1 Introduction

The insolubility of Fe³⁺ in aqueous environments severely limits the obtainment of this essential element for nearly all bacteria. To overcome iron starvation, bacteria generally produce and secrete low-molecule-weight siderophores to chelate Fe³⁺ with very high affinity^[1]. To date, hundreds of siderophores with different structures have been discovered, and they contain key functional groups for Fe³⁺ solubilization, such as the commonly found catecholates, hydroxamates, and (α-hydroxy-) carboxylates^[2]. The siderophore-iron complexes are transported into cells by the bacterial acquisition system (i.e., ABC transporters), and then Fe³⁺ is released via different mechanisms^[3-6]. Siderophores showed various bioactivities, and played important roles in pharmaceutical development. Desferrioxamine and nocardichelins showed strong growth inhibitory activities toward tumor cells^[7]. Desferrioxamine has been developed to a drug (trade name Desferal), which is administrated to patients suffering from iron overload^[8-10]. Enterobactin could promote cancer cell apoptosis^[11]. Interestingly, some natural product antibiotics, such as albomycin and ferrimycin A, consist of a siderophore part and a structural part with antibacterial activity. These antibiotics exhibited potent antibacterial activities, partially due to the presence of siderophore moieties that facilitated the cellular uptake of these antibiotics once the siderophore-iron complexes were formed^[12-14]. Inspired by the chemical features of these natural products, a trojan horse strategy was developed by integrating two functional molecule parts into one structural scaffold, to form the so-called siderophore-antibiotic conjugates^[1]. Many of these conjugates were synthesized and developed to fight membrane-associated antibiotic resistance^{[1, 15-16]}.

Bacterial siderophores are biosynthesized mainly through two routes. One involves nonribosomal peptide synthetases (NRPSs), while the other is NRPS-independent and catalyzed by siderophore synthetases^[17-19]. The NRPS-mediated assembly-line biosynthetic mechanism has been well studied^[20]. One of the most representative NRPS-dependent siderophores is enterobactin, the cyclic trimer of 2, 3-dihydroxybenzoyl-l-serine (2, 3-DHBS)^[21]. Enterobactin is mainly produced by bacteria of the family Enterobacteriaceae, and shows very high affinity for Fe³⁺ (i.e., a pFe³⁺ of 35.5 at pH 7.4)^[22-23]. The biosynthetic process of enterobactin includes the formation and activation of 2, 3-DHB, and the following condensation (between 2, 3-DHB and Ser), elongation and cyclization steps catalyzed by the iterative one-module NRPS enzyme EntF^[24]. In contrast to the “one-module” architecture of EntF, a group of “two-module” NRPS homologs are also identified in some siderophore biosynthetic gene clusters (BGCs), the products of which enrich the structural diversity of catecholate-type siderophores by having a spacer amino acid between 2, 3-DHB and the ester core (e.g., griseobactin)^{[21, 25-27]}.

The Gram-positive bacteria Streptomyces are well known for their complex life cycles and capability of producing various natural products with therapeutic applications^[28]. They can also excrete different siderophores to increase access to iron. Desferrioxamines are the typical siderophores of streptomycetes, whereas some Streptomyces strains are also able to produce other siderophores, such as griseobactin, coelichelin, albomycin and enterobactin^{[21, 29-33]}. Among them, enterobactin [(DHB-^LSer)₃] and griseobactin [(DHB-^LArg-^LThr)₃] belong to tricatecholate-type siderophores, which are biosynthesized by aforementioned NRPS pathways^[34-35].

In this study, we analyzed the distribution of siderophore BGCs in 308 annotated Streptomyces genomes, and identified the orphan enterobactin BGCs, which lacked the biosynthetic enzymes of 2, 3-DHB, in S. albofaciens JCM 4342 and other strains. We further identified four enterobactin-derived linear natural products from S. albofaciens JCM 4342. These results suggested an interesting functional cross-talk between the orphan enterobactin BGC and the griseobactin BGC in S. albofaciens JCM 4342.

2 materials and Methods 2.1 Bioinformatics analyses of Streptomyces genomes

The siderophore BGCs in Streptomyces genomes were predicted using standalone BLASTp program^[36] with the enzymes from the BGCs of desferrioxamine, coelichelin, enterobactin, griseobactin, and albomycin as query sequences^{[21, 29-33]}. Global protein sequence alignment was performed using EMBOSS 6.5.0^[37]. The substrate specificities of adenylation domains were predicted by NRPSpredictor2 and NRPSsp web servers^[25-26].

2.2 Strain cultivation and fermentation conditions

S. albofaciens JCM 4342 (genome accession number in GenBank: PDCM01000002.1) was purchased from CGMCC (No. 4.1655). This strain was cultivated on ISP2 agar plates for 3−4 days for sporulation. Then, fresh spores were collected and cultivated in TSB for 24 h to prepare seed culture. Next, the seed culture was inoculated (2%, v/v) to 50 mL of iron-deficient fermentation medium (M/v, 2.0% soluble starch, 1.0% glucose, 0.5% peptone, 0.5% yeast extract, 0.4% NaCl, 0.05% KH₂PO_4, 0.05% MgSO₄·7H₂O, 0.2% CaCO₃, pH 7.0), and cultivated at 28 ℃ and 200 r/min for 5 days. On day 2, 1 g of XAD-16 resins was added to the fermentation culture.

2.3 HPLC, LC-MS, and LC-HRMS analyses of the products of S. albofaciens JCM 4342

Once the fermentation of S. albofaciens JCM 4342 (50 mL) was done, the XAD-16 resins were harvested, washed with distilled water, and allowed to air dry. Then, the resin was extracted with methanol (5 mL). After centrifugation (13 000 r/min, 1 min), 40 µL supernatant was analyzed by LC-MS. LC-MS (ESI source) experiments were performed on a triple-quadrupole LC-MS (Agilent 1260/6460) equipped with a diode array detector. An Agilent Zorbax SB-Aq column (3.5 µm, 2.1 mm100 mm) and the following pump method were used: 0–2 min, 5% acetonitrile (0.1% formic acid); 2−27 min, a linear gradient to 80% acetonitrile (0.1% formic acid); 27−30 min, 95% acetonitrile (0.1% formic acid) at a flow rate of 0.25 mL/min. The UV absorptions, along with the molecular weights (MWs), suggested that one target compound might be enterobactin. To further identify the products, the aforementioned methanol extracts were evaporated in vacuo, and then subjected to Sephadex LH-20 to afford ten fractions, all of which analyzed by analytical HPLC with a diode array detector. HPLC analyses were performed on an Agilent 1260 Infinity LC system equipped with an Agilent ZORBAX SB-C₁₈ column (5 µm, 4.6 mm×250 mm). The following pump method was used: 0−5 min, 5% acetonitrile (0.1% formic acid); 5−40 min, a linear gradient to 80% acetonitrile (0.1% formic acid); 40−45 min, a linear gradient to 95% acetonitrile (0.1% formic acid); 45−50 min, 95% acetonitrile (0.1% formic acid) at a flow rate of 1 mL/min. The ninth fraction containing the target compounds was analyzed by LC-HRMS (high resolution electrospray ionization mass spectra), which was performed using an Agilent 1200 HPLC separation module and a qTOF-MS 6520 mass detector with a diode array detector. An Agilent SB-C₁₈ column (1.8 µm, 4.6 mm×100 mm) and the following pump method were used: 0−5 min, 5% acetonitrile (0.1% formic acid); 5−30 min, a linear gradient to 60% acetonitrile (0.1% formic acid) at a flow rate of 1.0 mL/min.

2.4 Isolation and characterization of enterobactin- related natural products

A 10 L fermentation was carried out with the above mentioned seed and fermentation medium. Similarly, the resins were harvested and extracted with methanol, and then the crude extracts were fractionated using Sephadex LH-20. Compounds 1 and 2 were obtained through HPLC purification. Semi-preparation was performed on a Shimadzu LC-20AT system equipped with a Silgreen C₁₈ column (5 μm, 10 mm×250 mm). The following pump method was used: 0−5 min, 5% acetonitrile; 5−10 min, a linear gradient to 35% acetonitrile; 10−30 min, 35% acetonitrile at a flow rate of 3 mL/min. Finally compounds 1 (2.5 mg, 24.5 min) and 2 (2.0 mg, 18.2 min) were obtained.

All NMR spectra were conducted on a Bruker Avance Ⅲ 500 MHz NMR spectrophotometer equipped with a cryoprobe. The compounds in this study were dissolved in the deuterated DMSO (Cambridge Isotope Laboratories Inc). Chemical shifts were reported in ppm, and the residual solvent signal was at 2.50 ppm.

Compound 1 (=dehydrated 2, 3-DHBS trimer): yellow amorphous powder. HRESIMS m/z 670.151 8 [M+H]⁺ (calcd for C₃₀H₂₈N₃O₁₅, 670.152 0, Δ= –0.4 ppm).

Compound 2 (=dehydrated-DHBS dimer): yellow amorphous powder. HRESIMS m/z 447.103 4 [M+H]⁺ (calcd for C₂₀H₁₉N₂O₁₀, 447.104 0, Δ= –1.3 ppm).

Compound 3 (=linear 2, 3-DHBS trimer): HRESIMS m/z 688.162 2 [M+H]⁺ (calcd for C₃₀H₃₀N₃O₁₆, 688.162 6, Δ= –0.6 ppm).

Compound 4 (=linear 2, 3-DHBS dimer): HRESIMS m/z 465.114 2 [M+H]⁺ (calcd for C₂₀H₂₁N₂O₁₁, 465.114 5, Δ= –0.7 ppm).

3 Results and Discussion 3.1 Bioinformatic analyses of siderophore BGCs in Streptomyces

To have an overview of the distribution of siderophore BGCs in Streptomyces, we analyzed 308 annotated Streptomyces genomes available in NCBI Genome database (as of Dec, 2020) using the enzymes from desferrioxamine, coelichelin, griseobactin, and albomycin biosynthetic pathways^{[21, 29-33]}, as query sequences. Overall, about 30% strains each contain more than one siderophore BGCs. The BGCs of desferrioxamines are widely distributed in Streptomyces (found in 83% analyzed genomes) (Figure 1A), further supporting that the desferrioxamine-type siderophores play a primary role in iron uptake for streptomycetes^[13]. In addition, 73 BGCs of coelichelin and 21 BGCs of griseobactin were found, while only one BGC was identified to encode the biosynthesis of albomycin in Streptomyces globisporus strain TFH56 (Figure 1A). Most of these newly identified BGCs showed similar genetic organizations and high sequence identities (70%−100%) to previously reported siderophore BGCs in Streptomyces.

3.2 The genome of Streptomyces albofaciens JCM 4342 encodes two BGCs that potentially produce two groups of catecholate-type siderophores

Although previous studies reported the production of enterobactin in several Streptomyces strains including S. tandae Tü 901/8c and Streptomyces sp. Tü 6125^[35], the corresponding BGCs of enterobactin have not been identified in Streptomyces. Using EntF from the enterobactin BGC of E. coli as a query sequence, 18 BGCs were found to contain the EntF homologs (sequence identities ranging in 36%−40%) surrounded by enzymes responsible for 2, 3-DHB biosynthesis^[21], indicating that they could produce enterobactin-like siderophores (Figures 1A and 1B). Interestingly, four unusual BGCs each harboring an EntF homolog were observed in Streptomyces albofaciens JCM 4342 (ATCC 23873: PDCM01000002.1) and three Streptomyces rimosus strains (ATCC 10970: CP023688.1; WT5260: CP025551.1; R6-500: CP045803.1) (Figure 1C). However, they lacked the enzymes responsible for the biosynthesis of 2, 3-DHB, and therefore, they were designed as orphan enterobactin BGCs. The orphan enterobactin BGC (eno cluster) in S. albofaciens JCM 4342 was annotated in detail, in which the one-module NRPS enzyme EnoE (with 38% sequence identity to EntF) was also predicted to activate L-Ser (Table 1)^[38]. In addition, a 2, 3-DHB carrier protein (EnoD), and the enzymes related to product hydrolysis (EnoF) and export (EnoABC) were identified (Figure 1C, Table 1).

Besides the orphan enterobactin BGC, each of these four strains also had another BGC proposed for the biosynthesis of griseobactin, in which the biosynthetic enzymes of 2, 3-DHB were intact. The predicted griseobactin BGC (gsb cluster) in S. albofaciens JCM 4342 was annotated, and it highly resembled the griseobactin BGC from Streptomyces sp. ATCC 700974 (accession No. FN545130.1) (Figure 1C, Table 1)^{[21, 33]}. The two-module NRPS enzyme GsbE (with 72% sequence identity to GriE) was predicted to activate Arg and Thr as expected^[33]. It was noteworthy that, in the middle of gsb cluster, there was an insertion of 10 CDSs without any sequence similarity to enzymes in known BGCs of triscatechol siderophores (Figure 1C). Based on these analyses, the gsb cluster very likely would produce griseobactin or its derivatives. Taken together, S. albofaciens JCM 4342 might be able to produce two groups of siderophores.

3.3 Discovery of dehydrated 2, 3-DHBS trimer and dimer from Streptomyces albofaciens JCM 4342

Based on the above analyses, we were interested to know the siderophores that the strain JCM 4342 produced. The small scale fermentation of JCM 4342 was performed, and the crude extracts were fractionated before subjected to LC-MS analyses. The MS analyses revealed a product peak (compound 1) with an m/z of 670.151 8 [M+H]⁺ which seemingly accounted for the production of enterobactin (Figure 2A), whereas no griseobactin-like natural products were detected. To isolate compound 1, a 10 L fermentation was carried out. After XAD-16 adsorption, LH-20 filtration (6 cm×19 cm, eluted with 70% acetonitrile in water) and HPLC purification, compound 1 as well as its congener 2 were isolated. Their structures were determined based on HRMS and 1D/2D-NMR spectra.

The molecular formulas of 1 and 2 were assigned as C₃₀H₂₇N₃O₁₅ and C₂₀H₁₈N₂O₁₀ by HR-ESIMS, which showed [M+H]⁺ ion peaks at m/z 670.151 8 (calcd 670.151 5, Δ= –0.4 ppm) and 447.1034 (calcd 447.103 4, Δ= –1.3 ppm), respectively (Figure 2A). The 1D-NMR data revealed that the planar structure of 1 consisted of six carbonyl carbons, three benzenes (nine are proton bearing and six are oxygenated), three methenes (in particular, one terminal double bond at δ_H 6.50/5.77 and δ_C 109.5) and two nitrogen-bearing methines at δ_H 4.97/4.80 (m) (Table 2). These data suggested that 1 was not enterobactin but a dehydrated product of linear 2, 3-DHBS trimer (=N, N′, N″-tris(2, 3-dihydroxybenzoyl)- O-seryl-O-seryl serine). The key HMBC correlations further supported the above structural elucidation (Figures 2B). Compared to 1, 1D-NMR spectra of 2 also showed the α-aminoacrylyl residue signals (δ_H 6.54/5.81 and δ_C 109.4), but possessed additional proton signals corresponding to one more 2, 3-DHBS monomer in the structure (Table 2). Based on bioinformatics analyses, the adenylation domain of EnoE was predicted to use l-serine as a substrate. EntF, the homolog of EnoE, also utilized l-serine as the precursor for the biosynthesis of enterobactin^[38]. Taken together, the compounds 1 and 2 were determined as N, N′, N″-tris(2, 3-dihydroxybenzoyl)- O-(α-aminoacrylyl)-O-l-seryl l-serine (dehydrated 2, 3-DHBS trimer) and N, N′-bis(2, 3-dihydroxybenzoyl)- O-(α-aminoacrylyl)-O-l-serine (dehydrated 2, 3-DHBS dimer), respectively (Figure 2C). Further analysis of the LC-MS data of JCM 4342 revealed the other two products, which were proposed to be 2, 3-DHBS trimer (3, MW=687.154 3) and 2, 3-DHBS dimer (4, MW=464.106 3), respectively, whereas enterobactin was not detected (Figure 2C).

3.4 Proposed biosynthetic routes for enterobactin- related natural products

Apparently, based on structural comparisons, compounds 1−4 are enterobactin-related natural products. Except lacking the enzymes for 2, 3-DHB biosynthesis, the eno cluster in JCM 4342 encodes adequate enzymes to catalyze the rest biosynthetic steps of enterobactin and its hydrolysis (Figure 2C). As the ghd cluster (proposed for griseobactin biosynthesis) retains the enzymes responsible for 2, 3-DHB biosynthesis, we propose that the precursor 2, 3-DHB biosynthesized by the ghd cluster might be hijacked and incorporated into the Eno pathway, leading to the formation of compounds 1−4 (Figure 2C). Specifically, 2, 3-DHB was first biosynthesized by GdhABC, and then activated and loaded to the carrier protein EnoD by GhdE. Next, the iterative one-module NRPS enzyme EnoE catalyzed the elongation, and cyclization reactions to release the product enterobactin^[24]. As compounds 3 and 4^{[21, 40]} were previously shown to be the FeS-catalyzed hydrolysis products of enterobactin, EnoF (homolog of FeS) was herein predicted to carry out the same hydrolysis reactions to form 3 and 4. Compounds 1 and 2 were proposed to be derived from 3 and 4, respectively, through dehydrations catalyzed by unknown dehydratase(s). In addition, compound 2 might also be formed through hydrolysis of 1 by EnoF.

Compounds 1 and 2 were first reported as hydrolysis products of enterobactin in chemical reactions^[41-43]. Both compounds were later detected as natural products in the crude extracts of Streptomyces longisporous JCM4261 and exhibited inhibition activity against hepatitis C virus protease, whereas no MS and NMR data were reported for structural elucidation^[44]. Our study provided detailed chemical information, which enabled us to elucidate the structures of compounds 1 and 2. Interestingly, we showed that the biosynthesis of compounds 1−4 was likely involving the functional cross-talk between eno and gdh clusters. As compounds 3 and 4 remained the ability of binding Fe³⁺ shown by a previous study^[45], compounds 1 and 2 might also possess the iron chelating capability.

4 Conclusion

In this study, we systematically analyzed the distribution and conservation of siderophore BGCs in Streptomyces, from which the orphan enterobactin BGCs (lacking 2, 3-DHB biosynthetic enzymes) were identified in several strains. S. albofaciens JCM 4342, which harbors an orphan enterobactin BGC (eno cluster) and a griseobactin BGC (gdh cluster), was found to be able to produce the enterobactin-derived linear compounds 3 and 4, and their dehydrated products 1 and 2. Based on bioinformatic analyses, the gdh cluster was proposed to biosynthesize the 2, 3-DHB unit, and deliver it to the biosynthetic machinery encoded by the eno cluster, eventually leading to the formation of compounds 1−4. These results suggested an interesting synergistic biosynthetic mechanism accomplished by two siderophore BGCs. As orphan enterobactin BGCs and griseobactin BGCs also coexist in S. rimosus strains, similar biosynthetic process might occur in these strains. In addition, the discovery and characterization of compounds 1 and 2 provides new substrates for the further structural diversification to generate novel enterobactin-related siderophores^[21].

Acknowledgments: We thank Drs. WANG Wenzhao and REN Jinwei for the assistance in collection of LC-HRMS and NMR data, respectively.