CLONING, SEQUENCING AND EXPRESSION OF THE ILVBNC GENE-CLUSTER FROM STREPTOMYCES-AVERMITILIS

The metabolism of the branched-chain amino acids (BCAA) isoleucine, le ucine and valine is correlated to the production of polyketide antibio tics in many streptomycetes. Despite its significance, this biosynthet ic pathway is poorly understood in Streptomyces. In order to develop a better understanding of Streptomyces BCCA biosynthesis, two genes, il vBN and ilvC, encoding acetohydroxy acid synthase (AHS) and acetohydro xy acid isomeroreductase (IR), respectively, were cloned from Streptom yces avermitilis, a strain producing avermectins, potent antiparasitic compounds. The genes were isolated by applying a combination of PCR a nd genomic library screening. The deduced amino-acid sequences reveale d 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 E c PS1283. Ec transformants produced high levels of AHS, whose activity was feedback inhibited by valine.