Cytosine Deaminase as a Negative Selection Marker for Gene Disruption and Replacement in the Genus
            <i>Streptomyces</i>
            and Other Actinobacteria

Dubeau, Marie-Pierre; Ghinet, Mariana Gabriela; Jacques, Pierre-Étienne; Clermont, Nancy; Beaulieu, Carole; Brzeziński, Ryszard

doi:10.1128/aem.02139-08

Cited by 45 publications

(41 citation statements)

References 24 publications

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“…typhimurium (Manuel et al 2011), S. lividans (Dubeau et al 2009), wheat (Mihálik et al 2015), transplastomic tobacco plants (chloroplast translation system) (Occhialini et al 2015), P. berghei (Singer et al 2015), S. frugiperda (Geisler et al 2015), S. pneumoniae (Overkamp et al 2013), A.…”

Section: Acc E P Ted P R E P R I Ntmentioning

confidence: 99%

Synthetic gene design—The rationale for codon optimization and implications for molecular pharming in plants

Webster

Teh

K.‐C.

2016

Biotech & Bioengineering

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Degeneracy in the genetic code allows multiple codon sequences to encode the same protein. Codon usage bias in genes is the term given to the preferred use of particular synonymous codons. Synonymous codon substitutions had been regarded as "silent" as the primary structure of the protein was not affected; however, it is now accepted that synonymous substitutions can have a significant effect on heterologous protein expression. Codon optimization, the process of altering codons within the gene sequence to improve recombinant protein expression, has become widely practised.Multiple inter-linked factors affecting protein expression need to be taken into consideration when optimizing a gene sequence. Over the years, various computer programmes have been developed to aid in the gene sequence optimization process. However, as the rulebook for altering codon usage to affect protein expression is still not completely understood, it is difficult to predict which strategy, if any, will design the 'optimal' gene sequence.In this review, codon usage bias and factors affecting codon selection will be discussed and the evidence for codon optimization impact will be reviewed for recombinant protein expression using plants as a case study. These developments will be relevant to all recombinant expression systems, however, molecular pharming in plants is an area which has consistently encountered difficulties with low levels of recombinant protein expression, and should benefit from an evidence based rational approach to synthetic gene design. This article is protected by copyright. All rights reserved

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Section: Acc E P Ted P R E P R I Ntmentioning

confidence: 99%

Synthetic gene design—The rationale for codon optimization and implications for molecular pharming in plants

Webster

Teh

K.‐C.

2016

Biotech & Bioengineering

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“…The blaL reporter gene originated from plasmid pDML619 (21). The blaL gene was subcloned into the SphI and HindIII restriction sites of pHM8aB⌬M, a modified version of the integrative vector pHM8a (14,35), generating pHM-blaL. The intergenic region (IR) upstream of csnA (IR-csnA) was amplified by PCR with the BamHI-IR-csnA and SphI-IR-csnA primers (see Table S1 in the supplemental material) from S. lividans TK24 genomic DNA.…”

Section: Methodsmentioning

confidence: 99%

“…Standard methods were used for E. coli transformation, plasmid isolation, and DNA manipulation (44). S. lividans TK24 (27) and the isogenic S. lividans ⌬csnR strain (formerly the ⌬2657h strain) (14) were used as recipients for transformation or conjugation. They were also used in quantitative PCR (qPCR) assays, while Streptomyces avermitilis MA-4680 was used in reverse transcription (RT)-PCR experiments.…”

Section: Methodsmentioning

confidence: 99%

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Properties of CsnR, the Transcriptional Repressor of the Chitosanase Gene, csnA , of Streptomyces lividans

et al. 2011

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A palindromic sequence is present in the intergenic region preceding the chitosanase gene csnA (SSPG_06922) of Streptomyces lividans TK24. This sequence was also found in front of putative chitosanase genes in several other actinomycete genomes and upstream genes encoding putative transcriptional regulators of the ROK family, including csnR (SSPG_04872) in S. lividans. The latter was examined as a possible transcriptional regulator (CsnR) of chitosanase gene expression. In vitro, purified CsnR bound strongly to the palindromic sequences of the csnA and csnR genes (equilibrium dissociation constant [K D ] ‫؍‬ 0.032 and 0.040 nM, respectively). Binding was impaired in the presence of chitosan oligosaccharides and D-glucosamine, and chitosan dimer was found to be the best effector, as determined by an equilibrium competition experiment and 50% inhibitory concentration (IC 50 ) determination, while glucose, N-acetyl-glucosamine, and galactosamine had no effect. In vivo, comparison of the S. lividans wild type and ⌬CsnR strains using ␤-lactamase reporter genes showed that CsnR represses the expression of csnA and of its own gene, which was confirmed by quantitative PCR (qPCR). CsnR is localized at the beginning of a gene cluster, possibly an operon, the organization of which is conserved through many actinomycete genomes. The CsnR-mediated chitosanase regulation mechanism seems to be widespread among actinomycetes.

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“…Transformation of the uracil analog 5-FU to 5-fluorouridine monophosphate (5-FUMP) by Upp and further conversion of 5-FUMP results in irreversible inhibition of thymidylate synthetase (19,20). An alternative counterselectable marker is cytosine deaminase (codA) (EC 3.5.4.1), which has been applied for various bacteria (21)(22)(23). CodA (EC 3.5.4.1) catalyzes the deamination of cytosine and its analog, 5-fluorocytosine (5-FC), to uracil and 5-FU, respectively, which are subsequently converted to UMP and 5-FUMP by Upp (24).…”

mentioning

confidence: 99%

Markerless Gene Deletion with Cytosine Deaminase in Thermus thermophilus Strain HB27

Wang

Hoffmann

Watzlawick

et al. 2016

Appl Environ Microbiol

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We developed a counterselectable deletion system for Thermus thermophilus HB27 based on cytosine deaminase (encoded by codA) from Thermaerobacter marianensis DSM 12885 and the sensitivity of T. thermophilus HB27 to the antimetabolite 5-fluorocytosine (5-FC). The deletion vector comprises the pUC18 origin of replication, a thermostable kanamycin resistance marker functional in T. thermophilus HB27, and codA under the control of a constitutive putative trehalose promoter from T. thermophilus HB27. The functionality of the system was demonstrated by deletion of the bglT gene, encoding a ␤-glycosidase, and three carotenoid biosynthesis genes, CYP175A1, crtY, and crtI, from the genome of T. thermophilus HB27.

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Cytosine Deaminase as a Negative Selection Marker for Gene Disruption and Replacement in the Genus Streptomyces and Other Actinobacteria

Cited by 45 publications

References 24 publications

Synthetic gene design—The rationale for codon optimization and implications for molecular pharming in plants

Synthetic gene design—The rationale for codon optimization and implications for molecular pharming in plants

Properties of CsnR, the Transcriptional Repressor of the Chitosanase Gene, csnA , of Streptomyces lividans

Markerless Gene Deletion with Cytosine Deaminase in Thermus thermophilus Strain HB27

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