Identification and analysis of the resorcinomycin biosynthetic gene cluster

<div><p>Resorcinomycin (1) is composed of a nonproteinogenic amino acid, (<i>S</i>)-2-(3,5-dihydroxy-4-isopropylphenyl)-2-guanidinoacetic acid (2), and glycine. A biosynthetic gene cluster was identified in a genome database of <i>Streptoverticillium roseoverticillatum</i> by searching for orthologs of the genes responsible for biosynthesis of pheganomycin (3), which possesses a (2)-derivative at its <i>N</i>-terminus. The cluster contained a gene encoding an ATP-grasp-ligase (<i>res5</i>), which was suggested to catalyze the peptide bond formation between 2 and glycine. A <i>res5</i>-deletion mutant lost 1 productivity but accumulated 2 in the culture broth. However, recombinant RES5 did not show catalytic activity to form 1 with 2 and glycine as substrates. Moreover, heterologous expression of the cluster resulted in accumulation of only 2 and no production of 1 was observed. These results suggested that a peptide with glycine at its <i>N</i>-terminus may be used as a nucleophile and then maturated by a peptidase encoded by a gene outside of the cluster.</p></div

A Glycopeptidyl-Glutamate Epimerase for Bacterial Peptidoglycan Biosynthesis

d-Glutamate (Glu) supplied by Glu racemases or d-amino acid transaminase is utilized for peptidoglycan biosynthesis in microorganisms. Comparative genomics has shown that some microorganisms, including Xanthomonas oryzae, perhaps have no orthologues of these genes. We performed shotgun cloning experiments with a d-Glu auxotrophic Escherichia coli mutant as the host and X. oryzae as the DNA donor. We obtained complementary genes, XOO_1319 and XOO_1320, which are annotated as a hypothetical protein and MurD (UDP-MurNAc-l-Ala-d-Glu synthetase), respectively. By detailed in vitro analysis, we revealed that XOO_1320 is an enzyme to ligate l-Glu to UDP-MurNAc-l-Ala, providing the first example of MurD utilizing l-Glu, and that XOO_1319 is a novel enzyme catalyzing epimerization of the terminal l-Glu of the product in the presence of ATP and Mg²⁺. We investigated the occurrence of XOO_1319 orthologues and found that it exists in some categories of microorganisms, including pathogenic ones

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

Identification and Characterization of Enzymes Catalyzing Pyrazolopyrimidine Formation in the Biosynthesis of Formycin A

Genome scanning of <i>Streptomyces kaniharaensis</i>, the producer of formycin A, reveals two sets of <i>purA</i>, <i>purB</i>, <i>purC</i>, and <i>purH</i> genes. The Pur enzymes catalyze pyrimidine assembly of purine nucleobases. To test whether enzymes encoded by the second set of <i>pur</i> genes catalyze analogous transformations in formycin biosynthesis, formycin B 5′-phosphate was synthesized and shown to be converted by ForA and ForB to formycin A 5′-phosphate. These results support that For enzymes are responsible for formycin formation

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

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

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

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

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