Predictable tuning of protein expression in bacteria

Bonde, Marianne; Pedersen, Martin Wæver; Klausen, Michael Schantz; Jensen, Sheila Ingemann; Wulff, Tune; Harrison, S. P.; Nielsen, Alex Toftgaard; Herrgård, Markus J.; Sommer, Morten Otto Alexander

doi:10.1038/nmeth.3727

Cited by 123 publications

(152 citation statements)

References 34 publications

Supporting

Mentioning

146

Contrasting

Unclassified

Order By: Relevance

“…Previous attempts to control gene expression in Escherichia coli cells have used degeneration of ribosome binding sequence2 (rbs) or spacer sequence between rbs sequence and starting AUG codon22, but did not focus on the coding sequence. To show that polyA tracks can be used to control gene expression in E. coli cells, we created a set of reporters with increasing length of polyA tracks under the arabinose-inducible promoter pBAD (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For instance, an approach tested for attenuating gene expression in E. coli involves mutagenesis of the Shine-Dalgarno sequence (ribosome binding sequence, rbs) in the gene of interest222. The expression levels from all possible six-mer Shine-Dalgarno sequences were experimentally determined and the information is available in the EMOPEC database2. However, this valuable resource would have to be generated anew to use this approach in other bacteria and it cannot be applied to eukaryotic systems.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, because of evolution, discovered alleles can be species specific. These difficulties and the importance of hypomorphic alleles have prompted the development of several methods to generate hypomorphic mutations directly in various model organisms234567891011. However, these methods again are usually specific to one organism, can have unpredictable alterations in gene activity (separation of function, gain of function), or can change other aspects of regulation that affect interpretation of phenotype, such as spatial-temporal gene expression.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Rapid generation of hypomorphic mutations

et al. 2017

View full text Add to dashboard Cite

Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Rapid generation of hypomorphic mutations

et al. 2017

View full text Add to dashboard Cite

show abstract

“…Moreover, computational tools have been developed to estimate protein expression and design optimal sequences, such as E x E n S o (Expression Enhancer Software) [78], RBS C alculator [79], RBS D esigner [80], UTR D esigner [81] and EMOPEC [82]. All calculators were designed for use with E. coli and have been shown to give good approximations of protein expression levels [83].…”

Section: Expression Vectors For High-throughput Protein Expressionmentioning

confidence: 99%

High-throughput recombinant protein expression in Escherichia coli : current status and future perspectives

Jia¹,

Jeon²

2016

Open Biol.

237

160

View full text Add to dashboard Cite

The ease of genetic manipulation, low cost, rapid growth and number of previous studies have made Escherichia coli one of the most widely used microorganism species for producing recombinant proteins. In this post-genomic era, challenges remain to rapidly express and purify large numbers of proteins for academic and commercial purposes in a high-throughput manner. In this review, we describe several state-of-the-art approaches that are suitable for the cloning, expression and purification, conducted in parallel, of numerous molecules, and we discuss recent progress related to soluble protein expression, mRNA folding, fusion tags, post-translational modification and production of membrane proteins. Moreover, we address the ongoing efforts to overcome various challenges faced in protein expression in E. coli, which could lead to an improvement of the current system from trial and error to a predictable and rational design.

show abstract

“…Heterologous protein expression in bacteria, such as E. coli, has become a very attractive way to produce proteins, quickly and at low cost, as well as in whole-cell biocatalysts. Good enzyme production depends on (a) the choice of culture conditions (Phillips, Park, & Huber, 2000), (b) recombinant strain design (Marschall, Sagmeister, & Herwig, 2017;Rosano & Ceccarelli, 2014;Terpe, 2006) that is the E. coli strain derivative, the promoter type, and codon optimization (Boël et al, 2016), and (c) the genetic regulation system, such as ribosome binding sites (RBS; Bonde et al, 2016) and untranslated regions (UTR; Mahalik, Sharma, & Mukherjee, 2014). Recently, in the context of an in cellulo multienzymatic system, the control of UTR (Song, Woo, Jung, Bornscheuer, & Park, 2016) or RBS (Kohl et al, 2018) elements has been successfully used to balance the production of enzymes.…”

Section: Systematic Construction and Comparison Of The Co-expressinmentioning

confidence: 99%

Tuning of the enzyme ratio in a neutral redox convergent cascade: A key approach for an efficient one‐pot/two‐step biocatalytic whole‐cell system

Ménil

Petit

Courvoisier-Dezord

et al. 2019

Biotech & Bioengineering

View full text Add to dashboard Cite

The efficiency of a versatile in vivo cascade involving a promiscuous alcohol dehydrogenase, obtained from a biodiversity search, and a Baeyer–Villiger monooxygenase was enhanced by the independent control of the production level of each enzyme to produce ε‐caprolactone and 3,4‐dihydrocoumarin. This goal was achieved by adjusting the copy number per cell of Escherichia coli plasmids. We started from the observation that this number generally correlates with the amount of produced enzyme and demonstrated that an in vivo multi‐enzymatic system can be improved by the judicious choice of plasmid, the lower activity of the enzyme that drives the limiting step being counter‐balanced by a higher concentration. Using a preconception‐free approach to the choice of the plasmid type, we observed positive and negative synergetic effects, sometimes unexpected and depending on the enzyme and plasmid combinations. Experimental optimization of the culture conditions allowed us to obtain the complete conversion of cyclohexanol (16 mM) and 1‐indanol (7.5 mM) at a 0.5‐L scale. The yield for the conversion of cyclohexanol was 80% (0.7 g ε‐caprolactone, for the productivity of 244 mg·L −1·h −1) and that for 1‐indanol 60% (0.3 g 3,4‐dihydrocoumarin, for the productivity of 140 mg·L −1·h −1).

show abstract

Predictable tuning of protein expression in bacteria

Cited by 123 publications

References 34 publications

Rapid generation of hypomorphic mutations

Rapid generation of hypomorphic mutations

High-throughput recombinant protein expression in Escherichia coli : current status and future perspectives

Tuning of the enzyme ratio in a neutral redox convergent cascade: A key approach for an efficient one‐pot/two‐step biocatalytic whole‐cell system

Contact Info

Product

Resources

About