Cloning, characterization and heterologous expression of the aspartokinase and aspartate semialdehyde dehydrogenase genes of cephamycin C-producer Streptomyces clavuligerus

Date

2004-09-01

Author

Tunca, S
Yilmaz, EI
Piret, J
Liras, P
Özcengiz, Gülay

Metadata

Show full item record

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Item Usage Stats

232
views

0
downloads

Carbon flow through the lysine branch of the aspartate biosynthetic pathway is a rate-limiting step in the formation of cephamycin C, a broad spectrum P-lactam antibiotic produced by Streptomyces clavuligerus. In this study, genes which encode the enzymes catalyzing the first two steps of the aspartate pathway, ask (aspartokinase) and asd (aspartate semialdehyde dehydrogenase), in S. clavuligerus NRRL 3585 were cloned and sequenced. Nucleotide sequencing and codon preference analysis revealed three complete open reading frames (ORFs). ORF2 starts within ORFI and terminates by utilizing the same stop codon as ORFI, an arrangement typical of many ask genes. ORF3 is located 2 nucleotides downstream of ORF1,2. Database comparisons with these proteins identified ORFI as the large (a) subunit of aspartokinase, ORF2 as the small (P) subunit of aspartokinase and ORF3 as the aspartate semialdehyde dehydrogenase. The cloned genes were functionally expressed in auxotrophic Escherichia coli strains, CGSC5074 (ask(-)) and E. coli CGSC5080 (asd(-)), the two enzymes were partially purified from E. coli cell extracts and their kinetic parameters were determined. The effects of end product amino acids and diaminopimelic acid on the activity of Ask and Asd enzymes were also described. (C) 2004 Elsevier SAS. All rights reserved.

Subject Keywords

Molecular Biology, Microbiology, General Medicine

URI

https://hdl.handle.net/11511/47603

Journal

RESEARCH IN MICROBIOLOGY

DOI

https://doi.org/10.1016/j.resmic.2004.03.007

Collections

Department of Biology, Article

Suggestions

OpenMETU
Core

Cloning, characterization and heterologous expression of the aspartokinase and aspartate semialdehyde dehydrogenase genes from the cephamycin C producer streptomyces clavuligerus Tunca, Sedef; Özcengiz, Gülay; Piret, Jacqueline; Department of Biotechnology (2002) The carbon flow through the lysine branch of aspartate pathway is a rate limiting step in the formation of cephamycin C, a broad spectrum P-lactam antibiotic produced by Streptomyces clavuligerus. In this study, the ask m(aspartokinase) and asd (aspartate semialdehyde dehydrogenase) genes which encode the enzymes catalyzing the first two steps of aspartate pathway were cloned and characterized as the first time from S. clavuligerus NRRL 3585. This was accomplished by constructing four mini-libraries of S. c...
Bioproduction, structure elucidation and in vitro antiproliferative effect of eumelanin pigment from Streptomyces parvus BSB49 Bayram, Sinan; Dengiz, Çağatay; GERÇEK, Yusuf Can; ÇETİN, İdil; TOPÇUL, Mehmet Rıfkı (Springer Science and Business Media LLC, 2020-01-01) In this study, the structure of the purified extracellular eumelanin pigment isolated from Streptomyces spp. was elucidated by detailed analysis via two different spectroscopic techniques (FT-IR and NMR). In vitro antiproliferative effects of eumelanin were evaluated on HeLa cell line. These experiments were carried out with the evaluation of the parameters including cell viability, cell index, and mitotic index. With the cell viability and cell index, IC50 concentration of eumelanin was determined as 10 μM...
Regulation of crystal protein biosynthesis by Bacillus thuringiensis: I. Effects of mineral elements and pH ICGEN, Y; Icgen, BÜLENT; Özcengiz, Gülay (Elsevier BV, 2002-11-01) Crystal protein synthesis by a local isolate of Bacillus thuringiensis was monitored and compared in association with growth and sporulation in media differing in mineral element content. Mg and Cu were the most important metals for the biosynthesis of 135 kDa and 65 kDa toxin components in that the former was essential and the latter was greatly stimulatory at 10(-6) to 10(-7) M concentration. Also the inclusion of Mn favored toxin production at concentrations ranging from 3 x 10(-4) to 10(-5) M. The omiss...
Analysis of a bac operon-silenced strain suggests pleiotropic effects of bacilysin in Bacillus subtilis Ertekin, Ozan; Taskin, Ash Aras; Demir, Mustafa; Karataş, Ayten; Özcengiz, Gülay (Springer Science and Business Media LLC, 2020-04-01) Bacilysin, as the simplest peptide antibiotic made up of only L-alanine and L-anticapsin, is produced and excreted by Bacillus subtilis under the control of quorum sensing. We analyzed bacilysin-nonproducing strain OGU1 which was obtained by bacA-targeted pMutin T3 insertion into the parental strain genome resulting in a genomic organization (bacA '::lacZ::erm::bacABCDEF) to form an IPTG-inducible bac operon. Although IPTG induction provided 3- to 5-fold increment in the transcription of bac operon genes, n...
Regulation of crystal protein biosynthesis by Bacillus thuringiensis: II. Effects of carbon and nitrogen sources. Içgen, Y; İçgen, Bülent; Özcengiz, Gülay (Elsevier BV, 2002-11-01) The regulation of crystal protein production in Bacillus thuringiensis 81 by sources of carbon and nitrogen was investigated. The highest titers of toxin were obtained on sucrose, lactose and inulin which also supported sporulation. Whey and molasses were also potential carbon substrates for toxin production. Other carbohydrates including glucose, glycerol, maltose, starch and dextrin yielded lower amounts of toxin. Nitrogen sources were found to exert the most profound controls. Peptone was the best organi...

Citation Formats

S. Tunca, E. Yilmaz, J. Piret, P. Liras, and G. Özcengiz, “Cloning, characterization and heterologous expression of the aspartokinase and aspartate semialdehyde dehydrogenase genes of cephamycin C-producer Streptomyces clavuligerus,” RESEARCH IN MICROBIOLOGY, pp. 525–534, 2004, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/47603.