Use of fed-batch cultivation for achieving high cell densities for the pilot-scale production of a recombinant protein (phenylalanine dehydrogenase) in Escherichia coli.
A fed-batch process for the high cell density cultivation of E. coli TG1 and the production of the recombinant protein phenylalanine dehydrogenase (PheDH) was developed. A model based on Monod kinetics with overflow metabolism and incorporating acetate utilization kinetics was used to generate simulations that describe cell growth, acetate production and reconsumption, and glucose consumption during fed-batch cultivation. Using these simulations a predetermined feeding profile was elaborated that would maintain carbon-limited growth at a growth rate below the critical growth rate for acetate formation (mu < mu(crit)). Two starvation periods are incorporated into the feed profile in order to induce acetate utilization. Cell concentrations of 53 g dry cell weight (DCW)/L were obtained with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the total cell protein. The yield of PheDH was 129 U/mL with a specific activity of 1.2 U/mg DCW and a maximum product formation rate of 0.41 U/mg DCW x h. The concentration of aectate was maintained below growth inhibitory levels until 3 h before the end of the fermentation when the concentration reached a maximum of 10.7 g/L due to IPTG induction of the recombinant protein.
Faulkner
Eilis
E
School of Chemical and Bioprocess Engineering, Centre for Synthesis and Chemical Biology, University College Dublin (UCD), Dublin 4, Ireland.
Barrett
Mark
M
Okor
Sola
S
Kieran
Patricia
P
Casey
Eoin
E
Paradisi
Francesca
F
Engel
Paul
P
Glennon
Brian
B
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000187:drug effects; D001431:Bacteriological Techniques / Q000379:methods; D018533:Biomass; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development*; D005285:Fermentation; D008954:Models, Biological; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects; D013997:Time Factors
D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase