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. 2026 Mar 3;6(3):1966–1976. doi: 10.1021/jacsau.6c00051

Targeted Discovery of β‑Branched Conjugated Polyketides from Bacteria Based on Genomic and Metabolomic Hallmarks

Jinsheng Cui †, Ilnam Kang ‡, Haewon Lee †, Bo Eun Heo §, Suhyun Ye ∥, Jayho Lee †,⊥, Dong Hyun Moon †, Eun Seo Bae †, Chae Won Ock †, Daniel Shin †, Joon Soo An †, Hahk-Soo Kang #, Sang Kook Lee †, Ki-Bong Oh ⊥, Illhwan Cho ∥, Hye Yun Kim ∥, YoungSoo Kim ∥, Jichan Jang §, Jang-Cheon Cho ‡, Dong-Chan Oh †,*
PMCID: PMC13014233  PMID: 41889741

Abstract

A logical discovery method based on metabologenomic targeting was designed to search for new β-branched conjugated polyketides. PCR screening of a bacterial DNA library (1,638 strains) targeting the key enzyme hydroxymethylglutaryl-CoA synthase (HCS) homolog-encoding gene as a genomic hallmark identified 67 hit strains. Phylogenetic analysis of HCS homolog-encoding gene amplicons prioritized strains potentially producing new β-branched conjugated polyketides. Vibronic fine structures in ultraviolet spectra as a metabolomic hallmark clearly identified β-branched conjugated polyketides in bacterial extracts, logically leading to the discovery of paenillaene (1), paeniformicins A–D (2–5) from two strains belonging to the relatively unexplored genus Paenibacillus, and pulvomycins E–F (6–7) from a Streptomyces strain. Combined analysis of spectroscopic data, chemical reactions, quantum mechanics-based calculations, and biosynthetic gene clusters led to the structure elucidations of 1–7 as new β-branched conjugated polyketides. Paenillaene (1) dissociated amyloid-β aggregates, paeniformicin D (5) showed significant anti-tubercular activity against Mycobacterium tuberculosis, and pulvomycin E (6) displayed remarkable anti-proliferative activity against several human cancer cells, including a docetaxel-resistant breast cancer cell line. This metabologenomic targeting method provides an efficient tool for systematic discovery of new bioactive β-branched conjugated polyketides.

Keywords: metabologenomic targeting, β-branched conjugated polyketides, hydroxymethylglutaryl-CoA synthase, vibronic fine structure, bioactive natural products

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Introduction

Polyketides are natural products that are biosynthesized using simple C-2 building blocks to manufacture diverse bioactive small molecules with complex structures. Among polyketides produced by microorganisms in nature, the major group consists of α-branched polyketides originating from side chain-bearing building blocks loaded by the acyltransferase (AT) domain. β-branched polyketides are much rarer because of the difficulty of introducing alkyl branches at electrophilic β-carbon. However, bacteria have developed a unique β-branching mechanism involving the aldol addition of acetate to the electrophilic β-carbon of the keto-thioester, which is catalyzed by a key enzyme, hydroxymethylglutaryl-CoA synthase (HCS) homolog. Subsequent dehydration by enoyl-CoA hydratase, which is always found together with HCS homolog in β-branched polyketide biosynthetic pathways, generates a conjugated double bond in most cases and often produces β-branched conjugated polyketides.

Even though β-branched conjugated polyketides have been occasionally discovered from bacteria, they often display significant bioactivity. One of the well-known bioactive examples is mupirocin, which is now being used as a topical ointment antibiotic drug (Bactroban) against bacterial skin infections. The mechanism of action of mupirocin involves the inhibition of bacterial isoleucyl-tRNA synthetase. Bryostatin-1, which bears a β-branching methyl acrylate moiety conjugated with a double bond, was initially isolated from the marine bryozoan Bugula neritina, and its actual origin was elucidated as a bacterial symbiont. This β-branched conjugated polyketide was designated as an orphan drug by the Food and Drug Administration for the treatment of Fragile X syndrome in 2015 (Figure a). Despite their significant pharmaceutical potential, β-branched conjugated polyketides have been randomly reported without a systematic search method, hampering their logical structural diversification. Occasional discoveries since the 1950s include the following examples. Pulvomycin, a 22-membered macrocyclic lactone that bears a triene and two trienone conjugated systems, was discovered in Streptomyces sp. in 1957 and structurally revised in 1985. It exhibits significant antibacterial activity through a unique mode of action by preventing bacterial translational elongation factor Tu·GTP from complexing with aminoacyl-tRNA. − Leinamycin, an 18-membered macrolactam bearing conjugated and β-branched moieties discovered in Streptomyces atroolivaceus, displays both antibacterial and antitumor activities. Kalimantacin, a β-branched conjugated polyketide antibiotic hybrid with a non-ribosomal peptide produced by Alcalgenes sp., exhibits cytotoxicity against two cancer cell lines, P388 mouse leukemic cells and HeLa S3 cells. , Virginiamycin M1, a β-branched conjugated macrolide antibiotic produced by Streptomyces pristinaespiralis, shows bioactivity against Micrococcus aureus (Figure a).

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(a) Representative β-branched polyketides. (β-branched carbons are highlighted in red). (b) β-branch formation through trans-AT PKS by key enzymes of HCS homolog and two ECHs. (c) Workflow of metabologenomic targeting discovery of structurally novel β-branched polyketides.

Because of the high pharmaceutical potential of β-branched conjugated polyketides but the limited logical exploration of their structural diversity, we desired to develop a metabologenomic targeting discovery method for new β-branched conjugated polyketides. We screened our in-house bacterial DNA library (over 1,600 strains mostly without full genome data) with a degenerate PCR primer set targeting the hallmark gene encoding HCS homolog, which plays a key role in biosynthesizing β-branched conjugated polyketides. In addition, vibronic fine structures in ultraviolet (UV) spectra were utilized as metabolomic hallmarks for the first time in natural product discovery, which substantially accelerates the discovery of new β-branched conjugated polyketides. Here, we report the method development and applications demonstrating the discovery of seven new bioactive β-branched conjugated polyketides, paenillaene (1), paeniformicins A–D (2–5), and pulvomycins E–F (6–7).

Results and Discussion

Design of a Degenerate PCR Primer Set Targeting HCS Homolog-Encoding Gene

β-branched modification in polyketides is an ingenious mechanism involving the aldol addition of acetate to the electrophilic β-carbon of the keto-thioester, which is catalyzed by a key enzyme, HCS-homolog. A freestanding T-domain and ketosynthase domain are also involved. Subsequent dehydration and decarboxylation proceed are followed by two enoyl-CoA hydratase (ECH)-mediating steps to generate a single α, β- or β, γ-unsaturated β-branched alkyl group (Figure b), which contributes to conjugated double bond formation. The HCS homolog-encoding gene, which is indispensable in the β-branching cassettes, was selected as a genomic hallmark to mine β-branched polyketide-producing bacterial strains.

Analysis of the four representative HCS homolog-encoding genes from β-branched conjugated polyketide biosynthetic gene clusters in kalimantacin-producing Streptomyces sp. SLBN-134, leinamycin-producing S. atroolivaceus S-140, pulvomycin-producing S. sp. HRS33, and the virginiamycin M1-producing strain S. virginiae enabled the design of a degenerate PCR primer set targeting the HCS homolog-encoding gene (Figure S1). The HCS homology gene primer set was validated using genomic DNA of S. sp. HRS33 (pulvomycin), S. sp. MBP16 (hamuramicin C), S. sp. DM28 (dumulmycin), and S. sp. BA01 (formicolide).

Genomic Hallmark-Based DNA Library Screening and Phylogenetic Analysis of PCR Amplicons

We applied the HCS-like enzyme-encoding gene primer set for screening our in-house DNA library (1,638 bacterial gDNA samples from various natural sources, including intertidal mudflats, volcanic islands, Siberian ice wedges, salterns, deep-sea sediment, and insects) and identified 67 hit strains possessing HCS homologs (hit rate: 4.1%) (Figure c). A phylogenetic tree was generated to analyze the relationships among the HCS homolog gene amplicon sequences (∼680 bp) of the 67 hit strains, along with 27 reference sequences extracted from reported β-branched conjugated polyketides (Figure and Figure S2). The degenerate primers used in this study were designed to target conserved motifs of HCS-like enzymes associated with canonical β-branching cassettes, which have been predominantly characterized in actinobacterial trans-AT PKS pathways. Accordingly, the phylogenetic analysis presented in Figure reflects a representative landscape of HCS homolog-encoding genes accessible through this targeted strategy, rather than the full natural diversity of HCS-like enzymes. Despite the actinobacterial gene-based design, the primer set successfully identified HCS homolog-encoding genes from phylogenetically diverse bacteria, including low-G+C Gram-positive genera (Bacillus and Paenibacillus: 9 strains) and Gram-negative proteobacteria (Pseudomonas and Alcaligenes: 3 strains) among the 67 hits, supporting the utility of this approach for uncovering β-branched polyketides from diverse phylogenetic taxa.

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Phylogenetic tree of HCS homolog gene amplicons and structures of β-branched compounds. Shown in bold and underlined are the 11 strains that actually produce β-branched compounds. 27 reference strains are marked in bold. Bootstrap values are shown on the branches. β-branched carbons are highlighted in red.

The amplicons were sorted into seven monophyletic clades. Clade 1 included the reference strain producing virginiamycin M1 and 26 hit strains. Most of these strains were Streptomyces, but two strains were identified as Bacillus. The reference strains whose metabolites were the exo-β-methylene or β-carboxymethyl group-bearing compounds guangnanmycin, weishanmycin, and largimycin, endo-β-methoxymethyl compound myxovirescin A, leinamycin with an unusual 1,3-dioxo-1,2-dithiolane moiety, ripostatin with a carboxymethyl-β-branch installed by a trans-acting HCS homolog were assigned to clade 2, together with 19 hit strains.

Clade 3 had only one strain, Marinospora sp., which yielded a unique dimeric macrolide with β-methylation, marinomycin. Clade 4 had a single hit strain of Bacillus sp. WA1CD. Clade 5 incorporated various β-branched polyketide-producing Streptomyces strains of kalimantacin, pulvomycins, hamuramicin C, dumulmycin, and formicolides which were mainly macrocyclic lactones with β-methylation, together with 15 hit strains. Clade 6 was composed of a Burkholderia sp., which produced spliceostatin A, and two Burkholderia gladioli strains that produced a macrocyclic lactone gladiolin and bongkrekic acid with a carboxymethyl-β-branch.

Clade 7 was massive with ten reference strains, but only six hit strains. First, two single hit strains of Paenibacillus sp. GAI108 and Pseudomonas sp. GIN12 were represented. Thereafter, in this large clade, one or two reference strains were identified at each descending taxonomic tier, including the reference strains of mupirocin, lasonolide A, β-methyl and β-methylene bearing leptolyngbyalide and phormidolide, oocydin (a chlorinated-branched polyketide natural product), curacin A, jamaicamide A, bryostatin, difficidin, and bacillaene. , Specifically, one hit strain, Paenibacillus sp. BYK1458, formed a small subclade with three more Paenibacillus strains in the core area of clade 7 (Figure ).

The phylogenetic tree of the HCS homolog gene amplicons (∼680 bp) enabled the prioritization of strains for further chemical analysis to discover structurally new β-branched polyketides. In the phylogenetic tree, most of the hit strains were actinobacteria; however, distinctive Paenibacillus strains existed (GAI108, BYK1456, BYK1457, BYK1458, and BYK1468), which were classified only in clade 7. Because the chemistry of Paenibacillus had not been extensively studied with respect to secondary metabolites, in contrast to actinobacteria, we primarily investigated the metabolites of these Paenibacillus strains along with the other hit strains.

Detection of β-Branched Polyene Polyketides Based on Vibronic Fine Structures in UV Spectra as Metabolomic Hallmarks

Conjugated systems in β-branched polyketides confer chromophores, providing UV absorbance usually in the range of 230–340 nm. The extended conjugations of polyenes exhibit strong π → π* electronic transitions at designated wavelengths (λmax) depending on the numbers of conjugations. Moreover, the UV spectra of polyenes display characteristic well-resolved vibronic fine structures, which are observed as line graphs with breaks or sharp multiple peaks in their UV spectra (Figure S3) because of the relatively high symmetry, minimal orbital mixing with heteroatoms, and clear Franck-Condon progressions coupled with an electron transition with a specific vibrational mode.

The vibronic fine structures in the UV spectra enable the unequivocal detection of conjugated double bond-bearing polyketides because they are distinct from those of aromatic chromophores, the general features of polyketide type II natural products, which are not well resolved owing to degenerate electronic states, ring conjugation effects, and more complicated vibrational modes (Figure S4). In addition, the olefinic double bonds in β-branched polyketides can be conjugated with carbonyl groups. The UV spectra of compounds with conjugated carbonyl systems can be easily identified from those of aromatic compounds because they typically show broadened vibronic fine structures modified from sharp multiple polyene features owing to electronic perturbation by the n → π* transition. Therefore, vibronic fine structures can be valuable metabolomic hallmarks of β-branched conjugated polyketides potentially produced from hit strains selected by the genomic hallmark of the HCS homolog-encoding gene.

Vibronic fine structure in UV spectra is not exclusive to β-branched polyketides and can also be observed in other conjugated metabolites, including generic polyenes. Accordingly, in the present workflow, vibronic fine structure is not used as a standalone diagnostic criterion but rather as an initial metabolomic prioritization feature. Selectivity for β-branched polyketides is achieved through orthogonal integration of this UV-based hallmark with genomic hallmark-based PCR screening targeting HCS homolog-dependent β-branching cassettes, such that only strains harboring the canonical β-branching machinery are advanced to LC-MS-based metabolomic analysis and UV spectral inspection.

To further evaluate the possibility of false positives, we surveyed the peer-reviewed literature for reports describing a single bacterial strain that produces both a β-branched conjugated polyene and a non-β-branched conjugated polyene. To the best of our knowledge, documented examples of such coincident production are rare or absent in the current literature. Consistent with this observation, our chemical analyses of the 67 hit strains and reference strains did not reveal concurrent production of β-branched conjugated polyenes and non-β-branched conjugated polyenes. Collectively, these results suggest that, although vibronic fine structure itself is not exclusive, coincident detection of generic conjugated polyenes within the present metabologenomic targeting framework is likely to be rare.

We cultivated all 67 hit strains in 50 mL liquid medium and analyzed the chemical profiles of the extracts by liquid chromatography-mass spectrometry (LC-MS) coupled with a diode array UV detector (see Supporting Information). LC-MS was used to acquire the full UV (200–700 nm) and MS (100–2000 Da) spectra of the chemical constituents in the extracts. We then searched for UV spectra displaying the hallmark vibronic fine structures originating from polyenes and identified the production of β-branched conjugated polyketides. This process also rapidly dereplicated the detected polyketides based on our in-house UV library with 1,803 microbial natural products.

Based on the phylogenetic tree showing the seven clades, we logically searched for new β-branched conjugated polyketides by utilizing the UV metabolomic hallmark. In clade 1, which contains the virginiamycin producer, three Streptomyces strains (ASAS0305, AMD38, and PSN02) were found to produce mainly virginiamycin M1 (Figure S5) by strong absorption at 230 nm in its UV spectrum and a positive molecular ion [M + H]+ at m/z 526 along with the NMR spectra of the compound isolated from ASAS0305 (Table S13 and Figures S88 and S89). These strains (ASAS0305, AMD38, and PSN02) originate from different niches such as mountains and intertidal mudflats. However, they are closely related in the phylogenetic tree of the HCS homolog gene amplicons (Figure ) and produce the identical compound, rationalizing their homologous chemistry and demonstrating the validity of our metabologenomic targeting method.

One strain in clade 2 (Streptomyces sp. C2-2_21) produced weishanmycin A2 (Figure S5), which belongs to the leinamycin family, the major family of clade 2. Clades 3 and 4 both contained only one strain. For clades represented by a single strain, the current sample size is insufficient to infer statistically meaningful trends. In clade 5, the vibronic fine structures in UV were detected in the extracts of the reference strains, confirming the production of pulvomycins, formicolides, dumulmycin, and hamuramicin C in Streptomyces sp. HRS33, Streptomyces sp. BA01, Streptomyces sp. DM28, and Streptomyces sp. MBP16, respectively (Figure S5). Furthermore, the extract of Streptomyces sp. YJD131, which is most closely related to the reference pulvomycin producer, was detected to produce metabolites with the UV vibronic fine structures analogous to those of pulvomycins (Figure S13) but different molecular ions in the MS spectra, indicating new members of the pulvomycin family and thus requiring full structure elucidation. Clade 6 showed only three reference strains without a hit, not allowing for further discovery of β-branched conjugated polyketides.

Interestingly, two Paenibacillus strains (GAI108 and BYK1458) in chemically diverse clade 7 produced metabolites displaying the hallmark vibronic fine structures of polyenes (Figure S13). Because the polyene compounds were not in our UV library and the genus Paenibacillus was chemically underexplored, these strains were prioritized for further scaling-up of the cultures and subsequent chemical analysis.

Structural Elucidation and Bioinformatic Analysis of Biosynthesis of New β-Branched Polyketides

The cultivation of Paenibacillus sp. BYK1458 yielded 1, which was named paenillaene (Figure ). Analysis of its HR-ESI-MS spectrum revealed a negative ion [M-H]− at m/z 608.3223; thus, we determined the chemical formula of 1 to be C35H47NO8, possessing 13 double bond equivalents. One-bond 1H–13C correlations were assigned based on 1H and HSQC NMR data (Table S4 and Figures S14–S19). Five six-membered aromatic ring protons [δH 7.82 (2H), 7.52, and 7.44 (2H)] and seven olefinic protons (δH 6.63, 6.58, 6.52, 5.93, 5.92, 5.65, and 5.22) were observed in the downfield region. One amide proton was identified at 8.30 ppm. Three oxygenated methine protons were detected at 4.75, 3.87, and 3.78 ppm, and nitrogenous methylene protons were identified at 3.52 and 3.44 ppm. Further interpretation revealed the existence of 25 aliphatic protons, including 12 methyl protons [δH 1.78 (3H), 1.70 (3H), 1.64 (3H), and 0.86 (3H)], 12 methylene protons [δH 2.60, 2.35, 2.28, 2.23, 2.22, 2.16, 2.14 (2H), 2.04 (2H), 1.63, and 1.48], and one methine proton (δH 2.25). Analysis of the 13C NMR data along with the 1H and HSQC spectra assigned one ketone (δC 210.9), two carbonyl carbons (δC 173.5 and 167.0), 16 double-bond carbons (δC 140.9–126.4), four oxygenated carbons (δC 98.6, 73.5, 65.9, and 65.3), one nitrogen-bearing carbon (δC 48.3), and 11 aliphatic carbons (δC 48.6–9.2). The partial structures of 1 were assembled using COSY and HMBC correlations (Figure ). First, the spin system from C-2 to C-5 was assigned by COSY correlations from H2-2 (δH 2.22 and 2.35) to H-5 (δH 4.75), and the second spin system from C-7 to C-10 was elucidated based on the consecutive COSY correlations from H-7 (δH 5.92) to H-10 (δH 5.93). The C-12 to C-16 fragment was constructed by a series of COSY cross-peaks from H-12 (δH 6.63) to H-16 (δH 5.22). A small partial structure of C-18–C-19–C-20–C-35 was confirmed by the COSY correlations of H2-18 (δH 2.23 and 2.16), H-19 (δH 3.87), H-20 (δH 2.25), and H3-35 (δH 0.86). The last spin system belonging to a six-membered aromatic ring was constructed by the COSY correlations among H-27, H-28, H-29 (δH 7.52), H-30 (δH 7.44), and H-31 (δH 7.82).

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Structures of paenillaene (1), paeniformicins A–D (2–5), and pulvomycins E–F (6–7).

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Key COSY, HMBC, and ROESY correlations of 1.

The carbonyl carbon C-1 was assigned next to C-2 based on the H2-2 (δH 2.35 and 2.22)/C-1 (δC 173.5) two-bond 1H–13C correlations. The HMBC correlation of H3-32 (δH 1.70) to C-5 (δC 65.9), C-6 (δC 140.9), and C-7 (δC 126.6) installed the C-32 methyl group at C-6, connecting the two spin systems to make the C-2 to C-10 chain. This chain structure was further extended to C-16 by the HMBC correlations from H3-33 (δH 1.78) to C-10 (δC 128.5), C-11 (δC 132.5), and C-12 (δC 127.1). The long-range heteronuclear correlations from H3-34 (δH 1.64) to C-16 (δC 126.4), C-17 (δC 132.3), and C-18 (δC 42.7) constructed the C-2 to C-35 linear structure. On the opposite side, a benzamide moiety was assembled from the aromatic spin system from C-27 to C-31 with the H-28 (δH 7.44)/C-26 (δC 133.6), H-27 (δH 7.82)/C-25­(δC 167.0), and 24-NH (δH 8.30)/C-25 (δC 167.0) HMBC correlations (Figure ).

H2-24 (δH 3.52 and 3.44) displayed COSY correlation with 24-NH (δH 8.30), and HMBC correlations to the amide carbonyl carbon C-25 (δC 167.0) and the dioxygenated carbon C-23 (δC 98.6), thus assigning this methylene between C-23 and 24-NH. C-23 was then connected to C-22 and C-24 based on the H2-22 (δH 2.60 and 2.28)/C-23 (δC 98.6), H2-24 (δH 3.52 and 3.44)/C-23 (δC 98.6) and 24-NH (δH 8.30)/C-23 (δC 98.6) HMBC correlations. H3-35 (δH 0.86) and H2-22 (δH 2.60 and 2.28) showed an HMBC correlation to the ketone carbon C-21 (δC 210.9), extending the chain from C-20 to the terminal benzamide.

The constructed structure contained all 35 carbons in the molecular formula, including five olefinic double bonds, one benzene ring, and three carbonyl groups, accounting for 12 out of 13 unsaturations. Therefore, paenillaene (1) must contain an additional ring. An HMBC correlation from H-19 to C-23 established the connectivity between C-19 and C-23 (Figure S21) through an ether bond and constructed the tetrahydropyranone moiety. The ROESY correlations of H-19 (δH 3.85)/H2-24 (δH 3.52 and 3.44) and H2-22 (δH 2.60, 2.28)/H2-24 further supported the formation of tetrahydropyranone, satisfying the last double bond equivalent. Subsequently, the four invisible −OHs were assigned to a carboxylic acid group at C-1 and three hydroxy groups at C-3, C-5, and C-23. The ROESY correlations shown in Figure determined the geometries of the double bonds and the relative configuration of the tetrahydropyranone. Therefore, paenillaene (1) was elucidated as a structurally novel polyketide incorporating two β-methyl groups at C-11 and C-17 (C-33 and C-35 methyl groups) and a tetraene showing the polyene UV hallmark, along with a benzamide and a tetrahydropyranone.

Whole-genome sequencing of Paenibacillus sp. BYK1458 yielded a draft genome approximately 6.09 Mb in size. We utilized Anti-SMASH 6.0.0 to identify BGCs in the genome (Table S5 and Figure S23). A trans-AT PKS and NRPS hybrid BGC containing 13 modules matched the structure of paenillaene (1). A detailed analysis of this gene cluster identified the β-branch cassette of PnlN–R, which was consistent with the elucidated structure. Furthermore, the absolute configuration of the hydroxy group-bearing stereogenic centers, which could not be determined by the modified Mosher’s method because of the instability of 1 during derivatization under basic conditions, could be predicted by analyzing the presence of a diagnostic Asp residue at the third position within an LDD signature motif in the KR domains. The B1-type KR domains (KR2, KR7, and KR8) supported 3R, 5S, and 19S configurations (Table S6). Thus, we proposed 3R, 5S, 19S, 20S, and 23S configurations for paenillaene (1). The double-bond geometry was also confirmed using the KR-type principle.

Chemical investigation of the culture of Paenibacillus sp. GAI108 led to the discovery of paeniformicins A–D (2–5). Paeniformicin A (2), a predicted β-branched conjugated polyketide (Figure ), was purified as a yellow powder. Based on HR-ESI-MS analysis, its molecular formula was determined to be C33H50O6, with an unsaturated number of 9. Combined analysis of 1H and HSQC NMR data revealed one-bond carbon–proton correlations (Table S7).

The structural fragments were connected using COSY and HMBC correlations. First, the spin system from C-2 to C-18 was assigned by the consecutive COSY correlations from H2-2 (δH 2.36 and 2.27) to H-18 (δH 5.27), and the second spin system from C-19 to C-23 was constructed by the corresponding COSY correlations from H-19 (δH 5.28) to H-23 (δH 5.29). The last spin system in the tail was identified by the COSY correlations of H-25 (δH 5.99) through H2-30 (δH 5.29 and 5.14). The linkage between C-18 and C-19 was confirmed by the H-17 (δH 3.80)/C-19 and H-20 (δH 2.25)/C-18 HMBC correlations. The four hydroxy groups were assigned to C-3 (δC 64.8), C-5 (δC 68.9), C-11 (δC 66.9), and C-17 (δC 71.5) by their corresponding HMBC correlations of 3-OH (δH 4.71)/C-3, 5-OH (δH 4.62)/C-5, 11-OH (δH 4.22)/C-11, and 17-OH (δH 4.64)/C-17. The methyl group C-33 (δC 24.1) was located at the fully substituted olefinic C-24 (δC 134.1), and the two fragments were merged into one by the key HMBC correlations of H3-33 (δH 1.86 (3H))/C-24, H-26 (δH 6.09)/C-24 (δC 134.1), and H2-22 (δH 2.23/2.13)/C-24 (δC 134.1) (Figure ).

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Key COSY, HMBC, and ROESY correlations of paeniformicin A (2).

The ester linkage was secured by the H-21 (δH 4.71)/C-1 (δC 170.9) HMBC correlation to elucidate the planar structure of paeniformicin A (2) as a new 22-membered macrocyclic lactone with one β-branched methyl group and a tetraene moiety displaying the characteristic vibronic fine structure in the UV spectrum (λmax = 310 nm) as a metabolomic hallmark (Figure S13c). The double-bond geometries were assigned as 6E, 14E, 18E, 23Z, 25Z, and 27E based on J-coupling constants and ROESY correlations of 2 (Table S7 and Figure ).

Chiral centers C-9/C-11 and C-20/C-21 were assigned as 9S*/11R* and 20R*/21S* by J-based configuration analysis with vicinal 1H–1H and two- or three-bond 1H–13C coupling constants, along with ROESY correlations (Figures S30 and S33). , The absolute configurations of alcoholic chiral centers C-3, C-5, C-11, and C-17 were determined as 3S, 5R, 11R, and 17R by the modified Mosher’s method (Figures S34–S40). For the stereochemistry of C-20 and C-21, conformational search and DP4 computational calculation were applied to propose 20R and 21S with 100% probability (Figure S41).

The UV spectrum of paeniformicin B (3) indicated the modification of the tetraene moiety in 2 to a triene moiety because it displayed a hypsochromic shift from 310 to 280 nm (Figures S7 and S8). HR-ESI-MS analysis revealed its molecular formula to be C37H54O9, possessing 11 double bond equivalents. Interpretation of the 1H, 13C, and 2D NMR data (Table S7) illustrated that paeniformicin B (3) shares the same macrocyclic skeleton from C-1 to C-21 as 2. Furthermore, based on the COSY correlations of methylene protons H2-28 (δH 2.94) with H-29 (δH 5.81) and H-27 (δH 5.48), we assigned aliphatic methylene carbon C-28 between C-27 and C-29. C-29 was then connected to the terminal olefinic methylene group, C-30, whereas the C-27 end was expanded to C-26 and C-25 via H-27/H-26 and H-26/H-25 homonuclear coupling. The side chain bearing a triene instead of a tetraene was identified using the H-21/H-22 and H-22/H-23 COSY correlations, along with the HMBC correlations from H3-33 to C-23, C-24, and C-25. An additional ester bond between C-11 and C-34 was identified by the key HMBC correlation H-11 (δH 4.87)/C-34 (δC 171.9). The last partial structure was determined to be a succinate side chain using COSY and HMBC correlations, completing the structure of 3 as a new β-branched polyketide bearing a triene (Figure ).

The molecular formula of paeniformicin C (4) was confirmed as C33H52O7 by the molecular ion in the HR-ESI-MS spectrum. The NMR spectroscopic analysis (Table S7) revealed that its carbon skeleton is very similar to that of 3 except for the absence of a succinic side chain. However, paeniformicin C (4) did not form a macrocycle (Figure ).

Paeniformicin D (5) possessed the molecular formula C37H56O10, and its carbon chain was the same as that of 3 according to the NMR spectroscopic analysis (Table S7). Further NMR analysis revealed that 5 has a linear structure without macrolactone formation based on the relatively upfield signal of H-21 (δH 3.99) along with additional H2O in the molecular formula compared to 3 (Figure ). Detailed structural elucidation of compounds 3–5 is provided in the Supporting Information.

Bacteria culture, extraction, and isolation of Streptomyces sp. YJD131 led to the discovery of pulvomycins E and F (6 and 7). Pulvomycin E (6), a yellow powder with the molecular formula C48H68O13 (15 degrees of unsaturation), was characterized using HR-ESI-MS and NMR (1H, 13C, COSY, HSQC, HMBC and ROESY) data. Its planar structure features two ketones, an ester, ten olefins, oxygenated carbons, and three methoxy groups. Key spin systems and HMBC correlations established connectivity, while ROESY and J-coupling constants confirmed geometry for most double bonds and relative configuration in the sugar moiety. J-based configuration analysis and ROESY correlations defined relative configurations. The CD spectrum of 6 matched that of pulvomycin A, confirming its absolute configuration (Figure S82). Pulvomycin F (7), with the same molecular formula of C48H68O13 as 6, differed in double-bond position and geometry (4Z instead of 4E), as supported by NMR and CD comparison with pulvomycin B (Figure S83). The methoxy group was also shifted from C-5 to C-3 compared with 6. (Figure )

Biological Activity

The antimicrobial activities of the new β-branched conjugated polyketides 1–7 were evaluated against human pathogenic bacterial and fungal strains (Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Klebsiella pneumoniae, Salmonella enterica, Escherichia coli, Candida albicans, Aspergillus fumigatus, Trichophyton rubrum, and Trichophyton mentagrophytes) (Tables S14 and S15). Paeniformicin B (3) showed high antibacterial activity against S. aureus (MIC = 0.5 μg/mL) and K. pneumoniae (MIC = 8.0 μg/mL), and paeniformicin A (2) displayed moderate antibacterial activity against S. aureus (MIC = 32 μg/mL). Paenillaene (1) showed mild bioactivity against several strains [S. aureus (MIC = 64.0 μg/mL), E. faecalis (MIC = 64.0 μg/mL), and E. faecium (MIC = 64.0 μg/mL)]. Based on the structural features of 1-5, the macrolactone scaffold bearing a limited number of hydroxyl groups, together with the succinate moiety, might contribute to the enhanced antibacterial activity of compound 3. Further investigation of their S. aureus sortase A (SrtA) inhibition assay was conducted, and paeniformicin A (2) showed moderate bioactivity (IC50 = 34.2 μg/mL) (Table S16). Especially paeniformicin D (5) exhibited significant bioactivity against Mycobacterium tuberculosis mc2 6230 (MIC50 = 0.27 μM), whereas the other compounds showed no anti-tuberculosis activity (Table S17).

The cytotoxicity of compounds 1–7 was evaluated against several human cancer cell lines. 1 showed mild cytotoxicity against colon cancer (HCT116) and stomach cancer (SNU638) cell lines (IC50 of 38.0 and 37.2 μM, respectively), whereas paeniformicins showed no cytotoxicity (Table S18). Pulvomycin E (6) showed high cytotoxic effects against all the tested cancer cell lines with IC50 values of 7.58, 1.28, 6.58, 4.17, 4.61, and 3.11 μM for A549, HCT116, SNU-638, SK-HEP-1, MDA-MB-231, and docetaxel-resistant MDA-MB-231 (MDA-DTX) cell lines, respectively. 7 also presented mild cytotoxicity against these human cancer cell lines (Table S19).

We further examined the biological effects of structurally novel paenillaene (1) against amyloid-β (Aβ) oligomers, which are the major pathological and biological hallmarks in the brains of patients with Alzheimer’s disease (AD). Because the most promising therapeutic strategy for AD is to remove preformed aggregates of Aβ, we utilized an Aβ42 aggregate dissociation assay to evaluate the dissociation ability of 1. We observed a significant and dose-dependent reduction of the fluorescence signal, indicating that 1 induced dissociation of Aβ oligomers, with a 29% reduction at 5 μM and 57% at 50 μM, suggesting that 1 may reduce preformed Aβ aggregates in the AD brain. Additional animal and clinical studies are warranted to determine whether 1 may translate into a therapeutic agent (Figure S90).

Conclusions

The genomic hallmark-based PCR screening, which specifically targets the homologous DNA sequence encoding HCS homolog, the essential enzyme biosynthesizing β-branched conjugated polyketides, enabled the selection of bacterial strains potentially producing β-branched conjugated polyketides without full genome analysis. By employing phylogenetic analysis of the partial HCS homolog-encoding gene sequences obtained through PCR amplification (∼680 bp), we successfully sorted various types of β-branched conjugated polyketides into seven clades to construct their clade-structure atlas in nature, which guided the selection of strains to be investigated further for the discovery of structurally novel β-branched polyketides. Vibronic fine structures in UV spectra were utilized as metabolomic hallmarks in natural product discovery and facilitated the rapid identification of β-branched conjugated polyketides in small-scale cultures.

Further cultivation and chemical analyses of the hit strains led to the identification of β-branched conjugated polyketides from four clades except for three clades containing only one strain or reference strains. Such clade-based chemical investigation efficiently discovered seven new β-branched polyketides: paenillaene (1), paeniformicins A–D (2–5), and pulvomycins E–F (6–7) from chemically prolific clades 5 and 7 and identified previously reported virginiamycin M1 and weishanmycin A1 from clades 1 and 2 as predicted by the phylogenetic clade-structure atlas (Figure ). The structures of the new compounds, including their stereochemistry, were elucidated using spectroscopic, genomic, and computational analyses. In particular, paenillaene (1), bearing a benzamide, tetrahydropyran, and tetraene chain, showed high structural novelty, possibly because of its unique starting unit, benzoic acid, which was subsequently connected to glycine by the trans-AT PKS and NRPS hybrid pathway.

Evaluation of the new β-branched polyketides revealed significant anti-tuberculosis activity of paeniformicin D (5) against Mycobacterium tuberculosis, accentuating that the uncyclized and succinylated form is important for antitubercular activity. In addition, paenillaene (1) dissociated amyloid-β (Aβ) aggregates, indicating potential related to AD. Pulvomycins E–F (6–7) showed anti-proliferative activity with several cancer cells, especially against the MDA-DTX cell line.

The discovery of these novel β-branched polyketides, along with our recent systematically-targeting discovery of natural products, − underscores the efficacy of the metabologenomic targeting method as a logical approach for discovering new bioactive compounds with targeted structural motifs.

Supplementary Material

au6c00051_si_001.pdf (11.3MB, pdf)

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2025-00553834, Republic of Korea) to D.-C. Oh. This research was also supported by Learning & Academic research institution for Master’s·PhD students, and Postdocs (LAMP) Program of the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education (RS-2023-00301974, Republic of Korea).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.6c00051.

  • Detailed descriptions of materials and methods, including genomic hallmark-based screening, cultivation of hit strains, purification of compounds, biological evaluation, and NMR spectra (PDF)

Conceptualization: D.-C.O., J.C.; investigation: J.C., H.L., B.E.H., S.Y., J.L., D.H.M., E.S.B., C.W.O.; project administration: D.-C.O.; formal analysis: J.C., I.K., D.S.; data curation: I.K., J.-C.C.; resources: J.C., H.L., D.S., J.S.A., D.-C.O.; writing-original draft: J.C., I.K., H.L., B.E.H., S.Y.; writing-review and editing: J.S.A., H.-S.K., S.K.L., K.-B.O., I.C., H.Y.K., Y.K., J.J., J.-C.C., D.-C.O.; supervision: D.-C.O.; funding acquisition: D.-C.O. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

∇.

Deceased on December 19, 2020.

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