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

Abstract

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.

Organization of the biosynthetic gene cluster for the polyketide macrolide mycinamicin in Micromonospora griseorubida

“…The proposed biosynthetic pathways for synthesis of the deoxysugars involved in mycinamicin synthesis. Similar routes to desosamine have been previously proposed in erythromycin biosynthesis [18], and routes to mycinose have been proposed in tylosin biosynthesis [16].…”

Effects of Pregnane Glycosides on Food Intake Depend on Stimulation of the Melanocortin Pathway and BDNF in an Animal Model

“…These include a bulky lipophilic cinnamoyl moiety attached at the C12 position, additional hydroxylation at C18 position, and four rare deoxysugar moieties (oleandrose, digitoxose, and cymarose) attached at the C3 position (Figure 3). A number of other bioactive compounds possess deoxysugars attached to their aglycones; in many cases, these deoxysugars are essential for their biological activity as in the case of cardiac glycosides, 30 antibiotics, 31 or antitumor agents. 32 Although incarnatin treatment achieved stronger suppression of food intake in females than in males (Figure 5), its effect on body weight and body weight gain was similar for both genders, indicating that female rats compensated for reduced food intake with an unknown mechanism.…”

Microbial aldo-keto reductases

“…The organization of co-expressed genes into biosynthetic clusters has provided some clues for the roles of enzymes involved in the biosynthesis of antibiotics, for example, in the synthesis of sugars required for the glycosylation of erythromycin and tylosin. The eryBII gene encoding an oxidoreductase from Sacharopolyspora erythraea (AKR12B) was identi¢ed as part of the erythromycin biosynthetic gene cluster in the pathway from NDP-4-keto-6deoxy-D-glucose to NDP-L-mycarose [42] which was substantiated through analysis of metabolites in a strain spe-ci¢cally deleted for eryBII [43]. By analogy, the tylCII gene identi¢ed in the tylosin biosynthetic gene cluster, encoding AKR12A from Streptomyces fradiae, and aveB-VIII/aveBI from the avermectin cluster encoding AKR12C from Streptomyces avermitilis, are also thought to play roles as reductases in L-mycarose and L-oleandrose biosynthesis respectively [44], though a role for these latter two enzymes through the use of gene mutation/deletion has not been proven.…”