Biosynthesis of the nosiheptide indole side ring centers on a cryptic carrier protein NosJ

Nosiheptide is a prototypal thiopeptide antibiotic, containing an indole side ring in addition to its thiopeptide-characteristic macrocylic scaffold. This indole ring is derived from 3-methyl-2-indolic acid (MIA), a product of the radical S-adenosylmethionine enzyme NosL, but how MIA is incorporated into nosiheptide biosynthesis remains to be investigated. Here we report functional dissection of a series of enzymes involved in nosiheptide biosynthesis. We show NosI activates MIA and transfers it to the phosphopantetheinyl arm of a carrier protein NosJ. NosN then acts on the NosJ-bound MIA and installs a methyl group on the indole C4, and the resulting dimethylindolyl moiety is released from NosJ by a hydrolase-like enzyme NosK. Surface plasmon resonance analysis show that the molecular complex of NosJ with NosN is much more stable than those with other enzymes, revealing an elegant biosynthetic strategy in which the reaction flux is controlled by protein–protein interactions with different binding affinities.

37 °C/200rpm until the OD 600 reached 0.7-0.9. IPTG was added to a final concentration of 0.5 mM and the culture was grown at 37°C for an additional 3 h. Cells were harvested by centrifugation at 4000 x g for 15 min at 4 °C, and the pellet was resuspended in 20 mL of Buffer A (20 mM NaH 2 PO 4 , 500 mM NaCl, 0.5 mM imidazole, 10% glycerol, pH 7.5 at 25°C) and lysed by sonication. The sample was centrifuged at 21000 x g for 1hour at 25°C. The pellet was resuspended in 20 mL of denaturing Buffer B (6 M guanidine hydrochloride, 20 mM NaH 2 PO 4 , 500 mM NaCl, 0.5 mM imidazole pH 7.5 at 25 o C). The insoluble portion was removed by centrifugation (21000 x g for 1hour) and the supernatant was clarified through 0.45-mm syringe filters. The 6 x His NosM peptide was purified by immobilized metal affinity chromatography (IMAC) approach at room temperature using a HiTrap chelating HP nickel affinity column (GE Healthcare). The desired NosM peptide was eluted using 3 x CV (column volume) of Elution Buffer (4M guanidine hydrochloride, 20 mM Tris, pH 7.5 at 25°C, 100 mM NaCl, 1M imidazole) before washing by 20 x CV of Washing Buffer (4M guanidine hydrochloride, 20 mM Tris, pH 7.5 at 25°C, 100 mM NaCl, 50 mM imidazole). Desalting was performed by solid phase extraction (SPE) using BioSelect TM reversed-phase C4 column (214SPE3000, Vydac). Collected fractions were lyophilized to afford white fluffy solid, which was stored at -20°C upon further use.

Preparation of the Reconstituted NosN
Production, purification and reconstitution of NosN were performed according to the previously reported protocol. 3 Protein purification was performed in an anaerobic glove box (Coy Laboratory Product Inc., USA) with less than 5 ppm of O 2 . The pellet was resuspended in 30 mL of lysis buffer (40mM Tris, 200 mM NaCl, 10mM imidazole, 10% glycerol, pH 8.0) and was lysed by sonication on ice. Cell debris was removed via centrifugation at 21000 x g for 1 hr at 4 °C. The supernatant was passed through a column containing 4 mL of high-affinity Ni-NTA resin (Qiagen Co. Ltd) pre-equilibrated with lysis buffer, and the column was then washed using 50 mL wash buffer (40 mM Tris, 200mM NaCl, 40 mM imidazole, 10% glycerol, pH 8.0). The protein fractions were collected using 10 mL of elution buffer (40 mM Tris, 200 mM NaCl, 500 mM imidazole, 10% glycerol, pH 8.0).
The desired fractions were combined and concentrated using an Amicon Ultra-15 Centrifugal Filter Unit and analyzed by SDS-PAGE (12% Tris-glycine gel).
For reconstitution of the NosN [4Fe-4S] cluster, dithiothreitol (DTT) was added to the purified protein solution to a final concentration of 5 mM. Fe(NH4) 2 (SO4) 2 solution was then added slowly to a final concentration of 800 µM. After 15min incubation at the room temperature, Na 2 S solution was added carefully to a final concentration of 800 µM. After further incubation on ice for 7-10 h, the resulting dark solution was subjected to desalt on a PD-10 (GE) column pre-equilibrated with the elution buffer (40mM Tris, 25mM NaCl, 10 mM DTT and 10% (v/v) glycerol, pH 8.0). The eluted protein fraction was collected and concentrated, and was used directly for in vitro assay or stored at -80°C upon further use.

Construction of the nosI Mutant
To inactivate nosI, a 2.2 kb upstream fragment and 1.8 kb downstream fragment were amplified separately from the S. actuosus genomic DNA by PCR using the primer pairs nosI-L-For and nosI-L-Rev, and nosI-R-For and nosI-R-Rev, respectively (Supplementary Table 1). The resulting fragments were purified using a PCR purification kit (CWbiotech Co.Ltd, China) and then cloned into the EcoRI /HindIII site of pKC1139 using ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech Co. Ltd, China) to give the in-frame deletion construct pFDU1711. This plasmid was then introduced into S. actuosus via E. coli-Streptomyces conjugation. The nosI in-frame deletion mutant (designated as mFDU1711) was screened and its genotype was confirmed by PCR and DNA sequencing.

Construction of the nosK mutant
To inactivate nosK, a 2.2 kb upstream fragment and 2.1 kb downstream fragment were amplified separately from the S. actuosus genomic DNA by PCR using the primer pairs nosK-L-For and nosK-L-Rev, and nosK-R-For and nosK-R-Rev, respectively (Supplementary Table 1). The resulting fragments were purified using a PCR purification kit (CWbiotech Co.Ltd, China) and then cloned into the EcoRI /HindIII site of pKC1139 using ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech Co. Ltd, China) to give the in-frame deletion construct pFDU1713. This plasmid was then introduced into S. actuosus via E. coli-Streptomyces conjugation. The nosK in-frame deletion mutant (designated as mFDU1713) was screened and its genotype was confirmed by PCR and DNA sequencing.

In trans Complementation of nosI in S. actuosus
A ~1.3kb PCR product containing nosI was amplified by PCR using the primer nosI-C-For and nosI-C-Rev (Supplementary Table 1), and the resulting DNA fragment was cloned into pMD19-T to yield pNosI-MD19. The 1.3 kb HindIII/XbaI fragment was recovered from pNosI-MD19, which was co-ligated with a 0.4 kb EcoRI/HindIII fragment harboring the promoter PermE* into the EcoRI/XbaI site of pSET152 to yield pFDU1712, in which the 1.3kb fragment containing nosI is under the control of the PermE* promoter. pFDU1712 was then introduced into mFDU1711 using E.
coli-Streptomyces conjugation to yield the recombinant strain mFDU1712.

In trans Complementation of nosK in S. actuosus
A ~0.8 kb PCR product containing nosK was amplified by PCR using the primer nosK-C-For and nosK-C-Rev (Supplementary Table 1), and the resulting DNA fragment was cloned into pMD19-T to yield pNosK-MD19. The 0.8 kb HindIII/XbaI fragment was recovered from pNosK-MD19, which was co-ligated with a 0.4 kb EcoRI/HindIII fragment harboring the promoter PermE* into the EcoRI/XbaI site of pSET152 to yield pFDU1714, in which the 0.8 kb fragment containing nosK is under the control of the PermE* promoter. pFDU1714 was then introduced into mFDU1713 using E.
coli-Streptomyces conjugation to yield the recombinant strain mFDU1714.

Nosiheptide Production and Analysis
Fermentation and production of nosiheptide was performed similarly to a previously reported procedure. 6 100 μl spore suspension the S. actuosus wild type or mutant strains was inoculated into a 2L flask containing 500 ml of seed medium (sucrose 2.0%, corn steep liquor 3.0%, peptone 0.5% and CaCO 3 0.5%) and grown for 36 h at 28 °C, 220 rpm. The culture was then transferred into 10 x 500 ml fermentation medium (L-glutamate 0.5%, L-arginine 0.1%, L-aspartate 0.1%, K 2 HPO 4 ·7H 2 O 0.05%, MnSO 4 ·H 2 O 0.002%, and glucose 4%, the pH was adjusted to 7.25 before autoclaving) for continuous cultivation at 28 °C, 220 rpm for 7 days. The mycelia cake was collected by centrifugation (4000 x g for 25 min), and was soaked with 3 L acetone overnight, whereas the culture was adjusted to a pH of ~4 before extracted by n-butanol. The organic solvent was removed on a rotary evaporator, and the residue was combined and mixed with 100 ml 100 mM sodium citrate (pH 4.0), and the resulting mixture was extracted three times with an equal volume of ethyl acetate. The organic layer was then combined and taken to dryness on a rotary evaporator, and the residue was redissolved in methanol and analyzed by HPLC. The column (Agilent 1100 with a Zorbax SB-C18 column (9.4 mm × 25 cm) was equilibrated with 85% solvent A (H 2 O containing 0.1% TFA) and 15% B (CH 3 CN), and developed with the following program: 0 to 3 min, constant 85% A/15% B; 3 to 6 min, a linear gradient from 85% A/15% B to 60% A/40% B; 6 to 12 min, constant 60%A/40% B; 12 to 19 min, a linear gradient from 60% A/40% B to 45% A/55% B; 19 to 22 min, a linear gradient from 45% A/55% B to 15% A/85% B; 22 to 28 min, constant 15% A/85% B; and 28 to 32 min, a linear gradient from 15% A/85% B to 85% A/15% B.

Preparation of Methyl N-acetyl-S-(3-methyl-indole-2-carbonyl)cysteinate (11)
Methyl cysteinate hydrochloride salt (MCA) (1.03 g, 6 mmol) was added to a pyridine-toluene solution (2 mL pyridine in 20 mL toluene), and acetic anhydride (0.56 mL, 7.2 mmol) was subsequently added in a dropwise manner at 0 o C. After a further stirring for 2 h, the reaction mixture was poured into 30 ml cold water, and toluene was removed by stratification. The aqueous layer was adjusted to pH 7 ~ 8 by saturated NaHCO 3 , and the resulting solution was then stirred at RT for another 30 min. The mixture was then adjusted to pH 5 ~ 6 by 1M HCl, extracted with AcOEt (20 mL×3), and the combine organic layer was dried by anhydrous NaSO 4 . The crude methyl N-acetylcysteinate (MACA, 0.85 g) was obtained as yellow oil, which was used for the next step

S3
Preparation of S3 was performed by following a previous report. 8 To a solution of (R)-pantothenic acid (S2) (9.5 g, 43.4 mmol) in acetone (250 mL), 2-methoxyprop-1-ene (13.5 mL,141 mmol) and pTsOH·H 2 O (0.45 g, 2.39 mmol) were added subsequently. The mixture was stirred for 3~4 hours at room temperature and then concentrated in vacuo to give a yellow solid, which was washed with water and acetone. After dried at room temperature, a pale yellow solid was obtained Hz, -NH-), 12.26 (s, 1H, -COOH).
The organic layer were dried over Na 2 SO4 and concentrated in vacuo to yield a colorless oil. The crude product was used directly without further purification.
The organic layer were dried over Na 2 SO 4 and concentrated in vacuo to yield yellow oil. The crude product was purified using preparative TLC plate (V(Petroleum) / V(ethyl acetate) = 1:3) to give

Preparation of Compound 5 (in the main text)
Compound S5 was dissolved in AcOH / H 2 O (2:1, 2 mL) and the mixture was stirred for 5 h. The

Synthesis of CoA thioesters
Under the protection of N 2 , 50 µmol MIA or DMIA, 30 mg PyBOP and 15 mg K 2 CO 3 were dissolved in 2 ml THF in a 15 mL serum bottle, and 39 µm CoA tri-lithium salt dissolved in 1 ml ddH 2 O was added to the solution. The reaction mixture was stirred at room temperature for 3 hrs, and was then filtered using 0.45µm filter membrane. The resulting CoA thioester was purified from the resulting solution using semi-preparative HPLC, which was carried out on a Thermo Scientific Dionex