MOLECULAR-CLONING OF THE ACTINOMYCIN SYNTHETASE GENE-CLUSTER FROM STREPTOMYCES-CHRYSOMALLUS AND FUNCTIONAL HETEROLOGOUS EXPRESSION OF THE GENE ENCODING ACTINOMYCIN SYNTHETASE II

The actinomycin synthetases ACMS I, TI, and III catalyze the assembly of the acyl peptide lactone precursor of actinomycin by a nonribosomal mechanism, me have cloned the genes of ACMS I (acmA) and ACMS II (acm B) by hybridization screening of a cosmid library of Streptomyces chry somallus DNA with synthetic oligonucleotides derived from peptide sequ ences of the two enzymes. Their genes were found to be closely linked and are arranged in opposite orientations. Hybridization mapping and p artial sequence analyses indicate that the gene of an additional pepti de synthetase, most likely the gene of ACMS III (acmC), is located imm ediately downstream of acmB in the same orientation. The protein seque nce of ACMS II, deduced from acmB, shows that the enzyme contains two amino acid activation domains, which are characteristic of peptide syn thetases, and an additional epimerization domain. Heterologous express ion of acmB from the mel promoter of plasmid PIJ702 in Streptomyces li vidans yielded a functional 280-kDa peptide synthetase which activates threonine and valine as enzyme bound thioesters, It also catalyzes th e dipeptide formation of threonyl-L-valine, which is epimerized to thr eonyl-D-valine. Both of these dipeptides are enzyme bound as thioester s. This catalytic activity is identical to the in vitro activity of AC MS II from S. chrysomallus.