Cloning, characterization and heterologous expression of the aspartokinase and aspartate semialdehyde dehydrogenase genes of cephamycin C-producer Streptomyces clavuligerus.
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 beta-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 ORF1 and terminates by utilizing the same stop codon as ORF1, an arrangement typical of many ask genes. ORF3 is located 2 nucleotides downstream of ORF1,2. Database comparisons with these proteins identified ORF1 as the large (alpha) subunit of aspartokinase, ORF2 as the small (beta) 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.
Tunca
Sedef
S
Department of Biology, Middle East Technical University, Ankara, Turkey.
Yilmaz
Ebru I
EI
Piret
Jacqueline
J
Liras
Paloma
P
Ozcengiz
Gülay
G
D001222:Aspartate Kinase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001223:Aspartate-Semialdehyde Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002513:Cephamycins / Q000096:biosynthesis*; D003001:Cloning, Molecular; D018387:Codon, Initiator; D018388:Codon, Terminator; D016366:Open Reading Frames / Q000235:genetics; D009876:Operon; D012294:Rifamycins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism
D002513:Cephamycins; D018387:Codon, Initiator; D018388:Codon, Terminator; D012294:Rifamycins; D001223:Aspartate-Semialdehyde Dehydrogenase; D001222:Aspartate Kinase; C024925:cephamycin C