title: Pichia pastoris fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement.
abstract: Fed-batch fermentation of a methanol utilization plus (Mut(+)) Pichia pastoris strain typically has a growth phase followed by a production phase (induction phase). In the growth phase glycerol is usually used as carbon for cell growth while in the production phase methanol serves as both inducer and carbon source for recombinant protein expression. Some researchers employed a mixed glycerol-methanol feeding strategy during the induction phase to improve production, but growth kinetics on glycerol and methanol and the interaction between them were not reported. The objective of this paper is to optimize the mixed feeding strategy based on growth kinetic studies using a Mut(+) Pichia strain, which expresses the heavy-chain fragment C of botulinum neurotoxin serotype C [BoNT/C(Hc)] intracellularly, as a model system. Growth models on glycerol and methanol that describe the relationship between specific growth rate ( micro ) and specific glycerol/methanol consumption rate ( nu(gly), nu(MeOH)) were established. A mixed feeding strategy with desired micro (gly)/ micro (MeOH) =1, 2, 3, 4 (desired micro (MeOH) set at 0.015 h(-1)) was employed to study growth interactions and their effect on production. The results show that the optimal desired micro (gly)/ micro (MeOH) is around 2 for obtaining the highest BoNT/C(Hc) protein content in cells: about 3 mg/g wet cells.
authors.affiliation: Department of Chemical Engineering, University of Nebraska-Lincoln, 207M Othmer Hall, Lincoln, NE 68583-0919, USA. wzhang1@unl.edu
authors.forename: Wenhui
authors.initials: W
authors.lastname: Zhang
authors.forename: Karen J
authors.initials: KJ
authors.lastname: Hywood Potter
authors.forename: Bradley A
authors.initials: BA
authors.lastname: Plantz
authors.forename: Vicki L
authors.initials: VL
authors.lastname: Schlegel
authors.forename: Leonard A
authors.initials: LA
authors.lastname: Smith
authors.forename: Michael M
authors.initials: MM
authors.lastname: Meagher
mesh_terms: D001905:Botulinum Toxins / Q000096:biosynthesis; D003014:Clostridium botulinum; D003470:Culture Media / Q000378:metabolism; D005285:Fermentation / Q000502:physiology*; D005990:Glycerol / Q000378:metabolism*; D007218:Industrial Microbiology / Q000379:methods; D000432:Methanol / Q000378:metabolism*; D010843:Pichia / Q000254:growth & development* / Q000378:metabolism*
chemical_list: D003470:Culture Media; D001905:Botulinum Toxins; C096324:botulinum toxin type C; D005990:Glycerol; D000432:Methanol