Modeling Pichia pastoris Growth on Methanol and Optimizing the Production of a Recombinant Protein, the Heavy-Chain Fragment C of Botulinum Neurotoxin, Serotype A

Authors

Wenhui Zhang, Biological Process Development Facility, Department of Food Science and Technology, University of Nebraska–Lincoln, Lincoln, Nebraska
Mark A. Bevins, Biological Process Development Facility, Department of Food Science and Technology, University of Nebraska–Lincoln, Lincoln, Nebraska
Bradley A. Plantz, Biological Process Development Facility, Department of Food Science and Technology, University of Nebraska–Lincoln, Lincoln, Nebraska
Leonard A. Smith, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, Maryland 21702-5012Follow
Michael M. Meagher, Department of Chemical Engineering, University of Nebraska-LincolnFollow

Date of this Version

October 2000

Comments

Originally Published in, BIOTECHNOLOGY AND BIOENGINEERING, VOL. 70, NO. 1, OCTOBER,2000;John Wiley & Sons, Inc. This article can be better viewed at the publishers site: http://www3.interscience.wiley.com/cgi-bin/fulltext/72514259/PDFSTART DOI: 10.1002/1097-0290(20001005)70:1<1::AID-BIT1>3.0.CO;2-Y

Abstract

An unstructured growth model for the recombinant methylotrophic yeast P. pastoris Mut+ expressing the heavy-chain fragment C of botulinum neurotoxin serotype A [BoNT/A(Hc)], was successfully established in quasi-steady state fed-batch fermentations with varying cell densities. The model describes the relationships between specific growth rate and methanol concentration, and the relationships between specific methanol and ammonium consump-tion rates and specific growth rate under methanol-limited growth conditions. The maximum specific growth rate (μ) determined from the model was 0.08 h−1 at a methanol concentration of 3.65 g/L, while the actual maximum μ was 0.0709 h−1. The maximum specific methanol consumption rate was 0.0682 g/g WCW/h. From the model, growth can be defined as either methanol-limited or metha-nol-inhibited and is delineated at a methanol concentration of 3.65 g/L. Under inhibited conditions, the observed biomass yield (YX/MeOH) was lower and the maintenance coefficient (mMeOH) was higher than compared to limited methanol conditions. The YX/MeOH decreased and mMeOH increased with increasing methanol concen-tration under methanolinhibited conditions. BoNT/A(Hc) content in cells (a) under inhibited growth was lower than that under limited growth, and decreased with increasing methanol concentration. A maximum a of 1.72 mg/g WCW was achieved at a μ of 0.0267 h−1 and induction time of 12 h.