Recombinant protein production provoked accumulation of ATP, fructose-1,6-bisphosphate and pyruvate in E. coli K12 strain TG1

Abstract

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.

Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures

“…This accumulation is probably due to the high energy demand imposed to the cell for the synthesis of the recombinant protein. Recently, it has been demonstrated that this ‘‘metabolic burden’’ is not caused by energy limitation; on the contrary, this is due to restrictions in anabolic functions (Weber et al, 2021 ). This energy excess due to RPP causes a reduction of catabolic carbon processing, hence, (i) affecting negatively the growth rate; (ii) enhancing acetate formation, and (iii) reducing the carbon substrate uptake, evidenced by the accumulation of glucose (Weber et al, 2002 , 2021 ).…”

Is energy excess the initial trigger of carbon overflow metabolism? Transcriptional network response of carbon-limited Escherichia coli to transient carbon excess

“…The transcriptomic response towards recombinant protein production was also mainly characterized by a vanishing influence of CRP-cAMP and an increasing influence of phosphorylated ArcA [25]. Energy-limiting conditions were also not detected, on the contrary, induced recombinant protein production was also initially accompanied by elevated ATP levels and an elevated adenylate energy charge [15,64]. Recent experiments corroborated that interference of recombinant protein production with normal host cell metabolism is not the result of competition for energy and precursor metabolites but mainly an inhibitory effect of toxic amounts of recombinant mRNA on well organized and structured anabolic processes which are needed for cell growth [68][69][70][71].…”

“…The reduction of respiratory activity and continued uptake of glucose leads to accumulation of metabolites and forces carbon flow towards side reactions, e.g., to acetate, the most prominent overflow metabolite of E. coli but also to less energy efficient pathways such as the methyl-glyoxal pathway [58]. As the initial metabolic responses occur immediately after the glucose pulse they are certainly also regulated at the allosteric level of enzyme activity through accumulating metabolites auch as ATP, fructose-1,6-bisphosphate and pyruvate (please refer also to [20] for additional data and [64] for discussion). These initial responses towards glucose excess can reduce respiratory energy generation but they are not sufficient to lead to balanced catabolic and anabolic carbon processing.…”

“…5, Additional file 2, see also https:// ecocyc. org/ [25,47,64]). Also, upon binding of pyruvate to the transcriptional repressor IclR, downregulation of the genes of the glyoxylate bypass occurs (e.g., aceA, aceB, see also https:// ecocyc.…”

Strategies for efficient production of recombinant proteins in Escherichia coli: alleviating the host burden and enhancing protein activity

“…Despite the availability of so many alternative expression systems, there is no guarantee that every type of protein will have a high yield or catalytic/functional activity. The occurrence of these phenomena can be attributed to two main aspects: (i) the host burden caused by the massive production of RPs [16] and (ii) the limited post-translational modification (PTM) capacity and generation of inclusion bodies (IBs) [17]. In fact, any production of RPs, especially toxic proteins, will inevitably compete with the host for resources, which are mainly reflected in the additional DNA replication burden, competition for transcription-and translation-related elements (RNAP, ribosomes, tRNA, and amino acids), and the additional energy and substrates consumed by PTMs [18].…”