A translation-coupling DNA cassette for monitoring protein translation in Escherichia coli

Mendez-Perez, Daniel; Gunasekaran, Suman; Orler, Victor J.; Pfleger, Brian F.

doi:10.1016/j.ymben.2012.04.005

Cited by 27 publications

(29 citation statements)

References 28 publications

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“…Although ols expression could be optimized to achieve active translation in E. coli, no targeted α-olefin product was detected in a heterologous expression experiment (Mendez-Perez et al, 2012). In our study, the iterative PKS SgcE, strictly controls seven rounds of PKS chain elongation to generate a specific hydrocarbon product.…”

Section: Discussionmentioning

confidence: 99%

Engineering an iterative polyketide pathway in Escherichia coli results in single-form alkene and alkane overproduction

Liu

Cheng

et al. 2015

Metabolic Engineering

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Section: Discussionmentioning

confidence: 99%

Engineering an iterative polyketide pathway in Escherichia coli results in single-form alkene and alkane overproduction

Liu

Cheng

et al. 2015

Metabolic Engineering

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“…Previous work has focused on coupling such promoters to the expression of luciferase 22 or betagalactosidase 23 , which both require chemical assays for assessing their activity. Several methods are available for monitoring the level of protein production, such as it has been demonstrated using a translation-coupling system in E. coli 24 . None of the current methods, however, are suited for highthroughput approaches with simultaneous but independent in vivo monitoring of both translation and protein folding.…”

Section: Introductionmentioning

confidence: 99%

A dual-reporter system for investigating and optimizing protein translation and folding inE. coli

Zutz

Hamborg

Pedersen

et al. 2020

Preprint

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Strategies for investigating and optimising the expression and folding of proteins for biotechnological and pharmaceutical purposes are in high demand. Here, we describe a dual-reporter biosensor system that simultaneously assesses in vivo protein translation and protein folding, thereby enabling rapid screening of mutant libraries. We have validated the dual-reporter system on five different proteins and find an excellent correlation between reporter intensity signals and the levels of protein expression and solubility of the proteins. We further demonstrate the applicability of the dual-reporter system as a screening assay for deep mutational scanning experiments. The system enables high throughput selection of protein variants with high expression levels and altered protein stability. Next generation sequencing analysis of the resulting libraries of protein variants show a good correlation between computationally predicted and experimentally determined protein stabilities. We furthermore show that the mutational experimental data obtained using this system may be useful for protein structure calculations.

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“…However, the codon-optimized ols gene still cannot be expressed in E. coli [15]. A possible solution could be separating this protein into short functional fragments.…”

Section: Resultsmentioning

confidence: 99%

Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement

et al. 2013

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The formation of fusion protein in biosynthetic pathways usually improves metabolic efficiency either channeling intermediates and/or colocalizing enzymes. In the metabolic engineering of biochemical pathways, generating unnatural protein fusions between sequential biosynthetic enzymes is a useful method to increase system efficiency and product yield. Here, we reported a special case. The malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate (3HP), and is a key enzyme in microbial production of 3HP, an important platform chemical. Functional domain analysis revealed that the N-terminal region of MCR (MCR-N; amino acids 1-549) and the C-terminal region of MCR (MCR-C; amino acids 550-1219) were functionally distinct. The malonyl-CoA was reduced into free intermediate malonate semialdehyde with NADPH by MCR-C fragment, and further reduced to 3HP by MCR-N fragment. In this process, the initial reduction of malonyl-CoA was rate limiting. Site-directed mutagenesis demonstrated that the TGXXXG(A)X(1-2)G and YXXXK motifs were important for enzyme activities of both MCR-N and MCR-C fragments. Moreover, the enzyme activity increased when MCR was separated into two individual fragments. Kinetic analysis showed that MCR-C fragment had higher affinity for malonyl-CoA and 4-time higher K cat/K m value than MCR. Dissecting MCR into MCR-N and MCR-C fragments also had a positive effect on the 3HP production in a recombinant Escherichia coli strain. Our study showed the feasibility of protein dissection as a new strategy in biosynthetic systems.

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A translation-coupling DNA cassette for monitoring protein translation in Escherichia coli

Cited by 27 publications

References 28 publications

Engineering an iterative polyketide pathway in Escherichia coli results in single-form alkene and alkane overproduction

Engineering an iterative polyketide pathway in Escherichia coli results in single-form alkene and alkane overproduction

A dual-reporter system for investigating and optimizing protein translation and folding inE. coli

Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement

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