Sequencing and mutagenesis of genes from the erythromycin biosynthetic gene cluster of Saccharopolyspora erythraea that are involved in L-mycarose and D-desosamine production

Abstract

The nucleotide sequence on both sides of the eryA polyketide synthase genes of the erythromycin-producing bacterium S0cch0rop0lysp0ra erythraea reveals the presence of ten genes that are involved in t-mycarose (eryB) and Ddesosamine ( e m biosynthesis or attachment. Mutant strains carrying targeted lesions in eight of these genes indicate that three (eryB/V, eryBVand eryBV/) act in L-mycarose biosynthesis or attachment, while the other five (eryC//, eryC///, eryC/V, e m and e m / ) are devoted to D-desosamine biosynthesis or attachment. The remaining two genes (eryB// and eryBV//) appear to function in L-mycarose biosynthesis based on computer analysis and earlier genetic data. Three of these genes, eryB//, eryC/// and eryC//, lie between the eryA/// and eryG genes on one side of the polyketide synthase genes, while the remaining seven, eryB/V, eryBV, e m / , eryBVI, erycIV, eryCV and eryBV// lie upstream of the eryAI gene on the other side of the gene cluster. The deduced products of these genes show similarities to: aldohexose 4-ketoreductases (eryB/V), aldoketo reductases (eryB//), aldohexose 5-epimerases (eryBV//), the dnml gene of the daunomycin biosynthetic pathway of Streptomyces peucetius (eryBvl), glycosyltransferases (eryBV and eryC///), the AscC 3,bdehydratase from the ascarylose biosynthetic pathway of Yeminis pseudotuberculosis (eryC/V), and mammalian N-methyltransferases ( e m / ) . The eryC// gene resembles a cytochrome P450, but lacks the conserved cysteine residue responsible for coordination of the haem iron, while the eryCV gene displays no meaningful similarity to other known sequences. From the predicted function of these and other known eryB and eryC genes, pathways for the biosynthesis of L-mycarose and D-desosamine have been deduced.

The mycarose-biosynthetic genes of Streptomyces fradiae, producer of tylosin The GenBank accession number for the sequence determined in this work is AF147704.

“…erythraea, eryBVII encodes a mycarose-biosynthetic enzyme and similar genes in other organisms are also associated with the production of -sugars, including -rhamnose (rfbC gene in Shigella ; Macpherson et al, 1994), -ascarylose (ascE in Yersinia ; Thorson et al, 1994) and -dihydrostreptose (strM in Streptomyces griseus ; Pissowotzky et al, 1991). Although direct experimental evidence is lacking, StrM (Pissowotzky et al, 1991) and EryBVII (Gaisser et al, 1997 ;Summers et al, 1997) are believed to be NDP-4-keto-6-deoxyglucose 3,5-(or 5-) epimerases and a similar role is proposed here for the orf6* product. Proof that Orf6* is involved in the biosynthesis of mycarose was obtained by fermentation analysis of a strain in which orf6* was specifically disrupted.…”

“…orf9* (tylCIV). The deduced product of orf9* (maximum size, 347 amino acids ; M r 3 6200) displays 49 % sequence identity to that of eryBIV (Gaisser et al, 1997 ;Summers et al, 1997). The only other significant end-to-end sequence match (46 % identity) was to an uncharacterized protein, PCZA 361.4, from vancomycin-producing Amycolatopsis orientalis (van Wageningen et al, 1998).…”

“…Such enzymes inactivate their substrates and are involved in antibiotic efflux and\or resistance. Less close matches (20-25 % sequence identity) were also seen with glycosyltransferases involved in antibiotic biosynthesis such as OleG2 (Olano et al, 1998), EryCIII and EryBV (Gaisser et al, 1997 ;Summers et al, 1997 ;Salah-Bey et al, 1998). Nevertheless, when the role of orf7* was addressed by targeted gene disruption it proved to be a mycarosebiosynthetic gene and not an mgt.…”

“…In particular, the Orf6* protein shares 44 % sequence identity with EryBVII (Gaisser et al, 1997 ;Summers et al, 1997). In Sa.…”

“…3 (compound 2). Alternatively, 2-deoxygenation might occur first (Summers et al, 1997), in which case the stereochemistry at C-3 following the enoylreductase reaction would dictate the mode of subsequent epimerization. Support for the latter route, at least in the biosynthesis of other 2,6-dideoxyhexoses, came from recent in vitro studies (Draeger et al, 1999) involving pairs of purified enzymes that catalyse 2-deoxygenation of dTDP-4-keto-6-deoxy--glucose (Fig.…”