Elucidation of salicylate attachment in celesticetin biosynthesis opens the door to create a library of more efficient hybrid lincosamide antibiotics

Combinatorial biosynthesis for more efficient antibiotics: 150 novel lincosamides prepared by combining lincomycin and celesticetin biosynthetic pathways.


Bacterial strains
The Streptomyces lincolnensis ∆lmbIH mutant strain was derived from lincomycin producer S. lincolnensis ATCC 25466 as decribed previously. 1 The strain was used for production of lincomycin intermediates 1a and 2a. Celesticetin producer Streptomyces caelestis ATCC 15084 was used for production of celesticetin and O-demethylcelesticetin and as a source of genomic DNA for gene

Cultivation of streptomycete strains in liquid media
The seed culture of S. lincolnensis ∆lmbIH or S. caelestis was prepared by inoculation of spores from MS plates into 50 mL of the YEME medium 2 without sucrose and incubated in 500 mL flat-bottom boiling flasks at 28 °C. Two mL of 24 h seed culture were inoculated into 40 mL of AVM 3 (S. lincolnensis) or GYM 4 medium (S. caelestis) and incubated in 500 mL flat-bottom boiling flasks at 28 °C for 120 h. The cells were centrifuged at 5000 g at 20 °C for 15 min and the supernatant was used for LC-MS analyses or purification of metabolites of interest.

In vivo incorporation experiments with benzoic acid derivatives
Plates with 10 mL of agar GYM medium supplemented with 2 mM benzoic acid derivative were inoculated with spores of S. caelestis. The plates were incubated at 28 °C for 8 days. Three discs 5 mm in diameter were cut from the agar medium, inserted into 500 µL methanol, vortexed 1 min and incubated in a sonication bath for 15 min. Then, 200 µL of the methanolic extract was removed, evaporated to dryness, reconstituted in 50 µL methanol:water 50:50 (v/v) and analyzed by LC-MS.

Preparation of desalicetin and O-demethydesalicetin from celesticetin and O-demethylcelesticetin and purification of lincomycin intermediates 1a and 2a, lincosamides CELIN and ODCELIN, and intermediate 47
Celesticetin and O-demethylcelesticetin were purified from the culture broth of S. caelestis. Intermediates 1a and 2a were purified from the culture broth of the S. lincolnensis ∆lmbIH mutant strain. The compounds were purified as follows. An Oasis HLB 6cc 200 mg cartridge (hydrophiliclipophilic balanced sorbent, Waters, USA) was conditioned with 5 mL methanol, equilibrated with 5 mL water, and then 50 mL cultivation broth was loaded. Subsequently, the cartridge was washed with 5 mL water and absorbed substances were eluted with 15 mL methanol. The eluent was evaporated to dryness, reconstituted in 2 mL methanol, and the extracts were injected into the HPLC apparatus equipped with flow controller 600, autosampler 717, and UV detector 2487 operating at 194 nm (Waters, USA). Data were processed with Empower 2 software (Waters, USA). The analytes were separated on the Luna C18 chromatographic column (250 × 15 mm I.D., particle size 5 µm, Phenomenex, USA) with the two component mobile phase, A and B, consisting of 0.1% formic acid and methanol, respectively. The analyses were performed under a linear gradient program (min/%B) 0/5, 31/27.5 followed by a 9-min column clean-up (100% B) and a 9-min equilibration (5% B), at a flow rate of 3 mL min -1 . The fractions containing celesticetin and O-demethylcelesticetin were treated with 0.1mM NaOH overnight, resulting in complete conversion of the compounds into desalicetin and O-demethydesalicetin, respectively. Intermediates 1a, 2a as well as desalicetin and O-demethydesalicetin were subjected to another HPLC purification using the XTerra Prep RP18 column (150 × 7.8 mm I.D., particle size 5.0 μm, Waters, USA). The analytes were eluted using the isocratic program with 1 mM ammonium formate (pH 9.0):acetonitrile (90:10 v/v) as mobile phase. Compounds CELIN, ODCELIN and 47 were purified from the scaled-up in vitro reactions -see below the paragraph Preparation of hybrid lincosamides from intermediates 1a and 2a; point (1) applies for compound 47 and points (1) and (2) apply for CELIN and ODCELIN. Proteins in the in vitro assays were precipitated by 98% formic acid (30 µL per 1 mL sample) and the supernatant was subjected to purification using Oases HLB cartridges and HPLC as described above for purification of intermediates 1a and 2a with the following exceptions. Two component mobile phase, A and B, consisting of 0.1% formic acid and acetonitrile, respectively, was used. The analyses were performed under a linear gradient program (min/%B) 0/30, 40/65 followed by a 9-min column clean-up (100% B) and a 9-min equilibration (30% B). Single purification step by HPLC was considered as sufficient. The fractions containing the separated compounds were checked for purity (>95% -LC with UV detection at 220 nm; no significant cross-contamination).
Heterologous production and purification of CcbF, Ccb1, Ccb2, Ccb4, and Ccb5 The ccbF, ccb1, ccb2, ccb4, and ccb5 genes were PCR-amplified from the genomic DNA of S. caelestis using the primer pairs listed in Table S1. The ccbF and ccb2 genes were inserted into the pET42b vector (Novagen), and the ccb1, ccb4, and ccb5 genes were inserted into the pET28b vector (Novagen). The resulting constructs were used to produce soluble C-terminally (CcbF and Ccb2) or N-terminally (Ccb1, Ccb4, and Ccb5) His-tagged fusion proteins in E. coli BL21 (DE3) with the GroES and GroEL chaperonins co-expressed. Overproduction was induced with 0.4 mM IPTG and was performed at 17 °C for 20 h. The cells were harvested by centrifugation (4200 rpm, 20 min, 4 °C), and cell-free extracts were prepared using ultrasonic homogenization in a binding buffer consisting of 20 mM TRIS pH 8, 100 mM NaCl, 10% glycerol, and 20 mM imidazole followed by centrifugation (9000 rpm, 25 min, 4 °C). The proteins were purified from the cell-free extracts using a 1 mL HiTrap TM Ni 2+ column (GE Healthcare). Ccb2 was eluted with binding buffer supplemented with 100 mM imidazole, Ccb4 and Ccb5 were eluted with binding buffer supplemented with 200 mM imidazole and CcbF and Ccb1 were eluted with binding buffer supplemented with 250 mM imidazole. Imidazole was eliminated from the purified proteins by buffer exchange using 30-kDa Amicon cartridges (Millipore) and binding buffer without imidazole. Ccb4 was stable for at least a week at 4 °C with no decrease in activity; CcbF, Ccb1, Ccb2, and Ccb5 were stable for at least a week at 4 °C or a month at -80 °C with no decrease in activity. Protein concentration was determined with a Nanodrop ND-1000 Spectrophotometer (Thermo Scientific, USA) at 280 nm using extinction coefficients calculated from the protein sequences (98 890 for CcbF; 91 440 for Ccb1, 70 360 for Ccb2, 28 420 for Ccb4, and 24 410 for Ccb5).  Table S1. Primers used for amplification of S. caelestis genes. Restriction sites used for insertion into the vector are underlined.

Gel filtration
The monomeric or oligomeric form of Ccb1 was determined by gel filtration with a Superose 12 HR 10/30 column (Pharmacia) in a buffer containing 20 mM Tris pH 8, 100 mM NaCl, 10% glycerol; the flow rate was 0.5 mL min -1 . BSA was used as a standard.

Blue native electrophoresis
This experiment was performed as described by Schägger et al. 5 in a linear acrylamide concentration gradient of 8-18%.

Bioinformatic tools
The BLASTX and BLASTP were used for prediction of functions of encoded proteins . The multiple sequence alignment of WS/DGAT proteins was generated using the MAFFT and visualized in the Geneious.

Preparation of hybrid lincosamides from desalicetin or
Reactions (1) and (2) were mixed and incubated at 30 °C for additional 2 h.

Fig. S1
Multiple sequence alignment of Ccb1 and characterized WS/DGAT enzymes. The alignment was generated using the MAFFT web tool and was visualized with Geneious software. Residues that are identical or similar are highlighted. The conserved HHxxxDG motif is in the rectangle.    -The benzoic acid derivative incorporated into the lincosamide is indicated by the number assigned to the derivative in Figure 3, e.g. CEL-2 contains the derivative 2.