title: Utility of an Escherichia coli strain engineered in the substrate uptake system for improved culture performance at high glucose and cell concentrations: an alternative to fed-batch cultures.
abstract: Overflow metabolism is an undesirable characteristic of aerobic cultures of Escherichia coli. It results from elevated glucose consumption rates that cause a high substrate conversion to acetate, severely affecting cell physiology and bioprocess performance. Such phenomenon typically occurs in batch cultures under high glucose concentration. Fed-batch culture, where glucose uptake rate is controlled by external addition of glucose, is the classical bioprocessing alternative to prevent overflow metabolism. Despite its wide-spread use, fed-batch mode presents drawbacks that could be overcome by simpler batch cultures at high initial glucose concentration, only if overflow metabolism is effectively prevented. In this study, an E. coli strain (VH32) lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) with a modified glucose transport system was cultured at glucose concentrations of up to 100 g/L in batch mode, while expressing the recombinant green fluorescence protein (GFP). At the highest glucose concentration tested, acetate accumulated to a maximum of 13.6 g/L for the parental strain (W3110), whereas a maximum concentration of only 2 g/L was observed for VH32. Consequently, high cell and GFP concentrations of 52 and 8.2 g/L, respectively, were achieved in VH32 cultures at 100 g/L of glucose. In contrast, maximum biomass and GFP in W3110 cultures only reached 65 and 48%, respectively, of the values attained by the engineered strain. A comparison of this culture strategy against traditional fed-batch culture of W3110 is presented. This study shows that high cell and recombinant protein concentrations are attainable in simple batch cultures by circumventing overflow metabolism through metabolic engineering. This represents a novel and valuable alternative to classical bioprocessing approaches.
authors.affiliation: Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, AP 510-3, Morelos 62250, Mexico.
authors.forename: Alvaro R
authors.initials: AR
authors.lastname: Lara
authors.forename: Luis
authors.initials: L
authors.lastname: Caspeta
authors.forename: Guillermo
authors.initials: G
authors.lastname: Gosset
authors.forename: Francisco
authors.initials: F
authors.lastname: Bolívar
authors.forename: Octavio T
authors.initials: OT
authors.lastname: Ramírez
mesh_terms: D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods*; D005947:Glucose / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D005947:Glucose