Elucidation of the ferrichrome siderophore biosynthetic pathway in albomycin-producing Streptomyces sp. ATCC 700974

Abstract

Sideromycins are a unique subset of siderophores comprising of a siderophore conjugated to an antimicrobial agent. The “Trojan horse” antibiotic albomycins are unique sideromycins consisting of a ferrichrome-type siderophore conjugated to a peptidyl nucleoside antibiotic. They exhibit potent antibacterial activities against many model bacteria and a number of clinical pathogens. Earlier studies have provided significant insight into the biosynthetic pathway of the peptidyl nucleoside moiety. We herein decipher the biosynthetic pathway of the ferrichrome-type siderophore in Streptomyces sp. ATCC 700974. Our genetic studies suggested that abmA, abmB, and abmQ are involved in the formation of the ferrichrome-type siderophore. Additionally, we performed biochemical studies to demonstrate that a flavin-dependent monooxygenase AbmB and an N-acyltransferase AbmA catalyze sequential modifications of L-ornithine to generate N⁵-acetyl-N⁵-hydroxyornithine. Three molecules of N⁵-acetyl-N⁵-hydroxyornithine are then assembled to generate the tripeptide ferrichrome through the action of a nonribosomal peptide synthetase AbmQ. Of special note, we found out that orf05026 and orf03299, two genes scattered elsewhere in the chromosome of Streptomyces sp. ATCC 700974, have functional redundancy for abmA and abmB, respectively. Interestingly, both orf05026 and orf03299 are situated within gene clusters encoding putative siderophores. In summary, this study provided new insight into the siderophore moiety of albomycin biosynthesis and shed light on the contingency of multiple siderophores in albomycin-producing Streptomyces sp. ATCC 700974.

Siderophores are low molecular weight iron chelating molecules that play a central role in maintenance of iron homeostasis. They are biosynthesized and secreted by various microorganisms to scavenge iron from the surrounding environment under iron-restricted conditions (1). Based on the characteristic functional group, siderophores are generally classified into four main families: hydroxamates, catecholates, carboxylates, and pyoverdines. Ferrichromes, one of the most common hydroxamate-based siderophores, are produced mainly by fungi and a few strains of bacteria (2). They are featured by a cyclic hexapeptide consisting of three N-acyl-N-hydroxyl-L-ornithine and three additional amino acids, with two variable residues (alanine, serine, or glycine) and a third residue being a glycine. The acyl groups in this family are acetyl, malonyl, cis-anhydromevalonyl, and trans-anhydromevalonyl, and trans-β-methylglutaconyl (3).

Sideromycins are a unique subset of siderophores that are comprised of an antibacterial moiety covalently linked to a siderophore. They are actively transported into bacterial cells through siderophore uptake pathway, and the antibacterial moiety was then delivered in a “Trojan horse” fashion (1). These siderophore-antibiotic conjugates are promising drug candidates for the treatment of various bacterial infections, especially those caused by multidrug-resistant pathogens. Over the past few decades, only a few naturally occurring sideromycins have been identified (1, 4). Among them, albomycins are the most widely known natural sideromycins. Albomycin consists of a ferrichrome-type siderophore and a 6′-amino-4′-thioheptose nucleoside that are linked via amide linkages to a serine residue. The ferrichrome-type siderophore consists of three tandem N⁵-acetyl-N⁵-hydroxyornithine residues. Different components of albomycins (δ₁, δ₂, and ε) differ only in the C₄ substituent (R) of the pyrimidine nucleoside (Fig. 1A). When albomycin δ₂ is transported into the bacterial cell, it will be hydrolyzed by host peptidases to release the thionucleoside SB-217452, which blocks protein synthesis by acting as a specific inhibitor of seryl-tRNA synthetase. Thus, albomycins have potent antibacterial activities against many model bacteria, as well as a number of clinical pathogens (5).

Figure 1.

Proposed biosynthetic pathway of albomycins.A, chemical structures of albomycins. B, biosynthetic gene cluster governing the biosynthesis of albomycins. C, pathway for the biosynthesis of albomycins. Part shaded in navy blue indicates the ferrichrome siderophore. The dashed line indicates the site of cleavage to release the SB-217452. The three naturally occurring albomycin congeners differ mainly in C₄ substituent (R) of the pyrimidine nucleoside as indicated. AbmB, AbmA, and AbmQ were proposed to participate in the biosynthetic process from 2 to 5. AbmQ was highlighted to show its unusual domain organization. Biosynthetic process from 6 to 11 proceeds through the actions of AbmH, AbmD, AbmK, AbmF, and AbmJ. AbmE and AbmI were responsible for the tailoring modifications. AbmC was proposed to catalyze the assembly of the siderophore moiety with SB-217452.

The gene cluster responsible for albomycin biosynthesis has been identified in Streptomyces sp. ATCC 700974 (6). The contiguous region consists of 25 complete ORFs including ORFs 1–7 and 18 genes from abmA to abmR (Fig. 1B). A few recent studies have been dedicated to biosynthetic process of the thionucleoside SB-217452 (6, 7, 8). These studies established that the formation of SB-217452 proceeds via multiple steps through tandem reactions of AbmH, AbmD, AbmI, AbmE, AbmK, AbmF, and AbmJ (Fig. 1C). It was hypothesized that three genes, abmA, abmB, and abmQ, might be involved in the formation of the ferrichrome siderophore (6, 9). However, there is no experimental evidence to support this assumption. In this study, we provided genetic evidence that abmA, abmB, and abmQ are responsible for the formation of the ferrichrome-type siderophore. Biochemical studies suggested that a flavin-dependent monooxygenase AbmB and an N-acyltransferase AbmA catalyze sequential hydroxylation and acetylation of L-ornithine to form the N⁵-acetyl-N⁵-hydroxyornithine building blocks. Interestingly, we also noticed functional redundancy for abmA and abmB by two genes scattered beyond the boundaries of albomycin gene cluster. This study provided significant insight into the concise biosynthetic pathway of ferrichrome-type siderophore during albomycin biosynthesis.

Results

Boundaries of the albomycin biosynthetic gene cluster

To facilitate characterization of the biosynthetic pathway, a DNA fragment of 34.3 kb was directly cloned from the chromosome of Streptomyces sp. ATCC 700974 into the capture vector pCAP01 via CRISPR/Cas9-mediated transformation-associated recombination to obtain pCAP01::LRabm (Figs. S1 and S2A). The 34.3 kb insert was then transferred into the Streptomyces integrating vector pSET152 via λ-mediated homologous recombination to generate pSET152::LRabm (Figs. S1 and S2B). Next, the recombinant plasmid was introduced into different surrogate hosts, such as Streptomyces coelicolor M1146, Streptomyces lividans TK24, and Saccharomyces albidoflavus J1074. The resulting recombinant strains, M1146-pSET152::LRabm, TK24-pSET152::LRabm, and J1074-pSET152::LRabm, were first subjected to antibacterial assay. The results showed that the recombinant strains displayed obvious zones of inhibition against the indicator strain Escherichia coli JM109. However, no antibacterial activity was observed with the negative control strains, including the hosts alone or hosts containing empty pSET152 vector (Figs. 2B and S3; Table S4). To further confirm albomycin production, culture filtrate from M1146-pSET152::LRabm was subjected to LC-MS analysis. A distinct ion that matches exactly to that of Streptomyces sp. ATCC 700974 was detected in the fermentation broth of M1146-pSET152::LRabm (Fig. 2C). These results demonstrated that we have successfully cloned the complete albomycin gene cluster, which is sufficient to confer albomycin production in three heterologous hosts.

Figure 2.

Boundary determination of albomycin gene cluster.A, schematic diagrams showing the 34.3 kb insert and its truncated derivatives. B, bioassay of albomycin produced by different recombinant strains. Fermentation broths were collected from cultures of 2 to 5 days. Escherichia coli JM109 was used as indicator strain. C, LC-MS analysis of albomycin δ₂ produced by Streptomyces sp. ATCC 700974, M1146-pSET152::LRabm, and M1146-pSET152::abm. 1a: albomycin δ₂ (calculated mass 1045.30).

To define the minimal gene cluster required for albomycin biosynthesis, deletion derivatives with truncations from both ends of the insert were generated and then transferred into S. coelicolor M1146 (Figs. 2A and S4). The effects of these deletions on albomycin production were then monitored by bioassay analyses. Like M1146-pSET152::LRabm, M1146-pSET152::abm, a recombinant strain lacking ORFs 1–7, still exhibited antibacterial activity (Fig. 2B and Table S4). Furthermore, a distinct ion that matches exactly to that of Streptomyces sp. ATCC 700974 was also detected in the culture filtrate of M1146-pSET152::abm (Fig. 2C). However, both abmA deletion mutant (M1146-pSET152::ΔabmA) and abmR deletion mutant (M1146-pSET152::ΔabmR) lost the ability to inhibit the growth of the indicator strain (Fig. 2B and Table S4). These results suggested that the minimal gene cluster contains 18 genes from abmA to abmR, spanning a contiguous DNA fragment of appropriate 23.8 kb.

Genes involved in the biosynthesis of ferrichrome-type siderophore

To provide genetic evidence for the participation of abmA, abmB, and abmQ in the ferrichrome formation, it is necessary to create inactivation mutants of abmA, abmB, and abmQ. To facilitate gene inactivation events, we set out to construct an albomycin-deficient mutant (Δabm) lacking the entire gene cluster. For this purpose, the gene cluster was removed from Streptomyces sp. ATCC 700974 by CRISPR/Cas9-mediated genome editing (Fig. S5). Next, abmA, abmB, and abmQ were deleted individually in pSET152::abm via λ-mediated homologous recombination to generate pSET152::ΔabmA, pSET152::ΔabmB, and pSET152::ΔabmQ, respectively. The resulting mutant clusters were then introduced into Δabm to obtain mutant strains ΔabmA, ΔabmB, and ΔabmQ. Antimicrobial assay showed that ΔabmQ lost the ability to inhibit the growth of the indicator strain. Furthermore, complementation of ΔabmQ with a functional copy of abmQ under the control of constitutive promoter restored antimicrobial activity (Fig. 3A and Table S4). These studies suggested that abmQ is indispensable for albomycin production. The culture filtrate from ΔabmQ was then subjected to LC-MS analysis. A distinct ion that matches exactly to SB-217452 was detected in the fermentation broth (Fig. 3B), while no production of albomycin was observed. The results suggested that abmQ is required for the formation of the ferrichrome-type siderophore. Unexpectedly, both ΔabmA and ΔabmB still retained antimicrobial activity (Fig. 3A and Table S4). To further confirm this phenomenon, a double mutant ΔabmAB was generated in a manner similar to that of ΔabmA and ΔabmB. Antimicrobial assay showed that ΔabmAB still retained antimicrobial activity, though the inhibition halo is significantly smaller than those of ΔabmA and ΔabmB (Fig. 3A and Table S4). To identify bioactive compounds present in ΔabmA and ΔabmB, the fermentation broths of these mutants were subjected to LC-MS analysis. Surprisingly, albomycin was detected in the fermentation broths of ΔabmA and ΔabmB (Fig. 3B), suggesting that both abmA and abmB might have functional redundancy.

Figure 3.

Genes involved in ferrichrome biosynthesis.A, bioassay of albomycin production in different mutants. Δabm-Cabm, the Δabm mutant complemented with the albomycin gene cluster; ΔabmAB, double deletion mutant of abmA and abmB; ΔabmQ, deletion mutant of abmQ; Δabm-CabmQ, ΔabmQ complemented with abmQ; ΔabmA, deletion mutant of abmA; ΔabmA-Δorf01157/01158, triple deletion mutant of abmA, orf01157, and orf01158; ΔabmA-Δorf05026, double deletion mutant of abmA and orf05026; Δorf05026-Cabm, the mutant strain Δorf05026 complemented with the albomycin gene cluster; ΔabmB, deletion mutant of abmB; ΔabmB-Δorf01157/1158, triple deletion mutant of abmB, orf01157, and orf01158; ΔabmB-Δorf02009, double deletion mutant of abmB and orf02009; ΔabmB-Δorf03299, double deletion mutant of abmB and orf03299; Δorf03299-Cabm, the mutant strain Δorf03299 complemented with the albomycin gene cluster. B, LC-MS analysis of SB-217452 produced by ΔabmQ and albomycin δ2 produced by ΔabmA and ΔabmB. 1a: albomycin δ2 (calculated mass 1045.30), 11: SB-217452 (calculated mass 476.13).

Genes that compensate for the loss-of-function of abmA and abmB

To pinpoint genes that have functional redundancy for AbmA and AbmB, we performed a local BLAST against the genome sequence of Streptomyces sp. ATCC 700974. The search retrieved several genes encoding homologs of AbmA and AbmB. Of note is that most homologs exhibit low similarity with AbmB and AbmA, except for ORF03299 with 57% identity and 70% similarity to AbmB (Tables 1 and 2). To this end, a transcriptomic analysis was performed with the WT and Δabm. An obvious expression of orf02009, orf03299, orf01158, orf05026, and orf01157 was observed in Δabm (Fig. S6), indicating that these genes are candidates for the compensation of AbmA and AbmB. Of note is that orf01157 is situated adjacent to orf01158 within the same genetic loci. Thus, these genes were then selected for the generation of inactivation mutants, such as Δorf02009, Δorf05026, Δorf03299, and Δorf01157/01158, in the albomycin-deficient mutant Δabm (Fig. S7). Next, pSET152::ΔabmA was transferred into Δorf05026 and Δorf01157/01158 to generate mutant strains ΔabmA-Δorf05026, ΔabmA-Δorf01157/01158. In the meantime, pSET152::ΔabmB was introduced into Δorf02009, Δorf03299, and Δorf01157/01158 to obtain mutant strains ΔabmB-Δorf02009, ΔabmB-Δorf03299, and ΔabmB-Δorf01157/01158. Antimicrobial assay showed that ΔabmA-Δorf05026 and ΔabmB-Δorf03299 lost the ability to inhibit the growth of the indicator strain, while the other mutants still retained antimicrobial activity (Fig. 3A and Table S4). These studies suggest that orf05026 and orf03299 compensate for the loss-of-function of abmA and abmB, respectively. Interestingly, further examinations found out that orf05026 and orf03299 are associated with genes encoding putative siderophores. The orf05026 was situated within a gene cluster encoding a yet unknown siderophore, while orf03299 was located within a gene cluster encoding a putative hydroxamate siderophore coelichelin (Table S5).

Table 1.

Homologs of abmA in the genome of Streptomyces sp. ATCC 700974

Gene ID (accession number)	Coverage (%)	Identity (%)	Similarity (%)	Putative product
orf05026 (WP_030802290)	75.13	33	46	acetyltransferase
orf01157 (OP822674)	47.2	30	45	acetyltransferase
orf00588 (OP822673)	55.96	28	40	GNAT family N-acetyltransferase

Table 2.

Homologs of abmB in the genome of Streptomyces sp. ATCC 700974

Gene ID (accession number)	Coverage (%)	Identities (%)	Similarity (%)	Putative product
orf03299 (WP_030807506)	96.22	57	70	L-lysine 6-monooxygenase
orf02009 (WP_044370950)	97.78	32	45	SidA/IucD/PvdA family monooxygenase
orf01158 (OP822675)	93.33	30	49	Lysine N (6)-hydroxylase/L-ornithine N (5)-oxygenase family protein
orf04526 (OP822672)	99.33	30	46	SidA/IucD/PvdA family monooxygenase

Biochemical characterization of N⁵-monooxygenase AbmB and N⁵-acyltransferase AbmA

Sequence analyses suggested that abmB and abmA encode a flavin-dependent ornithine monooxygenase and an N-acyltransferase, respectively. Further examination showed that abmB encodes a flavin-dependent ornithine monooxygenase with 56% identity and 68% similarity to CchB, which was previously identified as an L-ornithine monooxygenase responsible for the biosynthesis of siderophore coelichelin in S. coelicolor (10). To decipher its role in the biosynthetic pathway, abmB was overexpressed in E. coli and purified to homogeneity (Fig. S8). AbmB was then incubated with L-ornithine, NADPH, and FAD. Derivatizations with 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA) were performed to facilitate detection of the enzymatic products (Fig. S9). LC-MS analysis detected the presence of L-FDLA-N⁵-hydroxyornithine in the reaction mixture (Figs. 4B and S9). Of note is that CchB also catalyzes the formation of N⁵-hydroxyornithine with L-ornithine as the substrate (Fig. S10). When AbmA and acetyl-CoA were supplied in the reaction mixture, LC-MS analysis detected the presence of L-FDLA-N⁵-acetyl-N⁵-hydroxyornithine (Figs. 4B, S9 and S10). However, neither L-FDLA-N⁵-hydroxyornithine nor L-FDLA-N⁵-acetyl-N⁵-hydroxyornithine was detected when heat-inactivated CchB, AbmB, and AbmA were supplied (Figs. 4B and S10). To examine the possibility of acetylation prior to hydroxylation, AbmA was then incubated with acetyl-CoA and L-ornithine. However, no L-FDLA-N⁵-acetylornithine was detected in the reaction mixture. Similarly, no enzymatic product was observed when L-ornithine was replaced with D-ornithine in the reaction mixture (Figs. S9 and S11). Overall, these studies established that AbmB and AbmA catalyze the sequential modifications of L-ornithine to generate N⁵-hydroxyornithine and subsequently N⁵-acetyl-N⁵-hydroxyornithine.

Figure 4.

Biochemical analysis of AbmA, AbmB, and ORF05026.A, enzymatic catalysis of L-ornithine (2) to N⁵-hydroxyornithine (3) and N⁵-acetyl-N⁵-hydroxyornithine (4). B, LC-MS analysis of enzymatic products catalyzed by AbmB, AbmA, and ORF05026 with L-ornithine as the substrate. C, LC-MS analysis of enzymatic products catalyzed by AbmB with L-lysine as the substrate. 3′: L-FDLA-N⁵-hydroxyornithine (calculated mass 442.18); 4′: L-FDLA-N⁵-acetyl-N⁵-hydroxyornithine (calculated mass 484.19); 12′ and 12″ represent the two putative L-FDLA derivatives of L-lysine, i.e., N⁶-L-FDLA-lysine and N¹- L-FDLA-lysine (calculated mass 440.20). L-FDLA, 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide.

Typically, the hydroxamate functional groups are derived from diamino acids such as L-lysine and L-ornithine (11). Thus, we also tested substrate specificity of AbmB with alternative substrates, such as D-ornithine, L-lysine, D-lysine, and L-2,4-diaminobutyric acid. However, no enzymatic product was observed with these substrates (Figs. 4C and S11), suggesting that AbmB has substrate specificity for L-ornithine. Sequence analysis revealed that abmQ encodes a nonribosomal peptide synthetase with one adenylation (A) domain, two condensation (C) domains, and three thiolation (T) domains (6). AbmQ was proposed to be responsible for condensation of the N⁵-acetyl-N⁵-hydroxyornithine to generate the tripeptide ferrichrome (6, 8). Thus, we made great efforts to overexpress abmQ in E. coli and Streptomyces. Unfortunately, we are unable to obtain enough amount of high quality AbmQ protein for biochemical analysis. Further studies are needed to clarify the exact function of AbmQ in the formation of the siderophore moiety.

As mentioned earlier, genetic evidence shows that orf03299 and orf05026 have functional redundancy for abmB and abmA, respectively. Bioinformatics analysis showed that orf03299 encodes an L-lysine 6-monooxygenase with 82% identity and 90% similarity to CchB (Data not shown), indicating that ORF03299 most likely catalyzes the formation of N⁵-hydroxyornithine in a way similar to CchB and AbmB. However, a low similarity was observed between ORF5026 and AbmA. To further validate the biosynthetic role of ORF5026, biochemical experiment verified that ORF05026 catalyzed the generation of N⁵-acetyl-N⁵-hydroxyornithine in the presence of AbmB (Fig. 4B). The results reinforced the notion that orf03299 and orf05026 compensate the loss-of-function of abmB and abmA, respectively.

Wide distribution of albomycin gene cluster in various Streptomyces spp.

Previous studies suggested that AbmK plays dual role in the biosynthesis and immunity of albomycins. A recent study indicated that AbmK participates directly in the formation of SB-217452 during albomycin biosynthesis (8). In the meanwhile, it confers self-resistance to albomycins to ensure survival of the producer (12). Thus, a BlastP search was performed with AbmK as a query to search for homologs in the NCBI nonredundant protein database. AbmK homologs were widely found in the genera of actinomycete bacteria, such as Streptomyces, Actinoalloteichus, Dactylosporangium, Micromonospora, Salinispora, and Verrucosispora (Data not shown). For illustration purpose, analysis of abmK homologs and neighboring genes retrieved albomycin gene clusters from nine additional Streptomyces species (Fig. 5). In these strains, homologs of the 18 albomycin biosynthetic genes were arranged in the same order as that in Streptomyces sp. ATCC 700974. Further examination showed that genes within the clusters were highly conserved, with similarity higher than 79%, suggesting wide distribution of the gene cluster in various Streptomyces species. To the best of our knowledge, none of these strains has been reported to synthesize albomycin or its analogs. Our attention was then directed to phylogenetic analysis of AbmB and AbmA. To this end, amino acid sequences of monooxygenase and acyltransferase involved in siderophores biosynthesis were retrieved from the NCBI nonredundant protein database and used for phylogenetic analysis. The results showed that the monooxygenase family splits into three types, termed type I, II, and III. Each type of enzyme exhibits high preference for either L-lysine–, L-ornithine–, or L-lysine–derived substrates. Sid1 and SidA from fungi are representatives of the type Ⅰ group, which includes PvdA, CchB, AbmB, and VbsO. IcuD from E. coli is the representative of type Ⅱ group, which includes MbtG, MbsG, and FrbG from actinomycetes. It seems that monooxygenases of the same type are highly conserved among different species. Of note is that AbmB, CchB, and ORF03299 are clustered in a closer tree branch, suggesting that they are closely related and having equivalent functions (Fig. 6A). The acyltransferases divide into two subgroups (transacetylase and formyltransferase), depending on the substrates and acyl donors used. Similarly, AbmA and ORF05026 are clustered in a closer tree branch than other homologs from Streptomyces sp. ATCC 700974 (Fig. 6B). Taken together, these results suggested that gene clusters for the biosynthesis of albomycin or its analogs are widely distributed in the genera of actinomycete bacteria.

Figure 5.

Distribution of albomycin gene cluster in various Streptomyces species. Genetic organization of the gene cluster in Streptomyces sp. ATCC 700974 as well as nine additional strains of Streptomyces. Sequence identities of N-acetyltransferase, flavin-dependent monooxygenase, and NRPS are indicated. NRPS, nonribosomal peptide synthetase.

Figure 6.

Phylogenetic analysis of monooxygenases and acyltransferase.Black dots indicate the enzymes involved in mixed siderophore biosynthesis. Proteins in orange letters represent homologs of AbmA or AbmB in Streptomyces sp. ATCC 700974. A, phylogenetic tree of monooxygenase. Proteins shaded in light orange, light blue, and gray represent type Ⅰ, type Ⅱ, and type Ⅲ monooxygenases, respectively. The substrate for each type is L-ornithine, L-lysine, and L-lysine derivatives. B, phylogenetic tree of acyltransferase classified into formyltransferase and transacetylase. Proteins shaded with light blue are formyltransferase with N⁵-hydroxyornithine as the substrate. Transacetylase shaded with light orange have N⁵-hydroxyornithine as the substrate, while transacetylase shaded with gray have a variety of substrates.

Discussion

Under iron-limited conditions, iron chelating siderophores are biosynthesized and secreted by microorganisms to scavenge iron from the environment to ensure their nutritional requirement. In the bacterial genus Streptomyces, representative hydroxamate-based siderophores include desferrioxamine B, desferrioxamine E, and coelichelin (13, 14). Gene clusters for the biosynthesis of desferrioxamine B, desferrioxamine E, and coelichelin have been identified. The des gene cluster directs the production of desferrioxamines B and E (15), while the cch gene cluster is responsible for coelichelin production (13, 14). In the albomycin-producing Streptomyces sp. ATCC 700974, the gene cluster for the biosynthesis of griseobactin, a catecholate siderophore, has been identified (16). Moreover, antiSMASH analysis revealed the presence of three additional gene clusters encoding putative siderophores in the genome of Streptomyces sp. ATCC 700974 (Table S5). Of particular interest is that a ferrichrome-type siderophore was conjugated with a thionucleoside antibiotic to constitute the “Trojan horse” antibiotic albomycin. The presence of the ferrichrome-type siderophore allows active transport of albomycin into bacterial cells, thus exhibiting potent antibacterial activities against many model bacteria and a number of clinical pathogens (5). However, to our surprise, there are no reports on enzymatic mechanism of the ferrichrome formation in albomycin biosynthesis. In this study, we provided genetic evidence that abmA, abmB, and abmQ are responsible for the formation of the ferrichrome-type siderophore. Furthermore, biochemical studies established that a flavin-dependent monooxygenase AbmB and an N-acyltransferase AbmA catalyze sequential N⁵-hydroxylation and N⁵-acetylation of L-ornithine to form N⁵-acetyl-N⁵-hydroxyornithine, which was then assembled to form ferrichrome via iterative condensation through the action of a nonribosomal peptide synthetase AbmQ.

In previous studies, abmA, abmB, and abmQ were proposed to be responsible for the formation of the ferrichrome siderophore (6, 8). In an earlier study, an inactivation mutant of abmB was generated by replacing the coding region of abmB with apramycin resistance cassette. The mutant strain lost its antibacterial activity against E. coli BW25113 (6). In this study, ΔabmB and ΔabmA were created by in-frame deletion to avoid possible inactivation of downstream genes by polar effects. Surprisingly, both ΔabmB and ΔabmA still retained antibacterial activity (Fig. 3A and Table S4), suggesting that abmA and abmB have functional redundancy. Further genetic and biochemical studies revealed that orf05026 and orf03299 compensate for the loss-of-function of abmA and abmB, respectively. Of particular interest is that orf05026 and orf03299 are associated with genes encoding putative siderophores. The orf03299 was situated within a gene cluster encoding a putative coelichelin, while orf05026 was located within a gene cluster encoding a yet unknown siderophore. Considering genes from clusters encoding siderophores can compensate the loss-of-function for genes within the albomycin gene cluster, we speculate that albomycin may retain its canonical role as siderophores to acquire scarce iron from the external environment. It would be interesting to explore this possibility.

Unlike ΔabmB and ΔabmA, ΔabmQ lost the ability to inhibit the growth of the indicator strain, while the complementary strain restored antimicrobial activity (Fig. 3A and Table S4). Moreover, a distinct ion that matches exactly to SB-217452 was detected in the fermentation broth of ΔabmQ (Fig. 3B). These studies established that abmQ is definitely required for the formation of the ferrichrome-type siderophore. As mentioned earlier, sequence analysis suggested that abmQ encodes an unusual nonribosomal peptide synthetase enzyme catalyzing condensation of the N⁵-acetyl-N⁵-hydroxyornithine to generate the tripeptide ferrichrome. However, the enzymatic mechanism will remain a mystery unless there is biochemical evidence with purified AbmQ. Of interest is that AbmQ lacks a C-terminal thioesterase domain, indicating no free siderophore is released from AbmQ. A recent study suggested that AbmC is responsible for assembly of the siderophore moiety with the thionucleoside SB-217452 (8). Similarly, further biochemical evidence is needed to clarify the exact function of AbmC.

In recent years, resistance-guided genome mining has been proved to be a used successful strategy for the discovery of novel antimicrobial agents (17, 18, 19, 20). A BlastP search showed that AbmK homologs were widely found in the genus of Streptomyces, as well as genera of actinomycetes including Actinoalloteichus, Dactylosporangium, Micromonospora, Salinispora, and Verrucosispora. Further analysis of abmK homologs and neighboring genes will be helpful for the discovery of BGCs encoding novel analogs of albomycins. Genome mining of these underexplored biosynthetic potentials open up new opportunities for the discovery and development of novel antimicrobial drugs to address the growing global threat of antimicrobial resistance.

Experimental procedures

Strains, plasmids, primers, and culture conditions

Bacterial strains, plasmids, and primers used in this study were listed in the supplementary materials, respectively (Tables S1–S3). Streptomyces sp. ATCC 700974, a natural albomycin producer, was purchased from the American Type Culture Collection (ATCC). S. coelicolor M1146, S. lividans TK24, and S. albidoflavus J1074 were used for heterologous expression of the albomycin gene cluster. E. coli JM109 was used as an indicator strain for antimicrobial assay. E. coli DH5α was used as a general host for routine subcloning. E. coli BW25113 (pIJ790) was used for the construction of recombinant plasmids via λ-Red–mediated recombination technology (21). E. coli ET12567 (pUZ8002) was used as a host for transferring DNA from E. coli to Streptomyces by intergeneric conjugation (22).

For general purpose, Streptomyces strains were grown on soya flour mannitol (SFM) agar or in tryptic soy broth liquid medium (23) or yeast extract-malt extract liquid medium (22). All Streptomyces stains were cultivated at 28 °C. General approaches for E. coli or Streptomyces manipulations were performed according to standard protocols (22, 24). When necessary, antibiotics were used at the following concentrations: ampicillin, 100 μg/ml in LB medium for E. coli; hygromycin B 100 μg/ml in LB for E. coli; apramycin, 50 μg/ml in LB for E. coli; kanamycin, 100 μg/ml in LB for E. coli; chloramphenicol, 25 μg/ml in LB for E. coli; nalidixic acid, 25 μg/ml in SFM for Streptomyces; apramycin, 50 μg/ml in SFM for Streptomyces; hygromycin B 50 μg/ml in SFM for Streptomyces.

Direct cloning of albomycin biosynthetic gene cluster

A DNA fragment of 34.3 kb was directly captured from genomic DNA of Streptomyces sp. ATCC 700974 via CRISPR/Cas9-mediated transformation-associated recombination. For this purpose, two DNA fragments corresponding to approximately 1600 bp of sequences on both sides of albomycin gene cluster are amplified from genomic DNA of Streptomyces sp. ATCC 700974 by using primer pairs LRUp F/R and LRDn F/R. The PCR amplicons were ligated together by overlapping PCR and then inserted into XhoI/SpeI of the capture vector pCAP01 by Gibson assembly (25) to generate pCAP01::LR. Next, genomic DNA was extracted from Streptomyces sp. ATCC 700974 and subjected to digestion by Cas9 enzyme in vitro. The digested DNA was then cotransformed with EcoRI-linearized pCAP01::LR into Saccharomyces cerevisiae VL6-48 as described previously (26). PCR was then used for screening of positive clones. The insert was transferred into pSET152 via λ-Red–mediated recombination (21).

Production and analysis of albomycin

For albomycin production, spore suspensions (∼1.2 × 10⁶) were inoculated in liquid tryptic soy broth (Streptomyces sp. ATCC 700974 and its derivatives) or yeast extract-malt extract (S. coelicolor M1146 and its derivatives) and incubated for 48 h as a seed culture, and then 0.5 ml of each seed culture was transferred into a shake flask containing 50 ml APB media (27). The cultures were cultivated for different time intervals at 28 °C, and then fermentation broths were collected by centrifugation. For albomycin analysis, culture broths were centrifuged at 14,000g for 10 min to remove the mycelia. High-Resolution Mass Spectrometry analysis was performed on Agilent 1290 Infinity LC System/6230 Accurate-Mass Time-of-Flight (TOF) LC/MS system with a Rapid Resolution HD C18 column (1.8 μm pore size; 2.1 by 150 mm, Agilent, ZORBAX Eclipse Plus). HPLC conditions for albomycin analysis were as described previously (28).

Albomycin bioassays

Antibacterial assay was performed with E. coli JM109 as the indicator strain. For this purpose, E. coli JM109 was inoculated in 3 ml LB and cultured overnight. The overnight culture was then diluted 1:100 into 3 ml fresh LB and cultivated till OD₆₀₀ reached 0.4. A total of 800 μl cell suspension was well dispersed in 200 ml premelted LB agar and poured into a 20 cm plate. Holes of 7 mm in diameter were bored with a sterile borer. A total of 40 μl culture filtrates were then added into the holes. After an incubation of 8 to 10 h, antibacterial activity was evaluated by the size of inhibition haloes.

Construction of recombinant strains

Deletion constructs were created with truncations from both ends of the 34.3 kb insert via λ-Red–mediated recombination (21). For the construction of pSET152::tarAR, two DNA fragments corresponding to approximately 500 bp of abmA and abmR are amplified from genomic DNA of Streptomyces sp. ATCC 700974 with primer pairs abmUp F/R and abmDn F/R. The PCR amplicons were digested with the appropriate restriction enzymes and inserted into the corresponding sites of pSET152 to generate pSET152::tarAR. A similar strategy was used for the construction of pSET152::tarΔA, pSET152::tarΔR, and pSET152::tarΔAB, respectively. These plasmids were linearized with EcoRⅤ and then used to generate pSET152::abm, pSET152::ΔabmA, pSET152::ΔabmR, and pSET152::ΔabmAB via λ-Red–mediated recombination (Fig. S4). The resulting deletion derivatives were introduced into S. coelicolor M1146 by intergeneric conjugation to generate recombinant strains, M1146-pSET152::abm, M1146-pSET152::ΔabmA, and M1146-pSET152::ΔabmR.

To generate a mutant lacking the entire gene cluster (Δabm), an approximately 2.2 kb DNA fragment containing sgRNA of abmG, left and right homologous arm of the albomycin gene cluster, was obtained by overlapping-PCR amplifications. The fragment was digested with SpeI/HindIII and then inserted into SpeI/HindIII-linearized pKCCas9 backbone (29) to generate pKCCas9dabm. After restriction digestion analyses, pKCCas9dabm was transformed into E. coli ET12567/pUZ8002 and then transferred into Streptomyces sp. ATCC 700974 by E. coli-Streptomyces intergeneric conjugation. A similar strategy was used for the construction of pKCCas9dorf05026, pKCCas9dorf03299, pKCCas9dorf02009, and pKCCas9dorf01157/01158. For the construction of pSET152::ΔabmB and pSET152::ΔabmQ, the spectinomycin resistance cassette (aadA) was PCR amplified from pIJ778 by using the primer pairs dabmB F/R and dabmQ F/R. The mutant clusters were obtained following the instructions as described previously (21). Next, the mutant clusters were then introduced into Δabm to generate mutant stains ΔabmB and ΔabmQ, respectively.

Protein overexpression and purification

The coding sequences of abmA, abmB, and orf05026 were PCR amplified from the genomic DNA of Streptomyces sp. ATCC 700974, and the coding sequence of cchB was PCR amplified from the genomic DNA of S. coelicolor. The amplicons were digested with NdeI/XhoI and ligated into corresponding sites of pET28a to generate pET28a::abmA, pET28a::abmB, pET28a::cchB, and pET28a::orf05026, respectively. The constructs were verified by sequencing and then transformed into E. coli BL21 (DE3). The strains were used for protein overexpression following standard protocols (24). In brief, the strains were cultured in LB medium supplemented with kanamycin (100 μg/ml) at 37 °C with 200 rpm shaking until OD₆₀₀ reached 0.6. The culture was supplemented with 0.2 mM IPTG and then grown at 16 °C with 200 rpm shaking for 20 h.

For protein purification, cell pellets were collected by centrifugation (10,000g for 2 min at 4 °C) and resuspended in 60 ml buffer A (20 mM Tris–HCl pH 8.0, 150 mM NaCl) per liter of bacterial culture. The cells were then lysed by sonication on ice, and the cellular debris were removed by centrifugation (13,000g for 30 min at 4 °C). The supernatants were filtered with 0.22 μm filter (Titan, TYLQ-0009) and then passed through a column containing high-affinity Co²⁺-NTA agarose resin. The column was washed sequentially with 18 ml buffer A and 15 ml washing buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 20 mM imidazole). The His-tagged proteins were eluted with 6 ml elution buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 250 mM imidazole). The purified proteins were desalted with PD-10 columns (GE Healthcare), eluted with 3.5 ml buffer C (20 mM Tris–HCl, 50 mM NaCl, 10% glycerol, pH 8.0), and followed by treatment with ultrafiltration centrifugal tube (Millipore Amicon Ultra-15 ml). Protein concentration was determined with the BCA Protein Assay Kit following instructions of the supplier (Sangon Biotech).

Biochemical assays

To measure the N-hydroxylase activity, a 100 μl reaction mixture was prepared as follows: Tris–HCl (100 mM, pH 8.0), L-ornithine (1 mM), FAD (20 μM), NADPH (2 mM), AbmB, or CchB (5 μM). The acetyltransferase activity of AbmA and ORF05026 was measured at 25 °C in a 100 μl reaction mixture containing AbmA (or ORF05026, 5 μM) and AbmB (or CchB, 5 μM), Tris–HCl (100 mM, pH 8.0), L-ornithine (1 mM), FAD (20 μM), NADPH (2 mM), and acetyl-CoA (1 mM). The acetyltransferase activity of AbmA to L-ornithine and D-ornithine was measured at 25 °C in a 100 μl reaction mixture containing AbmA (5 μM), Tris–HCl (100 mM, pH 8.0), L-ornithine or D-ornithine (1 mM), and acetyl-CoA (1 mM). The enzymes were boiled at 100 °C for 10 min to serve as negative controls. All reaction mixtures were incubated at 25 °C for 2 h and then stopped by adding 100 μl acetonitrile. To facilitate detection of the products, derivatizations were carried out according to the advanced Marfey's method (30). In brief, reaction mixture was dried by vacuum rotary evaporation and then dissolved in 50 μl ddH₂O. Next, derivation solution containing 10 μl NaHCO₃ (1 M) and 50 μl 1% L-FDLA was added, and the mixture was then incubated at 45 °C for 2 h. After cooling to room temperature, the mixture was quenched by adding 5 μl HCl (2 N) and then dried and redissolved in 300 μl 50% aqueous acetonitrile. The derivatives were analyzed by LC-MS TOF on Agilent 1290 Infinity LC System/6230 Accurate-Mass TOF LC/MS system using Rapid Resolution HD column (1.8 μm pore size; 2.1 by 150 mm, Agilent, ZORBAX Hilic Plus) under the following conditions: anion mode, solvent A (H₂O in 0.1% formic acid) and solvent B (acetonitrile). Solvent B was increased from 2% to 15% in 10 min, 15% to 100% in 5 min, 100% for 3 min, and 100% to 2% in 1 min at a flow rate of 0.3 ml/min. The UV detection wavelength was set at 340 nm.

Bioinformatic analysis

Amino acid sequences of hydroxylases and acyltransferases were collected referring to previous literatures (31, 32). A local BLAST was carried out with the genome of Streptomyces sp. ATCC 700974 to search for homologs of AbmA and AbmB. A Muscle alignment was then performed with MEGA 7 using the collected sequences. A rooted tree was generated using MEGA 7 based on the Muscle alignment.

Data availability

All data are contained within the article.

Supporting information

This article contains supporting information (21, 29, 33, 34, 35, 36, 37, 38, 39, 40).

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Reviewed by members of the JBC Editorial Board. Edited by Chris Whitfield