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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.
Metab Eng. 2006 May; 8(3):281-90.ME

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+Show Affiliations

Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), A.P. 510-3, Cuernavaca, Morelos 62250, México.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16517196

Citation

De Anda, Ramón, et al. "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." Metabolic Engineering, vol. 8, no. 3, 2006, pp. 281-90.
De Anda R, Lara AR, Hernández V, et al. 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. Metab Eng. 2006;8(3):281-90.
De Anda, R., Lara, A. R., Hernández, V., Hernández-Montalvo, V., Gosset, G., Bolívar, F., & Ramírez, O. T. (2006). 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. Metabolic Engineering, 8(3), 281-90.
De Anda R, et al. 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. Metab Eng. 2006;8(3):281-90. PubMed PMID: 16517196.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - De Anda,Ramón, AU - Lara,Alvaro R, AU - Hernández,Vanessa, AU - Hernández-Montalvo,Verónica, AU - Gosset,Guillermo, AU - Bolívar,Francisco, AU - Ramírez,Octavio T, Y1 - 2006/03/06/ PY - 2005/08/19/received PY - 2005/12/23/revised PY - 2006/01/05/accepted PY - 2006/3/7/pubmed PY - 2006/8/22/medline PY - 2006/3/7/entrez SP - 281 EP - 90 JF - Metabolic engineering JO - Metab. Eng. VL - 8 IS - 3 N2 - 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. SN - 1096-7176 UR - https://www.unboundmedicine.com/medline/citation/16517196/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_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1096-7176(06)00005-X DB - PRIME DP - Unbound Medicine ER -