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.

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

Frontiers in Microbiology ◽

10.3389/fmicb.2021.682001 ◽

2021 ◽

Vol 12 ◽

Author(s):

Gema Lozano Terol ◽

Julia Gallego-Jara ◽

Rosa Alba Sola Martínez ◽

Adrián Martínez Vivancos ◽

Manuel Cánovas Díaz ◽

...

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Protein Expression ◽

Recombinant Proteins ◽

Copy Number ◽

Recombinant Protein Production ◽

Protein Production ◽

Recombinant Protein Expression ◽

Expression System ◽

E Coli

Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB1′ and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.

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Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems

10.21203/rs.2.19057/v2 ◽

2020 ◽

Author(s):

Artur Schuller ◽

Monika Cserjan-Puschmann ◽

Christopher Tauer ◽

Johanna Jarmer ◽

Martin Wagenknecht ◽

...

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Recombinant Protein Production ◽

Protein Production ◽

Expression System ◽

Cellular Level ◽

Lac Repressor ◽

Expression Systems ◽

E Coli ◽

Basal Expression

Abstract Background The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy of. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ 70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators ( lacO ) into the constitutive T5 and A1 promoter sequences. Results We showed that, in genome-integrated E. coli expression systems that used σ 70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacI Q , on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level. Conclusions Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

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Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems

10.21203/rs.2.19057/v1 ◽

2019 ◽

Author(s):

Artur Schuller ◽

Monika Cserjan-Puschmann ◽

Christopher Tauer ◽

Johanna Jarmer ◽

Martin Wagenknecht ◽

...

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Recombinant Protein Production ◽

Protein Production ◽

Expression System ◽

Lac Repressor ◽

Expression Systems ◽

E Coli ◽

Basal Expression ◽

T7 Expression System

Abstract Background The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy of. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ 70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators ( lacO ) into the constitutive T5 and A1 promoter sequences.Results We showed that, in genome-integrated E. coli expression systems that used σ 70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacI Q , on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level.Conclusions Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

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Development of an Autoinducible Plasmid for Recombinant Protein Production

Protein and Peptide Letters ◽

10.2174/0929866528666211105113750 ◽

2021 ◽

Vol 28 ◽

Author(s):

Young Kee Chae ◽

Hakbeom Kim

Keyword(s):

Recombinant Protein ◽

Stationary Phase ◽

Recombinant Protein Production ◽

Protein Production ◽

Binding Protein ◽

Maltose Binding Protein ◽

Target Protein ◽

Expression Vectors ◽

Lethal Gene ◽

E Coli

Background: The production of recombinant proteins in E. coli involves such factors as host strains, expression vectors, culture media, and induction methods. The typical procedure to produce heterologous proteins consists of the following: (1) insertion of the target gene into a suitable vector to construct an overexpression plasmid, (2) transformation of a strain specialized for protein production with the constructed plasmid DNA, (3) growth of the host in a suitable medium and induction of the protein production at a right moment, and (4) further growth to get the maximum yield. There are hurdles involved in each of these steps, and researchers have developed many materials or methods, which often require special recipes or procedures. Objective: To eliminate the special requirements for the recombinant protein production by using readily available materials. Also to save time and effort in the routine protein production work. Method: We started with a vector capable of producing a target protein fused to the C-terminus of the maltose binding protein (MBP). The mCherry (red fluorescent protein) gene was fused to MBP. It acted as a reporter in the initial screening procedure. The original lethal gene (barnase) was replaced with sacB. We chose 3 stationary phase promoters, and made hybrids of them by mixing halves from each one. The T5 promoter was replaced with these stationary phase promoters or their hybrids. The best plasmid was selected by the color intensity of the cell pellet. MBP and GST genes were inserted in place of sacB, and their production yields were compared with the original plasmid in the conventional way of expression. Results: We constructed an expression plasmid with an autoinducible promoter working in a host that was not specially designed for protein production and in a TB medium which did not contain any secret ingredient, nor was difficult to prepare unlike Studier’s defined medium. This plasmid also contains a color indicator which turns red when protein production is successful. We tested our system with the maltose binding protein (MBP) and the glutathione S-transferase (GST), and showed that both proteins were produced to a level comparable to what the commercial medium and/or the specialized strain yielded. Conclusion: We developed a plasmid equipped with an autoinducible promoter, a hybrid of the two promoters which were activated at the stationary phase. This plasmid does not need a special E. coli strain nor a sophisticated nor an expensive medium. It produces an intense red (or pink) color, which can be used as an indicator of a successful production of the target protein and as a predictive measure of the amount of the produced target protein. We speculate that this plasmid will have its greatest advantage when growing cells at low temperatures which would inevitably take a long time.

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