SHORT COMMUNICATION S. Torkkell á K. Ylihonko á J. Hakala á M. Skurnik P. MaÈntsaÈlaÈ Characterization of Streptomyces nogalater genes encoding enzymes involved in glycosylation steps in nogalamycin biosynthesis Received: 26 November 1996 / Accepted: 5 June 1997 Abstract The sno gene cluster in Streptomyces nogalater ATCC 27451 contains the nogalamycin biosynthesis genes. A set of plasmid constructions carrying fragments of the sno cluster that lie downstream of snoD were used to complement the S. galilaeus mutant H039, which is blocked in rhodosamine and 2-deoxyfucose biosynthesis in the aclacinomycin pathway. Sequence analysis of this cluster revealed three contiguous open reading frames (ORFs) that were designated snoF, snoG, and snoH. Only those plasmid constructs that expressed SnoG were able to complement H039. SnoG shows similarity to GalE, a UDP-glucose-4-epimerase catalyzing the epimerization of UDP-glucose to UDP-galactose. The putative SnoF protein is similar to 3,5-epimerases in- volved in rhamnose biosynthesis. The deduced product of snoH is a 489-amino acid polypeptide. It is similar to the product of dau ORF3 found in the daunomycin cluster. However its function is still unclear. Based on the complementation experiments and sequence analy- sis, this part of the sno cluster is suggested to be involved in the biosynthesis of the sugar portion of nogalamycin. Interestingly, SnoA, a transcriptional activator for the sno minimal polyketide synthase, is also needed to ex- press this cluster. Key words Streptomyces nogalater á Anthracycline á Nogalamycin á 3,5-Epimerase á 4-Epimerase Introduction Nogalamycin (Fig. 1) is an antitumour anthracycline antibiotic, which was discovered in 1965 by Bhuyan and Dietz. The structure of this extraordinarily glycosylated anthracycline was determined by Wiley et al. (1977). Nogalamycin diers from anthracyclines of the dauno- mycin group (e.g. Fujiwara et al. 1986) in the aglycone moiety, in positions of glycosylation and in its attached sugar residues, which are nogalamine and nogalose. Studies on anthracycline biosynthesis have mainly fo- cused on the daunomycin group. Based on the use of well documented mutants blocked in daunomycin bio- synthesis (Bartel et al. 1990) and the techniques of mo- lecular cloning in Streptomyces, the biosynthetic pathway for daunomycin, particularly with respect to the aglycone moiety, has been almost completely re- solved. Daunomycin gene clusters have been cloned from S. peucetius (Grimm et al. 1994; Otten et al. 1995; Madduri and Hutchinson 1995) and from the mutant Streptomyces sp. C5 (Ye et al. 1994; Dickens et al. 1995, 1996), and the part of the cluster involved in the bio- synthesis of the sugar portion of daunomycin has been isolated from S. griseus (KruÈgel et al. 1993). Almost all the genes responsible for forming of daunomycin have been identi®ed. We are carrying out molecular genetic studies on the biosynthesis of anthracyclines with a view to the rational construction of strains that can produce hybrid anthra- cyclines (Niemi et al. 1994; Ylihonko et al. 1996a). In these studies, mutants derived from the aclacinomycin (Fig. 1) producer S. galilaeus (Ylihonko et al. 1994) are used as host strains. Biosynthetic genes for the nogalamycin chromophore were recently reported (Ylihonko et al. 1996b). Here, we describe the analysis of a 4-kb DNA fragment from the sno cluster that most probably encodes enzymes involved in biosynthesis of the sugar portion of nogalamycin. A putative scheme for the biosynthetic route from NDP-glucose precursors to the sugars found in anthracyclines is outlined in Fig. 1. Mol Gen Genet (1997) 256: 203±209 Ó Springer-Verlag 1997 Communicated by A. Kondorosi S. Torkkell á K. Ylihonko (&) á P. MaÈntsaÈ laÈ Department of Biochemistry and Food Chemistry, University of Turku, Arcanum, Vatselantie 2, FIN-20014 Turku, Finland Fax: +358-2-333-6860; e-mail: kylihonk@®nabo.abo.® J. Hakala Galilaeus Oy, P.O. Box 113, FIN-20781 Kaarina, Finland M. Skurnik Centre for Biotechnology, Biocity, TykistoÈkatu 6, FIN-20520 Turku