Strategies for enhancing gene expression in Escherichia coli

Kondo, Tomo; Yumura, Shigehiko

doi:10.1007/s00253-020-10430-4

Cited by 22 publications

(13 citation statements)

References 79 publications

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“…Kondo et al . six described six genetics elements of E. coli which contribute to an increased gene expression levels and result in higher yields of the desired protein, namely: promoter, translation enhancer, Shine‐Dalgarno sequence, spacer, encoding gene, and terminator of the gene [48] . Our results additionally demonstrated that the presence of a terminator region along with the “Best” enhancer fragment can result in further improvement of secretion through the Hly secretion system.…”

Section: Discussionmentioning

confidence: 53%

Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region

Cui

Khosa

et al. 2022

ChemBioChem

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Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering.Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2-fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5-fold.

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Section: Discussionmentioning

confidence: 53%

Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region

Cui

Khosa

et al. 2022

ChemBioChem

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“…15−17 Specifically, the 48 bp insertion disrupts the translation enhancer sequence located between the RBS and the promoter that is involved in ribosome interactions and mRNA stabilization (Figure S1A). 16,17 The source of the insertion was the pUC57-Kan vector (Genscript) in which the cap BGC had been cloned after synthesis. 7 The 48 bp insertion consists of inverted sequence repeats originating from the pUC57-Kan cloning vector (Figure S1B), which are not present in pSK020 from the overproducer.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Whole-plasmid sequencing of the pSK020 plasmid harboring the cap BGC revealed a 48 bp insertion between the ribosome binding site (RBS) and the −10 and −35 promoter regions in the low-producer pSK020 plasmid (hereafter termed “pSK020 + 48”), whereas pSK020 from the overproducer was as designed without the insertion. The 48 bp insertion increases the distance between the P BAD promoter and the RBS and alters the sequence context for transcription and translation initiation, which is known to affect gene expression and subsequent production yield. − Specifically, the 48 bp insertion disrupts the translation enhancer sequence located between the RBS and the promoter that is involved in ribosome interactions and mRNA stabilization (Figure S1A). , The source of the insertion was the pUC57-Kan vector (Genscript) in which the cap BGC had been cloned after synthesis .…”

Section: Resultsmentioning

confidence: 99%

High-Yield Lasso Peptide Production in a Burkholderia Bacterial Host by Plasmid Copy Number Engineering

Fernandez,

Kretsch,

Kunakom

et al. 2024

ACS Synth. Biol.

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The knotted configuration of lasso peptides confers thermal stability and proteolytic resistance, addressing two shortcomings of peptide-based drugs. However, low isolation yields hinder the discovery and development of lasso peptides. While testing Burkholderia sp. FERM BP-3421 as a bacterial host to produce the lasso peptide capistruin, an overproducer clone was previously identified. In this study, we show that an increase in the plasmid copy number partially contributed to the overproducer phenotype. Further, we modulated the plasmid copy number to recapitulate titers to an average of 160% relative to the overproducer, which is 1000-fold higher than previously reported with E. coli, reaching up to 240 mg/L. To probe the applicability of the developed tools for lasso peptide discovery, we targeted a new lasso peptide biosynthetic gene cluster from endosymbiont Mycetohabitans sp. B13, leading to the isolation of mycetolassin-15 and mycetolassin-18 in combined titers of 11 mg/L. These results validate Burkholderia sp. FERM BP-3421 as a production platform for lasso peptide discovery.

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“…As for the synthesis of bioactive nanomaterials, it is realized by edition and modification of genome of E. coli by exogenous editing plasmid, thus developing a system suitable for the efficient synthesis of bioactive nanomaterials in E. coli in large scale, which is considered as the most promising microbe factory for further industrial and commercial application. [ 147 ]…”

Section: Genetic Manipulation Technology For Synthesis Of Bc‐based Ma...mentioning

confidence: 99%

Microbe‐Mediated Biosynthesis of Multidimensional Carbon‐Based Materials for Energy Storage Applications

Shen

Chen

Zhou

et al. 2023

Advanced Energy Materials

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Biosynthesis methods are considered to be a promising technology for engineering new carbon‐based materials or redesigning the existing ones for specific purposes with the aid of synthetic biology. Lots of biosynthetic processes including metabolism, fermentation, biological mineralization, and gene editing have been adopted to prepare novel carbon‐based materials with exceptional properties that cannot be realized by traditional chemical methods, because microbes evolved to possess special abilities to modulate components/structure of materials. In this review, the recent development on carbon‐based materials prepared via different biosynthesis methods and various microbe factories (such as bacteria, yeasts, fungus, viruses, proteins) are systematically reviewed. The types of biotechniques and the corresponding mechanisms for the synthesis of carbon‐based materials are outlined. This review also focuses on the structural design and compositional engineering of carbon‐based nanostructures (e.g., metals, semiconductors, metal oxides, metal sulfides, phosphates, Mxenes) derived from biotechnology and their applications in electrochemical energy storage devices. Moreover, the relationship of the architecture–composition–electrochemical behavior and performance enhancement mechanism is also deeply discussed and analyzed. Finally, the development perspectives and challenges on the biosynthetic carbons are proposed and may pave a new avenue for rational design of advanced materials for the low‐carbon economy.

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Strategies for enhancing gene expression in Escherichia coli

Cited by 22 publications

References 79 publications

Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region

Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region

High-Yield Lasso Peptide Production in a Burkholderia Bacterial Host by Plasmid Copy Number Engineering

Microbe‐Mediated Biosynthesis of Multidimensional Carbon‐Based Materials for Energy Storage Applications

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