Organization of a cluster of erythromycin genes in Saccharopolyspora erythraea

Abstract

We used a series of gene disruptions and gene replacements to mutagenically characterize 30 kilobases of DNA in the erythromycin resistance gene (ermE) region of the Saccharopolyspora erythraea chromosome. Five previously undiscovered loci involved in the biosynthesis of erythromycin were found, eryBI, eryBII, eryCl, eryCII, and eryH; and three known loci, eryAl, eryG, and ermE, were -further characterized. The new Ery phenotype, EryH, was marked by (i) the accumulation of the intermediate 6-deoxyerythronolide B (DEB), suggesting a defect in the operation of the C-6 hydroxylase system, and (ii) a block in the synthesis or addition reactions for the first sugar group. Analyses of ermE mutants indicated that erinE is the only gene required for resistance to erythromycin, and that it is not required for production of the intermediate erythronolide B (EB) or for conversion of the intermediate 3-a-mycarosyl erythronolide B (MEB) to erythromycin. Mutations in the eryB and eryC loci were similar to previously reported chemically induced eryB and eryC mutations blocking synthesis or attachment of the two erythromycin sugar groups. Insertion mutations in eryAI, the macrolactone synthetase, defined the largest (at least 9-kilobase) transcription unit of the cluster. These mutants help to define the physical organization of the erythromycin gene cluster, and the eryH mutants provide a source for the production of the intermediate DEB.Erythormycin A is a medically important macrolide antibiotic produced by the gram-positive, sporeforming bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus NRRL 2338) (29). As is the case for other antibiotic-producing organisms, the genes for the biosynthesis of erythromycin (ery) are thought to be clustered about the gene for resistance to erythromycin, ermE (22,27,29); however, much remains to be learned about the organization and location of the many individual genes predicted to be involved in the pathway (6, 18).The ermE gene was originally cloned by Thompson et al. (24) and later sequenced by Uchiyama and Weisblum (25); its promoter and the promoter of an adjacent upstream open reading frame were characterized by Bibb et al. (3,4). The adjacent open reading frame has since been sequenced by Dhillon et al. (7), inactivated by gene disruption, and found to be involved in erythromycin biosynthesis as an eryC-type gene. Independently, the open reading frame was shown by Vara et al. (25a) to be an eryC-type gene through complementation of the eryC160 allele, and we obtained the same results as Dhillion et al. (7) by using linker insertion mutagenesis (see below). Other landmarks in the ery gene cluster are (i) eryG, the O-methyltransferase located 6.5 kilobase pairs (kb) downstream of the ermE promoter region (1Sa, 28); (ii) eryAI, the macrolactone synthetase, a large locus beginning 3 kp upstream of eryG (J. Tuan, J. M. Weber, M. Staver, J. 0. Leung, and L. Katz, Gene, in press); and (iii) the eryB25, eryB26, and eryD24 genes, mapped within an 18-kb region upstream o...

An acyl‐carrier‐protein – thioesterase domain from the 6‐deoxyerythronolide B synthase of Saccharopolyspora erythraea

“…About 40% of the polypeptide present had lost the N-terminal methionine, presumably by post-translational processing in E. coli. The sequence exactly matches that expected for the product of the eryCZZ gene which is apparently essential for the addition of desosamine to 3-0-mycarosylerythronolide B [7]. The synthesis of this polypeptide in induced cells suggests that the eryA and eryCZZ genes are translationally coupled.…”

Engineering of the methylmalonyl-CoA metabolite node of Saccharopolyspora erythraea for increased erythromycin production

“…erythraea wild-type and the mutB mutant strain FL2302 were transformed with plasmid DNA using standard protoplast transformation procedures (Weber et al, 1990). Confirmation of plasmid integration was performed by PCR using sequence-specific primers that amplify a 1.04 kb region of the thiostrepton-resistance (tsr) gene contained on pFL8 (Reeves et al, 2004).…”

Identification of DEBS 1, DEBS 2 and DEBS 3, the multienzyme polypeptides of the erythromycin‐producing polyketide synthase from Saccharopolyspora erythraea

“…The nature of the polyketide synthase that catalyses this multistep process, 6-deoxyerythronolide B synthase (DEBS), has been extensively investigated by genetic analysis [3][4][5] and by nucleotide sequencing of the structural genes encoding the synthase [6][7][8]. These eryA genes are found clustered near e~'mE, the erythromycin resistance gene [9], and extend over approximately 45 kilobase-pairs (kbp) of DNA.…”