Genomics-driven discovery of taiwachelin, a lipopeptide siderophore from Cupriavidus taiwanensis

Abstract

A genome mining study led to the identification of a previously unrecognised siderophore biosynthesis gene cluster in the nitrogen-fixing bacterium Cupriavidus taiwanensis LMG19424. Based upon predicted structural residues, a convenient strategy for an NMR-assisted isolation of the associated metabolite was designed. The structure of the purified siderophore, taiwachelin, was fully characterized by spectroscopic methods and chemical derivatisation.

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Introduction

The development of cost-effective high-throughput sequencing technology and the resulting surge in whole genome sequences has promoted a paradigm shift in natural product research in the past few years. Rather than relying on bioactivity-guided screening for the discovery of useful molecules, many research groups nowadays exploit the information that derives from genomic data to identify new biosynthetic pathways and their associated chemical compounds.¹ This strategy, which is commonly referred to as genome mining, is particularly useful in the case of siderophores, small molecules that are secreted by microorganisms and plants to mobilize ferric iron from the environment under conditions when this transient metal becomes scarce.² Several siderophores exhibit a peptidic structure, which is assembled through the concerted action of nonribosomal peptide synthetases (NRPSs) or NRPS-independent siderophore synthetases (NIS).³ The multifunctional NRPS complexes typically inhere a co-linear logic, in which the arrangement of catalytic domains determines the sequence of enzymatic reactions.^3a Through analyses of their substrate-specifying domains, it is often possible to predict the amino acid building blocks that are utilised during the assembly process.⁴ This knowledge, together with the iron-responsive production of siderophores, facilitates the isolation and structure elucidation of new members of this compound class.⁵

We recently reported the genomics-inspired discovery of an NRPS-derived siderophore from the freshwater bacterium Cupriavidus necator H16 (syn. Ralstonia eutropha H16).⁶ The isolated compound, cupriachelin, is unusual in that it exhibits structural and physicochemical features that are typically associated with siderophores from oceanic bacteria, but only rarely found in other microbial groups, namely a lipopeptide backbone and photoreactivity.⁷ We were hence interested in whether the production of cupriachelin is specific to C. necator H16, or whether other taxonomically related strains also depend on such compounds for iron sequestration. To clarify this, we analysed available genomic sequences from soil-derived and plant-associated members of this bacterial group for the presence of siderophore biosynthesis pathways that bear genetic hallmarks of fatty acid incorporation. In the case of cupriachelin, the attachment of the fatty acid side-chain was proposed to be catalysed by the starter condensation domain of an NRPS.⁶ Alternative mechanisms, which are also conceivable for lipidation reactions, involve the combined action of a fatty acyl-AMP ligase and an acyl carrier protein (ACP) or require a single ACP alone.⁸ By integrating the search for fatty acid-loading enzymes in the sequence-based screening for novel siderophore biosynthesis pathways, it should hence be possible to identify loci that code for the production of lipopeptide iron(III) ligands.

Here, we report the successful application of this genome mining strategy resulting in the discovery of the taiwachelin biosynthesis gene cluster in the nitrogen-fixing bacterium Cupriavidus taiwanensis LMG19424. The product of the identified pathway represents a previously unknown lipopeptide siderophore, which was isolated and physicochemically characterized as part of this study.

Results and discussion

In silico screening for pathways to lipopeptide siderophores

For the bioinformatic analysis, we selected 20 environmental strains of the family Burkholderiaceae whose genomes had been fully assembled, annotated and deposited in GenBank. The chosen strains represent the genera Burkholderia, Cupriavidus, Ralstonia and Polynucleobacter.⁹ Their genomic sequences were initially screened for the presence of NRPS and NIS encoding genes, using biochemically characterised representatives as driver sequences in BLASTP analyses according to a previously described method.¹⁰ To identify putative siderophore loci, the genomic regions surrounding the primary hits were subsequently analysed for the presence of open reading frames involved in siderophore export and uptake, as well as hydroxamate, salicylate and catecholate formation.

Except for the endosymbiotic Polynucleobacter species, which possess comparatively small genomes, all investigated strains were found to harbor siderophore biosynthesis genes that were distributed over one or two independent loci (Table S1†). While the analysed Burkholderia genomes feature only NRPS-dependent siderophore pathways, NIS genes appear to be quite common in the genera Ralstonia and Cupriavidus. In many cases, the identified loci could be associated with the production of known siderophores based upon genetic precedence. The ornibactin assembly line appears thus to be one of the most prevalent siderophore biosynthetic machineries among environmental Burkholderia strains, which is consistent with previous studies on human pathogenic Burkholderia isolates.¹¹ In contrast, gene clusters for the biosynthesis of micacocidin, yersiniabactin or staphyloferrin B are commonly encountered in Ralstonia solanacearum genomes.¹² Among the gene clusters, which could not be correlated with a known product, we identified one candidate locus that met our search criteria for the biosynthesis of a lipopeptide siderophore. The corresponding gene cluster was detected on chromosome 2 of the rhizobium C. taiwanensis LMG19424,¹³ and includes 18 ORFs between RALTA_B1222 and RALTA_B1239 covering 41.9 kb of contiguous DNA; the genes of the cluster, which is in the following referred to as taiwachelin or tai cluster, have been renamed in this study from taiA to taiR (Table S2†).

Architecture of the tai gene cluster and proposed biosynthesis

The gene cluster comprises four successive NRPS genes (taiDEFG), which are proposed to assemble the peptidic backbone of the siderophore (Fig. 1). The MbtH-like protein TaiB, the type-II thioesterase TaiC and the phosphopantetheinyl transferase TaiQ have possible roles in activating and tuning the NRPS biosynthetic machinery,¹⁴ whereas the lipase TaiN and the monooxygenase TaiP are likely involved in supplying a fatty acid starter unit and the amino acid building block N^δ-hydroxy-Orn, respectively. The cyclase TaiA and the acyltransferase TaiO can be envisioned to catalyze tailoring reactions. Five genes within the cluster (taiH, taiI, taiK, taiM, and taiR) are associated with siderophore-mediated iron transport and turnover.

Fig. 1 Organisation of the tai biosynthesis gene cluster (top). Molecular processing line deduced from taiD–G (bottom). Domain notation: FAAL, fatty acyl-AMP ligase; ACP, acyl carrier protein; C, condensation; TauD, hydroxylase; A, adenylation; PCP, peptidyl carrier protein; E, epimerase; TE, thioesterase.

Sequence alignments suggest a functional relatedness of the N-terminal AMP-binding domain of TaiD to acyl-AMP ligases that prime lipid starter units.¹⁵ TaiD is therefore proposed to initiate the biosynthesis by loading an unspecified fatty acid onto the NRPS assembly line. The consecutive incorporation of monomeric amino acids is then carried out by the six NRPS extension modules that are encoded by taiE, taiF and taiG. The respective building blocks were predicted from the specificity-conferring code of adenylation domains,⁴ and their configuration in the final natural product was deduced from the presence or absence of epimerisation domains in the NRPS modules.¹⁶ According to these analyses, the fully assembled siderophore should feature L-Asp, D-allo-Thr, L-Asp, D-N^δ-hydroxy-Orn, L-Ser and L-N^δ-hydroxy-Orn as amino acid constituents. The C-terminal TauD domain in TaiD may account for the hydroxylation of either or both predicted Asp residues. TaiO is closely related to CucL, an enzyme implicated in the acylation of N^δ-hydroxy-Orn with a 3-hydroxybutanoyl (Hbu) moiety.⁶ We assumed a similar reaction in taiwachelin biosynthesis, as the resulting hydroxamate function could serve as a bidentate ligand for the coordination of ferric iron. It was unclear, however, whether either or both N^δ-hydroxy-Orn moieties are converted by TaiO. The termination module of TaiG features a thioesterase domain and should thus catalyse the hydrolytic offload of the acyl-S-enzyme intermediate from the assembly line.

A search for the proposed peptide sequence in a natural product database¹⁷ suggested that the product from the tai gene cluster is likely to be novel, thereby minimising the chance to retrieve a known compound in a subsequent fermentation study.

Genomics-guided isolation of taiwachelin

Select changes in media composition are known to increase or even trigger natural product biosynthesis.¹⁸ In order to induce the production of the predicted siderophore, we cultivated C. taiwanensis LMG19424 in an iron-deficient H-3 minimal medium. This approach appeared highly promising, since the tai gene cluster represents the only putative siderophore biosynthesis locus that was identified on the C. taiwanensis genome during our in silico screening. To recover the siderophore from the fermentation broth, we resorted to the adsorber resin XAD-2, which has proven useful for the adsorption of amphiphilic siderophores.¹⁹ The extract that was eluted from the adsorber resin gave a strong response in the chrome azurol S (CAS) assay,²⁰ confirming the presence of iron(III)-chelating molecules. In contrast, the extract from an iron-saturated C. taiwanensis culture did not decolourise the iron dye complex. From the chromatographic comparison of the two extracts, it was evident that the observed CAS activity in the former was likely due to one peak (Fig. 2). To obtain structural evidence that the identified peak may actually represent the sought molecule, the crude extract from the iron-deficient culture was subjected to NMR analyses prior to starting the isolation process. The assignment of amino acid-related proton resonances is generally difficult in extracts of heterotrophically grown microorganisms, because of signal overlapping with media components. We thus decided to look for signals that would indicate the predicted incorporation of the Hbu residue. Previous studies on Hbu-featuring siderophores had revealed that the methylene protons of acylated Hbu exhibit resonances in the ¹H NMR spectrum, which can be easily distinguished from those of media-derived amino acids within the same chemical shift range based upon their distinctive double doublet splitting pattern.^6,21 Furthermore, we evaluated the presence of hydroxamate groups by a ¹H,¹⁵N HMBC experiment, as the corresponding correlations are quite specific and only rarely detected in extracts from bacterial cultures. Both NMR-based analyses supported the assumption that the extract contained the product of the tai gene cluster. While the ¹H NMR spectrum showed discrete dd signals between 2.40 and 3.00 ppm, we also detected ¹H,¹⁵N HMBC-derived connectivities that could be ascribed to long-range couplings with hydroxamate nitrogens in addition to typical amine and amide proton correlations (Fig. S11†). HPLC clean-up was then guided by CAS activity and NMR analyses, respectively, as the UV profile of the identified peak lacked a conspicuous chromophore. This approach yielded 20 mg of taiwachelin, which was isolated as a white crystalline powder.

Fig. 2 Chromatographic profiles of extracts from C. taiwanensis LMG19424 grown in an H-3 medium under iron-deficient conditions (A) and in the presence of 190 μM Fe(NH₄) citrate (B).

Structure elucidation of taiwachelin

The FT-IR spectrum of taiwachelin (1, Fig. 3) indicated the presence of hydroxyl and amide functions, as expected from the bioinformatic prediction. High resolution ESI-MS yielded m/z 963.4879 for the [M + H]⁺ ion, which is consistent with a molecular formula of C₄₁H₇₀O₁₈N₈ and corresponds to 11 degrees of unsaturation. The double bond equivalents were assigned to 10 carbonyl moieties and one ring structure based upon an inspection of the resonances in the ¹³C spectrum. ¹H,¹H COSY data enabled an elucidation of the amino acid constituents of taiwachelin by resolving the individual spin systems of β-hydroxy-Asp, Thr, Asp, Ser as well as two discrete N^δ-hydroxy-Orn residues. One of the latter two had to be cyclic, as its δ-methylene protons showed strong long-range interactions in the HMBC spectrum with the associated carbonyl carbon of the same spin system. The second N^δ-hydroxy-Orn residue was found to be N-acylated with a 3-hydroxybutanoyl moiety. The combined results from the ¹H,¹H COSY, DEPT135, and HSQC spectra eventually revealed a dodecanoyl moiety, thus concluding the assignment of the 41 carbon resonances of taiwachelin. The sequence of the six amino acids and the fatty acid was initially established from HMBC correlations between the amide protons and their ²J-coupled carbonyl carbons. MALDI-TOF/TOF fragmentation then proved the deduced planar structure (Fig. S1†). To resolve the configuration of the single amino acids in taiwachelin, we applied Marfey's method following an acid-catalysed hydrolysis of the native natural product.²² This study unequivocally confirmed the exclusive presence of the single enantiomers L-threo-β-hydroxy-Asp, D-allo-Thr, L-Asp and L-Ser in the acid hydrolysate. In the case of N^δ-hydroxy-Orn, both the D- and the L-form were identified (Table S4†). Although Marfey's analysis provided no information on the position of the two N^δ-hydroxy-Orn enantiomers in the natural product, it is reasonable to assume the sequence depicted in Fig. 3, which is fully consistent with the arrangement of E domains in the taiwachelin assembly line. The 3-hydroxybutanoyl (Hbu) moiety was determined to be in the S configuration by chiral GC analysis following an established protocol.⁶

Fig. 3 Structure of taiwachelin (1).

Complexing properties and photoreactivity of taiwachelin

It is obvious that the structure of taiwachelin features three bidentate coordination sites for iron(III). The six oxygen ligands come from the two hydroxamate functions and the β-hydroxy-Asp residue, allowing the formation of an octahedral complex. MS studies were carried out to test the coordination ability of taiwachelin for divalent and trivalent metal ions, respectively. Similar to cupriachelin, the siderophore from C. taiwanensis selectively chelated Fe³⁺, but did not coordinate Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺. The stoichiometry of the complex formed between iron(III) and taiwachelin was 1 : 1 (Fig. S3†).

The structural features of taiwachelin, particularly the presence of a β-hydroxy-Asp moiety, suggested a susceptibility to UV light when complexed to iron(III). Marine lipopeptide siderophores and also cupriachelin were shown to undergo photoreductive dissociation under these conditions, which is accompanied by the release of ferrous iron.^6,7 To explore whether taiwachelin is likewise prone to photochemical reactions, we monitored the shifting of the iron(II) level in an aqueous solution of its ferric-ion complex after exposure to sunlight. For quantification, Fe²⁺ was trapped with the specific ligand bathophenanthroline disulfonate (BPDS) and the absorbance of the developing coordination complex was measured at 535 nm. Control reactions that were kept in the dark remained at very low absorption values of 0.003 ± 0.002 during the entire assay, whereas the absorbance of sunlight-treated samples increased from 0.001 ± 0.001 to 0.073 ± 0.006 in the same period. MS analyses confirmed the loss of the intact iron(III)–taiwachelin complex in the latter.

Conclusions

We successfully identified a novel iron-chelating natural product from the nitrogen-fixing bacterium C. taiwanensis LMG19424 by a combined genomic and analytical screening approach. The structure of the metabolite was predicted with sufficient accuracy from the molecular processing line to enable an NMR-assisted isolation. Furthermore, the knowledge about the domain architecture of the taiwachelin biosynthesis enzymes expedited the determination of the absolute configuration, underlining the great potential of genome mining techniques.¹ Taiwachelin shares many structural features and physicochemical properties with siderophores from oceanic bacteria.⁷ This resemblance is particularly evident in comparison to loihichelin D, a photoreactive lipopeptide siderophore from a marine Halomonas sp., which also features a dodecanoate moiety and a terminal cyclic N^δ-hydroxy-Orn residue.¹⁹ The production of photoreactive, lipopeptide iron(III) ligands appears thus not to be a characteristic of the previously investigated C. necator strain H16, but is probably more common among taxonomically related bacteria. Further investigation on the role of taiwachelin's photoreactivity as well as cellular uptake experiments are in progress in our laboratory.

Experimental

General experimental procedures

LC-MS experiments for metabolic profiling and Marfey's analysis were conducted on an Agilent 1100 series HPLC-DAD system coupled with an MSD trap (Agilent) operating in alternating ionization mode and an Antek 8060 HPLC-CLN-detector (Antek Instruments GmbH) using a C8 column (Zorbax Eclipse XDB C8, 150 × 4.6 mm, 5 μm; Agilent). A linear gradient of methanol in water + 0.1% trifluoroacetic acid (10% → 90% methanol within 15 min; flow rate 1 mL min⁻¹) was used for metabolic profiling. Analytical HPLC for Marfey's analysis of serine was conducted on a Shimadzu UFLC liquid chromatography system equipped with a Nucleosil 100 C18 column (250 × 4.6 mm, 5 μm; CS Chromatographie Service). The separation was accomplished by using a linear gradient from solvent A (1% methanol, 5% acetonitrile in 10 mM ammonium formate) to solvent B (1% methanol, 60% acetonitrile in 10 mM ammonium formate) over 40 min with a flow rate of 1 mL min⁻¹ and wavelength monitoring at 365 nm.²³ High resolution mass determination was carried out using an Exactive Mass Spectrometer (Thermo-Scientific). One- and two-dimensional MALDI-TOF MS data using post-source decay were acquired on a Bruker Ultraflex spectrometer (Bruker Daltonics). NMR spectra were recorded at 300 K on a Bruker Avance III 500 MHz spectrometer with dimethylsulfoxide-d₆ as a solvent and internal standard. ¹H,¹⁵N HMBC spectra were referenced externally to urea. IR spectra were obtained with a benchtop FT/IR 4100 spectrometer (JASCO).

Bioinformatic studies

Genomic sequences of bacteria were retrieved from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov). These sequences were screened for putative pathways to lipopeptide siderophores using homology-based alignments against a handmade library of conserved biosynthesis genes.¹⁰ Initial hits were grouped by physical proximity and mapped onto the respective genome using Vector NTI (Invitrogen).

Growth conditions

For the isolation of taiwachelin, C. taiwanensis LMG19424 was grown in an H-3 mineral medium lacking ammonium ferric citrate: 1 g L⁻¹ aspartic acid, 2.3 g L⁻¹ KH₂PO₄, 2.57 g L⁻¹ Na₂HPO₄, 1 g L⁻¹ NH₄Cl, 0.5 g L⁻¹ MgSO₄·7H₂O, 0.5 g L⁻¹ NaHCO₃, 0.01 g L⁻¹ CaCl₂·2H₂O, and 5 mL L⁻¹ SL-6 trace element solution.⁷ The strain was shaken (160 rpm) at 30 °C for 4 days. The influence of Fe³⁺ on taiwachelin production was tested in the same medium containing 190 μM ammonium ferric citrate.

Isolation of taiwachelin

The supernatant from a 12 L culture was separated from the cells by centrifugation at 9500g. Siderophores that had been secreted into the culture broth during cultivation were recovered by adsorption onto 150 g L⁻¹ Amberlite XAD-2 (Supelco) overnight. After two washing steps with distilled water, the adsorbed metabolites were eluted from the resin with 3 × 200 mL of methanol. The eluate was concentrated under vacuum to dryness prior to resuspension in 2 mL of methanol. Crude taiwachelin was isolated from this extract on a Shimadzu UFLC liquid chromatography system equipped with a Nucleodur C18 HTec column (VP 250 × 10 mm, 5 μm; Macherey-Nagel) using an isocratic flow of 75% methanol in water + 0.1% trifluoroacetic acid at a flow rate of 3.5 mL min⁻¹ with wavelength monitoring at 210 nm. Final purification was accomplished with successive HPLC separation on a Nucleodur PFP column (VP 250 × 10 mm, 5 μm; Macherey-Nagel) using the same conditions as described above.

Taiwachelin (1). White powder: FT-IR ν_max/cm⁻¹ 3291, 2924, 2845, 1644, 1537. ¹H-NMR (500 MHz, dimethylsulfoxide-d₆) δ_H [ppm] (J [Hz]) 0.85 (3 H, t, J 7.0, H-41), 1.03 (3 H, d, J 6.4, H-25), 1.07 (3 H, d, J 6.3, H-17), 1.23 (14 H, m, H-33–H-39), 1.26 (2 H, m, H-40), 1.46 (2 H, m, H-32), 1.47 (2 H, m, H-11), 1.55 (2 H, m, H-12), 1.70 (1 H, m, H_b-4), 1.90 (2 H, m, H-3), 1.92 (1 H, m, H_a-4), 2.14 (2 H, t, J 7.3, H-31), 2.37 (1 H, dd, J 15.0, 6.3, H_b-15), 2.52 (1 H, dd, J 15.0, 6.3, H_a-15), 2.53 (1 H, dd, J 16.5, 7.2, H_b-20), 2.70 (1 H, dd, J 16.5, 6.8, H_a-20), 3.42 (1 H, m, H_b-13), 3.48 (2 H, m, H-2), 3.50 (1 H, m, H_a-13), 3.59 (2 H, d, J 7.2, H-8), 3.82 (1 H, quint, J 6.4, H-24), 4.00 (1 H, sext J 6.3, H-16), 4.19 (1 H, dd, J 8.0, 6.4, H-23), 4.29 (1 H, m, H-10), 4.31 (1 H, m, H-7), 4.34 (1 H, ddd, J 10.7, 8.4, 5.1, H-5), 4.48 (1 H, d J 2.7, H-28), 4.56 (1 H, ddd, J 7.8, 7.2, 6.8, H-19), 4.74 (1 H, dd, J 9.2, 2.7, H-27), 7.73 (1 H, d, J 8.0, NH), 7.85 (1 H, d, J 9.2, NH), 7.91 (1 H, d, J 7.6, NH), 7.95 (1 H, d, J 7.8, NH), 8.09 (1 H, d, J 8.4, NH), 8.15 (1 H, d, J 7.8, NH). ¹³C-NMR (125 MHz, dimethylsulfoxide-d₆), δ_C [ppm] 14.0 (C-41), 19.7 (C-25), 20.3 (C-3), 22.2 (C-40), 22.8 (C-12), 23.4 (C-17), 25.3 (C-32), 27.3 (C-4), 28.7 (C-33), 28.8 (C-34), 28.9 (C-38), 29.0 (C-35), 29.1 (C-11), 29.1 (C-36), 29.1 (C-37), 31.4 (C-39), 35.3 (C-31), 36.1 (C-20), 41.5 (C-15), 46.7 (C-13), 49.7 (C-19), 49.9 (C-5), 51.2 (C-2), 52.7 (C-10), 55.1 (C-7), 55.5 (C-27), 58.8 (C-23), 62.1 (C-8), 63.2 (C-16), 67.2 (C-24), 70.5 (C-28), 164.9 (C-1), 169.4 (C-26), 169.9 (C-7), 170.0 (C-22), 170.3 (C-18), 171.4 (C-9), 171.4 (C-14), 171.9 (C-21), 172.7 (C-30), 173.0 (C-29). HR-ESI-MS: m/z 963.4879 [M + H]⁺ (calcd for C₄₁H₇₁O₁₈N₈, 963.4881).

Assignment of the absolute configuration

The configurations of the amino acid constituents of taiwachelin were determined following acid hydrolysis and derivatisation with Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide, L-FDAA, Sigma-Aldrich) by coelution experiments with L-FDAA-derivatised amino acid standards. To this end, 500 μg purified taiwachelin was dissolved in 400 μL concentrated HI and heated at 110 °C for 3 h. The solution was lyophilised, and the dried hydrolysate was resuspended in 10 μL of water and 20 μL of 1 M NaHCO₃. Derivatisation was carried out with 170 μL of 1% L-FDAA in acetone at 37 °C for 1 h. The products were lyophilised and prepared for LC-MS analysis by dissolving in 1 mL of 50% acetonitrile. Standards for co-chromatography were prepared by reacting 50 μL of 50 mM aqueous amino acid solution with 20 μL of 1 M NaHCO₃ and 100 μL of 1% L-FDAA in acetone at 37 °C for 1 h. The lyophilised products were then dissolved in 1 mL of 50% acetonitrile. LC-MS analysis was performed under the aforementioned conditions with a detection wavelength of 365 nm. The configuration of Hbu was determined after hydrolysing 500 μg taiwachelin with 1 mL of 0.3 N HCl. The reaction was carried out at room temperature for 4 days after which excess reagent was removed under vacuum. The hydrolysate was resuspended in water and purified over a Sep-Pak RP-18 cartridge. Following the evaporation of the eluent, 100 μL of trifluoroacetic anhydride–methylene chloride (1 : 1, v/v) was added, and the mixture heated at 100 °C for 10 min. Excess reagents were removed with a stream of argon. Methylene chloride, 100 μL, was added, and the resulting trifluoroacetyl ester analysed using a Trace GC Ultra (Thermo) coupled in parallel to an FID detector and a Polaris-Q ion trap mass detector (Thermo). Separation was established on a chiral silica column (25 m × 0.25 mm ID) coated with Chir-D-Val (Varian) using a continuous He gas flow of 1.5 mL min⁻¹ and a temperature gradient from 40 to 200 °C at a rate of 5 °C min⁻¹. Co-chromatography of the derivatised taiwachelin hydrolysate was conducted against a derivatised Hbu standard in S configuration as well as a mixture containing both enantiomers.

Photoreactivity of taiwachelin

Reduction of the complexed ferric iron to ferrous iron was examined using a previously reported protocol.²⁴ Briefly, each reaction contained 100 μM taiwachelin, 10 μM FeCl₃ and 40 μM of the trapping agent BPDS (Fluka) in a PBS buffer (pH 7.5). The reactions were either exposed to sunlight or kept in the dark for 4 h. The formation of Fe(BPDS)₃²⁺ was recorded before and after exposure to sunlight/darkness by measuring the absorption at 535 nm using a Genesys 10 UV spectrophotometer (Thermo). All experiments were run in triplicate.

Acknowledgements

We gratefully acknowledge financial support from the Bundesministerium für Bildung und Forschung (BMBF GenoMik-Transfer programme; grant# 0315591A). We thank A. Perner and T. Neuwirth (HKI Jena, Department of Biomolecular Chemistry) for recording high resolution ESI-MS data and for assistance with GC-MS measurements, respectively. We also thank M. Poetsch (HKI Jena, Department of Molecular and Applied Microbiology) for MALDI-TOF/TOF measurements.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c2ob26296g

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