Search

Additional file 3: Figure S3. Inactivation of nysB in S.ahygroscopicus S91. a. Construction of the recombinant plamid pDNB; b. Double crossover validation of the recombinant strain S91-ΔNB; c.Verification of sequencing in the recombinant strain S91-ΔNB.

Additional file 4: Figure S4. Inactivation of ttmD in S.ahygroscopicus S91-ΔNB. a. Construction of the recombinant plasmid pDTD; b. Double crossover validation of the recombinant strain S91-ΔNBΔTD; c. Verification of sequencing in the recombinant strain S91-ΔNBΔTD.

Additional file 8: Table S2. Strains, plasmids and primers used in this study.

Additional file 5: Figure S5. Cloning and overexpression of ttmRIV. a. Construction of the recombinant plamid pETRIV; b. PCR analysis of the recombinant strain S91-ΔNBΔTD::TRIV, M. DL2000 (2.0k, 1.0k, 0.75k, 0.5k, 0.25k, 0.1k), 1. S91-ΔNBΔTD::pETRIV/PB-1& TRIV-R (1.1k), 2. S91-ΔNBΔTD/PB-1&TRIV-R, 3. S91-ΔNBΔTD::pSET152/PB-1&TRIV-R.

Additional file 7: Table S1. Production analysis in S.ahygroscopicus S91 and its mutants.

Additional file 6: Figure S6. Cloning and overexpression of ttmD. a. Construction of the recombinant plamid p2ETD; b. PCR analysis of the multicope ttmD recombinant strains, M. DL2000, 1. S91-ΔNB::pETD/PB-1&TD-R (1.7k), 2. S91-ΔNB::p2ETD/PB-1&TD-R (1.7k), 3. S91-ΔNB::p3ETD/PB-1&TD-R (1.7k), 4. S91-ΔNB/PB-1&TD-R, 5. S91-ΔNB::pSET152/PB-1&TD-R.

Additional file 1: Figure S1. Biosynthesis of tetramycin.

Additional file 2: Figure S2. LC-MS analysis of tetramycin and nystatin in Streptomyces ahygroscopicus S91. a. The HPLC profile of Streptomyces ahygroscopicus S91 fermentation products; b. The UV absorption spectra and MS of tetramycin B; c. The UV absorption spectra and MS of nystatin; d. The UV absorption spectra and MS of tetramycin A.

Additional file 9: Figure S9.1H NMR and 13C NMR spectroscopic analyses of compound (8) reduced with NaBH4 or NaBD4. (a) 1H NMR spectroscopic analysis of compound (8) reduced with NaBD4. 1H NMR (600 MHz, D2O) δ 5.40 (d, J = 3.5 Hz, 1H), 5.01 (d, J = 3.7 Hz, 1H), 4.14 (dd, J = 10.9, 3.7 Hz, 1H), 3.95–3.92 (m, 1H), 3.91 (d, J = 12.9 Hz, 1H), 3.81–3.76 (m, 1H), 3.72 – 3.66 (m, 3H), 3.56 (d, J = 1.3 Hz, 1H), 3.56 – 3.53 (m, 1H), 3.48 (ddd, J = 11.9, 6.2, 3.3 Hz, 4H), 3.44–3.37 (m, 2H), 2.83 (s, 3H), 2.45 (dt, J = 12.6, 4.2 Hz, 1H), 1.96–1.92 (m, 1H), 1.88 (dd, J = 12.6, 4.1 Hz, 1H), 1.84 (t, J = 8.7 Hz, 1H), 1.72 (dd, J = 14.0, 2.8 Hz, 1H), 1.48–1.41 (m, 1H), 1.26 (s, 3H). (b) 13C NMR spectroscopy analysis of the compound (8) reduced with NaBD4. 13C NMR (151 MHz, D2O) δ 101.14, 96.49, 83.43, 80.38, 73.63, 72.02, 71.10, 69.83, 64.76 (d, J = 432.03 Hz), 63.32, 62.44, 49.50, 49.06, 48.81, 34.42, 27.80, 23.78, 21.23, 20.82.

Additional file 1: Figure S1. In-frame deletion of genB4 in M. echinospora. (a) Schematic representation of the in-frame deletions in wild-type M. echinospora; (b) Confirmation of M. echinospora △genB4 by PCR with the primers, B4Y1 and B4Y4. The arrows indicate the expected size of the PCR fragments in the wild type and mutants. (c) Confirmation of M. echinospora △genK△genB4 by PCR with the primers, B4Y1 and B4Y4. The arrows indicate the expected size of the PCR fragments in the wild type and mutants.

Additional file 2: Figure S2. (1) and (6) reduced with NaBH4. (1) and (6) had the same retention times in HPLC-ELSD when separated by cation-exchange chromatography. (1) was reduced by NaBH4, whereas (6) was not.

Additional file 8: Figure S8. Characterization of purified recombinant GenB4. (a) SDS-PAGE analysis of purified GenB4 (51.9 kDa). The production of N-His6-tagged GenB4 was carried out in E. coli BL21(DE3). The acrylamide percentage of the SDS-PAGE gels was 12%. (b) UV–vis absorption spectrum of the purified recombinant proteins, His6-GenB4.

Additional file 5: Figure S5.1H NMR and 13C NMR spectroscopic analyses of intermediate (1). (a) 1H NMR spectroscopic analysis of intermediate (1). 1H NMR (600 MHz, D2O) δ 6.30 (t, J = 4.1 Hz, 1H), 5.49 (s, 1H), 4.96 (d, J = 3.7 Hz, 1H), 4.10 (dd, J = 10.9, 3.6 Hz, 1H), 3.94–3.89 (m, 1H), 3.86 (d, J = 12.7 Hz, 2H), 3.69–3.61 (m, 2H), 3.46–3.39 (m, 2H), 3.39–3.32 (m, 2H), 2.79 (d, J = 5.3 Hz, 3H), 2.59 (s, 2H), 2.49 (dt, J = 20.1, 4.2 Hz, 1H), 2.43 (dt, J = 12.6, 4.2 Hz, 1H), 2.26 (s, 3H), 1.82 (q, J = 12.7 Hz, 1H), 1.22 (s, 3H). (b) 13C NMR (151 MHz, D2O) δ 195.94 (s), 146.75 (s), 113.70 (s), 101.19 (s), 97.10 (s), 83.14 (s), 80.08 (s), 73.35 (s), 69.83 (s), 67.62 (s), 66.24 (s), 63.36 (s), 48.15 (s), 45.69 (s), 38.65 (s), 34.38 (s), 27.52 (s), 24.45 (s), 23.99 (s), 20.80 (s).

Additional file 4: Figure S4. Mass-spectra analysis of the intermediates in the mutants.

Additional file 10: Figure S10. Mass-spectra analysis of the transamination product of 6′-deamino-6′-oxogentamincin C1a (8).

Additional file 3: Figure S3. Mass-spectra analysis of the intermediates in the mutants.

Additional file 7: Figure S7. HPLC-ELSD analysis of fermentation production by complementation strains and the original strains.

Additional file 14: Table S1. Primers used in the present study.

Additional file 6: Figure S6. Complementation of the genB4 disrupting strain. (a) Map of the genetic complementation vector, pEAP1B4. The phrdb1 and B4dn are primers used to check the complementation strains. (b) Confirmation of the complementation strain by PCR. The arrows indicate the expected size of the PCR fragments in the original strain and the mutants.

Additional file 12: Figure S12. Analysis of the effect of different aminotransferases on transamination of 6′-deamino-6′-oxogentamincin C1a (8) to gentamicin C1a (9). GenB1, GenB2, GenB3, and GenB4 were separately added after 1 h of incubation of the GenB4 reaction system containing exogenous PLP and the amino donor, L-Glu.

Additional file 13: Figure S13. Mass-spectra analysis of (11) and (10).

Additional file 2: Figure S2. 1H NMR spectrum of the new compound from kanJK expression strains.

Additional file 1: Figure S1. Schematic representation and confirmation of recombination strains. (A) Gene disruption of genK and genP. (B) Expression of kanJ and kanK by site specific intergration. (C) Expression of kanJ and kanK with the promoter PermE*. (D) Expression of kanJ and kanK with the native promoter of genP. (E) Expression of kanJ and kanK with promoter PhrdB.

Additional file 3: Table S1. Sequence of primers used in this study.