Figure 1.
Biosynthetic pathway of the final Er product, Er-A.
Three DEBSs, EryAI-AIII, are responsible for the generation of the 16-membered lactone, 6-dEB; Tailoring enzymes catalyze sequential reactions, including two hydroxylations, two glycosylations, and one methylation, to obtain the final product, Er-A.
Figure 2.
The identified Er metabolites in Ac. erythraea YIM90600.
EB and Er-C are normal Er intermediates, while 3′-N-demethyl-Er-C, EH, and EI are novel Er congeners that have rarely been reported.
Table 1.
Bacterial strains and plasmids used and constructed in this study.
Figure 3.
Comparative analysis of the two gene clusters of Sa. erythraea NRRL2338 and Ac. erythraea YIM90600.
The genes share high identity (82–93 %) and similarity (88–96 %). Most of the genes exhibit the same order and direction as their homologues, except for eryBI and eryG, which are absent in the newly identified gene cluster.
Figure 4.
Proposed biosynthetic pathways of the Er metabolites in Ac. erythraea YIM90600 modeled on erythromycin biosynthesis in Sa. erythraea [25].
The EryAIII TE domain is supposed dual functional. The TE-catalyzed intramolecular cyclization releases the 14-membered 6-dEB via path a, and a 6-membered lactone via path b. Both serve as substrates for further modifications.
Figure 5.
HPLC-ESI-MS analyses of the fermentation cultures of Sa. erythraea EX101 and EX102.
(A) Total ion current chromatogram (i), and reconstructed base peak chromatogram for 6-dEB (ii) of the fermentation products of EX101. ESI-MS data recorded at the retention time of 27.06 min (iii). (B) Total ion current chromatogram (iv), and reconstructed base peak chromatogram for EB (v) of the fermentation products of EX102. ESI-MS data recorded at the retention time of 15.96 min (vi).
Figure 6.
HPLC-ESI-MS analyses of the in vitro enzymatic reactions catalyzed by EryFSa and EryFAc, respectively.
(A) Total ion current chromatograms indicating the in vitro conversion of 6-dEB to EB in the absence of active EryFAc (i), in the presence of active EryFAc (ii), or in the presence of active EryFSa (iii). (B) Total ion current chromatogram (iv) and reconstructed base peak chromatogram for 6, 18-epoxy-EB (v) of the EryFAc reaction mixture. (C) Total ion current chromatogram (vi) and reconstructed base peak chromatogram for 6, 18-epoxy-EB (vii) of the EryFSa reaction mixture.
Figure 7.
HPLC-ESI-MS analysis of the fermentation culture of Sa. erythraea EX103.
Total ion current chromatogram (i), and reconstructed base peak chromatograms for 6-deoxy-Er-A (ii), Er-A (iii), 6-deoxy-Er-B (iv), Er-B (v), 6-deoxy-Er-C (vi), Er-C (vii), 6-deoxy-Er-D (viii), and Er-D (ix) are recorded. Note that 6-deoxy-Er-A and Er-B, as well as 6-deoxy-Er-C and Er-D share the same molecular weights and similar polarities, their base peaks are thus overlapping.
Figure 8.
HPLC-ESI-MS analyses of the in vitro enzymatic reactions catalyzed by EryKSa and EryKAc, respectively.
Total ion current chromatograms indicating standard Er-B (red circle, i) and Er-A (blue lozenge, ii), the in vitro conversion of Er-B to Er-A in the absence of active EryKSa (iii), in the presence of active EryKSa (iv), in the absence of active EryKAc (v), or in the presence of active EryKAc (vi).