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Expanding our understanding of sequence-function relationships of type II polyketide biosynthetic gene clusters: bioinformatics-guided identification of Frankiamicin A from Frankia sp. EAN1pec.

Ogasawara Y, Yackley BJ, Greenberg JA, Rogelj S, Melançon CE - PLoS ONE (2015)

Bottom Line: 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.Our findings provide new information linking this set of Frankia 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, New Mexico, United States of America.

ABSTRACT
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 Frankia species, a group of Actinobacteria with underexploited natural product biosynthetic potential. Through LC-MS profiling of extracts from several Frankia species grown under various conditions, we identified Frankia 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 Frankia 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.

No MeSH data available.


Related in: MedlinePlus

Dendrogram of KSα/β sequences showing the relationship between dendrogramatic position, polyketide subclass, and poly-β-ketone structure.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 Frankia 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: E. coli FAS—E. coli fatty acid synthase, which was used as the outgroup.
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pone.0121505.g003: Dendrogram of KSα/β sequences showing the relationship between dendrogramatic position, polyketide subclass, and poly-β-ketone structure.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 Frankia 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: E. coli FAS—E. coli fatty acid synthase, which was used as the outgroup.

Mentions: The ketosynthase α/β dendrogram was generated as follows: The amino acid sequences of all ketosynthase α and ketosynthase β enzymes identified by Dynamite were compiled as two separate multi-fasta files using a custom script. Each set was then aligned using Clustal Omega [22] and unconserved N- and C-terminal regions were trimmed based on the multiple sequence alignments to minimize their effects on tree building after constructing the concatenated sequence. Residues corresponding to positions 6–420 of the 424 amino acid actinorhodin KSα, and to positions 1–403 of the 407 amino acid actinorhodin KSβ were retained. Trimmed ketosynthase α/β sequence pairs were concatenated and aligned again using Clustal Omega. A bootstrapped maximum likelihood dendrogram was generated from the alignment using FastTree 2.[23] The dendrogram was visualized and color coded using the Interactive Tree of Life (iTOL) [24] web interface. Ketosynthase I (FabB) from the Escherichia coli fatty acid biosynthetic pathway was treated similarly and used to construct a pseudo-dimer sequence that was used as the outgroup. The identities of the 64 studied type II polyketide systems and their starter and extender unit specificities were compiled manually by cross referencing Dynamite results with literature, and were color coded by type in iTOL. A high resolution version of the dendrogram in Fig. 3, including bootstrap values, species names, and training set compound names, is available in the S2 Fig.


Expanding our understanding of sequence-function relationships of type II polyketide biosynthetic gene clusters: bioinformatics-guided identification of Frankiamicin A from Frankia sp. EAN1pec.

Ogasawara Y, Yackley BJ, Greenberg JA, Rogelj S, Melançon CE - PLoS ONE (2015)

Dendrogram of KSα/β sequences showing the relationship between dendrogramatic position, polyketide subclass, and poly-β-ketone structure.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 Frankia 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: E. coli FAS—E. coli fatty acid synthase, which was used as the outgroup.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383371&req=5

pone.0121505.g003: Dendrogram of KSα/β sequences showing the relationship between dendrogramatic position, polyketide subclass, and poly-β-ketone structure.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 Frankia 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: E. coli FAS—E. coli fatty acid synthase, which was used as the outgroup.
Mentions: The ketosynthase α/β dendrogram was generated as follows: The amino acid sequences of all ketosynthase α and ketosynthase β enzymes identified by Dynamite were compiled as two separate multi-fasta files using a custom script. Each set was then aligned using Clustal Omega [22] and unconserved N- and C-terminal regions were trimmed based on the multiple sequence alignments to minimize their effects on tree building after constructing the concatenated sequence. Residues corresponding to positions 6–420 of the 424 amino acid actinorhodin KSα, and to positions 1–403 of the 407 amino acid actinorhodin KSβ were retained. Trimmed ketosynthase α/β sequence pairs were concatenated and aligned again using Clustal Omega. A bootstrapped maximum likelihood dendrogram was generated from the alignment using FastTree 2.[23] The dendrogram was visualized and color coded using the Interactive Tree of Life (iTOL) [24] web interface. Ketosynthase I (FabB) from the Escherichia coli fatty acid biosynthetic pathway was treated similarly and used to construct a pseudo-dimer sequence that was used as the outgroup. The identities of the 64 studied type II polyketide systems and their starter and extender unit specificities were compiled manually by cross referencing Dynamite results with literature, and were color coded by type in iTOL. A high resolution version of the dendrogram in Fig. 3, including bootstrap values, species names, and training set compound names, is available in the S2 Fig.

Bottom Line: 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.Our findings provide new information linking this set of Frankia 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, New Mexico, United States of America.

ABSTRACT
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 Frankia species, a group of Actinobacteria with underexploited natural product biosynthetic potential. Through LC-MS profiling of extracts from several Frankia species grown under various conditions, we identified Frankia 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 Frankia 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.

No MeSH data available.


Related in: MedlinePlus