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Published in final edited form as: ACS Chem Biol. 2021 Feb 8;16(2):270–276. doi: 10.1021/acschembio.0c00982

Natural occurrence of hybrid polyketides from two distinct biosynthetic pathways in Streptomyces pactum

Wei Zhou 1, Priyapan Posri 1, Taifo Mahmud 1,*
PMCID: PMC7932018  NIHMSID: NIHMS1671386  PMID: 33601889

Abstract

Nature has always been seemingly limitless in its ability to create new chemical entities. It provides vastly diverse natural compounds through a biomanufacturing process that involves myriads of biosynthetic machineries. Here, we report a case of unusual formations of hybrid natural products, which are derived from two distinct polyketide biosynthetic pathways, the NFAT-133 and the conglobatin pathways, in Streptomyces pactum ATCC 27456. Their chemical structures were determined by NMR, mass spectrometry, and chemical synthesis. Genome sequence analysis and gene inactivation experiments uncovered the biosynthetic gene cluster of conglobatin in S. pactum. Biochemical studies of the recombinant thioesterase (TE) domain of the conglobatin polyketide synthase (PKS) as well as its S74A mutant revealed that the formation of these hybrid compounds requires an active TE domain. We propose that NFAT-133 can interfere with conglobatin biosynthesis by reacting with the TE domain-bound intermediates in the conglobatin PKS assembly-line to form hybrid NFAT-133/conglobatin products.

Graphical Abstract

graphic file with name nihms-1671386-f0001.jpg

The soil bacterium Streptomyces pactum ATCC 27456 is known to produce a number of secondary metabolites. Some of them are structurally intriguing and highly bioactive with distinct mechanisms of action.1-3 The most well-studied metabolite from this strain is pactamycin (1) (Figure 1), a broad-spectrum aminocyclitol-derived antitumor antibiotic that has antibacterial, antifungal, anti-plasmodial, and anti-tumor activities.1,4-7 The strain also produces NFAT-133 (2),2 an aromatic polyketide compound with immunosuppressive, antidiabetic, and anti-trypanosomal activities.8-11 It suppresses interleukin-2 (IL-2) expression and T cell proliferation by inhibiting transcription mediated by nuclear factor of activated T cells (NFAT).8 It also activates the AMPK pathway and increases glucose uptake in L6 myotubes.11 NFAT-133 is derived from modular type-I polyketide synthases (PKSs) whose genes in S. pactum ATCC 27456 are highly disorganized and inconsistent with the co-linearity of modular PKS gene clusters seen in many other secondary metabolites.12

Figure 1.

Figure 1.

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Secondary metabolites from Streptomyces pactum ATCC 27456. (a) Chemical structures of known metabolites; (b) TLC analysis of SiO2 column chromatography fractions from the EtOAc extract of S. pactum culture broths; (c) HPLC traces of NFAT-133, TM-127 and TM-128 detected at 254 nm; (d) chemical structures of TM-127 and TM-128.

S. pactum ATCC 27456 also produces conglobatin (3), a C2-symmetrical macrodiolide antitumor compound. This cyclic dimeric polyketide was first isolated from Streptomyces conglobatus,13 but later was found in several other strains of Streptomyces.10,14,15 It binds to heat shock protein 90 (Hsp90) that results in the inhibition of cell proliferation and the induction of apoptosis.14 Conglobatin is synthesized by modular type-I PKSs, involving a cyclase/thioesterase (TE) domain that acts iteratively, couples two monomers head-to-tail, rebinds the dimer product, and then cyclizes it.16

During our biosynthetic study of NFAT-133, we created a mutant strain, S. pactum ΔptmTDQ/ΔBGC-1.2, in which both the pactamycin and the NFAT-133 pathways have been inactivated. These genetic modifications abolished the production of pactamycin and NFAT-133, as well as some other metabolites whose chemical structures were unknown. To isolate the unknown metabolites that are related to NFAT-133, culture broths of S. pactum ΔptmTDQ were partitioned with EtOAc and H2O, and the resulting EtOAc extract was chromatographed on SiO2 column and HPLC (Figure 1c). S. pactum ΔptmTDQ was used because it does not produce pactamycin and its congeners, simplifying the isolation procedure for the NFAT-133 derivatives. As NFAT-133 gives a yellow color with p-anisaldehyde on SiO2 thin layer chromatography (TLC), the investigation was focused on compounds that showed a similar yellow color on TLC (Figure 1b). Consequently, two compounds, TM-127 (4) and TM-128 (5), were isolated from the extract (Figure 1d).

TM-127 (4) was isolated as a pink/purple solid, and its high-resolution ESI-MS (m/z 526.3168 [M+H]+) suggested a molecular formula of C31H43NO6. 1H NMR spectrum of TM-127 showed a number of resonances that are similar to those of NFAT-133, while other resonances are similar to those of conglobatin (Supplementary Table 1). 13C NMR spectrum of TM-127 showed the presence of seven methyls, two methylenes, four methines, three oxygenated methylenes or methines, as well as 13 olefinic/aromatic and two carbonyl carbons (Supplementary Table 2). 1H–1H COSY data showed correlations between H-1 and H-2, H-2 and H-3, H-7 and H-8, H-16 and H-10, H-10 and H-11, H-11 and H-12, H-12 and H-17 (Supplementary Figure 1a), which fit the patterns that one would expect for NFAT-133.

1H–1H COSY correlations were also observed between H-3´ and H-4´, H-4´ and H-5´, H-4´ and H-13´, H-5´ and H-6´, H-6´ and H-7´, H-6´ and H-14´, H-7´ and H2-8´. Moreover, HMBC correlations between H2-8´ and C-9´, H2-8´ and C-11´, H-11´ and C-10´, H-10´ and C-11´, H-10´ and C-9´ established the presence of an oxazole ring at the end of the alkyl chain (Supplementary Figure 1a). Other HMBC correlations between the olefinic proton H-3´ (δH 7.09, d, J = 15.5 Hz) and C-1´ (δC 170.0), C-2´, C-12´, and between H-12´ (δH 1.90, s) and C-1´ suggested the presence of a methyl-substituted α,β-unsaturated ester moiety in NFAT-133. Together, the alkyl chain, the oxazole ring and the methyl-substituted α,β-unsaturated ester moieties form a structure that is identical to a monomer of conglobatin. Finally, the HMBC correlation between H2-1 (4.81, d, J = 6 Hz) and C-1´ unites the two polyketide structures (Supplementary Figure 1a), establishing TM-127 as a hybrid product of NFAT-133 and conglobatin-monomer.

To confirm that TM-127 is indeed derived from NFAT-133 and conglobatin-monomer, and to establish the absolute configuration of TM-127, the compound was synthesized and spectroscopically compared with the natural product. NFAT-133 was isolated from the culture broth of S. pactum, whereas conglobatin-monomer was obtained by treating conglobatin with 0.4 N LiOH·H2O. Coupling reaction between NFAT-133 and conglobatin-monomer was performed using hydroxybenzotriazole (HOBt) and 4-dimethylaminopyridine (DMAP) (Supplementary Figure 2a). The HPLC retention time (Supplementary Figure 2b), the 1H and 13C NMR spectra (Supplementary Figures 3 and 4), the mass spectrum (Supplementary Figure 5), and the optical rotation of the synthetic product are consistent with those of the natural product, confirming the chemical structure and the absolute configuration of TM-127 as depicted in Figure 1.

The second compound TM-128 (5) was also isolated as a pink/purple solid. Its high-resolution ESI-MS (m/z 775.4526 [M+H]+) suggested a molecular formula of C45H62N2O9. Similar to TM-127, 1H NMR spectrum of TM-128 showed a number of resonances that are similar to those of NFAT-133, while other resonances are similar to those of conglobatin, except that there are two similar sets of resonances for conglobatin polyketide chains (Supplementary Table 1). 13C NMR spectrum of TM-128 showed the presence of 10 methyls, four methylenes, six methines, four oxygenated methylenes or methines, 18 olefinic/aromatic and three carbonyl carbons (Supplementary Table 2). These data suggest that TM-128 contains NFAT-133 and two conglobatin-monomers. 1H–1H COSY and HMBC data for TM-128 showed correlations that are consistent with the patterns seen in TM-127 with an additional conglobatin-monomer in the molecule (Supplementary Figure 1b). This extra polyketide chain was predicted to form an ester bond with the C-7´ hydroxy group, as indicated by a significant change in the chemical shift of H-7´ from 3.77 ppm in TM-127 to 5.14 ppm in TM-128. Finally, HMBC correlation between H-7´ and C-1´´ (167.5 ppm) established the chemical structure of TM-128 as a hybrid product of NFAT-133 and two conglobatin-monomers. The typical production ratio of NFAT-133, conglobatin, TM-127, and TM-128 by the ΔptmTDQ mutant cultivated in BTT medium under our laboratory condition is estimated to be 100:20:2:1.

As reported previously, we have obtained a draft genome sequencing of S. pactum ATCC 27456, resulted in 33 putative biosynthetic gene clusters, among which nine were identified as putative PKS gene clusters, including the NFAT-133 BGC (Figure 2a).12 Bioinformatic analysis of the PKS clusters showed that locus BGC-1.23 fits with the expected number of PKS modules for conglobatin. This BGC contains five modular type-I PKS genes (congA, congB, congC1, congC2, and congD), a cyclodehydratase (congE) and some other genes with unknown functions. The cyclodehydratase CongE has been proposed to catalyze the formation of the oxazole ring in conglobatin. However, the BGC-1.23 locus has only 36% overall similarity to the conglobatin BGC from S. conglobatus.16 Furthermore, the BGC-1.23 locus contains a total of five PKS genes, as oppose to four modular type-I PKS genes (congA – congD) present in S. conglobatus (Figure 2b). The congC gene in S. conglobatus exists as two separate genes, congC1 and congC2, in S. pactum (Figure 2c). This divergence was confirmed by amplifying congC1-congC2 DNA region by PCR using S. pactum ATCC 27456 chromosome as template and sequencing the DNA product.

Figure 2.

Figure 2.

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Biosynthetic gene clusters (BGCs) of NFAT-133 and conglobatin in strains of Streptomyces. (a) BGC of NFAT-133 in S. pactum; (b) BGC of conglobatin in S. conglobatus; (c) BGC of conglobatin in S. pactum; (d) proposed formation of conglobatin polyketide backbone; (e) partial mass spectra (selected for conglobatin) of the EtOAc extracts from culture broths of S. pactum mutants; (f) partial mass spectra (selected for TM-127) of the EtOAc extracts from culture broths of S. pactum mutants; (g) partial mass spectra (selected for TM-128) of the EtOAc extracts from culture broths of S. pactum mutants.

To confirm that the BGC-1.23 locus is involved in conglobatin biosynthesis, the congC1 and congC2 genes were inactivated by in-frame deletion. DNA fragments of congC1 and congC2 were amplified from the genome of S. pactum ATCC 27456 (Supplementary Tables 3 and 4) and cloned into plasmid pTMN002.17 The constructed plasmid, pTMN002-congC1-C2, was subsequently transferred into S. pactum ΔptmTDQ strain by conjugation with Escherichia coli ET12567 (pUZ8002) (Supplementary Table 5 and Supplementary Figure 6). The single and double crossover mutants were screened as previously reported,18,19 and the mutant (S. pactum ΔptmTDQ/ΔcongC) was cultured in a liquid BTT medium for 7 days at 30 °C. The culture broth was extracted with EtOAc and analyzed by LC-MS (Figure 2e-g). The results showed that no conglobatin as well as TM-127 and TM-128 were produced by the mutant, indicating that BGC-1.23 is necessary for their biosynthesis. In addition, we also re-analyzed the metabolites of S. pactum ΔptmTDQ/ΔnftCD, in which the NFAT-133 PKS genes have been inactivated. The results also showed that no NFAT-133 as well as TM-127 and TM-128 were produced by this mutant, confirming that TM-127 and TM-128 are produced by two independent biosynthetic machineries, the NFAT-133 and the conglobatin pathways. Inspections of the genome sequence of S. pactum ATCC 27456 revealed that both the NFAT-133 BGC and the conglobatin BGC reside in the same 7.28 Mb contig, but their loci are separated from each other by 5.65 Mb.

While conglobatin is formed from two identical polyketide chains through two consecutive head-to-tail couplings,16 the monomer has never been isolated as a natural product. Therefore, the formation of TM-127 and TM-128 may occur while the conglobatin polyketide chains are still attached to the PKS protein. We propose that the conglobatin PKS assembly line is disrupted by NFAT-133 by reacting with a TE-bound conglobatin-monomer or a TE-bound uncyclized dimer to give TM-127 and TM-128, respectively (Figure 3).

Figure 3.

Figure 3.

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Proposed formation of conglobatin, TM-127, and TM-128. (a) a head-to-tail coupling in conglobatin biosynthesis; (b) coupling reaction between NFAT-133 and a TE-bound conglobatin-monomer; (c) coupling reaction between NFAT-133 and a TE-bound uncyclized dimer.

To test this hypothesis, we cloned the CongD-TE domain gene (Supplementary Figure 7) and expressed it in E. coli. The purified recombinant protein (Figure 4a) was then incubated with synthetically prepared N-acetylcysteamine (NAC) thioester of conglobatin-monomer in the presence of NFAT-133 (Supplementary Figure 8). HPLC, MS, and MS/MS analysis of the reaction mixture showed the formation of TM-127 (m/z 548.12 [M+Na]+), which is absent in the reaction with boiled CongD-TE (Figures 4c-d, and Supplementary Figure 9). Incubation of CongD-TE with NFAT-133 and conglobatin-monomer did not give any product (Fig. 4c), indicating the need of an acyl carrier protein (ACP)-bound substrate in the reaction. A mutant of CongD-TE, in which the catalytic Ser74 residue (Figure 4b) has been replaced by Ala, was also produced and tested. The result showed that the mutant was not able to produce TM-127 (Figures 4c-d), suggesting that conglobatin-monomer needs to be transferred to the TE domain before a coupling reaction with NFAT-133 can take place. In addition to TM-127, a small amount of conglobatin was observed in the mixture containing CongD-TE, NFAT-133, and conglobatin-monomer-SNAC (Figure 4e), indicating that dimerization of conglobatin-monomers also occurred under this condition, albeit less efficiently.

Figure 4.

Figure 4.

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Biochemical conversion of NFAT-133 and conglobatin-monomer-SNAC to TM-127. (a) SDS-PAGE of purified recombinant CongD-TE and CongD-TE S74A; (b) a modeled structure of CongD-TE based on the structure of pikromycin thioesterase (2H7X.A). The catalytic triad residues Ser74, Asp101, and His197 are indicated in red; (c) Partial HPLC chromatograms of CongD-TE, CongD-TE S74A, and boiled CongD-TE incubated with NFAT-133 and conglobatin-monomer-SNAC or conglobatin-monomer detected at 230 nm. (i) CongD-TE + NFAT-133 and conglobatin-monomer-SNAC, (ii) CongD-TE S74A + NFAT-133 and conglobatin-monomer-SNAC, (iii) Boiled CongD-TE + NFAT-133 and conglobatin-monomer-SNAC, (iv) CongD-TE + NFAT-133 and conglobatin-monomer, (v) TM-127 standard; (d) partial mass spectra (selected for TM-127) of CongD-TE, CongD-TE S74A, and boiled CongD-TE incubated with NFAT-133 and conglobatin-monomer-SNAC; (e) partial mass spectra (selected for conglobatin) of CongD-TE, CongD-TE S74A, and boiled CongD-TE incubated with NFAT-133 and conglobatin-monomer-SNAC.

While there are many examples of polyketide-peptide hybrid natural products and to some extent polyketide-isoprenoid hybrid compounds (e.g., furaquinocins),20,21 hybrid natural products from two unrelated polyketide pathways are relatively rare. One of the only few examples of secondary metabolites from two different PKS gene clusters is dalmanol from mantis-associated fungus Daldinia eschscholzii.22 Dalmanol and its analog, acetodalmanol, are formed through hybridization of chromane-related precursors and 1,3,6,8-tetrahydroxynaphthalene derivatives, which are biosynthesized by iterative type I PKSs. The discovery of TM-127 and TM-128 as well as the characterization of their modes of formation in S. pactum further validate the myriad of ways nature produces complex chemical entities.

Experimental Section

General experimental procedures.

For details of the general experimental procedures and the instruments used, see the Supplementary Information.

Bacterial strains and media.

All the strains and plasmids used in this study are listed in Supplementary Table 5. S. pactum ATCC 27456 was purchased from American Type Culture Collection (ATCC). S. pactum ΔptmTDQ triple mutant was constructed according to procedure described in our previous papers.17-19 For details of the bacterial strains and media used, see the Supplementary Information.

General DNA manipulations.

Genomic DNA from S. pactum ATCC 27456 was isolated using the DNease Tissue Kit (QIAGEN) according to the manufacturer’s and other standard protocols.23 PCR was performed in 30 cycles using Platinum Taq DNA polymerase (Invitrogen) or Platinum Pfx DNA polymerase (Invitrogen) on a Mastercycler gradient thermocycler (Eppendorf). Oligodeoxyribonucleotides for PCR primers were synthesized by Sigma-Genosys and are shown in Supplementary Table 4. For details of other general DNA manipulations, see the Supplementary Information.

Isolation of TM-127 and TM-128.

Strain S. pactum ΔptmTDQ was cultured in BTT liquid medium (5 L) at 30 °C with shaking for 7 days. The culture broth was collected by centrifugation, and equivoluminal EtOAc was used to extract the products from the culture broth twice. The crude extracts were dried by vacuum rotary evaporator and then were seperated by SiO2 column chromatography using step gradient elution with CHCl3 – MeOH (100-0%). The target products were detected in the eluted fractions by TLC. TLC analysis was performed as follows: the extract was developed on silica gel 60 F254 using CHCl3 – MeOH (95:5) as the solvent system and p-anisaldehyde as the staining reagent (the target compounds undergo chemical reactions with p-anisaldehyde and appear yellow on TLC plates). The eluted fractions containing the target compounds were combined and dried in vacuo by rotary evaporation. The fraction was directly separated by semi-preparative HPLC on a Shimadzu LC-20AD with a SPD-M20A detector. For details of the HPLC condition, see the Supplementary Information.

TM-127:

a pink/purple solid; [α]D = −14.8° (c = 0.17, 25 °C, MeOH); UV (MeOH) λmax (log ε) 217 (4.55), 246 (4.08), 290 (3.11) nm. 1H NMR: Supplementary Table 1; 13C NMR: Supplementary Table 2; High-resolution ESI MS: m/z 526.3159 [M+H]+, calcd for C31H44NO6+ 526.3163

TM-128:

a pink/purple solid; [α]D = −91.1° (c = 0.07, 25 °C, MeOH); UV (MeOH) λmax 217 (4.69), 243 (4.13), 290 (3.12). 1H NMR: Supplementary Table 1; 13C NMR: Supplementary Table 2; High-resolution ESI MS: m/z 775.4526 [M+H]+, calcd for C45H63N2O9+ 775.4528

Hydrolysis of conglobatin.

To a solution of conglobatin (12.4 mg, 0.025 mmol) in THF/H2O (3:1, 0.5 mL) was added lithium hydroxide monohydrate (8.4 mg, 0.2 mmol). The mixture was stirred for 48 h at 60 °C. For details, see the Supplementary Information.

Synthesis of TM-127.

4-Dimethylaminopyridine (DMAP) (0.5 mg, 0.004 mmol) and hydroxybenzotriazole (HOBt) (1.1 mg, 0.008 mmol) were added to a mixture of conglobatin-monomer (2.0 mg, 0.004 mmol), NFAT-133 (1.1 mg, 0.004 mmol) and N,N-dimethylformamide (1.0 mL). The reaction mixture was stirred for 1 h at room temperature and then extracted with EtOAc and H2O. The organic extract was dried with Na2SO4 and concentrated. The residue was separated by HPLC to obtain TM-127-CS (2.0 mg, 97.4% yield).

TM-127-CS:

a pink/purple solid; [α]D = −15.3° (c = 0.5, 25 °C, MeOH). 1H NMR: Supplementary Table 1; 13C NMR: Supplementary Table 2; ESI MS: m/z 526.09 [M+H]+, 548.04 [M+Na]+.

Inactivation of conglobatin PKS genes.

The in-frame gene deletion of congC1-C2 was carried out according to procedure described in our previous papers.12 For details, see the Supplementary Information.

Analysis of metabolites from S. pactum ΔptmTDQ/ΔcongC1C2 mutant.

For details, see the Supplementary Information.

Construction of expression plasmids for protein CongD-TE and CongD-TE S74A.

The fragment encoding CongD-TE was amplified from the genome of S. pactum by using primers CongTE-F(NdeI)/CongTE-R(HindIII). The fragment was cloned into plasmid pET28a using the NdeI and HindIII sites to give the plasmid pET28a-CongTE. In order to enhance the congD-TE gene expression in E. coli, the first three codons (AGC TCG GGG) were replaced by TCT TCT GGT, which were introduced into the primer Cong-TE-F (NdeI) (italics). To generate the plasmid for CongD-TE S74A production, the Ser74 residue of the TE domain was replaced by Ala using the point-mutation method. Briefly, the plasmid was generated by PCR amplification using the primers CongTEmu-F/CongTEmu-R and pET28a-CongTE as a template. The PCR products were digested with DpnI to remove the template and transferred into E. coli DH5α. Positive clones (pET28a-CongTE S74A) were confirmed by DNA sequencing.

Production and purification of recombinant CongD-TE and CongD-TE S74A.

For details, see the Supplementary Information.

Biochemical characterizations of recombinant CongD-TE and CongD-TE S74A.

Enzymatic assays were carried out in a final volume of 100 μL containing potassium phosphate buffer (100 mM, pH 8.2), NFAT-133 (2 mM), conglobatin-monomer-SNAC (2 mM) and the purified protein (20 μg). The mixtures were incubated at 20 °C for 12 h. The products of the enzymatic reactions were extracted with EtOAc (1 mL) and were dried with vacuum. Finally, the extracts were dissolved in MeOH (0.5 mL), the solutions (5 μL) were analyzed by HPLC, LC-MS and MS/MS.

Accession Number.

The draft genome sequence of Streptomyces pactum ATCC 27456 has been deposited in GenBank under the accession number JACYXC000000000.

Supplementary Material

Supporting Information

ACKNOWLEDGEMENTS

The authors thank S. Burade, A. Samadi, T. Tsunoda, and B. Philmus for technical assistance. This work was supported by grant AI129957 (to T.M.) from the National Institute of Allergy and Infectious Diseases. The content is solely the responsibility of the authors and does not represent the official views of the National Institute of Allergy and Infectious Diseases, or the National Institutes of Health (NIH). PP was supported by a Royal Golden Jubilee Ph.D. scholarship (grant number PHD/0007/2558) from the Thailand Science Research and Innovation. We acknowledge the support of the Oregon State University NMR Facility funded in part by the National Institutes of Health, HEI Grant 1S10OD018518, and by the M. J. Murdock Charitable Trust grant #2014162.

Footnotes

Supporting Information

Supplementary Methods, Supplementary Tables 1–5, Supplementary Figures 1–30, and Supplementary References.

This material is available free of charge via the internet at http://pubs.acs.org.

The authors declare no competing financial interest.

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Supporting Information
NIHMS1671386-supplement-Supporting_Information.pdf (4.2MB, pdf)

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