Kinetic metabolic modelling for the control of plant cells cytoplasmic phosphate.

A previously developed kinetic metabolic model for plant metabolism was used in a context of identification and control of intracellular phosphate (Pi) dynamics. Experimental data from batch flask cultures of Eschscholtiza californica cells was used to calibrate the model parameters for the slow dynamics (growth, nutrition, anabolic pathways, etc.). Perturbation experiments were performed using a perfusion small-scale bioreactor monitored by in vivo(31)P NMR. Parameter identification for Pi metabolism was done by measuring the cells dynamic response to different inputs for extracellular Pi (two pulse-response experiments and a step-response experiment). The calibrated model can describe Pi translocation between the cellular pools (vacuole and cytoplasm). The effect of intracellular Pi management on ATP/ADP and phosphomonoesters concentrations is also described by the model. The calibrated model is then used to develop a control strategy on the cytoplasmic Pi pool. From the identification of the systems dynamics, a proportional-integral controller was designed and tuned. The closed-loop control was implemented in the small-scale NMR bioreactor and experimental results were in accordance with model predictions. Thus, the calibrated model is able to predict cellular behaviour for phosphate metabolism and it was demonstrated that it is possible to control the intracellular level of cytoplasmic Pi in plant cells.

Identifying conditions for inducible protein production in E. coli: combining a fed-batch and multiple induction approach.

BACKGROUND: In the interest of generating large amounts of recombinant protein, inducible systems have been studied to maximize both the growth of the culture and the production of foreign proteins. Even though thermo-inducible systems were developed in the late 1970's, the number of studies that focus on strategies for the implementation at bioreactor scale is limited. In this work, the bacteriophage lambda PL promoter is once again investigated as an inducible element but for the production of green fluorescent protein (GFP). Culture temperature, induction point, induction duration and number of inductions were considered as factors to maximize GFP production in a 20-L bioreactor. RESULTS: It was found that cultures carried out at 37 degrees C resulted in a growth-associated production of GFP without the need of an induction at 42 degrees C. Specific production was similar to what was achieved when separating the growth and production phases. Shake flask cultures were used to screen for desirable operating conditions. It was found that multiple inductions increased the production of GFP. Induction decreased the growth rate and substrate yield coefficients; therefore, two time domains (before and after induction) having different kinetic parameters were created to fit a model to the data collected. CONCLUSION: Based on two batch runs and the simulation of culture dynamics, a pre-defined feeding and induction strategy was developed to increase the volumetric yield of a temperature regulated expression system and was successfully implemented in a 20-L bioreactor. An overall cell density of 5.95 g DW l(-1) was achieved without detriment to the cell specific production of GFP; however, the production of GFP was underestimated in the simulations due to a significant contribution of non-growth associated product formation under limiting nutrient conditions.

Na-butyrate sustains energetic states of metabolism and t-PA productivity of CHO cells.

The effects of Na-butyrate on the physiological behaviour and on the specific productivity of recombinant tissue plasminogen activator (t-PA) Chinese Hamster Ovary (CHO) cells were characterized. Batch cultures were performed in a 3.5-L bioreactor. Na-butyrate was added either at the mid-exponential growth phase (48 h) or at the end of the exponential growth phase (74 h). The cultures with Na-butyrate showed higher net specific productivity of t-PA and lower final cell density and viability. Maximum specific productivity of t-PA for all cultures coincided with the early plateau phase (84 h). The cell's specific oxygen uptake rate (qO2) increased after the Na-butyrate addition and remained higher than that of the controlled culture. Triphosphate nucleotides, ADP, AMP and UDP-sugars all increased after 84 h in the cultures with Na-butyrate, showing different behaviours when Na-butyrate was added at 48 h or 74 h. Na-butyrate did not affect the cell's adenylate energy charge until the cell's viability started to decrease (156-168 h). The controlled culture and the culture with Na-butyrate addition, showed at 74 h, similar time trends as for purine and nucleotide ratios ((ATP+GTP)/(UTP+CTP) and UTP/ATP) with clear shifts in behaviour at 84 h and 168 h. However, the addition of Na-butyrate at 48 h resulted in damped variations of purine and nucleotide ratios in comparison to both the control culture and the culture with Na-butyrate addition at 74 h.