File S1 - Identification and Characterization of a New Erythromycin Biosynthetic Gene Cluster in Actinopolyspora erythraea YIM90600, a Novel Erythronolide-Producing Halophilic Actinomycete Isolated from Salt Field

This file contains Figure S1-Figure S14 and Table S1. Figure S1. Construction and genotype verification of the eryFSa-deleting mutant, Sa. erythraea EX101. (A) Sa. erythraea EX101 with a 771 bp deletion within eryFSa is constructed via a double-crossover event. (B) Gel electrophoresis analysis of the PCR products amplified from the genomic DNAs of Sa. erythraea ZL2001 (lane 1), EX101 (lane 2), and the single-crossover exconjugant (lane 3), using primer pair pFf/pFr. Figure S2. Construction and genotype verification of the eryBVSa-deleting mutant, Sa. erythraea EX102. (A) Sa. erythraea EX102 with a 786 bp deletion within eryBVSa is constructed via a double-crossover event. (B) Gel electrophoresis analysis of the PCR products amplified from the genomic DNAs of Sa. erythraea ZL2001 (lane 1), EX102 (lane 2), and the single-crossover exconjugant (lane 3), using primer pair pBVf/pBVr. Figure S3. Construction of the gene complementation mutant, Sa. erythraea EX103. The gene fragment PermE*-eryFAc is introduced into the artificial attB sites of Sa. erythraea EX101 via the actinophage ΦC31 integrase-mediated site-specific recombination. Figure S4. SDS-PAGE analysis of the purified recombinant proteins with 6 x His-tag at the N terminus. Recombinant EryFSa (lane 1) exhibited a molecular mass of 47.4 kDa, recombinant EryFAc (lane 2) exhibited a molecular mass of 47.6 kDa, recombinant EryKSa (lane 3) exhibited a molecular mass of 46.0 kDa, and recombinant EryKAc (lane 4) exhibited a molecular mass of 46.3 kDa. Figure S5. CO difference spectra of the cytochrome P450 oxidases, EryFSa, EryFAc, EryKSa and EryKAc. UV-vis absorbance of both EryFSa (A) and EryFAc (B) exhibits a Soret peak at 423 nm under reducing condition, which shifts to 448 nm after binding of CO. UV-vis absorbance of both EryKSa (C) and EryKAc (D) exhibits a Soret peak at 420 nm under reducing condition, which shifts to 448 nm after binding of CO. Figure S6. HR-ESI-MS analyses of 6-dEB, EB, and the proposed 6, 18-epoxy-EB. A, purified 6-dEB with a molecular formula as C21H38O6, showing [M+Na]+ at m/z 409.2579, B, purified EB with a molecular formula as C21H38O7, showing [M+Na]+ at m/z 425.2517, C, an EryFAc-catalyzed enzymatic reaction containing a compound with a molecular formula as C21H36O7, showing [M+Na]+ at m/z 423.2342. Figure S7. Proposed fragmentation scheme for 6-dEB and the ESI-MS-MS product ion spectrum of 6-dEB. Figure S8. Proposed fragmentation scheme for EB and the ESI-MS-MS product ion spectrum of EB. Figure S9. Substrate binding spectra for 6-dEB bound to the cytochrome P450 oxidases, EryFSa and EryFAc. UV-vis absorbance of both EryFSa (A) and EryFAc (B) exhibits a Soret peak at 423 nm (green), which shifts to 392 nm after the addition of 6-dEB (blue). The Soret peaks at 392 nm increase with higher concentration of 6-dEB dissolved in the protein solutions (red). Figure S10. HPLC-ESI-MS analysis of the fermentation culture of Sa. erythraea ZL2001. Total ion current chromatogram (i), and reconstructed base peak chromatograms for Er-A (ii), Er-B (iii), Er-C (iv), and Er-D (v) are recorded. Figure S11. HPLC-ESI-MS analysis of the fermentation culture of Sa. erythraea EX101. Total ion current chromatogram (i), and reconstructed base peak chromatograms for 6-deoxy-Er-A (ii), 6-deoxy-Er-B (iii), 6-deoxy-Er-C (iv), and 6-deoxy-Er-D (v) are recorded. Figure S12. HPLC-ESI-MS analysis of the fermentation culture of Sa. erythraea EX102. Total ion current chromatogram (i), and reconstructed base peak chromatograms for 5-O-desosaminyl-EB (ii), 12-hydroxyl-5-O-desosaminyl-EB (iii) are recorded. Figure S13. HPLC-ESI-MS analysis of the fermentation culture of Sa. erythraea EX103. Total ion current chromatogram (i), and reconstructed base peak chromatograms for EH (ii), 6, 18-epoxy-EB (iii) are recorded. Figure S14. HPLC-ESI-MS analyses of the in vitro enzymatic reactions catalyzed by EryFAc and EryKAc, and by EryFSa and EryKSa, respectively. (A) Total ion current chromatogram (i) and reconstructed base peak chromatogram for EB (ii), 6, 18-epoxy-EB (iii), 12-hydroxyl-EB (iv), EH (v) of the EryFAc and EryKAc reaction mixture. (B) Total ion current chromatogram (i) and reconstructed base peak chromatogram for EB (ii), 6, 18-epoxy-EB (iii), 12-hydroxyl-EB (iv), EH (v) of the EryFSa and EryKSa reaction mixture. Table S1. Primers used for genetic manipulation and protein expression in this study. (DOC)