Ribosome-binding site interference caused by Shine-Dalgarno-like nucleotide sequences in Escherichia coli cells

Nishizawa, Akito; Nakayama, Miki; Uemura, Takuya; Fukuda, Y.; Kimura, Shigenobu

doi:10.1093/jb/mvp187

Cited by 6 publications

(5 citation statements)

References 23 publications

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“…when the activity of DhaT is lower than that of DhaB1. Such a situation often occurs in polycistronic expression systems where the mRNA lacks internal ribosome entry sites for the next cistrons (Nishizawa et al, 2010).That was not the case in our expression system, as we cloned the whole dha operon with all the sequences between individual genes. However, in a heterologous expression system the expression of individual genes may differ from that in the natural host (C. butyricum).…”

Section: Days After Induction Withmentioning

confidence: 96%

1,3-propanediol production by Escherichia coli expressing genes of dha operon from Clostridium butyricum 2CR371.5.

et al. 2012

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1,3-propanediol is used as a monomer in the production of some polymers e.g. polytrimethylene terephthalate used in the production of carpets and textile fibers and in the thermoplastics engineering. However, the traditional chemical synthesis is expensive, generates some toxic intermediates and requires a reduction step under high hydrogen pressure. Biological production of 1,3-propanediol could be an attractive alternative to the traditional chemical methods. Moreover, crude glycerol which is a by-product of biodiesel production, can be used. We constructed a recombinant Escherichia coli strain producing 1,3-propanediol from glycerol by introducing genes of the dha operon from Clostridium butyricum 2CR371.5, a strain from our collection of environmental samples and strains. The E. coli strain produced 3.7 g of 1,3-propanediol per one litre of culture with the yield of 0.3 g per 1 g of glycerol consumed.

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Section: Days After Induction Withmentioning

confidence: 96%

1,3-propanediol production by Escherichia coli expressing genes of dha operon from Clostridium butyricum 2CR371.5.

et al. 2012

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“…The prokaryotic RBS contains a purine-rich sequence named Shine-Dalgarno (SD) sequence (e.g. 5′-AGGAGGU-3′ for E. coli ), which is generally located 6–8 bases upstream of the AUG start codon [ 20 ]. The SD sequence can mediate ribosomal recruitment by forming strong base-pairing interactions with the 16S ribosomal RNA (rRNA) in the small (30S) ribosomal subunit.…”

Section: Introductionmentioning

confidence: 99%

“…The SD sequence can mediate ribosomal recruitment by forming strong base-pairing interactions with the 16S ribosomal RNA (rRNA) in the small (30S) ribosomal subunit. Thus, the RBS is a critical determinant of the translation initiation rate [ 7 , 20 ]. Since protein expression levels can be tuned by introducing mutations in the RBS sequence, RBS engineering has been widely applied for pathway optimization in prokaryotic hosts [ 21 – 23 ].…”

Section: Introductionmentioning

confidence: 99%

Fine-tuning the expression of pathway gene in yeast using a regulatory library formed by fusing a synthetic minimal promoter with different Kozak variants

Feng

et al. 2021

Microb Cell Fact

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Background Tailoring gene expression to balance metabolic fluxes is critical for the overproduction of metabolites in yeast hosts, and its implementation requires coordinated regulation at both transcriptional and translational levels. Although synthetic minimal yeast promoters have shown many advantages compared to natural promoters, their transcriptional strength is still limited, which restricts their applications in pathway engineering. Results In this work, we sought to expand the application scope of synthetic minimal yeast promoters by enhancing the corresponding translation levels using specific Kozak sequence variants. Firstly, we chose the reported UASF-E-C-Core1 minimal promoter as a library template and determined its Kozak motif (K0). Next, we randomly mutated the K0 to generate a chimeric promoter library, which was able to drive green fluorescent protein (GFP) expression with translational strengths spanning a 500-fold range. A total of 14 chimeric promoters showed at least two-fold differences in GFP expression strength compared to the K0 control. The best one named K528 even showed 8.5- and 3.3-fold increases in fluorescence intensity compared with UASF-E-C-Core1 and the strong native constitutive promoter PTDH3, respectively. Subsequently, we chose three representative strong chimeric promoters (K540, K536, and K528) from this library to regulate pathway gene expression. In conjunction with the tHMG1 gene for squalene production, the K528 variant produced the best squalene titer of 32.1 mg/L in shake flasks, which represents a more than 10-fold increase compared to the parental K0 control (3.1 mg/L). Conclusions All these results demonstrate that this chimeric promoter library developed in this study is an effective tool for pathway engineering in yeast.

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“…The translation coupling mechanism was first found in the Escherichia coli galactose operon. A hard-to-translate fore-cistron with embedded Shine-Dalgarno (SD)-like sequences can reduce the accumulation level of the second cistron, which promotes protein folding and increases production of the protein in its soluble form [4]. The introduction of the fore-cistron into the ribosome prevents the formation of a stable mRNA structure, Correspondence: Prof. Zhonghu Bai (baizhonghu@jiangnan.edu.…”

Section: Introductionmentioning

confidence: 99%

“…cn), National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, P. R. China Abbreviations: 5 -UTR, 5 untranslated region; BEP, bicistronic expression part; MEP, monocistronic expression part; SD sequence, Shine-Dalgarno sequence; scFv, single-chain variable fragment facilitating the translation initiation of the second cistron [3]. A hard-to-translate fore-cistron with embedded Shine-Dalgarno (SD)-like sequences can reduce the accumulation level of the second cistron, which promotes protein folding and increases production of the protein in its soluble form [4]. Bicistronic architecture can also include various functional peptides such as a first-cistronic peptide, which stabilizes the second-cistronic protein [5].…”

Section: Introductionmentioning

confidence: 99%

Bicistronic expression strategy for high‐level expression of recombinant proteins in Corynebacterium glutamicum

Liu

Zhao

Zhang

et al. 2017

Engineering in Life Sciences

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Directly using the promoter associated with 5′‐untranslated region of a high‐protein‐abundance gene from the genome may cause low expression activity of an expression system. A bicistronic expression part containing the short 5′ coding sequence of the source gene and an embedded Shine–Dalgarno sequence can cause higher expression levels of the recombinant gene in a bicistronic cassette. Here, we evaluated two methods to construct expression parts and exploited genomic sequence sources to provide specific functional sequences to complete the expression system. The architecture of the bicistronic part increased the expression levels of target genes and performed more reliably than conventional expression parts with the same promoter and 5′ untranslated region. For Corynebacterium glutamicum, the strongest bicistronic part, HP‐BEP4, was obtained from a heterologous sequence source, leading to a 2.24‐fold increase in the expression level of fluorescent protein over constitutively expressed pXMJ19 or the production of more than 100 mg/L single‐chain variable fragment (scFv). It could meet the needs of overexpressing key genes in C. glutamicum.

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Ribosome-binding site interference caused by Shine-Dalgarno-like nucleotide sequences in Escherichia coli cells

Cited by 6 publications

References 23 publications

1,3-propanediol production by Escherichia coli expressing genes of dha operon from Clostridium butyricum 2CR371.5.

1,3-propanediol production by Escherichia coli expressing genes of dha operon from Clostridium butyricum 2CR371.5.

Fine-tuning the expression of pathway gene in yeast using a regulatory library formed by fusing a synthetic minimal promoter with different Kozak variants

Bicistronic expression strategy for high‐level expression of recombinant proteins in Corynebacterium glutamicum

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