Abstract
This chapter provides rational approaches to design and optimize fed-batch and continuous fermentations of both Mut+ and Muts (methanol utilization plus and slow) Pichia pastoris strains. The methods are described in detail for glycerol batch, glycerol fed-batch, transition, and methanol fed-batch/mixed feed/continuous stirred tank reactor (CSTR) phases of the process based on glycerol and methanol consumption models. Cell density, broth volume, substrate feed rate, and the length of each phase are rationally designed to conduct runs with selected parameters for optimizing a process. The optimization is anchored by the impact of a specific growth rate/dilution time (for CSTRs) on productivity. Equations for simulation of a process with optimal parameters are derived for an optimal process design. This protocol can be used as a practical manual for process development of a P. pastoris recombinant fermentation, and also as a reference for fermentation of other microorganisms.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Cereghino, G. P. and Cregg, J. M. (1999) Applications of yeast in biotechnology: protein production and genetic analysis. Curr. Opin. Biotechnol. 10, 422–427.
Cregg, J. M., Madden, K. R., Barringer, K. J., Thill, G. P., and Stillman, C. A. (1989) Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol. Cell. Biol. 9, 1316–1323.
Romanos, M. A., Scorer, C. A., and Clare, J. J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423–488.
Cregg, J. M., Cereghino, J. L., Shi, J., and Higgins, D. R. (2000) Recombinant protein expression in Pichia pastoris. Mol. Biotechnol. 16, 23–52.
Invitrogen Co. (2002) Pichia Fermentation Process Guidelines, http://www.invitrogen.com . Invitrogen Co., San Diego, CA.
Stratton, J., Chiruvolu, V., and Meagher, M. M. (1998) High cell-density fermentation, in Pichia Protocols, (Higgins, D. R. and Cregg, J. M., eds.), Humana, Totowa, NJ, pp. 107–120.
Shimizu, H., Kozaki, Y., Kodama, H., and Shioya, S. (1999) Maximum production strategy for biodegradable copolymer P(HB-co-HV) in fed-batch culture of Alcaligenes eutrophus. Biotechnol. Bioeng. 62, 518–525.
Chim-Anage, P., Shioya, S., and Suga, K. (1991) Maximum histidine production by fed-batch culture of Brevibacterium flavum. J. Ferment. Bioeng. 71, 186–190.
Lim, H. C., Tayeb, Y. J., Modak, J. M., and Bonte, P. (1986) Computational algorithms for optimal feed rates for a class of fed-batch fermentation: Numerical results for penicillin and cell mass production. Biotechnol. Bioeng. 28, 1408–1420.
Modak, J. M., Lim, H. C., and Tayeb, Y. J. (1986) General characteristics of optimal feed rate profiles for various fed-batch fermentation processes. Biotechnol. Bioeng. 28, 1396–1407.
Parulekar, S. J. and Lim, H. C. (1985) Modeling, optimization and control of semibatch bioreactors, in Advances in biochemical engineering/biotechnology, vol. 32 (Fiechter, A., ed.), Springer, Berlin, pp. 207–258.
O’connor, G. M., Sanchez-Riera, F., and Cooney, C. L. (1992) Design and evaluation of control strategies for high cell density fermentations. Biotechnol. Bioeng. 39, 293–304.
Yamane, T. and Shimizu, S. (1984) Fed-batch techniques in microbial processes in Advances in biochemical engineering biotechnology, vol. 30, (Fiechter, A., ed.), Springer, Berlin, pp. 147–194.
Shioya, S. (1992) Optimization and control in fed-batch bioreactors, in Advances in biochemical engineering biotechnology, vol. 46, (Fiechter, A., ed.), Springer, Berlin, pp. 111–142.
Fishman, V. M. and Biryukov, V. V. (1974) Kinetic model of secondary metabolite production and its use in computation of optimal conditions. Biotechnol. Bioeng. Symp. 4, Pt. 2, 647–662.
Yamane, T., Kume, T., and Sada, E. (1977) A simple optimization technique for fed-batch culture. J. Ferment. Technol. 55, 587–598.
Ohno, H. and Nakanishi, E. (1976) Optimal control of a semibatch fermentation. Biotechnol. Bioeng. 18, 847–864.
Yamane, T. and Tsukano, M. (1977) Effect of several substrate-feeding modes on production of extracellular beta-amylase by fed-batch culture of Bacillus megaterium. J. Ferment. Technol. 55, 233–242.
Zhang, W., Bevins, M. A., Plantz, B. A., Smith, L. A., and Meagher, M. M. (2000) Modeling Pichia pastoris growth on methanol and optimizing the production of a recombinant protein, the heavy-chain fragment C of botulinum neurotoxin, serotype A. Biotechnol. Bioeng. 70, 1–8.
Zhang, W., Inan, M., and Meagher, M. M. (2000) Fermentation strategies for recombinant protein expression in the methylotrophic yeast Pichia pastoris. Biotechnol. Bioprocess Eng. 5, 275–287.
Hellwig, S., Robin, F., Drossard, J., Raven, N. P. G., Vaquero-Martin, C., Shively, J. E., and Fischer, R. (1999) Production of carcinoembryonic antigen (CEA) N-A3 domain in Pichia pastoris by fermentation. Biotechnol. Appl. Biochem. 30, 267–275.
Hellwig, S., Emde, F., Raven, N. P. G., Henke, M., Van der Logt, P., and 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, 344–352.
Laroche, Y., Storme, V., De Meutter, J., Messens, J., and Lauwereys, M. (1994) High-level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris. Biotechnology (N Y) 12, 1119–1124.
Brierley, R. A. (1998) Secretion of recombinant human insulin-like growth factor I (IGF-I), in Pichia Protocols, vol. 103, (Cregg, J. M., ed.), Humana Press, Totowa, New Jersey, pp. 149–177.
Siegel, R. S., and Brierley, R. A. (1989) Methylotrophic yeast Pichia pastoris produced in high-cell-density fermentations with high cell yields as vehicle for recombinant protein production. Biotechnol. Bioeng. 34, 403–404.
Waterham, H. R., Digan, M. E., Koutz, P. J., Lair, S. V., and Cregg, J. M. (1997) Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene 186, 37–44.
Zhang, W., Smith, L. A., Plantz, B. A., and Meagher, M. M. (2002) Design of Methanol Feed Control in Pichia pastoris Fermentations Based upon a Growth Model. Biotechnol Prog. 18, 1392–1399.
Zhang, W., Sinha, J., Smith, L. A., Inan, M., and Meagher, M. M. (2005) Maximization of production of secreted recombinant proteins in Pichia pastoris fed-batch fermentation. Biotechnol. Prog. 21, 386–393.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Zhang, W., Inan, M., Meagher, M.M. (2007). Rational Design and Optimization of Fed-Batch and Continuous Fermentations. In: Cregg, J.M. (eds) Pichia Protocols. Methods in Molecular Biology, vol 389. Humana Press. https://doi.org/10.1007/978-1-59745-456-8_4
Download citation
DOI: https://doi.org/10.1007/978-1-59745-456-8_4
Publisher Name: Humana Press
Print ISBN: 978-1-58829-429-6
Online ISBN: 978-1-59745-456-8
eBook Packages: Springer Protocols