Oxygen transfer as a tool for fine-tuning recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter

Abstract

Effects of oxygen transfer on recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter were investigated. Recombinant glucose isomerase was chosen as the model protein. Two groups of oxygen transfer strategies were applied, one of which was based on constant oxygen transfer rate where aeration rate was Q O/V = 3 and 10 vvm, and agitation rate was N = 900 min(-1); while the other one was based on constant dissolved oxygen concentrations, C DO = 5, 10, 15, 20 and 40 % in the fermentation broth, by using predetermined exponential glucose feeding with μ o = 0.15 h(-1). The highest cell concentration was obtained as 44 g L(-1) at t = 9 h of the glucose fed-batch phase at C DO = 20 % operation while the highest volumetric and specific enzyme activities were obtained as 4440 U L(-1) and 126 U g(-1) cell, respectively at C DO = 15 % operation. Investigation of specific enzyme activities revealed that keeping C DO at 15 % was more advantageous with an expense of relatively higher by-product formation and lower specific cell growth rate. For this strategy, the highest oxygen transfer coefficient and oxygen uptake rate were K L a = 0.045 s(-1) and OUR = 8.91 mmol m(-3) s(-1), respectively.

Physiological state as transferable operating criterion to improve recombinant protein production in Pichia pastoris through oxygen limitation

“…Hence, a pre‐programmed exponential feeding rate profile for substrate addition derived from mass balance equations to maintain a constant specific growth rate ( μ ) would be implemented . In the simplest scenario proposed in previous work, if the concentration of ethanol is taken as indirect physiological indicator, its control does not guarantee keeping either q ethanol or μ constant . Only in a chemostat, where the concentration of a component for a given dilution rate at steady‐state is unvarying, are the specific rate and productivity constant.…”

Hybrid fed‐batch bioreactor operation design: control of substrate uptake enhances recombinant protein production in high‐cell‐density fermentations

“…The cells from the pre‐cultivation medium were centrifuged for 10 min at 1500× g at +4 °C, and then inoculated into the BSM medium for GB‐phase fermentation. The bioreactor operation conditions were: T = 30 °C, pH = 5.5 and dissolved oxygen (DO) concentration was kept constant at pO 2 = 15% of air saturation using an air‐flow controller in cascade starting at Q O / V = 1vvm and increasing during the course of fermentations at constant agitation speed N = 900 min −1 . The DO concentration in the fermentation broth was measured by Hamilton VisiFerm DO 120 probe (Switzerland), with a measuring range of 4 ppb to 25 ppm.…”

“…The bioreactor operation conditions were: T = 30 ∘ C, pH = 5.5 25 and dissolved oxygen (DO) concentration was kept constant at pO 2 = 15% of air saturation using an air-flow controller in cascade starting at Q O /V = 1vvm and increasing during the course of fermentations at constant agitation speed N = 900 min −1 . 26 The DO concentration in the fermentation broth was measured by Hamilton VisiFerm DO 120 probe (Switzerland), with a measuring range of 4 ppb to 25 ppm. For pH control at pH = 5.5, 28-30% ammonia solution, and to prevent foaming, 10% antifoam solution (Y-30 emulsion, Sigma), were supplied in small amounts by positive-displacement pumps.…”

Carbon metabolism influenced for promoters and temperature used in the heterologous protein production using Pichia pastoris yeast

“… 29 Effects of oxygen transfer on recombinant protein production by Pichia pastoris under GA3P promoter producing recombinant glucose isomerase were investigated by Güneş and Çalık. 30 Two groups of oxygen transfer strategies were applied, one of which was based on constant oxygen transfer rate where the aeration rate ( Q O/V ) = 3 and 10 vvm, and the agitation rate was N = 900 min −1 ; the other one was based on constant dissolved oxygen concentrations ( C DO ) = 5, 10, 15, 20 and 40% in the fermentation broth, by using predetermined exponential glucose feeding with μ o = 0.15 h −1 . The highest cell concentration was obtained as 44 g L −1 at t = 9 h of the glucose fed-batch phase at C DO = 20% operation while the specific enzyme activities revealed that keeping C DO at 15% was more advantageous at the expense of relatively higher by-product formation and lower specific cell growth rate.…”