Cloning, sequencing and expression of the ilvBNC gene cluster from Streptomyces avermitilis.
The metabolism of the branched-chain amino acids (BCAA) isoleucine, leucine and valine is correlated to the production of polyketide antibiotics in many streptomycetes. Despite its significance, this biosynthetic pathway is poorly understood in Streptomyces. In order to develop a better understanding of Streptomyces BCCA biosynthesis, two genes, ilvBN and ilvC, encoding acetohydroxy acid synthase (AHS) and acetohydroxy acid isomeroreductase (IR), respectively, were cloned from Streptomyces avermitilis, a strain producing avermectins, potent antiparasitic compounds. The genes were isolated by applying a combination of PCR and genomic library screening. The deduced amino-acid sequences revealed significant homology to the AHS and IR proteins from other bacterial species. The ilvBN gene, expressed in Escherichia coli (Ec) by using the expression vector pGEX-4T-1, complemented the ilv- mutation of Ec PS1283. Ec transformants produced high levels of AHS, whose activity was feedback inhibited by valine.
De Rossi
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E
Department of Genetics and Microbiology, University of Pavia, Italy.
Leva
R
R
Gusberti
L
L
Manachini
P L
PL
Riccardi
G
G
D000095:Acetolactate Synthase / Q000235:genetics*; D000429:Alcohol Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D000597:Amino Acids, Branched-Chain / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D050540:Ketol-Acid Reductoisomerase; D008969:Molecular Sequence Data; D005810:Multigene Family; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000235:genetics*
D000597:Amino Acids, Branched-Chain; D017931:DNA Primers; D004269:DNA, Bacterial; D000429:Alcohol Oxidoreductases; D050540:Ketol-Acid Reductoisomerase; D000095:Acetolactate Synthase