High copy number mutants derived from Corynebacterium glutamicum cryptic plasmid pAM330 and copy number control

Hashiro, Shuhei; Mitsuhashi, Mayu; Yasueda, Hisashi

doi:10.1016/j.jbiosc.2018.10.012

Cited by 15 publications

(18 citation statements)

References 26 publications

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“…Quantification of produced RNA was carried out by PAGE analysis using DynaMarker dsRNA (BioDynamics Lab., Tokyo, Japan) as a standard for the calculation (Hashiro et al 2019b). The copy number of plasmid and the ratio of segregates in culture broth after fermentation were determined as described previously (Hashiro and Yasueda 2018; Hashiro et al 2019a).…”

Section: Methodsmentioning

confidence: 99%

“…In previous study, we have succeeded in overproducing recombinant RNA molecules in Corynebacterium glutamicum having an efficient RNA expression system equipped with strong promoter F1, derived from corynephage BFK20 (Hashiro et al 2019a, b). Although the model RNA overexpressed was single-stranded (ss) with double-stranded structures in part, accumulation of the target RNA reached about 300 mg per liter of culture.…”

Section: Introductionmentioning

confidence: 99%

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Construction of Corynebacterium glutamicum cells as containers encapsulating dsRNA overexpressed for agricultural pest control

Hashiro

Mitsuhashi

Chikami

et al. 2019

Appl Microbiol Biotechnol

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Double-stranded RNA (dsRNA) inducing RNA interference (RNAi) is expected to be applicable to management of agricultural pests. In this study, we selected a ladybird beetle, Henosepilachna vigintioctopunctata, as a model target pest that devours vegetable leaves, and examined the effects of feeding the pest sterilized microbes highly accumulating a target dsRNA for RNAi induction. We constructed an efficient production system for diap1*-dsRNA, which suppresses expression of the essential gene diap1 (encoding death-associated inhibitor of apoptosis protein 1) in H. vigintioctopunctata, using an industrial strain of Corynebacterium glutamicum as the host microbe. The diap1*-dsRNA was overproduced in C. glutamicum by convergent transcription using a strong promoter derived from corynephage BFK20, and the amount of dsRNA accumulated in fermented cells reached about 75 mg per liter of culture. In addition, we developed a convenient method for stabilizing the dsRNA within the microbes by simply sterilizing the diap1*-dsRNA-expressing C. glutamicum cells with ethanol. When the sterilized microbes containing diap1*-dsRNA were fed to larvae of H. vigintioctopunctata, diap1 expression in the pest was suppressed, and the leaf-feeding activity of the larvae declined. These results suggest that this system is capable of producing stabilized dsRNA for RNAi at low cost, and it will open a way to practical application of dsRNA as an environmentally-friendly agricultural insecticide.Electronic supplementary materialThe online version of this article (10.1007/s00253-019-10113-9) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Construction of Corynebacterium glutamicum cells as containers encapsulating dsRNA overexpressed for agricultural pest control

Hashiro

Mitsuhashi

Chikami

et al. 2019

Appl Microbiol Biotechnol

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“…In the case of aroB, a single integration of the aroB gene increased the shikimic acid titer over that of the plasmid-based overexpression system (SA-2/aroB; Figure 2B). Although we used medium copy number plasmid (about 30; Tauch, 2005), these results suggest that the production of large amounts of shikimic acid seems to depend on the plasmid copy number (Hashiro et al, 2019).…”

Section: Construction Of C Glutamicum Strains With Integrated Shikimmentioning

confidence: 82%

Metabolic Engineering of Shikimic Acid-Producing Corynebacterium glutamicum From Glucose and Cellobiose Retaining Its Phosphotransferase System Function and Pyruvate Kinase Activities

Sato

Kishida

Nakano

et al. 2020

Front. Bioeng. Biotechnol.

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The production of aromatic compounds by microbial production is a promising and sustainable approach for producing biomolecules for various applications. We describe the metabolic engineering of Corynebacterium glutamicum to increase its production of shikimic acid. Shikimic acid and its precursor-consuming pathways were blocked by the deletion of the shikimate kinase, 3-dehydroshikimate dehydratase, shikimate dehydratase, and dihydroxyacetone phosphate phosphatase genes. Plasmid-based expression of shikimate pathway genes revealed that 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, encoded by aroG, and DHQ synthase, encoded by aroB, are key enzymes for shikimic acid production in C. glutamicum. We constructed a C. glutamicum strain with aroG, aroB and aroE3 integrated. This strain produced 13.1 g/L of shikimic acid from 50 g/L of glucose, a yield of 0.26 g-shikimic acid/gglucose, and retained both its phosphotransferase system and its pyruvate kinase activity. We also endowed β-glucosidase secreting ability to this strain. When cellobiose was used as a carbon source, the strain produced shikimic acid at 13.8 g/L with the yield of 0.25 g-shikimic acid/g-glucose (1 g of cellobiose corresponds to 1.1 g of glucose). These results demonstrate the feasibility of producing shikimic acid and its derivatives using an engineered C. glutamicum strain from cellobiose as well as glucose.

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“…A strain of Corynebacterium glutamicum used in industrial production of amino acids was shown to carry a R1-like vector, named pVC7, engineered to be used as a shuttle vector in E. coli. A recent study aimed to select ideal vectors with high copy number in order to produce more of the desired molecules, it was shown that a naturally acquired, single point mutation in pCV7, increased significantly the copy number [28]. This mutation is located in the region of complementarity between the sense and the antisense RNAs changing the secondary structure of the 'kissing complex' leading to a weaker interaction.…”

Section: Replicationmentioning

confidence: 99%

Physiological roles of antisense RNAs in prokaryotes

2019

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Prokaryotes encounter constant and often brutal modifications to their environment. In order to survive, they need to maintain fitness, which includes adapting their protein expression patterns. Many factors control gene expression but this review focuses on just one, namely antisense RNAs (asRNAs), a class of non-coding RNAs (ncRNAs) characterized by their location in cis and their perfect complementarity with their targets. asRNAs were considered for a long time to be trivial and only to be found on mobile genetic elements. However, recent advances in methodology have revealed that their abundance and potential activities have been underestimated. This review aims to illustrate the role of asRNA in various physiologically crucial functions in both archaea and bacteria, which can be regrouped in three categories: cell maintenance, horizontal gene transfer and virulence. A literature survey of asRNAs demonstrates the difficulties to characterize and assign a role to asRNAs. With the aim of facilitating this task, we describe recent technological advances that could be of interest to identify new asRNAs and to discover their function.

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High copy number mutants derived from Corynebacterium glutamicum cryptic plasmid pAM330 and copy number control

Cited by 15 publications

References 26 publications

Construction of Corynebacterium glutamicum cells as containers encapsulating dsRNA overexpressed for agricultural pest control

Construction of Corynebacterium glutamicum cells as containers encapsulating dsRNA overexpressed for agricultural pest control

Metabolic Engineering of Shikimic Acid-Producing Corynebacterium glutamicum From Glucose and Cellobiose Retaining Its Phosphotransferase System Function and Pyruvate Kinase Activities

Physiological roles of antisense RNAs in prokaryotes

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