Stimulation of polyketide metabolism in Streptomyces fradiae by tylosin and its glycosylated precursors

Abstract

Three glycosyltransferases are involved in tylosin biosynthesis in Streptomyces fradiae. The first sugar to be added to the polyketide aglycone (tylactone) is mycaminose and the gene encoding mycaminosyltransferase is oH2* (tyhW2). However, targeted disruption of od2* did not lead to the accumulation of tylactone under conditions that normally favour tylosin production; instead, the synthesis of tylactone was virtually abolished. This may, in part, have resulted from a polar effect on the expression of genes downstream of oH2*, particularly od4* (ccr) which encodes crotonyl-CoA reductase, an enzyme that supplies 4-carbon extender units for polyketide metabolism. However, that cannot be the entire explanation, since tylosin production was restored a t about 10% of the wild-type level when oH2* was re-introduced into the disrupted strain. When glycosylated precursors of tylosin were fed to the disrupted strain, they were converted to tylosin, confirming that two of the three glycosyltransferase activities associated with tylosin biosynthesis were still intact. Interestingly, however, tylactone also accumulated under such conditions and, to a much lesser extent, when tylosin was added to similar fermentations. It is concluded that glycosylated macrolides exert a pronounced positive effect on polyketide metabolism in S. fradiae. ;ity OfKeywords : tylosin production, glycosyltransferase, polyketide metabolism, Streptomyces fradiae INTRODUCTIONThe macrolide antibiotic, tylosin (Fig. l ) , is produced by Streptomyces fradiae via a combination of polyketide and 6-deoxyhexose metabolism. Glycosylation of tylactone, the cyclized polyketide product, always begins with the addition of mycaminose followed, in a preferred but not obligatory order, by deoxyallose and then mycarose to generate demethyl-macrocin. Stepwise bis 0-methylation then converts the deoxyallose moiety to mycinose as macrocin is produced and converted to tylosin (Baltz et al., 1983). Characterization of the tylosin biosynthetic pathway (Fig. 2) was facilitated by the availability of non-producing mutants of S. fradiae, generated using NTG (Baltz & Seno, 1981). Collectively, these exhibited nine distinct phenotypes in cross-feeding Abbreviations: OMT, 0-mycaminosyl-tylonolide; DMT, demycinosyltyl osi n. experiments and allowed 13 genetic loci (tylA-M) to be mapped (Fig. 3) following complementation studies using cloned fragments of tyl DNA (Beckmann et al.,

Autoregulation of antibiotic biosynthesis by binding of the end product to an atypical response regulator

“…Autoregulatory phenomena were also reported in donorubicin and tylosin biosynthesis (38,39). However, JadR1 is the first example of a primary pathway-specific activator whose activity is controlled by the end product.…”

Feedback control of polyketide metabolism during tylosin production

“…Plasmids were manipulated in Escherichia coli DH5α using standard protocols (Sambrook et al, 1989). DNA was introduced into S. fradiae by conjugal transfer from E. coli S17-1 as described elsewhere (Fish & Cundliffe, 1997) using pOJ260 (Bierman et al, 1992) and pLST9828 . pOJ260 is incapable of replication in Streptomyces spp.…”

“…The complete insert was then excised using BglII sites which flank the pIJ2925 polylinker and ligated into the BamHI site of pOJ260. Following conjugal transfer into S. fradiae, transconjugants were selected on hygromycin B and then screened for apramycin sensitivity (Fish & Cundliffe, 1997) to identify those in which chromosomal target genes had been replaced with disrupted constructs via double recombination. The gene disruption constructs were assembled as follows.…”

“…The present work arises from earlier studies (Fish & Cundliffe, 1997) in which tylMII (inappropriately referred to as ' tylM2 ' at that time) was disrupted in the genome of S. fradiae. This gene (orf2* in the systematic nomenclature, see Fig.…”

SsaA, a Member of a Novel Class of Transcriptional Regulators, Controls Sansanmycin Production in Streptomyces sp. Strain SS through a Feedback Mechanism

“…For example, in tylosin biosynthesis, glycosylated macrolides were proposed to exert a pronounced positive effect on polyketide metabolism in S. fradiae (41). Rhodomycin D, the first glycosylated intermediate in donorubicin biosynthetic pathways, can interact with the TetR-like pathway-specific regulator DnrO to relieve its self-repression, thereby positively enhancing end production (42).…”