Promoters and Plasmid Vectors of Corynebacterium glutamicum

Pátek, Miroslav; Nešvera, Jan

doi:10.1007/978-3-642-29857-8_2

Cited by 28 publications

(19 citation statements)

References 150 publications

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“…Analysis of the P vanABK sequence indicated the TTGACA sequence as a perfect Ϫ35 box, whereas the Ϫ10 box (CAATAT) was less conserved. Although the CAATAT sequence was proposed previously as the Ϫ10 box of P vanABK (44), TATAT or AATATA (a and b lines; Fig. 2A) also were predicted as the probable Ϫ10 boxes of P vanABK (12,13).…”

Section: Resultsmentioning

confidence: 82%

“…During the preliminary studies, the strength of P vanABK was optimized by altering the promoter core elements and removing the GlxR binding site. Improvement of the P vanABK Ϫ10 box tremendously enhanced the P vanABK activity, indicating that the recently published P vanABK core elements were correctly predicted (44). Since C. glutamicum does not show diauxic growth on glucose together with protocatechuate or vanillate, it is presumed that the vanillate metabolism does not underlie a glucose-mediated carbon catabolite repression (13).…”

Section: Discussionmentioning

confidence: 83%

“…2A). In addition to Ϫ10 modifications, mutation of the conserved Ϫ35 box drastically decreased the expression level of eGFP, as shown by C. glutamicum pJOE8054.9, although it is described that C. glutamicum promoters do not share a highly conserved Ϫ35 region in general (44). In addition to promoter core elements, a GlxR binding sequence has been found inside the spacer sequence of P vanABK ( Fig.…”

Section: Resultsmentioning

confidence: 92%

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Transcriptional Regulation of the Vanillate Utilization Genes ( vanABK Operon) of Corynebacterium glutamicum by VanR, a PadR-Like Repressor

et al. 2015

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P lant biomass is the main carbon supply in the soil. The plant cell wall is a lignocellulosic complex consisting of hydrophilic polymers, i.e., cellulose, hemicellulose, and pectin, along with the hydrophobic aromatic heteropolymer lignin (1, 2). The integrity of the plant cell wall depends on its lignin content, which is crosslinked to polysaccharides by hydroxycinnamic acids bridges, mainly ferulate (3-5). These feruloylated polysaccharides, especially the ferulate-arabinoxylan complex, serve as initiation sites for lignification in the cell walls (6, 7). Lignin is degraded by lignolytic enzymes, including lignin peroxidase, manganese peroxidase, or laccase, into ␤-aryl ether, di-aryl ether, and biphenyl, which are further catabolized to other aromatic compounds, such as vanillin and vanillate. Likewise, ferulate bound through ester linkages to hemicellulose is released by esterases and degrades to vanillin and vanillate (for a review, see references 4, 5, 8, 9, and 10).Phenolic acids released by degradation of lignin, e.g., ferulate or p-coumarate, are toxic for many Gram-positive bacteria, such as Bacillus subtilis, at low pH. Thus, there is a system for phenolic acid stress response which detoxifies phenolic acid by decarboxylation and generation of vinyl phenol derivatives (11). In contrast to B. subtilis, Corynebacterium glutamicum utilizes ferulate, vanillin, and vanillate derived from lignin degradation as a carbon source. Generally, aromatic compounds are channeled via gentisate, catechol, protocatechuate, 1,2,4-trihydroxybenzene, or phenylacetyl coenzyme A (phenylacetyl-CoA) intermediates into the central carbon metabolism of C. glutamicum (12). Ferulate is catabolized via vanillin and vanillate as the intermediate products to protocatechuate (3,4-dihydroxybenzoate) (see Fig. S1 in the supplemental material) (13). Protocatechuate is further metabolized in the aerobic ␤-ketoadipate pathway and finally flows into the carbon and energy cycle (14). So far, only the genes for degradation of vanillate are identified in C. glutamicum. Conversion of vanillate to protocatechuate is carried out by vanillate O-demethylase (see Fig. S1) (13). The vanillate O-demethylase enzyme has two subunits, which are encoded by vanA (NCgl2300) and vanB (NCgl2301) (13,15). In addition to vanillate O-demethylase, the vanillate utilization system consists of a vanillate transporter encoded by vanK (NCgl2302), which forms the vanABK operon along with vanA and vanB (16). Transcription of the vanABK operon is regulated by VanR (NCgl2299), which forms a divergon with the vanABK operon (12).VanR belongs to the PadR-like transcriptional regulator family (17). The PadR-like protein family (Pfam accession no. PF03551) contains 26 reported structures deposited in the Protein Data Bank (http://www.rcsb.org/). Generally, PadR-like regulators play an important role in their bacterial host, especially concerning virulence and stress. Structurally, PadR-type regulators contain a highly conserved N-terminal winged helix-turn-helix (wHTH)

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Section: Resultsmentioning

confidence: 82%

Section: Discussionmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 92%

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Transcriptional Regulation of the Vanillate Utilization Genes ( vanABK Operon) of Corynebacterium glutamicum by VanR, a PadR-Like Repressor

et al. 2015

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“…◀ pEKEx2 (Eikmanns et al, 1991) contains the replicon from plasmid pBL1 (Santamaria et al, 1984), whereas pJC1 (Cremer et al, 1990) contains the replicon from plasmid pHM1519 (Miwa et al, 1984), which presumably is identical with the one from plasmids pCG1 (Ozaki et al, 1984), pSR1 (Yoshihama et al, 1985) and pCG100 (Trautwetter and Blanco, 1991), as described previously (Nešvera and Pátek, 2008). Both replicons mediate replication in the rolling circle mode, but the pBL1 replicons belong to pIJ101/pJV1 family, whereas the pHM1519 replicon belongs to pNG2 family (Pátek and Nešvera, 2013). The copy number of pBL1 and similar plasmids was estimated to be between 10 and 30 copies per chromosome (Miwa et al, 1984;Santamaria et al, 1984), and that of pCG100 was also reported to be about 30 copies per chromosome (Trautwetter and Blanco, 1991).…”

Section: Characterization Of T7 Rnap-dependent Expression System In Cmentioning

confidence: 99%

A chromosomally encoded T7 RNA polymerase‐dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single‐cell level

Kortmann

Kuhl

Klaffl

et al. 2014

Microbial Biotechnology

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Corynebacterium glutamicum has become a favourite model organism in white biotechnology. Nevertheless, only few systems for the regulatable (over)expression of homologous and heterologous genes are currently available, all of which are based on the endogenous RNA polymerase. In this study, we developed an isopropyl-β-d-1-thiogalactopyranosid (IPTG)-inducible T7 expression system in the prophage-free strain C. glutamicum MB001. For this purpose, part of the DE3 region of Escherichia coli BL21(DE3) including the T7 RNA polymerase gene 1 under control of the lacUV5 promoter was integrated into the chromosome, resulting in strain MB001(DE3). Furthermore, the expression vector pMKEx2 was constructed allowing cloning of target genes under the control of the T7lac promoter. The properties of the system were evaluated using eyfp as heterologous target gene. Without induction, the system was tightly repressed, resulting in a very low specific eYFP fluorescence (= fluorescence per cell density). After maximal induction with IPTG, the specific fluorescence increased 450-fold compared with the uninduced state and was about 3.5 times higher than in control strains expressing eyfp under control of the IPTG-induced tac promoter with the endogenous RNA polymerase. Flow cytometry revealed that T7-based eyfp expression resulted in a highly uniform population, with 99% of all cells showing high fluorescence. Besides eyfp, the functionality of the corynebacterial T7 expression system was also successfully demonstrated by overexpression of the C. glutamicum pyk gene for pyruvate kinase, which led to an increase of the specific activity from 2.6 to 135 U mg−1. It thus presents an efficient new tool for protein overproduction, metabolic engineering and synthetic biology approaches with C. glutamicum.

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“…Analysis of C. glutamicum promoter sequences revealed high similarity between -10 motifs of promoters of housekeeping genes (supposed to be recognized by r A ) and promoters recognized by r B [4,10,16,17] which suggests possible overlapping promoter specificity of r A and r B . The data on core elements (-35 and -10 motifs) of C. glutamicum promoters recognized by ECF sigma factors are still very limited [16,17]. Only the high similarity between core elements (-35 and -10) of C. glutamicum promoters recognized by r H [2,5] or r M [12] was found so far.…”

Section: Introductionmentioning

confidence: 96%

Use of In Vitro Transcription System for Analysis of Corynebacterium glutamicum Promoters Recognized by Two Sigma Factors

et al. 2016

Self Cite

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Promoter activities in Corynebacterium glutamicum strains with deletions of genes encoding sigma factors of RNA polymerase suggested that transcription from some promoters is controlled by two sigma factors. To prove that different sigma factors are involved in the recognition of selected Corynebacterium glutamicum promoters, in vitro transcription system was applied. It was found that a typical housekeeping promoter Pper interacts with the alternative sigma factor σ(B) in addition to the primary sigma factor σ(A). On the other way round, the σ(B)-dependent promoter of the pqo gene that is expressed mainly in the stationary growth phase was active also with σ(A). Some promoters of genes involved in stress responses (P1clgR, P2dnaK, and P2dnaJ2) were found to be recognized by two stress-responding sigma factors, σ(H) and σ(E). In vitro transcription system thus proved to be a useful direct technique for demonstrating the overlap of different sigma factors in recognition of individual promoters in C. glutamicum.

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Promoters and Plasmid Vectors of Corynebacterium glutamicum

Cited by 28 publications

References 150 publications

Transcriptional Regulation of the Vanillate Utilization Genes ( vanABK Operon) of Corynebacterium glutamicum by VanR, a PadR-Like Repressor

Transcriptional Regulation of the Vanillate Utilization Genes ( vanABK Operon) of Corynebacterium glutamicum by VanR, a PadR-Like Repressor

A chromosomally encoded T7 RNA polymerase‐dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single‐cell level

Use of In Vitro Transcription System for Analysis of Corynebacterium glutamicum Promoters Recognized by Two Sigma Factors

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