Libraries of synthetic stationary-phase and stress promoters as a tool for fine-tuning of expression of recombinant proteins in Escherichia coli

Miksch, Gerhard; Bettenworth, Frank; Friehs, Karl; Flaschel, Erwin; Saalbach, Axel; Twellmann, Thorsten; Nattkemper, Tim Wilhelm

doi:10.1016/j.jbiotec.2005.04.027

Cited by 45 publications

(31 citation statements)

References 53 publications

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“…Generally, with respect to the spacer between the Ϫ10 and Ϫ35 elements of bacterial promoters, the focus has been on the importance of the length rather than nucleotide sequence (23,36). It has been shown, however, that the nucleotide sequence of the spacer can influence promoter strength (14,17,21), which is consistent with the results obtained here. The Ϫ10 element of Pm has experimentally been determined to be the hexamer TAGGCT (9), corresponding to nucleotide positions Ϫ6 to Ϫ11 in Fig.…”

Section: Resultssupporting

confidence: 87%

Random Mutagenesis of the Pm Promoter as a Powerful Strategy for Improvement of Recombinant-Gene Expression

Bakke

Berg

Aune

et al. 2009

Appl Environ Microbiol

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The inducible Pm-xylS promoter system has proven useful for production of recombinant proteins in several gram-negative species and in high-cell-density cultivations of Escherichia coli. In this study we subjected a 24-bp region of Pm (including the ؊10 element) to random mutagenesis, leading to large mutant libraries in E. coli. Low-frequency-occurring Pm mutants displaying strongly increased promoter activity (up-mutants) could be efficiently identified by using ␤-lactamase as a reporter. The up-mutants typically carried multiple point mutations positioned throughout the mutagenized region, combined with deletions around the transcription start site. Mutants displaying up to about a 14-fold increase in ␤-lactamase expression (relative to wild-type Pm) were identified without loss of the inducible phenotype. The mutants also strongly stimulated the expression of two other reporter genes, luc (encoding firefly luciferase) and celB (encoding phosphoglucomutase), and were found to significantly improve (twofold) a previously optimized process for high-level recombinant production of the medically important granulocyte-macrophage colony-stimulating factor in E. coli under high-cell-density conditions. These results demonstrate the potential of using random mutagenesis of promoters to improve protein expression at industrial levels and indicate that targeted modifications of individual functional elements are not sufficient to obtain optimized promoter sequences.The Pm-xylS promoter system drives the expression of the meta-cleavage operon carried by the Pseudomonas putida TOL plasmid pWW0. The gene products of this operon are involved in the catabolism of alkylbenzoates and are expressed in response to meta-pathway substrates (25). XylS positively regulates Pm when forming an activated complex with effectors like benzoate or its derivatives (13,26). Transcriptional activation occurs through binding of the activated XylS to two direct imperfect repeats located directly upstream of the Ϫ35 region of Pm (12).Pm-xylS has been shown to be useful for high-level expression of recombinant proteins in a wide range of gram-negative bacterial species (3,4,20,24). The uninduced expression level is low, and the use of different effector compounds at various concentrations can be used to regulate the level of induced expression (35). Many of the inducers are low-cost compounds that enter the cell by passive diffusion. Previously, we reported the use of this system in the construction of broad-host-range expression vectors based on the RK2 minimal replicon (2, 3). One of these vectors, pJB658, has proven useful for tightly regulated recombinant-gene expression in several gram-negative species (2,3,5,31,35). Industrial levels of production of human proteins under high-cell-density conditions (HCDC) of Escherichia coli has also been demonstrated with the Pm-xylS system coupled to the RK2 replicon (30,31). By studying the functionality of different secretion signal sequences, the effects of various vector copy numbers, and the condit...

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Section: Resultssupporting

confidence: 87%

Random Mutagenesis of the Pm Promoter as a Powerful Strategy for Improvement of Recombinant-Gene Expression

Bakke

Berg

Aune

et al. 2009

Appl Environ Microbiol

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“…However, the largest category of regulated genes in our study was that of unknown and hypothetical genes. Despite their unknown functions, the promoters of these genes are expected to serve useful roles in synthetic biology studies (15,19).…”

Section: Sppsmentioning

confidence: 99%

Upregulation of Plasmid Genes during Stationary Phase in Synechocystis sp. Strain PCC 6803, a Cyanobacterium

Berla¹,

Pakrasi

2012

Appl Environ Microbiol

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We analyzed DNA microarrays to identify highly expressed genes during stationary-phase growth of Synechocystis sp. PCC 6803. Many identified genes are on endogenous plasmids, with copy numbers between 0.4 and 7 per chromosome. The promoters of such genes will be useful for synthetic biology applications with this phototrophic host. Bacterial cultures enter stationary phase when either nutrient limitation or a buildup of growth by-products ceases cell division. However, this does not necessarily imply that cells become metabolically inactive. Artificial "leaves" have been constructed from Rhodopseudomonas palustris cells embedded in latex that can produce H 2 photoheterotrophically for over 5 months without cell growth (5). Pfic in Escherichia coli was recently used to produce a high titer of a bacteriotoxin at stationary phase without any inducer and during exponential phase without detectable growthlimiting toxin during exponential phase (1).Cultures and microarray analysis. We used DNA microarray analysis to identify genes and promoters active during stationary phase in Synechocystis sp. PCC 6803. This naturally competent cyanobacterium is a widely used host organism for autotrophic synthetic biology (3,4,13,18). Synthetic biology is an emerging field in which genes, promoters, and other units of genetic code either taken from across the diversity of life or created entirely from scratch are mixed and matched in a host organism (chassis) to improve existing cellular functions or create entirely new ones. Such studies demand the use of a wide variety of promoters that are active under different conditions in the chassis organism.We grew replicate cultures of Synechocystis PCC 6803 in BG11 medium bubbled with air plus 5% CO 2 (autotrophic) or with air plus 5 mM glucose (mixotrophic). The temperature was maintained at 30°C, and light intensity was 100 E m Ϫ2 s Ϫ1 from cool white fluorescent lamps. Cell growth was monitored by measurement of the optical density at 730 nm on a BioTek Quant plate reader (BioTek, VT). Cultures were sampled for microarrays in exponential phase and twice during stationary phase ( Fig. 1) and analyzed as described previously (20). Briefly, 2 replicate microarrays were analyzed for each of 2 replicate cultures for each nutritional condition. Data were LOWESS normalized by using the MATLAB bioinformatics toolbox. Normalized probe intensities were grouped by genes and t tested to determine significant up-or downregulation (P Ͻ 0.05). SPPS.To quantify the activity of potential promoters at stationary phase, we calculated a stationary-phase promoter score (SPPS) for each open reading frame (ORF), based on the following equation: SPPS ϭ log 2 (fold change) ϩ log 2 (normalized expression).The changes were averaged across nutritional conditions. Nor-

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“…We identified that YojI binds to the interferon-alpha receptor (IFNAR2) on the surface of human brain microvascular endothelial cells (HBMECs) and mediates E. coli adhesion to the host cells, and it is an important virulence factor for E. coli invasion of HBMEC. It is reported that YojI is also a chemical induced bio-sensor for regulating it's expression, a leucine-responsive regulatory protein, which Lrp controls the expression of YojI, which regulates exporting of toxin J25 [28][29][30] . Therefore, the strong promoter can be indentified and cloned before chimeric gene YojI-mCherry gene to highly initiate the gene expression to strongly incorporate YojI-mCherry chimeric protein onto the cell surface.…”

Section: Integration Of Both Promoter-based Bio-sensor and Bacterium-mentioning

confidence: 99%

The Applications of Promoter-Gene Engineered Bio-Sensors

Feng¹,

Xie²,

Jiang³

et al. 2018

Preprint

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Abstract:Promoter is a small region of DNA sequence in response to various transcription factors, which initiates a particular gene expression. The promoter-engineered bio-sensor can activate or repress gene expression through transcription factor recognizing specific molecules, such as polyamine, sugars, lactams, amino acids, organic acids or redox molecule, however, the reported applications of promoter enhanced bio-sensor are not too much. This review paper highlights the strategies of construction of promoter-gene engineered bio-sensor with human and bacteria's genetic promoter array for high-throughput screening (HTS) molecular drugs, study of membrane protein's localization and nucleocytoplasmic shuttling mechanism of regulating factor, enzyme activity, detection of the toxicity of intermediate chemicals, and probing bacteria density to improve value-added product titer. These bio-sensors's sensitivity and specificity can be further improved by proposed approaches of Mn 2+ and Mg 2+ added random Error-prone PCR and site-directed mutagenesis which is applied for construction of bacteria's "mutant library". It is expected to establish flexible HTS platform (Bio-sensor array) to large-scale screen transcription factor-acting drugs, reducing the toxicity of intermediate compounds, and constructing gene dynamic regulatory system in "push and pull" mode to effectively regulate the valuable medicinal product production. This proposed novel promoter-engineered biosensors aided synthetic genetic circuit construction will maximize the efficiency of bio-synthesis of medicinal compound, which will greatly promote the development of microbial metabolic engineering and biomedical science.

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Libraries of synthetic stationary-phase and stress promoters as a tool for fine-tuning of expression of recombinant proteins in Escherichia coli

Cited by 45 publications

References 53 publications

Random Mutagenesis of the Pm Promoter as a Powerful Strategy for Improvement of Recombinant-Gene Expression

Random Mutagenesis of the Pm Promoter as a Powerful Strategy for Improvement of Recombinant-Gene Expression

Upregulation of Plasmid Genes during Stationary Phase in Synechocystis sp. Strain PCC 6803, a Cyanobacterium

The Applications of Promoter-Gene Engineered Bio-Sensors

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