Date

2002

Author

Tunca, Sedef

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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. clavuligerus genomic DNA on two different E. coli vectors and screening recombinant colonies with radioactively labelled (P32) ask-asd genes of Nocardia lactamdurans. Two colonies giving positive signal with the probe were obtained and the recombinant plasmids from these putative clones could transform E. coli CGSC 5074 {ask -), E. coli CGSC 5080 {asd -) and E. coli CGSC 5081 {asd ~) to prototrophy. Nucleotide sequencing and codon preference analysis revealed three complete open reading frames (ORFs). ORF2 starts within ORF1 and terminates by using the same stop codon of ORF1. ORF3 and ORFl,2 are separated by 2 nucleotides and they were encoding proteins of 355 aa (Mr 37 550), 421 aa (Mr 44 366) and 172 aa (Mr 18 129), respectively. By comparing the amino acid sequences of these proteins to those available in the database, we identified ORF1 as the large (a) subunit of aspartokinase, ORF2 as the small (P) subunit of aspartokinase and ORF3 as the aspartate semialdehyde dehydrogenase. The results of present study showed that unlike Streptomyces akiyoshiensis in which ask and asd genes are well separated in genome, S. clavuligerus ask and asd genes are clustered in an operon as in Mycobacterium, Bacillus, Corynebacterium and Amycolatopsis species. Moreover, in addition to a putative Streptomyces promoter consensus region similar to E. co//-like Ea70 promoters in upstream region of the askafi, an IVinverted repeat sequence which probably serves as a transcriptional terminator was determined at the downstream of the asd gene. Homologous expression of ask-asd cluster in S. clavuligerus and determination of the effects of multiple copies of these genes on cephamycin C biosynthesis were also among the goals of the present research. A streptomyces plasmid vector suitable for subcloning of ask-asd cluster was constructed for this aim and designated pNSTlOO. The ask-asd genes were next ligated to this vector which was used to transform S. lividans as an intermediate transformable host. In spite of all our attempts to obtain stable recombinants, there occurred plasmid rearrangement and loss of insert in transformants. The recombinant plasmid instability in streptomycete host is thought to be due to overexpression of the cloned genes under two tandem promoters.

Subject Keywords

Streptomyces, Aspartate aminotransferase, Molecular cloning, Molecular genetics, Streptomyces clavuligerus, Gene cloning, Aspartokinase, Aspartate semialdehyde dehydrogenase, Cephamycin C.

URI

https://hdl.handle.net/11511/12865

Collections

Graduate School of Natural and Applied Sciences, Thesis