Frankiamicin A bioactivity assay results.

<p><b>[a]</b> Assessed after 6 h incubation.</p><p><b>[b]</b> Assessed after 18 h incubation.</p><p>Frankiamicin A bioactivity assay results.</p

Structural analysis and elucidation of frankiamicin A (4).

<p>a) HMBC correlations and ¹³C-¹³C couplings observed through [1,2-¹³C₂]acetate feeding. b) structure of frankiamicin A.</p

General summary of type II polyketide biosynthesis.

<p>The key steps in type II polyketide biosynthesis—priming of the minimal polyketide synthase, extension of the polyketide chain by the ketosynthase α/β heterodimer to generate the poly-β-ketone intermediate, cyclization and aromatization of the poly-β-ketone by the immediate tailoring enzymes (aromatase/cyclase and cyclases) to form the cyclized core structure, and tailoring by various polyketide tailoring enzymes—are shown, using the elloramycin biosynthetic pathway as an example. Structural elements of the intermediates and final product are color-coded according to which enzymes catalyze their formation.</p

Expanding our Understanding of Sequence-Function Relationships of Type II Polyketide Biosynthetic Gene Clusters: Bioinformatics-Guided Identification of Frankiamicin A from <i>Frankia</i> sp. EAN1pec

<div><p>A large and rapidly increasing number of unstudied “orphan” natural product biosynthetic gene clusters are being uncovered in sequenced microbial genomes. An important goal of modern natural products research is to be able to accurately predict natural product structures and biosynthetic pathways from these gene cluster sequences. This requires both development of bioinformatic methods for global analysis of these gene clusters and experimental characterization of select products produced by gene clusters with divergent sequence characteristics. Here, we conduct global bioinformatic analysis of all available type II polyketide gene cluster sequences and identify a conserved set of gene clusters with unique ketosynthase α/β sequence characteristics in the genomes of <i>Frankia</i> species, a group of Actinobacteria with underexploited natural product biosynthetic potential. Through LC-MS profiling of extracts from several <i>Frankia</i> species grown under various conditions, we identified <i>Frankia</i> sp. EAN1pec as producing a compound with spectral characteristics consistent with the type II polyketide produced by this gene cluster. We isolated the compound, a pentangular polyketide which we named frankiamicin A, and elucidated its structure by NMR and labeled precursor feeding. We also propose biosynthetic and regulatory pathways for frankiamicin A based on comparative genomic analysis and literature precedent, and conduct bioactivity assays of the compound. Our findings provide new information linking this set of <i>Frankia</i> gene clusters with the compound they produce, and our approach has implications for accurate functional prediction of the many other type II polyketide clusters present in bacterial genomes.</p></div

NMR spectroscopic data (DMSO-<i>d</i>_<i>6</i>) for frankiamicin A (4).

<p>[a] Coupling constants in Hz, observed by [1,2-¹³C₂]acetate feeding.</p><p>[b] Obscured by overlapping.</p><p>NMR spectroscopic data (DMSO-<i>d</i>_<i>6</i>) for frankiamicin A (4).</p

Structures of prototypical type II polyketides.

<p>Structures of chlortetracycline (<b>1</b>), doxorubicin (<b>2</b>), R1128A (<b>3</b>), and the pentangular polyketide frankiamicin A (<b>4</b>) identified in this study.</p

Multiple sequence alignment of training set and <i>Frankia</i> KSα/β active site residues.

<p>Eight regions of KSα/β protein sequence from the 64 KSα/β training set members and eleven <i>Frankia</i> KSα/β sequences that are predicted to be in the closest proximity to the active site based on the X-ray crystal structure of the actinorhodin (<i>act</i>) KSα/β are shown. The five regions that lie within KSα and the three that lie within KSβ are noted by labeled black bars at the top of the figure. Predicted proximity to the active site is shown as a heat map at the top of the figure (red residues line the active site pocket, orange residues are within 4Å of the residues that line the active site, yellow residues are within 6Å, and green residue are within 8Å. Black squares immediately below the heat map mark the seven residues previously proposed to be responsible for product specificity. Residues are numbered using <i>act</i> numbering. Training set product names and <i>Frankia</i> cluster names are given to the left. Starter unit and number of extender units of training set systems appear on the far left. Ac—acetyl, Pr—propionyl, Mal—malonamyl, Gly—glycyl, Bu—butyryl, iBu—isobutyryl, Azd—aziridinyl, Hxd—hexadienyl, Hex—hexanoyl, MeBu—2-methylbutyryl, Bz—benzoyl.</p

Proposed frankiamicin A biosynthetic pathway.

<p>The minimal polyketide synthase FkmABC catalyze conversion of 12 malonyl-CoA units to the 24 carbon poly-β-ketone <b>6</b>; TcmN-like aromatase/cyclase FkmC1 catalyzes closure and aromatization of rings A and B; FkmC2, C3, O1, and O2 catalyze closure of the C, D, and E rings, aromatization of the C and E rings, and oxygenation of the B ring; FkmD catalyzes reduction of the C-6 ketone to form G-2A (<b>5</b>); and a P450 monooxygenase catalyzes C-5 hydroxylation to generate frankiamicin A (<b>4</b>).</p

Dendrogram of KSα/β sequences showing the relationship between dendrogramatic position, polyketide subclass, and poly-β-ketone structure.

<p>Dendrogram based on multiple alignment of 296 concatenated KSα/β protein sequences illustrating the large uncharacterized clade (left, shaded purple) in which KSα/β pairs from <i>Frankia</i> type II polyketide clusters that are the subject of this study (marked with purple bar) are found. KSα/β pairs from previously characterized type II polyketide clusters are colored according to their starter unit and number of extender units (see bottom figure legend, starter/extender colors are listed clockwise as they first appear in the figure). Type II polyketide subclasses are labeled and bracketed. Subclass abbreviations: REM—resistomycin; SP—spore pigment; PEN—pentangular; TCM—tetracenomycin; ANT—anthracycline; HED—hedamycin; R1128—R1128; ENT—enterocin; BIQ—benzoisochromanequinone; TET—tetracycline; AUR—aureolic acid; ANG—angucycline. Other abbreviations: <i>E</i>. <i>coli</i> FAS—<i>E</i>. <i>coli</i> fatty acid synthase, which was used as the outgroup.</p

UV-visible and mass spectral analysis of <i>Frankia</i> extracts and metabolites.

<p>(a) HPLC analysis of extracts from the three <i>Frankia</i> species grown using different carbon sources, and showing the presence of the major compound (labeled A) and the minor compound (labeled B). (b-g) ESI-MS analysis in positive and negative ionization modes and photodiode array (PDA) spectra of the major and minor peaks (data collected from 9.4–9.7 min, 12.7–12.9 min, respectively). (b) major peak, positive mode (M + H − H₂O and M + H − 2 H₂O); (c) minor peak, positive mode (M + H, M + H − H₂O); (d) major peak, negative mode (M − H, M − H − CO₂); (e) minor peak, negative mode (M − H, M − H − CO₂); (f) PDA spectrum of the major peak; (g) PDA spectrum of the minor peak.</p