methylotrophic growth. Arch Microbiol 154:566–575Google Scholar
  18. 18.
    Sibirny AA, Titorenko VI, Gonchar MV, Ubiyvovk VM, Ksheminskaya GP, Vitvitskaya OP (1988) Genetic control of methanol utilizing in yeast. J Basic Microbiol 28:293–319Google Scholar
  19. 19.
    Douma AC, Veenhuis M, de Koning W, Evers M, Harder W (1985) Dihydroxyacetone syntase is localized in the peroxisomal matrix of methanol-grown Hensenula polymorpha. Arch Microbiol 143:237–243Google Scholar
  20. 20.
    Ketudat-Cairns JR, Champattanachai V, Srisomsap C, Wittman-Liebold B, Thiede B, Svasti J (2000) Sequence and expression of Thai Rosewood β-glucosidase/β-fucosidase, a family 1 glycosyl hydrolase glycoprotein. J Biochem 128:999–1008Google Scholar
  21. 21.
    Bradford MM (1976) A rapid and sensitive method for the quantitative of microgram quantities of protein utilizing the principal of protein-dye binding. Anal Biochem 72:248–254Google Scholar
  22. 22.
    Evans CS (1985) Properties of the β-glucosidase (cellobiase) from the wood-rotting fungus Coriolus versicolor. Appl Microbiol Biotechnol 22:128–131Google Scholar
  23. 23.
    Hellwig S, Emde F, Raven NPG, 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. Biotechnol Bioeng 74:344–352Google Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Theppanya Charoenrat
    • 1
    • 2
  • Mariena Ketudat-Cairns
    • 2
  • Helle Stendahl-Andersen
    • 1
  • Mehmedalija Jahic
    • 1
  • Sven-Olof Enfors
    • 1
    Email author
  1. 1.School of Biotechnology, Royal Institute of TechnologyAlbaNova University CentreStockholmSweden
  2. 2.School of Biotechnology, Institute of Agricultural TechnologySuranaree University of TechnologyNakhon RatchasimaThailand

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