Elucidation of anthracyclinone biosynthesis by stepwise cloning of genes for anthracyclines from three different Streptomyces spp

Department of Biochemistry, University of Turku, Vatselantie 2, FIN-20014 Turku, Finland 1 Galilaeus Oy, PO BOX 113, FIN-20781 Kaarina, Finland 2 Author for correspondence: Kristiina Ylihonko. Tel: +358 2 3336879. Fax: +358 2 3336860. e-mail: kristiina.ylihonko{at}finabo.abo.fi The anthracycline skeleton is biosynthesized by aromatic (type II) polyketide synthases. Furthermore, three post-polyketide steps are needed to form the basic aglycone of anthracyclines. Auramycinone was produced in Streptomyces lividans by introducing nine structural genes from three different anthracycline-producing Streptomyces species. The genes used to construct the auramycinone biosynthesis cluster were derived from nogalamycin-, daunomycin- and aclacinomycin-producing Streptomyces strains. The biosynthetic stages were divided into polyketide and post-polyketide steps on the assumption that the first stable intermediate would be nogalonic acid, named analogously to aklanonic acid, the precursor of several anthracyclines. Single genes were cloned in the expression construct in the order determined by the proposed biosynthetic pathway. This facilitated investigation of the products formed in the heterologous host after addition of each separate gene to the construct. The results thus elucidate the biosynthesis steps, products and the genes responsible for the reactions needed to build up an anthracyclinone. Keywords: anthracyclines, auramycinone, biosynthesis, heterologous expression, Streptomyces Abbreviations: AAME, aklanonic acid methyl ester; minPKS, minimal PKS; PKS, polyketide synthase The GenBank accession number for acmA reported in this paper is AF043550 .