Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sun, 03 Feb 2019 22:22:13 Microbiology (1 999), 145, 855-868 Printed in Great Britain The tylosin biosynthetic cluster from Streptomyces fradiae: genetic organization of the left region Roberto Fouces, Encarnacion Mellado, Bruno Diez and Jose Luis Barredo Author for correspondence: Jose Luis Barredo. Tel: +34 987 895826. Fax: +34 987 895810. e-mail : jbarredo@antibioticos.it Laboratorio de lngenieria Genetics, Antibidticos SA, Avenida de Antibidticos 59-61, 24080 Ledn, Spain The genetic organization of the left edge (ty/€DHFJ region) of the tylosin biosynthetic gene cluster from Streptomyces fradiae has been determined. Sequence analysis of a 12.9 kb region has revealed the presence of 11 ORFs, 10 of them belonging to the biosynthetic cluster. The putative functions of the proteins encoded by these genes are as follows: peptidase (ORF1, ddd), tylosin resistance determinant (ORFZ, tlrB), glycosyltransferase (ORF3, ty//U), methyltransferase (ORF4, tyl€), ketoreductase (ORF5, tylD), ferredoxin (ORF6, tylH2), cytochrome P450 (ORF7, tylH7), methyltransferase (ORF8, tylF), epimerase (ORF9, tylJ), acyl-CoA oxidase (ORF10, tylP) and receptor of regulatory factors (ORF11, tylQ). The functional identification of the genes in the proposed tylosin biosynthetic pathway has been deduced by database searches and previous genetic complementation studies performed with tylosin idiotrophic mutants blocked at various stages in tylosin biosynthesis. The tlrB gene has been shown to be useful as a tylosin resistance marker in Streptomyces liwidans, Streptomyces parvulus and Streptomyces coelicolor and the effect of tylF on macrocin depletion has been confirmed. A pathway for the biosynthesis of 6-deoxy-~-allose, the unmethylated mycinose precursor, involving the genes tylD, tylJ and tylN is proposed. Keywords : glycosyltransferase, ketoreductase, cytochrome P4.50, methyltransferase, my c i n o s e INTRODUCTION Tylosin is a macrolide antibiotic used in veterinary medicine to treat infections caused by Gram-positive bacteria and as an animal growth promoter in the swine industry. It is produced by several Streptomyces species including S. fradiae (Seno et al., 1977), S. rimosus (Pape & Brillinger, 1973) and S. hygroscopicus (Jensen et al., 1964), but S. fradiae is the micro-organism of choice for its industrial production. As with other macrolides, the antibiotic activity of tylosin is due to the inhibition of protein hiosynthesis by a mechanism that involves the binding of tylosin to the ribosome, preventing the for ma ti on of the mRNA-aminoac y l- tR N A-r ibosome complex (Kageyama et al., 1971). . , , . . .. . . . . . . . . .. . . . . .. . . . .. . . . . . . . . , .. . . . . . ,,, . . . . . . . . . . .. , . . . .. . . . . . . .. . , . . . . . , . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .. . . .. . . . .. . . . . . . . . . . . . . .. . . . . Abbreviations: MLS, macrolide-lincosamide-streptogramin B; PBP, peni- cillin-binding protein. The GenBank accession number for the 12905 bp sequence reported in this paper is AF055922. The presence of gene clusters is a common phenomenon in antibiotic-producing micro-organisms. The genes involved in the biosynthetic pathway of antibiotics such as erythromycin, clavulanic acid, cephamycin, actino- rhodin, tylosin, nogalamycin, puromycin and dauno- rubicin are clustered in the chromosome of different Streptomyces species (Diez et al., 1997). Tylosin bio- synthesis has been extensively studied by both physio- logical (Fishman et al., 1987; Baltz & Seno, 1981, 1988) and genetic (Merson-Davies & Cundliffe, 1994; Gandecha et al., 1997; Cox et af., 1997) approaches. From the results of co-synthesis studies with idiotrophic mutants blocked at different steps in tylosin bio- synthesis, 13 different loci (tylA to tyfM) have been mapped. As a result, the most probable pathway for tylactone conversion to tylosin has been deduced (Baltz et al., 1983) (Fig. 1) : the biosynthetic pathway proceeds from two acetate, one butyrate and five propionate units to the tylactone moiety to which sugar residues are attached (Baltz & Seno, 1981). The tylosin gene cluster extends over about 85 kb in the genome of S. fradiae; it - 0002-2923 0 1999 SGM 855