Cloning and Expression of Key Enzyme Gene GalAT in Ramie Pectin Biosynthesis

Chorismate mutase (CM) catalyses the rearrangement of chorismate to prephenate and is also the first and the key enzyme that diverges the shikimate pathway to either tryptophan (Trp) or phenylalanine (Phe) and tyrosine (Tyr). Corynebacterium glutamicum is one of the most important amino acid producers for the fermentation industry and has been widely investigated. However, the gene(s) encoding CM has not been experimentally identified in C. glutamicum. In this study, the ncgl0819 gene, which was annotated as ‘conserved hypothetical protein’ in the C. glutamicum genome, was genetically characterized to be essential for growth in minimal medium, and a mutant deleted of ncgl0819 was a Phe and Tyr auxotroph. Genetic cloning and expression of ncgl0819 in Escherichia coli resulted in the formation of a new protein (NCgl0819) having CM activity. It was concluded that ncgl0819 encoded the CM of C. glutamicum (CM0819). CM0819 was demonstrated to be a homodimer and is a new member of the monofunctional CMs of the AroQ structural class. The CM0819 activity was not affected by Phe, Tyr or Trp. Two 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthases (DS0950 and DS2098, formerly NCgl0950 and NCgl2098) had been previously identified from C. glutamicum. CM0819 significantly stimulated DAHP synthase (DS2098) activity. Physical interaction between CM0819 and DS2098 was observed. When CM0819 was present, DS2098 activity was subject to allosteric inhibition by chorismate and prephenate. Conserved hypothetical proteins homologous to CM0819 were identified in all known Corynebacterium genomes, suggesting a universal occurrence of CM0819-like CMs in the genus Corynebacterium.

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A novel thermostable sulfite oxidase from Thermus thermophilus: characterization of the enzyme, gene cloning and expression in Escherichia coli

Extremophiles ◽

10.1007/s00792-006-0534-z ◽

2006 ◽

Vol 10 (6) ◽

pp. 587-598 ◽

Cited By ~ 15

Author(s):

Anna Di Salle ◽

Giovanni D’Errico ◽

Francesco La Cara ◽

Raffaele Cannio ◽

Mosè Rossi

Keyword(s):

Escherichia Coli ◽

Gene Cloning ◽

Thermus Thermophilus ◽

Sulfite Oxidase ◽

Cloning And Expression ◽

Gene Cloning And Expression ◽

Enzyme Gene ◽

Expression In Escherichia Coli

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Cloning and Expression of Staphylococcus Simulans Lysostaphin Enzyme Gene in Bacillus Subtilis WB600

10.21203/rs.3.rs-122682/v1 ◽

2020 ◽

Author(s):

Babak Elyasi Far ◽

Mehran Ragheb ◽

Reza Rahbar ◽

Ladan Mafakher ◽

Neda Yousefi Nojookambari ◽

...

Keyword(s):

Bacillus Subtilis ◽

Enzyme Stability ◽

Expression System ◽

Residual Activity ◽

Maximum Activity ◽

Cloning And Expression ◽

Gene Fragment ◽

Alkaline Lysis ◽

Enzyme Gene ◽

Staphylococcus Simulans

Abstract Background: Lysostaphin is a glycylglycine endopeptidase, secreted by Staphylococcus simulans, capable of specifically hydrolyzing pentaglycine crosslinks present in the peptidoglycan of the Staphylococcus aureus cell wall. In this paper, we describe the cloning and expression of the lysostaphin enzyme gene in Bacillus subtilis WB600 host using pWB980 expression system.Results: Plasmid pACK1 of S. simulans was extracted using alkaline lysis method. Lysostaphin gene was isolated by PCR and cloned into pTZ57R/T-Vector, then transformed into Escherichia coli DH5α. The amplified gene fragment and uncloned pWB980 vector were digested using PstI and XbaІ enzymes and purified. The restricted gene fragment was ligated into the pWB980 expression vector by the standard protocols, then the recombinant plasmid was transformed into B. subtilis WB600 using electroporation method. The recombinant protein was evaluated by the SDS-PAGE method and confirmed by western immunoblot. Analysis of the target protein showed a band corresponding to an about 27-kDa r-lysostaphin. Protein content was estimated 91µg/ml by Bradford assay.Conclusions: The recombinant lysostaphin represented 90% of its maximum activity at 40℃ and displayed good thermostability by keeping about 80% of its maximum activity at 45℃. Heat residual activity assay of recombinant lysostaphin demonstrated that the enzyme stability was up to 40℃ and showed good stability at 40℃ for 16 h incubation.

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Cloning and expression of the branching enzyme gene (glgB) from the cyanobacterium Synechococcus sp. PCC7942 in Escherichia coli

Gene ◽

10.1016/0378-1119(89)90309-0 ◽

1989 ◽

Vol 78 (1) ◽

pp. 9-17 ◽

Cited By ~ 18

Author(s):

J.A.K.W. Kiel ◽

H.S.A. Elgersma ◽

G. Beldman ◽

J.P.M.J. Vossen ◽

G. Venema

Keyword(s):

Escherichia Coli ◽

Cloning And Expression ◽

Branching Enzyme ◽

Enzyme Gene ◽

Synechococcus Sp

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Bifunctional CTP:Inositol-1-Phosphate Cytidylyltransferase/CDP-Inositol:Inositol-1-Phosphate Transferase, the Key Enzyme for Di-myo-Inositol-Phosphate Synthesis in Several (Hyper)thermophiles

Journal of Bacteriology ◽

10.1128/jb.00465-07 ◽

2007 ◽

Vol 189 (15) ◽

pp. 5405-5412 ◽

Cited By ~ 31

Author(s):

Marta V. Rodrigues ◽

Nuno Borges ◽

Mafalda Henriques ◽

Pedro Lamosa ◽

Rita Ventura ◽

...

Keyword(s):

Pyrococcus Furiosus ◽

Inositol Phosphate ◽

Thermotoga Maritima ◽

Single Gene ◽

Cloning And Expression ◽

Aquifex Aeolicus ◽

Inositol 1 ◽

Cell Extracts ◽

Genomic Approach ◽

Key Enzyme

ABSTRACT The pathway for the synthesis of di-myo-inositol-phosphate (DIP) was recently elucidated on the basis of the detection of the relevant activities in cell extracts of Archaeoglobus fulgidus and structural characterization of products by nuclear magnetic resonance (NMR) (N. Borges, L. G. Gonçalves, M. V. Rodrigues, F. Siopa, R. Ventura, C. Maycock, P. Lamosa, and H. Santos, J. Bacteriol. 188:8128-8135, 2006). Here, a genomic approach was used to identify the genes involved in the synthesis of DIP. Cloning and expression in Escherichia coli of the putative genes for CTP:l-myo-inositol-1-phosphate cytidylyltransferase and DIPP (di-myo-inositol-1,3′-phosphate-1′-phosphate, a phosphorylated form of DIP) synthase from several (hyper)thermophiles (A. fulgidus, Pyrococcus furiosus, Thermococcus kodakaraensis, Aquifex aeolicus, and Rubrobacter xylanophilus) confirmed the presence of those activities in the gene products. The DIPP synthase activity was part of a bifunctional enzyme that catalyzed the condensation of CTP and l-myo-inositol-1-phosphate into CDP-l-myo-inositol, as well as the synthesis of DIPP from CDP-l-myo-inositol and l-myo-inositol-1-phosphate. The cytidylyltransferase was absolutely specific for CTP and l-myo-inositol-1-P; the DIPP synthase domain used only l-myo-inositol-1-phosphate as an alcohol acceptor, but CDP-glycerol, as well as CDP-l-myo-inositol and CDP-d-myo-inositol, were recognized as alcohol donors. Genome analysis showed homologous genes in all organisms known to accumulate DIP and for which genome sequences were available. In most cases, the two activities (l-myo-inositol-1-P cytidylyltransferase and DIPP synthase) were fused in a single gene product, but separate genes were predicted in Aeropyrum pernix, Thermotoga maritima, and Hyperthermus butylicus. Additionally, using l-myo-inositol-1-phosphate labeled on C-1 with carbon 13, the stereochemical configuration of all the metabolites involved in DIP synthesis was established by NMR analysis. The two inositol moieties in DIP had different stereochemical configurations, in contradiction of previous reports. The use of the designation di-myo-inositol-1,3′-phosphate is recommended to facilitate tracing individual carbon atoms through metabolic pathways.

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