Predictive design of mRNA translation initiation region to control prokaryotic translation efficiency

Seo, Sang Woo; Yang, Jun; Kim, Inhae; Yang, Joseph; Min, Byung Eun; Kim, Sanguk; Jung, Gyoo Yeol

doi:10.1016/j.ymben.2012.10.006

Cited by 257 publications

(251 citation statements)

References 30 publications

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“…In recent years, a number of studies aiming to optimize the synthesis of recombinant proteins have indicated that one strategy to increase the translation initiation rate is to make substitutions in the region surrounding the initiation codon that generates unstructured RNA regions [Care et al, 2008;Komarova et al, 2005;Seo et al, 2013]. As an alternative to codon substitutions and based on the results shown in our research, we propose that the expression of any gene could be notably improved by performing a fine scanning of codon deletions for the first 15 codons of the gene, using 15 site-specific oligonucleotides synthesized independently and combined before the cloning process, or using a library of combined deletions such as that created by the COBARDE mutagenesis approach, which generates all possible combinations of single to multiple amino acid deletions on a focused region of the gene.…”

Section: Discussionmentioning

confidence: 99%

A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression

Rodríguez-Mejía¹,

Roldán‐Salgado²,

Osuna³

et al. 2016

Microb Physiol

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Recombinant protein expression is one of the key issues in protein engineering and biotechnology. Among the different models for assessing protein production and structure-function studies, green fluorescent protein (GFP) is one of the preferred models because of its importance as a reporter in cellular and molecular studies. In this research we analyze the effect of codon deletions near the amino terminus of different GFP proteins on fluorescence. Our study includes Gly4 deletions in the enhanced GFP (EGFP), the red-shifted GFP and the red-shifted EGFP. The Gly4 deletion mutants and their corresponding wild-type counterparts were transcribed under the control of the T7 or Trc promoters and their expression patterns were analyzed. Different fluorescent outcomes were observed depending on the type of fluorescent gene versions. In silico analysis of the RNA secondary structures near the ribosome binding site revealed a direct relationship between their minimum free energy and GFP production. Integrative analysis of these results, including SDS-PAGE analysis, led us to conclude that the fluorescence improvement of cells expressing different versions of GFPs with Gly4 deleted is due to an enhancement of the accessibility of the ribosome binding site by reducing the stability of the RNA secondary structures at their mRNA leader regions.

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Section: Discussionmentioning

confidence: 99%

A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression

Rodríguez-Mejía¹,

Roldán‐Salgado²,

Osuna³

et al. 2016

Microb Physiol

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“…C. glutamicum ΔAPE6937R42 (Jiang et al 2013a) was used for an L-ornithine producing strain. After codon optimization for C. glutamicum codon usage by the combined application of Optimizer (Puigbò et al 2007) and UTR Designer (Seo et al 2013), M. succiniciproducens sacC (Supplementary Table) was synthesized by Suzhou GENEWIZ, Inc (Suzhou, China) and ligated into pQE30 to obtain pQE30-sacC. The sac fragment was digested from pQE30-sacC and inserted into the EcoRI/KpnI sites of pEC-XK99E (Kirchner and Tauch 2003) to obtain pEC-sacC.…”

Section: Methodsmentioning

confidence: 99%

Production of L-ornithine from sucrose and molasses by recombinant Corynebacterium glutamicum

Zhang

Liu

2014

Folia Microbiol

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Sucrose and molasses are attractive raw materials for industrial fermentation. Although Corynebacterium glutamicum shows sucrose-utilizing activity, sucrose or molasses is only a fraction of carbon source used in the fermentation medium in most works. An engineered C. glutamicum strain was constructed for producing L-ornithine with sucrose or molasses as a sole carbon source by transferring Mannheimia succiniciproducens β-fructofuranosidase gene (sacC). The engineered strain, C. glutamicum ΔAPE6937R42 (pEC-sacC), produced 22.0 g/L of L-ornithine with sucrose as the sole carbon source, which is on par with that obtained by the parent strain C. glutamicum ΔAPE6937R42 with glucose as the sole carbon. The resulting strain C. glutamicum ΔAPE6937R42 (pEC-sacC) produced 27.0 g/L of L-ornithine with molasses as the sole carbon source, which is higher than that obtained by the parent strain C. glutamicum ΔAPE6937R42 with glucose as the sole carbon. This strategy can be applied for developing sucrose- or molasses-utilizing industrial strains.

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“…The Salis Lab at Penn State University and others have championed the groundwork for understanding the RBS sequence to RBS strength relationship [37][38][39]. Their development and continuous improvement of the RBS Calculator is a useful tool for all metabolic engineers and systems biologists attempting to optimize a pathway via translational balancing [40,41].…”

Section: Protein-level Optimizationmentioning

confidence: 99%

Metabolic pathway balancing and its role in the production of biofuels and chemicals

Jones

Toparlak

Koffas

2015

Current Opinion in Biotechnology

179

119

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In the last decade, metabolic engineering benefited greatly from systems and synthetic biology due to substantial advancements in those fields. As a result, technologies and methods evolved to be more complex and controllable than ever. In this review, we highlight up-to-date case studies using these techniques, examine their potential, and stress their importance for production of compounds such as fatty acids, alcohols, and high value chemicals. Beginning with basic rational control techniques and continuing with advanced level modern approaches, we review the vast number of possibilities for controlling metabolic fluxes. Our aim is to give a brief and informative insight about commonly used tools and universalized methodologies for metabolic pathway balancing and optimization.

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Predictive design of mRNA translation initiation region to control prokaryotic translation efficiency

Cited by 257 publications

References 30 publications

A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression

A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression

Production of L-ornithine from sucrose and molasses by recombinant Corynebacterium glutamicum

Metabolic pathway balancing and its role in the production of biofuels and chemicals

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