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

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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

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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...
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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.