title: Replacement of the glucose phosphotransferase transport system by galactose permease reduces acetate accumulation and improves process performance of Escherichia coli for recombinant protein production without impairment of growth rate.
abstract: Acetate accumulation under aerobic conditions is a common problem in Escherichia coli cultures, as it causes a reduction in both growth rate and recombinant protein productivity. In this study, the effect of replacing the glucose phosphotransferase transport system (PTS) with an alternate glucose transport activity on growth kinetics, acetate accumulation and production of two model recombinant proteins, was determined. Strain VH32 is a W3110 derivative with an inactive PTS. The promoter region of the chromosomal galactose permease gene galP of VH32 was replaced by the strong trc promoter. The resulting strain, VH32GalP+ acquired the capacity to utilize glucose as a carbon source. Strains W3110 and VH32GalP+ were transformed for the production of recombinant TrpLE-proinsulin accumulated as inclusion bodies (W3110-PI and VH32GalP+-PI) and for production of soluble intracellular green fluorescent protein (W3110-pV21 and VH32GalP+-pV21). W3110-pV21 and VH32GalP+-pV21 were grown in batch cultures. Maximum recombinant protein concentration, as determined from fluorescence, was almost four-fold higher in VH32GalP+-pV21, relative to W3110-pV21. Maximum acetate concentration reached 2.8 g/L for W3110-pV21 cultures, whereas a maximum of 0.39 g/L accumulated in VH32GalP+-pV21. W3110-PI and VH32GalP+-PI were grown in batch and fed-batch cultures. Compared to W3110-PI, the engineered strain maintained similar production and growth rate capabilities while reducing acetate accumulation. Specific glucose consumption rate was lower and product yield on glucose was higher in VH32GalP+-PI fed-batch cultures. Altogether, strains with the engineered glucose uptake system showed improved process performance parameters for recombinant protein production over the wild-type strain.
authors.affiliation: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), A.P. 510-3, Cuernavaca, Morelos 62250, México.
authors.forename: Ramón
authors.initials: R
authors.lastname: De Anda
authors.forename: Alvaro R
authors.initials: AR
authors.lastname: Lara
authors.forename: Vanessa
authors.initials: V
authors.lastname: Hernández
authors.forename: Verónica
authors.initials: V
authors.lastname: Hernández-Montalvo
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: D000085:Acetates / Q000378:metabolism*; D001693:Biological Transport, Active / Q000502:physiology; 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*; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis*
chemical_list: D000085:Acetates; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; C043910:galactose permease; D010770:Phosphotransferases; D005947:Glucose