Quantitative characterization of a recombinant Pichia pastoris Mut+ strain secreting avidin using transient continuous cultures

The objective of this thesis was to characterize the growth stoichiometry, the specific recombinant protein productivity and the regulation of the alcohol oxidase (AOX) enzyme of a Pichia pastoris Mut+ strain, performing conventional chemostat cultures and transient changes of culture parameters in continuous cultures. A P. pastoris strain secreting avidin was chosen as case study. The investigation focused on an analysis of the influence of specific growth rate, culture temperature and on use of mixed substrates. First, a chemically defined medium was designed for the production of recombinant biotin-free avidin in continuous cultures. Indeed, one problem with heterologous expression systems secreting avidin is that biotin, which is an essential vitamin for most microorganisms, binds strongly to the produced recombinant avidin. The addition of low amounts of biotin (20 µg L-1 biotin for a cell density of 8 g L-1) resulted in stable chemostat cultures on methanol with the concomitant production of biotin-free avidin. The influence of the specific growth rate on growth stoichiometry, recombinant avidin productivity and specific alcohol oxidase activity was studied with chemostat cultures on glycerol and on methanol. Results showed that the substrate consumption rate for maintenance purposes was low: 0.007 and 0.011 C-mol C-mol-1 h-1 for growth on glycerol and methanol, respectively. No recombinant avidin was detected in cultures growing on glycerol, but a partial derepression of the synthesis of AOX was observed. During chemostat cultures on methanol, the specific AOX activity was 20 to 100-fold higher than in chemostat cultures on glycerol and the specific avidin production rate was growth-associated, increasing linearly with dilution rate. No relationship between the specific avidin production rate and the specific AOX activity could be established, although avidin was expressed under the control of the AOX1 promoter. The applicability of a new and dynamic cultivation method, which consists in increasing linearly the temperature during continuous culture, was investigated. Comparison of culture characteristics determined during pseudo-steady state continuous cultures with linear increase of temperature at a rate of 0.1°C h-1 and during chemostat cultures showed that this technique can be used as a fast and accurate tool for the characterization of host cells according to cultivation temperature. Results showed that cultivation at a lower temperature than the optimal growth temperature of 30°C did not influence significantly the recombinant avidin productivity. The regulation of the AOX enzyme was investigated in continuous cultures on glycerol, methanol or mixed substrate cultures on glycerol and methanol with sudden changes of nutrient supply or pulses of methanol. Results showed that use of mixed feeds of glycerol and methanol allowed faster adaptation of cellular metabolism after growth on glycerol due to faster synthesis of methanol dissimil