Recombinant protein production‐associated metabolic burden reflects anabolic constraints and reveals similarities to a carbon overfeeding response

Abstract

A comparison of the metabolic response of Escherichia coli BL21 (DE3) towards the production of human basic fibroblast growth factor (hFGF-2) or towards carbon overfeeding revealed similarities which point to constraints in anabolic pathways. Contrary to expectations, neither energy generation (e.g., ATP) nor provision of precursor molecules for nucleotides (e.g., uracil) and amino acids (e.g., pyruvate, glutamate) limit host cell and plasmid-encoded functions. Growth inhibition is assumed to occur when hampered anabolic capacities do not match with the ongoing and overwhelming carbon catabolism. Excessive carbon uptake leads to by-product secretion, for example, pyruvate, acetate, glutamate, and energy spillage, for example, accumulation and degradation of adenine nucleotides with concomitant accumulation of extracellular hypoxanthine. The cellular response towards compromised anabolic capacities involves downregulation of cAMP formation, presumably responsible for subsequently better-controlled glucose uptake and resultant accumulation of glucose in the culture medium. Growth inhibition is neglectable under conditions of reduced carbon availability when hampered anabolic capacities also match with catabolic carbon processing. The growth inhibitory effect with accompanying energy spillage, respectively, hypoxanthine secretion and cessation of cAMP formation is not unique to the production of hFGF-2 but observed during the production of other proteins and also during overexpression of genes without transcript translation.

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

“…Recently it was shown that the growth inhibitory effect of IPTG-induced human hFGF-2 production in E. coli BL21(DE3) using a pET-based expression vector reflects similarities to a carbon overfeeding response and is the result of constraints in anabolic pathways and accompanied by accumulation of ATP and ATP degradation products as well as primary metabolites such as pyruvate [ 4 ]. Those studies are complemented here by analyzing the cellular response towards IPTG-induced production of hFGF-2 in the E. coli K12 strain TG1.…”

“…Until lately it was common understanding that the recombinant protein production associated metabolic burden is the result of redirection of metabolites and energy away from host cell towards plasmid encoded functions. More recently, it was shown for the E. coli BL21(DE3)/T7 promoter expression vector combination that the growth inhibitory effect of recombinant protein production is not elevated energy and precursor withdrawal for plasmid encoded functions but, on the opposite, ample catabolic generation and insufficient withdrawal of energy and precursor for anabolic purposes [ 4 ]. The growth inhibitory effect of recombinant protein production has been shown to be mainly attributable to recombinant gene transcription but also partly to recombinant protein synthesis in particular when difficult-to-fold proteins are being produced [ 5 , 6 ].…”

“…For the E. coli BL21(DE3)/T7 promoter expression vector combination it has been shown that induced recombinant gene expression leads to ATP accumulation which is being further subjected to degradation pathways finally leading to the extracellular accumulation of hypoxanthine [ 4 ]. This has been shown for a variety of recombinant genes and proteins including also for the production of human basic fibroblast growth factor (hFGF-2).…”

Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21

“…The metabolic burden is associated with energetic and precursor constraints due to the transcription and translation of non-essential proteins for the host cell. This limitation is reflected in the alteration of physiological parameters, such as growth rate, and in the downregulation of several essential metabolic pathways for the cell ( Mairhofer et al, 2013 ; Tan et al, 2020 ; Li and Rinas, 2021 ). In order to minimize metabolic imbalance, several commercially available plasmids have been engineered to design each biotechnological process, choosing between different promoters and origins of replication, both responsible for the expression level of the gene/s of interest ( Zaslaver et al, 2006 ; De Mey et al, 2007 ; Wang et al, 2009 ; Rosano and Ceccarelli, 2014 ; Yang et al, 2016 ; Jervis et al, 2019 ; Rosano et al, 2019 ).…”

Regulating the T7 RNA Polymerase Expression in E. coli BL21 (DE3) to Provide More Host Options for Recombinant Protein Production

“…However, it has been shown that T7 RNAP-mediated expression of genes encoding membrane proteins is usually toxic, which causes damage to cell [13]. There is another explanation that one of the reasons for this phenomenon was that the stronger P LacUV5 produced a large amount of T7 RNAP, which was toxic to cells [19,20]. Other studies had shown that this phenomenon was caused by the resource allocated limitation between cell growth and protein production, rather than the toxicity of T7 RNAP.…”