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Sequence-guided mining of metagenomic libraries provides a means of recovering specific natural product gene clusters of interest from the environment. In this study, we use ketosynthase gene (KS) PCR amplicon sequences (sequence tags) to explore the structural and biosynthetic diversities of pentangular polyphenols (PP). In phylogenetic analyses, eDNA-derived sequence tags often fall between closely related clades that are associated with gene clusters known to encode distinct chemotypes. We show that these common “intermediate” sequence tags are useful for guiding the discovery of not only novel bioactive metabolites but also collections of closely related gene clusters that can provide new insights into the evolution of natural product structural diversity. Gene clusters corresponding to two eDNA-derived KS β sequence tags that reside between well-defined KS β clades associated with the biosynthesis of (C24)-pradimicin and (C26)-xantholipin type metabolites were recovered from archived soil eDNA libraries.
Heterologous expression of these gene clusters in Streptomyces albus led to the isolation of three new PPs (compounds 1– 3). Calixanthomycin A ( 1) shows potent antiproliferative activity against HCT-116 cells, whereas arenimycins C ( 2) and D ( 3) display potent antibacterial activity. By comparing genotypes and chemotypes across all known PP gene clusters, we define four PP subfamilies, and also observe that the horizontal transfer of PP tailoring genes has likely been restricted to gene clusters that encode closely related chemical structures, suggesting that only a fraction of the “natural product-like” chemical space that can theoretically be encoded by these secondary metabolite tailoring genes has likely been sampled naturally. The analysis of DNA sequence is increasingly being used to guide the discovery of natural product biosynthetic gene clusters capable of encoding for novel metabolites. When studying cultured bacteria, it is possible to fully sequence a bacterial genome and therefore carry out detailed analyses of complete biosynthetic gene clusters to identify those likely to encode for novel metabolites. Similar full gene cluster analyses are not usually possible when exploring complex environmental microbiomes because the immense size of most metagenomes precludes full sequencing and assembly of complete gene clusters.
To circumvent this limitation, we and others have explored the use of the detailed phylogenetic analysis of individual, highly conserved natural product biosynthetic genes as markers for guiding the discovery of functionally novel natural product biosynthetic gene clusters from complex metagenomes. In this approach, individual biosynthetic genes are PCR-amplified from environmental DNA (eDNA) and sequenced in bulk.
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The resulting amplicon sequences (i.e., natural product sequence tags) are then aligned with reference sequences from functionally characterized gene clusters. In previous studies, we have shown that sequence tags that are only distantly related to any known sequence (e.g., sequence tag A in Figure ) can guide the identification of gene clusters that encode structurally novel bioactive natural products. We have also shown that sequence tags that are very closely related (e.g., sequence tag B in Figure ) to known genes can guide the discovery of gene clusters that encode congeners of known molecules.
While the utility of sequence tags that reside at the phylogenetic boundaries is now clear, many sequence tags fall at a more ambiguous intermediate distance from known sequences (e.g., sequence tag C in Figure ). We hypothesized that such “intermediate” sequence tags, especially those that fall between clades containing sequences known to encode for structurally distinct subclasses of natural products, might be useful for guiding the discovery of gene clusters that both encode novel bioactive metabolites and provide insights into the evolution of natural products structural diversity. Here, we explore these concepts through the comparison of the genotypes (i.e., gene content) and chemotypes (i.e., molecules encoded by) of eDNA-derived aromatic polyketide (PK) pentangular polyphenols (PPs).
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Aromatic PKs are a large group of secondary metabolites generated by iterative (i.e., type II) PKS biosynthetic systems. The initial step in aromatic PK biosynthesis involves the action of a minimal polyketide synthase (min-PKS). The min-PKS is composed of the three proteins KS α, KS β and ACP, which together catalyze the iterative condensation of malonyl-CoA into polyketides ranging from 16 to 30 carbons in length. PPs are constructed from the largest nonreduced PK precursors known (24–30 carbons in length) and are, therefore, potentially the most structurally diverse subclass of metabolites in this family. Mining of soil eDNA for pentangular polyphenols. (a) Phylogeny-guided strategy for the discovery of new natural products. (b) The maximum likelihood tree of partial KS β gene sequences is shown.
AZ, AB and TX denote amplicon sequences derived from Arizona, California and Texas desert soil eDNA libraries, respectively. A number of eDNA-derived PP KS β sequence tags fall between clades associated with PPs derived from polyketide precursors of different lengths (C24 and C26). The specific group of intermediate eDNA sequence tags used in this study are highlighted in orange. The highly conserved KS α and KS β genes from the min-PKS have proved particularly useful as sequence tags for guiding the discovery of new aromatic polyketides from metagenomes. While eDNA KS α and KS β sequencing studies suggested the presence of large numbers of unique PP clusters in the environment, the biosynthetic landscape of PPs from culture-based studies remained quite limited. In this study PP gene clusters associated with eDNA-derived KS α and KS β sequence tags that fall between well-defined clades containing genes from the biosynthesis of pradimicin and xantholipin type PP metabolites were recovered from archived soil eDNA libraries. The molecules encoded by these PP gene clusters were accessed through heterologous expression in Streptomyces albus and found to encode new bioactive pentangular polyphenols with potent antiproliferative and antibacterial activities.
By comparing the gene cluster genotypes and chemotypes across this expanded pentangular polyphenol biosynthetic landscape, we gain specific insights into pentangular polyphenol biosynthesis and general insights into potential limitations of the evolution of natural product structural diversity. To identify novel gene clusters encoding PP PKs, archived soil eDNA libraries were screened with degenerate primers targeting either the KS α or KS β genes of the min-PKS. KS α and KS β phylogenetic trees were constructed with the resulting eDNA amplicon sequences and sequences from previously characterized type II PKS gene clusters deposited in GenBank. In this phylogenetic analysis we identified two sequence tags of interest (KS β tag AB1692 and KS α tag AB1414) from our Anza Borrego Desert (AB) eDNA library. Both sequence tags fall between well-defined clades containing KS genes from gene clusters that encode structurally distinct PP subfamilies, pradimicins and xantholipin (Figure b). Sequence tags AB1692 and AB1414 were used to guide the recovery of eDNA cosmid clones containing PP biosynthetic gene clusters associated with these KS sequences. The recovered cosmids were PGM (Personal Genome Machine)-sequenced and this sequence data was subsequently used to guide the recovery of additional cosmid clones overlapping each end of the two min-PKS containing cosmids.
Sequencing and bioinformatics annotation of each set of three overlapping cosmid clones revealed a biosynthetic gene cluster flanked by collections of genes predicted to encode primary metabolic enzymes, suggesting that both gene clusters were recovered in their entirety on three overlapping cosmids. To access the metabolites encoded by the AB1692 and AB1414 PP gene clusters, each set of three overlapping eDNA cosmid clones (AB1692, AB916 and AB170; AB1442, AB1414 and AB561) was assembled into bacterial artificial chromosomes (BAC-AB1692/916/170 and BAC-AB1442/1414/561) using pathway specific pTARa ( E. Coli:yeast: Streptomyces) shuttle capture vectors and transformation-associated recombination (TAR) in yeast (Figure ). These approximately 90 kb BAC constructs were PGM-sequenced to ensure correct assembly and transferred into Streptomyces albus by intergenic conjugation and integrated into the S.
Albus genome using the ΦC31 integrase. For heterologous expression purposes, the resulting strains, S. Albus BAC-AB1692/916/170 and S. Albus BAC-AB1442/1414/561, were grown at 30 °C in R5A liquid media for 9 days. LC–MS analysis of EtOAc extracts derived from these cultures showed the presence of one and two major clone-specific peaks, respectively (Figure ). The three clone-specific metabolites were purified from the extracts of 1 L cultures using, in each case, two rounds of C 18 reversed-phase HPLC.
This gave calixanthomycin A ( 1) (12 mg/L) from S. Albus BAC-AB1692/916/170, and arenimycins C ( 2) (4.6 mg/L) and D ( 3) (2.5 mg/L) from S. Albus BAC-AB1442/1414/561. Heterologous expression and the structures of calixanthomycin A ( 1), and arenimycins C ( 2) and D ( 3).
Two PP gene clusters were recovered from California desert soil eDNA library on three overlapping eDNA cosmid clones (AB1692/916/170 and AB1442/1414/561). They were each reconstructed into BAC clones using TAR. The heterologous expression of these two PP gene clusters by S.
Albus led to the production of three clone-specific metabolites, compound 1 from the strain S. Albus BAC-AB1692/916/170, and compounds 2 and 3 from the strain S. Albus BAC-AB1442/1414/561.
Detailed HPLC trace analysis can be found in the (Figure S2). The structure of calixanthomycin A ( 1) features a xanthone-containing PP core structure. The polycyclic xanthone core is tailored by oxidation, O-methylation and glycosylation.
Rare structural features include the F ring lactone, which is more commonly seen as a lactam in xanthone-containing PPs, and the ortho-dimethoxy functionality on the A ring. Calixanthomycin A ( 1) is most closely related to IB-00208 from an unsequenced marine Actinomadura sp. They differ by the position of methoxy groups around the A ring, the presence or absence of the double bond in the D ring and the oxidation state of the C ring. The structures of arenimycins C ( 2) and D ( 3) produced by S.
Albus BAC-AB1442/1414/561 feature a highly oxidized benzo anaphthacene quinone core (rings A through E). This core structure is appended with a disaccharide ( OMe- l-rhamnose-OMe- l-Olivose) in 2 and a monosaccharide ( l-rhamnose) in 3 via a rare N-glycosidic linkage. These structures share an aglycone with the SF2446s from a soil actinomycete Streptomyces sp. SF2446, and the recently reported arenimycins A and B from a marine actinomycete Salinispora sp. Compounds 1– 3 were evaluated for antibacterial and antiproliferative activities (Table ).
Calixanthomycin A ( 1) is extremely toxic to HCT-116 cancer cells (IC 50 0.43 nM); however, it shows no antibacterial activity against MRSA (Methicillin-resistant S. Aureus) and VRE (Vancomycin-resistant Enterococcus faecium) at the highest concentrations tested (50 μg/mL), and weak activity against B. Subillis (MIC = 3.1 μg/mL). This is quite interesting as most reported xanthone-containing PPs display a broad spectrum of activity with both potent human cell cytotoxicity and antibacterial activities. Arenimycin C ( 2) shows potent Gram-positive antibacterial activity (MIC = 98 ng/mL against MRSA; 1.5 ng/mL against B. Subtilis) and moderate cytotoxicity against HCT-116 cells (IC 50= 0.17 μM). BAC-AB1692/916/170 is predicted to harbor the 50 kb Clx (calixanthomycin) gene cluster (GenBank KM881706) containing 47 genes involved in the biosynthesis, regulation and resistance of calixanthomycin A ( 1).
In our biosynthetic proposal, the predicted min-PKS (Clx9–11), cyclases (Clx8, 30 and 31) and a ketoreductase (Clx1) are responsible for generating a benzo anaphthacene quinone intermediate using an acetate starter unit and 12 malonyl CoA extension steps. The quinone could then be transformed to a xanthone via an oxidative rearrangement catalyzed by the predicted Bayer-Villiger oxidase (BVO) Clx27 (51% identity to pnxO4 from the FD-594 gene cluster). The final tailoring steps in our proposed calixanthomycin A ( 1) biosynthesis scheme involve formation of the ortho-dimethoxy functionality in the A ring and glycosylation in the E ring.
Formation of the rare ortho-dimethoxy functionality would require reduction at C-13 and hydroxylation at C-12 followed by two O-methylations. The mechanism of C-13 reduction is not clear at this point, but the hydroxylation at C-12 is predicted to be catalyzed by the cytochrome-P450 hydroxylase Clx28 due to its sequence similarity to PnxO5 (61% sequence identity) from the FD-594 gene cluster. Methylation of the ortho-hydroxyl groups could then occur by the action of the predicted O-methyltransferases Clx2 and Clx43. In our proposed biosynthesis, the resulting hexacyclic xanthone aglycone is appended with a d-quinovose sugar moiety by the predicted glycosyltransferase Clx40. BAC-AB1442/1414/561 harbors the 40 kb Arn (arenimycin) gene cluster (GenBank KJ440489) containing genes predicted to be involved in the biosynthesis, regulation and resistance of arenimycins C ( 2) and D ( 3).
In our proposed biosynthetic scheme the benzo anaphthacene core is synthesized via a min-PKS (Arn31, 32 and 36), an aromatase (Arn21), two cyclases (Arn19 and 20) and a ketoreductase (Arn33) (Figure ). On the basis of their high sequence identity to pdmH from the pradimicin gene cluster, Arn 22 and 37 are predicted to oxidize C-6 and C-10 to form the two 1,4-benzoquinone moieties (A and C rings) seen in 2 and 3. Arn 17, which shows 54 and 43% sequence identity to GrhO8 from the griseorhodin gene cluster and XanO5 from the xantholipin gene cluster, respectively, is likely to be involved in hydroxylations at the two angular positions (C-5 and C-18). Finally, this aglycone is predicted to be glycosylated by two glycosyltransferases Arn11 and Arn14, completing the biosynthesis of 2 and 3. The mechanism of N-glycosidic bond formation is not clear at this point. Different PP chemistries are organized according to the phylogenetic analysis of KS β gene sequences (Figure ). This analysis reveals four different PP scaffolds with distinct polyketide chain lengths.
We have designated these scaffolds PP-A, PP-B, PP-C and PP-D. Each different PP scaffold and the compounds arising form the scaffold are shown in different colors. Structures shown in black are hypothetical intermediates.
The structures of sugar moieties were not shown due to the limited space. The Arn gene cluster harbors all genes predicted to be required for the biosynthesis of the sugar moieties (rhamnose and olivose) found in 2 and 3 (Figure ). Interestingly, it also contains three additional predicted sugar biosynthesis genes, whose functions could not be assigned to a specific transformation in the biosynthesis of the sugar moieties found on 2 or 3.
These include genes predicted to encode for an N, N-dimethyltransferase (Arn6), an aminotransferase (Arn7) and a 3,4-dehydratase (Arn8). These observations along with the recently reported structure of arenimycin B, which contains the dimethylated amino deoxysugar forosamine in place of the OMe- l-olivose seen in 2, led us to re-examine culture broth extracts obtained from BAC-AB1442/1414/561 by LC–MS. The selective ion chromatogram for m/ z = 809 revealed the presence of a minor clone specific compound with a mass corresponding to that of arenimycin B. It appears that the eDNA-derived Arn gene cluster has the potential to encode a number of different glycosylated compounds, with arenimycin C ( 2) being the major product. The annotated genome of Salinispora arenicola CNB527, the arenimycin B producer, was recently made publicly available (GenBank: NZAZXI01000002). A comparison of the arenimycin B and the Arn gene clusters reveals that these two gene clusters are very closely related, showing 90 to 95% sequence identity between most biosynthetic genes.
In total, 12 PP biosynthetic gene clusters have now been sequenced and functionally characterized, including eight clusters from culture-based studies and four from culture-independent studies. In addition to the eDNA-derived Clx and Arn clusters described here, we previously reported eDNA gene clusters that encode for fasamycin and arixanthomycin type PPs. KS α and KS β sequence tags associated with these four eDNA clusters were selected by us for detailed analyses because they all fall between well characterized KS clades (i.e., intermediate sequence tags). In the following analyses, we explore this closely related collection of biosynthetic gene clusters to gain insights into the evolution of natural product structural diversity. The individual proteins that makeup min-PKS (KS α, KS β, and ACP) are highly conserved across type II PKS gene clusters. KS α and KS β phylogenetic trees show very similar topologies, both of which correlate closely with differences in the core polyketide structure (e.g., chain length and cyclization pattern) encoded by the gene cluster from which the min-PKS arises. KS β and, to a lesser extent, KS α genes have thus proved to be useful phylogenetic markers for predicting differences in polyketide core structures encoded by type II PKS gene clusters.
While PPs have so far been considered a single class of type II polyketides, based on both KS phylogeny and natural product structure, it appears that they arise from four distinct min-PKS lineages. These lineages differ in starter unit selectivity (acetate or hexanoate) and the number of malonyl CoA chain extension steps they carry out (11 or 12). PP polyketide precursors range from 24 to 30 carbons in length and generate four unique PP scaffolds that we have designated PP-A (C24–acetate and 11 extensions), PP-B (C26–acetate and 12 extensions), PP-C (C28–hexanoate and 11 extensions) and PP-D (C30–hexanoate and 12 extensions) (Figure ). The 30-carbon D scaffold is the largest polyketide chain observed in aromatic polyketide biosynthesis. With more than 30 predicted B scaffold-based natural products reported in the literature, this 26-carbon scaffold, which arises from an acetate starter unit and 12 malonyl CoA extension steps is responsible for most of the structural diversity seen in known PPs (Figure ). The only reported gene clusters predicted to encode the A, C, and D scaffolds are the pradimicins (PP-A), arenimycins (PP-A), benastatins (PP-C), FD-594s (PP-C) and fredericamycins (PP-D).
Although both KS α and KS β gene phylogenies indicate that the four PP scaffolds (A–C) have evolved independently from a common ancestor, similar tailoring modifications are observed across scaffolds from different lineages. This is especially interesting in light of the fact that some of modifications seen in multiple different PP lineages (e.g., the xanthone core and the gem-dimethyl functionality) are in fact quite rare outside of PP biosynthesis. This suggests that functionally successful horizontal transfer of the tailoring genes responsible for these modifications has not occurred globally throughout bacterial secondary metabolism but instead “locally” within the limited universe of PP biosynthesis. To evaluate evolutionary relationships between the genes responsible for the common modifications found across different PP scaffolds, we carried out detailed phylogenetic analyses of the four common tailoring genes seen in PP gene clusters including Baeyer–Villiger oxidase, geminal bis-methyltransferase, Asn-synthetase homologue and glycosyltransferase (Figure ). The most notable tailoring modification observed in PP biosynthesis is an oxidative rearrangement of the PP scaffold to generate either a xanthone or a spiroketal.
These oxidative rearrangements involve the action of FAD-dependent Baeyer–Villiger oxidase (BVO). BVOs are also found in some angucycline gene clusters (GilOI, JadH and UrdM) and in the mithramycin gene cluster (MtmOIV). To determine the phylogenetic relationship between these genes, a maximum likelihood phylogenetic tree was generated using full-length BVO gene sequences (Figure ).
In this analysis, the PP BVO genes form a monophyletic clade that is distinct from other BVO genes, suggesting that the PP BVO gene was acquired once in PP biosynthesis and diverged to enable the production of distinct xanthone and spiroketal functionalities. Interestingly, the BVO genes Clx27 and PnxO4 from the calixanthomycin A and FD-594 gene clusters, respectively, show the highest sequence identity even though their KS β genes belong to distinct PP lineages (Figure ). This suggests that once the BVO gene successfully entered the PP biosynthetic universe it was not only passed down vertically through the PP-B lineage, but also horizontally transferred between PP lineages. Curiously, while it appears that this BVO gene was capable of horizontal transfer between PP lineages with related core structures, similar, more global transfer to numerous other sequenced type II PKS gene cluster families is not observed, as the BVO genes found in other type II PKS gene clusters appear to be of distinct phylogenetic lineages. Geminal bis-methylation is another rare tailoring reaction that is seen in PP biosynthesis. The introduction of this functional group is known to occur through the action of a single, SAM-dependent methyltransferase (geminal bis-methyltransferase, GBM) via two rounds of C-methylation.
Known aromatic polyketides with this functionality include two PPs, the fasamycins and the benastatins, a pentacyclic polyketide resistomycin, and the tetracyclic quinone tetarimycin. In resistomycin biosynthesis, two methyl transferases are predicted to be required to introduce the dimethyl functionality, neither of which is closely related to the PP GBMs.
The three tetracyclic structures upon which the PP-like GBMs act (fasamycins, benastatins and tetarimycins) are closely related (Figure ), once again suggesting that horizontal transfer of these tailoring genes has been limited to interchange between gene clusters that encode closely related core structures. Overview of PP biosynthesis gene phylogenies. (a) Phylogenetic relationships between KS α, KS β, Baeyer–Villiger oxidase, Asn synthase homologue, geminal bis-methyltransferase and glycosyltransferase genes from PP biosynthetic gene clusters are reconstructed from the maximum likelihood phylogenetic trees to highlight the relationships between genes from clusters of different PP lineages. (b) The parent maximum likelihood phylogenetic trees for BVO genes, GBM genes and Asn synthase homologue genes are shown.