Recombinant protein production provoked accumulation of ATP, fructose-1,6-bisphosphate and pyruvate in E. coli K12 strain TG1
Creators: name: Weber, Jan
name: Li, Zhaopeng
name: Rinas, Ursula
Publisher: name: London : Biomed Central
Year: 2021
Subjects: subject: Escherichia coli
subject: Metabolic burden
subject: Recombinant protein production
subject: acetic acid
subject: adenosine diphosphate
subject: adenosine phosphate
subject: adenosine triphosphate
subject: ampicillin
subject: carbon dioxide
subject: dihydroxyacetone phosphate
subject: edetic acid
subject: fibroblast growth factor 2
subject: fructose 1,6 bisphosphate
subject: glucose
subject: glucose 6 phosphate
subject: glyceraldehyde 3 phosphate
subject: glycerol
subject: isopropyl thiogalactoside
subject: phosphoenolpyruvate
subject: phosphogluconate dehydrogenase
subject: phosphoglycerate kinase
subject: pyruvic acid
subject: recombinant protein
subject: thiamine
subject: bacterial growth
subject: bacterium culture
subject: biomass
subject: cell growth
subject: cell proliferation
subject: centrifugation
subject: controlled study
subject: enzyme analysis
subject: Escherichia coli K-12
subject: expression vector
subject: filtration
subject: gene expression system
subject: glycolysis
subject: growth inhibition
subject: human
subject: metabolite
subject: nonhuman
subject: optical density
subject: pentose phosphate cycle
subject: plasmid
subject: polyacrylamide gel electrophoresis
subject: promoter region
subject: Dewey Decimal Classification::500 | Naturwissenschaften::570 | Biowissenschaften, Biologie
subject: Dewey Decimal Classification::600 | Technik::610 | Medizin, Gesundheit

description: Background: Recently it was shown that production of recombinant proteins in E. coli BL21(DE3) using pET based expression vectors leads to metabolic stress comparable to a carbon overfeeding response. Opposite to original expectations generation of energy as well as catabolic provision of precursor metabolites were excluded as limiting factors for growth and protein production. On the contrary, accumulation of ATP and precursor metabolites revealed their ample formation but insufficient withdrawal as a result of protein production mediated constraints in anabolic pathways. Thus, not limitation but excess of energy and precursor metabolites were identified as being connected to the protein production associated metabolic burden. Results: Here we show that the protein production associated accumulation of energy and catabolic precursor metabolites is not unique to E. coli BL21(DE3) but also occurs in E. coli K12. Most notably, it was demonstrated that the IPTG-induced production of hFGF-2 using a tac-promoter based expression vector in the E. coli K12 strain TG1 was leading to persistent accumulation of key regulatory molecules such as ATP, fructose-1,6-bisphosphate and pyruvate. Conclusions: Excessive energy generation, respectively, accumulation of ATP during recombinant protein production is not unique to the BL21(DE3)/T7 promoter based expression system but also observed in the E. coli K12 strain TG1 using another promoter/vector combination. These findings confirm that energy is not a limiting factor for recombinant protein production. Moreover, the data also show that an accelerated glycolytic pathway flux aggravates the protein production associated “metabolic burden”. Under conditions of compromised anabolic capacities cells are not able to reorganize their metabolic enzyme repertoire as required for reduced carbon processing. © 2021, The Author(s).