The new pLAI (lux regulon based auto-inducible) expression system for recombinant protein production in Escherichia 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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1P140 A novel expression system for high-level recombinant protein production in Escherichia coli(4. Protein engineering,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Seibutsu Butsuri ◽

10.2142/biophys.46.s181_4 ◽

2006 ◽

Vol 46 (supplement2) ◽

pp. S181

Author(s):

Eri Hojo ◽

Masakatsu Kamiya ◽

Tomoyasu Aizawa ◽

Mitsuhiro Miyazawa ◽

Yusuke Kato ◽

...

Keyword(s):

Escherichia Coli ◽

Protein Engineering ◽

Recombinant Protein ◽

Recombinant Protein Production ◽

Protein Production ◽

Poster Session ◽

Expression System ◽

Meeting Program ◽

High Level

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Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm Bombyx mori

Transgenic Research ◽

10.1007/s11248-009-9328-2 ◽

2009 ◽

Vol 19 (3) ◽

pp. 473-487 ◽

Cited By ~ 56

Author(s):

Ken-ichiro Tatematsu ◽

Isao Kobayashi ◽

Keiro Uchino ◽

Hideki Sezutsu ◽

Tetsuya Iizuka ◽

...

Keyword(s):

Gene Expression ◽

Recombinant Protein ◽

Bombyx Mori ◽

Recombinant Protein Production ◽

Protein Production ◽

Expression System ◽

Silk Gland ◽

Gene Expression System ◽

Silkworm Bombyx Mori

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

Microbial Cell Factories ◽

10.1186/s12934-021-01661-9 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Jan Weber ◽

Zhaopeng Li ◽

Ursula Rinas

Keyword(s):

Recombinant Protein ◽

Recombinant Protein Production ◽

Protein Production ◽

Expression System ◽

Limiting Factors ◽

Expression Vectors ◽

Metabolic Enzyme ◽

E Coli ◽

Metabolic Burden ◽

Fructose 1,6 Bisphosphate

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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