Analysis of genes involved in 6-deoxyhexose biosynthesis and transfer in Saccharopolyspora erythraea.

Glycosylation represents an attractive target for protein engineering of novel antibiotics, because specific attachment of one or more deoxysugars is required for the bioactivity of many antibiotic and antitumour polyketides. However, proper assessment of the potential of these enzymes for such combinatorial biosynthesis requires both more precise information on the enzymology of the pathways and also improved Escherichia coli-actinomycete shuttle vectors. New replicative vectors have been constructed and used to express independently the dnmU gene of Streptomyces peucetius and the eryBVII gene of Saccharopolyspora erythraea in an eryBVII deletion mutant of Sac. erythraea. Production of erythromycin A was obtained in both cases, showing that both proteins serve analogous functions in the biosynthetic pathways to dTDP-L-daunosamine and dTDP-L-mycarose, respectively. Over-expression of both proteins was also obtained in S. lividans, paving the way for protein purification and in vitro monitoring of enzyme activity. In a further set of experiments, the putative desosaminyltransferase of Sac. erythraea, EryCIII, was expressed in the picromycin producer Streptomyces sp. 20032, which also synthesises dTDP-D-desosamine. The substrate 3-alpha-mycarosylerythronolide B used for hybrid biosynthesis was found to be glycosylated to produce erythromycin D only when recombinant EryCIII was present, directly confirming the enzymatic role of EryCIII. This convenient plasmid expression system can be readily adapted to study the directed evolution of recombinant glycosyltransferases.

Targeted gene inactivation for the elucidation of deoxysugar biosynthesis in the erythromycin producer Saccharopolyspora erythraea.

The production of erythromycin A by Saccharopolyspora erythraea requires the synthesis of dTDP-D-desosamine and dTDP-L-mycarose, which serve as substrates for the transfer of the two sugar residues onto the macrolactone ring. The enzymatic activities involved in this process are largely encoded within the ery gene cluster, by two sets of genes flanking the eryA locus that encodes the polyketide synthase. We report here the nucleotide sequence of three such ORFs located immediately downstream of eryA, ORFs 7, 8 and 9. Chromosomal mutants carrying a deletion either in ORF7 or in one of the previously sequenced ORFs 13 and 14 have been constructed and shown to accumulate erythronolide B, as expected for eryB mutants. Similarly, chromosomal mutants carrying a deletion in either ORF8, ORF9, or one of the previously sequenced ORFs 17 and 18 have been constructed and shown to accumulate 3-alpha-mycarosyl erythronolide B, as expected for eryC mutants. The ORF13 (eryBIV), ORF17 (eryCIV) and ORF7 (eryBII) mutants also synthesised small amounts of macrolide shunt metabolites, as shown by mass spectrometry. These results considerably strengthen previous tentative proposals for the pathways for the biosynthesis of dTDP-D-desosamine and dTDP-L-mycarose in Sac. erythraea and reveal that at least some of these enzymes can accommodate alternative substrates.

Stress-activated expression of a Streptomyces pristinaespiralis multidrug resistance gene (ptr) in various Streptomyces spp. and Escherichia coli.

A promoter which controls expression of the pristinamycin multidrug resistance gene (ptr) in Streptomyces pristinaspiralis could be induced by physiological stresses in both Streptomyces spp. and Escherichia coli. In S. pristinaspiralis, the ptr promoter (Pptr) was induced by pristinamycin I (PI) or pristinamycin II (PII). Streptomyces lividans was adopted as a convenient heterologous host for studies of Pptr regulation since it has no known pristinamycin biosynthetic genes. Two key regulatory features were documented in these studies: many (19 of 70) antibiotics and chemicals with no common targets or structural features induced the Pptr; induction with PI was most efficient during a transition phase when antibiotic biosynthetic genes are switched on. In Streptomyces coelicolor, Pptr activity was similarly inducible by PI and not dependent on sigma factors HrdA, HrdC, or HrdD. In E. coli, Pptr cloned in the bifunctional promoter probe vector pIJ2839 was functional and activated upon entry into stationary phase in the absence of exogenous inducer. Finally, gel-retardation studies demonstrated a Pptr-binding protein in S. lividans (where its activity was PI-inducible), S. coelicolor and S. pristinaespiralis. The fact that this activity was not detected in E. coli suggested the existence of another regulatory system perhaps also present in Streptomyces.

Molecular characterization and transcriptional analysis of a multidrug resistance gene cloned from the pristinamycin-producing organism, Streptomyces pristinaespiralis.

A multidrug resistance gene (mdr) has been cloned from Streptomyces pristinaespiralis, a producer of two antibiotics having synergistic activities together known as pristinamycin. This gene, ptr, provides resistance not only to two structurally dissimilar compounds (pristinamycin I, PI; pristinamycin II, PII) and the natural pristinamycin mixture but also to rifampicin. Mutagenesis and subcloning of ptr localized it to a 2 kb region which was sequenced and analyzed. It contained an open reading frame of 1506 bp which encoded a putative membrane protein with 14 hydrophobic domains, and showed sequence similarity to a superfamily of bacterial proteins that employ transmembrane electrochemical gradients to catalyse active efflux of various antibiotics and toxic compounds. Ptr was most similar to a subfamily which included other mdr genes and antibiotic transport genes associated with antibiotic biosynthetic gene clusters in actinomycetes. In vitro coupled transcription-translation experiments were used to identify the ptr gene product. Analysis of the upstream region did not reveal a divergently transcribed repressor gene, as is the case for several related resistance determinants involved in antibiotic transport, suggesting that ptr is regulated by a different mechanism. Transcriptional analyses of this gene, carried out in both S. pristinaespiralis and Streptomyces lividans, indicated the same transcriptional start point and predicted -10 and -35 hexamers which were somewhat similar to Streptomyces vegetative-type promoters.

Unusual regulatory mechanism for a Streptomyces multidrug resistance gene, ptr, involving three homologous protein-binding sites overlapping the promoter region.

A promoter controlling expression of the pristinamycin multidrug resistance gene (ptr), originally isolated from Streptomyces pristinaespiralis, is inducible by many toxic compounds in various Streptomyces species. Studies of ptr promoter control were carried out in the heterologous host, Streptomyces lividans. In S. lividans, a regulatory protein or a protein complex (Pip), identified by its ability to bind to the ptr promoter in gel-retardation experiments, was induced by pristinamycin I (PI). In situ copper-phenanthroline footprinting analysis identified three (A, B, and C) similar Pip-binding sites having the sequence GTACA(C/G)CGTA(C/T). These sites overlapped with functionally important regions of the promoter: the 'A' site overlapped with the -35 hexamer, 'B' overlapped with the -10 hexamer and 'C' was located between the transcription start site and the Shine-Dalgarno sequence. A GT-AG dinucleotide mutation was introduced at positions 8-9 of the consensus sequence to generate seven variant promoters: three mutated in one of the three sites, three mutated in two sites, and one mutated in all three sites. Whereas these promoters had reduced antibiotic (PI)-induced activity, their levels of expression in the absence of PI was higher. This suggested an unusual regulatory mechanism in which Pip could act either as an activator or repressor. Gel shift experiments revealed Pip or its homologues in many other Streptomyces species, suggesting that it is widely employed in the regulation of antibiotic resistance genes and perhaps secondary metabolism.