Abstract
Advances in genome sequencing have revitalized natural product discovery efforts, revealing the untapped biosynthetic potential of fungi. While the volume of genomic data continues to expand, discovery efforts are slowed due to the time-consuming nature of experiments required to characterize new molecules. To direct efforts toward uncharacterized biosynthetic gene clusters most likely to encode novel chemical scaffolds, we took advantage of comparative metabolomics and heterologous gene expression using fungal artificial chromosomes (FACs). By linking mass spectral profiles with structural clues provided by FAC-encoded gene clusters, we targeted a compound originating from an unusual gene cluster containing an indoleamine 2,3-dioxygenase (IDO). With this approach, we isolate and characterize R and S forms of the new molecule terreazepine, which contains a novel chemical scaffold resulting from cyclization of the IDO-supplied kynurenine. The discovery of terreazepine illustrates that FAC-based approaches targeting unusual biosynthetic machinery provide a promising avenue forward for targeted discovery of novel scaffolds and their biosynthetic enzymes, and it also represents another example of a biosynthetic gene cluster "repurposing" a primary metabolic enzyme to diversify its secondary metabolite arsenal.IMPORTANCE Here, we provide evidence that Aspergillus terreus encodes a biosynthetic gene cluster containing a repurposed indoleamine 2,3-dioxygenase (IDO) dedicated to secondary metabolite synthesis. The discovery of this neofunctionalized IDO not only enabled discovery of a new compound with an unusual chemical scaffold but also provided insight into the numerous strategies fungi employ for diversifying and protecting themselves against secondary metabolites. The observations in this study set the stage for further in-depth studies into the function of duplicated IDOs present in fungal biosynthetic gene clusters and presents a strategy for accessing the biosynthetic potential of gene clusters containing duplicated primary metabolic genes.
Highlights
IMPORTANCE Here, we provide evidence that Aspergillus terreus encodes a biosynthetic gene cluster containing a repurposed indoleamine 2,3-dioxygenase (IDO) dedicated to secondary metabolite synthesis
Fungal genomic DNA fragments containing intact biosynthetic gene clusters (BGCs) are inserted into fungal artificial chromosomes (FACs) and are used to transform a fungal host to discover new chemical scaffolds [10,11,12]
To determine the structure of the target compound, ϳ1.5 mg of material was purified from FAC-transformed A. nidulans extracts and subjected to tandem mass spectrometry (MS2) analysis, 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, and two-dimensional (2D) correlation approaches, including correlation spectroscopy (COSY), heteronuclear single quantum correlation (HSQC), and heteronuclear multiple bond correlation (HMBC)
Summary
IMPORTANCE Here, we provide evidence that Aspergillus terreus encodes a biosynthetic gene cluster containing a repurposed indoleamine 2,3-dioxygenase (IDO) dedicated to secondary metabolite synthesis. Recent studies spanning nearly 600 fungal genomes suggest that a mere 3% of molecules encoded by fungal biosynthetic gene clusters (BGCs) have been explored [8] To access this biosynthetic potential, an innovative discovery pipeline was recently developed to systematically annotate the biosynthetic abilities of fungi using comparative metabolomics and heterologous gene expression [9,10,11,12]. With this platform, fungal genomic DNA fragments containing intact BGCs are inserted into fungal artificial chromosomes (FACs) and are used to transform a fungal host to discover new chemical scaffolds [10,11,12]. Using structural clues provided by BGC data, it is possible to target compounds originating from BGCs containing unusual biosynthetic machinery (Fig. 1)
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