The expression of recombinant genes in <i>Escherichia coli</i> can be strongly stimulated at the transcript production level by mutating the DNA‐region corresponding to the 5′‐untranslated part of mRNA

Berg, Laila; Lale, Rahmi; Bakke, Ingrid; Burroughs, Nigel John; Valla, Svein

doi:10.1111/j.1751-7915.2009.00107.x

Cited by 39 publications

(74 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Finally, one of the pJBn expression vectors has been shown to give rise to production of industrial levels of several recombinant proteins in Escherichia coli (36,37). Recently, we have found that the bla gene (encoding ␤-lactamase) expression from the Pm promoter can be strongly stimulated in E. coli (up to 20-fold at the protein product level) by introducing mutations at the DNA level in its cognate 5Ј untranslated region (UTR) (5). The inducible promoter phenotype was intact in all of the UTR variants.…”

mentioning

confidence: 99%

Continuous Control of the Flow in Biochemical Pathways through 5′ Untranslated Region Sequence Modifications in mRNA Expressed from the Broad-Host-Range Promoter Pm

Lale

Berg

Stüttgen

et al. 2011

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

The inducible Pm promoter integrated into broad-host-range plasmid RK2 replicons can be fine-tuned continuously between the uninduced and maximally induced levels by varying the inducer concentrations. To lower the uninduced background level while still maintaining the inducibility for applications in, for example, metabolic engineering and synthetic (systems) biology, we report here the use of mutations in the Pm DNA region corresponding to the 5 untranslated region of mRNA (UTR). Five UTR variants obtained by doped oligonucleotide mutagenesis and selection, apparently reducing the efficiency of translation, were all found to display strongly reduced uninduced expression of three different reporter genes (encoding ␤-lactamase, luciferase, and phosphoglucomutase) in Escherichia coli. The ratio between induced and uninduced expression remained the same or higher compared to cells containing a corresponding plasmid with the wild-type UTR. Interestingly, the UTR variants also displayed similar effects on expression when substituted for the native UTR in another and constitutive promoter, P1 (P antitet ), indicating a broad application potential of these UTR variants. Two of the selected variants were used to control the production of the C 50 carotenoid sarcinaxanthin in an engineered strain of E. coli that produces the precursor lycopene. Sarcinaxanthin is produced in this particular strain by expressing three Micrococcus luteus derived genes from the promoter Pm. The results indicated that UTR variants can be used to eliminate sarcinaxanthin production under uninduced conditions, whereas cells containing the corresponding plasmid with a wild-type UTR produced ca. 25% of the level observed under induced conditions.The initially used methods of deleting or overexpressing genes have been demonstrated to be inadequate for many applications in metabolic engineering (21,31,32). For example, when the goal is to optimize the expression level of a desired protein by engineering the relevant metabolic pathway, it might be necessary to change the expression of multiple enzymes simultaneously and to different levels (30,38). Also, reducing the formation of particular by-products can increase the flux of the desired product (29). In addition, low basal expression is critical for applications such as the expression of toxic genes, metabolic engineering, and control analysis (2, 14, 27, 34). This has led to an increased focus on development of genetic tools to fine-tune gene expression to the desired levels. A commonly used strategy is to make so-called promoter libraries of constitutive promoters (1,15,17). Such promoters seem to be preferred over the corresponding inducible ones for industrial scale productions because of factors such as inducer costs, sensitivity to inducer concentration, and heterogeneity of expression caused by an all-or-none effect of induction (1,19). However, the all-or-none induction effect may be eliminated if the inducer enters the cell interior by passive diffusion (20). Thus, regulatable promoter s...

show abstract

mentioning

confidence: 99%

Continuous Control of the Flow in Biochemical Pathways through 5′ Untranslated Region Sequence Modifications in mRNA Expressed from the Broad-Host-Range Promoter Pm

Lale

Berg

Stüttgen

et al. 2011

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, the fact that larger amounts of the optimized parA mRNA were observed both in the one-copy and 14-copy parA systems (Fig. 5) may indicate that the new 5′-UTR mRNA structure is probably (Hsu et al 2006;Berg et al 2009;Goldman et al 2009) (i) increasing the stability of the complex RNA polymerase-transcript and thus reducing the amounts of abortive transcripts, (ii) decreasing the susceptibility to degradation of the transcript, and (iii) decreasing the availability to degradation because the transcript binds longer to rRNA. Thus, the final amount of the resolvase in the system is a compromise between transcriptional and translational rates.…”

Section: Discussionmentioning

confidence: 96%

Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase

Šimčíková

Alves

Brito

et al. 2016

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

show abstract

“…Growth media were supplemented with 200 μg/mL ampicillin (Sigma-Aldrich) if required. Standard DNA manipulations were performed as described elsewhere (Berg et al 2009). …”

Section: Methods and Experimentsmentioning

confidence: 99%

1H, 13C, 15N resonance assignment of the chitin-active lytic polysaccharide monooxygenase BlLPMO10A from Bacillus licheniformis

et al. 2014

View full text Add to dashboard Cite

The chitin-active 19.2 kDa lytic polysaccharide monooxygenase BlLPMO10A from Bacillus licheniformis has been isotopically labeled and recombinantly expressed. In this paper, we report the 1 H, 13 C, 15 N resonance assignment of BlLPMO10A. Keywords: lytic polysaccharide monooxygenase (LPMO), AA10, chitin, cellulose Biological contextCellulose and chitin are linear insoluble polymers composed of β-(1,4)-linked D-glucose and N-acetylglucosamine, respectively. These polysaccharides form crystalline structures, which makes them resistant to enzymatic hydrolysis and poses an obstacle for efficient biomass conversion. In recent years, a new family of copper-dependent redox enzymes with the ability to increase the rate of enzymatic degradation of these recalcitrant polysaccharides has been identified. (Vaaje-Kolstad et al. 2005a;Moser et al. 2008;Harris et al. 2010;Vaaje-Kolstad et al. 2010;Forsberg et al. 2011;Quinlan et al. 2011;Horn et al. 2012). These enzymes, which are abundantly present in biomass-degrading microbes, are today collectively referred to as lytic polysaccharide monooxygenases (LPMOs). In the CAZy database, they are classified as auxiliary activity (AA) families 9 (AA9, formerly GH61), 10 (AA10, formerly CBM33) and 11 (AA11) (see www.cazy.org and (Levasseur et al. 2013)). LPMOs have been shown to catalyze the cleavage of glycosidic bonds in chitin and cellulose through hydroxylation of either carbon within the scissile bond (Vaaje-Kolstad et al. 2010;Quinlan et al., 2011; Phillips et al., 2011; Kim et al., 2014).Whereas the structures of several LPMOs have been solved by X-ray crystallography (Vaaje-Kolstad et al. 2005b;Karkehabadi et al. 2008;Harris et al. 2010;Quinlan et al. 2011;Vaaje-Kolstad et al. 2012;Li et al. 2012;Hemsworth et al. 2013b;Wu et al. 2013;Hemsworth et al. 2014), the structure of the chitin-active AA10-type LPMO CBP21 from Serratia marcescens (PDB ID: 2LHS; correct name SmLPMO10A) is the only one that has been solved by NMR spectroscopy (Aachmann et al. 2012). The core LPMO structure has been described as a compact β-sandwich with a flat surface that includes the protein N-terminus and binds chitin or cellulose through a combination of polar and hydrophobic interactions (Vaaje-Kolstad et al. 2005a;Aachmann et al. 2012;Hemsworth et al. 2013a). The (putative) substrate-binding surfaces of LPMOs in all three families show considerable variation, but contain a conserved catalytic center with a copper-binding site composed of two histidines that bind the metal ion in a histidine brace (Vaaje-Kolstad et al. 2010;Quinlan et al. 2011;Hemsworth et al. 2013b;Hemsworth et al. 2013a). The metal binding site has high affinity for copper(II) and copper(I), and binding of copper has been shown to increase protein stability (Aachmann et al. 2012;Hemsworth et al. 2013b). 2The discovery of LPMOs has fundamentally changed our understanding of the degradation of recalcitrant polysaccharides and has opened up novel opportunities for development of cheaper and more efficient enzymatic tools for ...

show abstract

The expression of recombinant genes in Escherichia coli can be strongly stimulated at the transcript production level by mutating the DNA‐region corresponding to the 5′‐untranslated part of mRNA

Cited by 39 publications

References 46 publications

Continuous Control of the Flow in Biochemical Pathways through 5′ Untranslated Region Sequence Modifications in mRNA Expressed from the Broad-Host-Range Promoter Pm

Continuous Control of the Flow in Biochemical Pathways through 5′ Untranslated Region Sequence Modifications in mRNA Expressed from the Broad-Host-Range Promoter Pm

Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase

1H, 13C, 15N resonance assignment of the chitin-active lytic polysaccharide monooxygenase BlLPMO10A from Bacillus licheniformis

Contact Info

Product

Resources

About