Search

Gliotoxin and related epidithiodiketopiperazines (ETP) from diverse fungi feature highly functionalized hydroindole scaffolds with an array of medicinally and ecologically relevant activities. Mutation analysis, heterologous reconstitution, and biotransformation experiments revealed that a cytochrome P450 monooxygenase (GliF) from the human-pathogenic fungus Aspergillus fumigatus plays a key role in the formation of the complex heterocycle. In vitro assays using a biosynthetic precursor from a blocked mutant showed that GliF is specific to ETPs and catalyzes an unprecedented heterocyclization reaction that cannot be emulated with current synthetic methods. In silico analyses indicate that this rare biotransformation takes place in related ETP biosynthetic pathways., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

The human gut microbiome harbors hundreds of bacterial species with diverse biochemical capabilities. Dozens of drugs have been shown to be metabolized by single isolates from the gut microbiome, but the extent of this phenomenon is rarely explored in the context of microbial communities. Here, we develop a quantitative experimental framework for mapping the ability of the human gut microbiome to metabolize small molecule drugs: Microbiome-Derived Metabolism (MDM)-Screen. Included are a batch culturing system for sustained growth of subject-specific gut microbial communities, an ex vivo drug metabolism screen, and targeted and untargeted functional metagenomic screens to identify microbiome-encoded genes responsible for specific metabolic events. Our framework identifies novel drug-microbiome interactions that vary between individuals and demonstrates how the gut microbiome might be used in drug development and personalized medicine., Competing Interests: Declaration of Interests M.S.D. is a member of the scientific advisory board of DeepBiome Therapeutics. A patent is being filed by Princeton University for the use of quantitative MDM-Screen to measure inter-individual variability in drug metabolism., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Extensive progress has been made in determining the effects of the microbiome on human physiology and disease, but the underlying molecules and mechanisms governing these effects remain largely unexplored. Here, we combine a new computational algorithm with synthetic biology to access biologically active small molecules encoded directly in human microbiome-derived metagenomic sequencing data. We discover that members of a clinically used class of molecules are widely encoded in the human microbiome and that they exert potent antibacterial activities against neighboring microbes, implying a possible role in niche competition and host defense. Our approach paves the way toward a systematic unveiling of the chemical repertoire encoded by the human microbiome and provides a generalizable platform for discovering molecular mediators of microbiome-host and microbiome-microbiome interactions., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Aspirochlorine is an unusual antifungal cyclopeptide produced by Aspergillus oryzae, an important mold used for food fermentation. Whereas its structure suggested that a non-ribosomal peptide synthetase assembles the cyclopeptide from phenylalanine and glycine building blocks, labeling studies indicated that one Phe moiety is transformed into Gly after peptide formation. By means of genetic engineering, heterologous expression, biotransformations, and in vitro assays, we dissected and reconstituted four crucial steps in aspirochlorine biosynthesis, which involve two cytochrome P450 monooxygenases, (AclL and AclO), a methyltransferase (AclU), and a halogenase (AclH). We found that the installation of the N-methoxylation of the peptide bond sets the stage for a retro-aldol reaction that leads to the Phe-to-Gly conversion. The substrate scopes of the dedicated enzymes as well as bioassays revealed that the peptide editing has evolved to optimize the antifungal action of the natural product., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The regioselective functionalization of non-activated carbon atoms such as aliphatic halogenation is a major synthetic challenge. A novel multifunctional enzyme catalyzing the geminal dichlorination of a methyl group was discovered in Aspergillus oryzae (Koji mold), an important fungus that is widely used for Asian food fermentation. A biosynthetic pathway encoded on two different chromosomes yields mono- and dichlorinated polyketides (diaporthin derivatives), including the cytotoxic dichlorodiaporthin as the main product. Bioinformatic analyses and functional genetics revealed an unprecedented hybrid enzyme (AoiQ) with two functional domains, one for halogenation and one for O-methylation. AoiQ was successfully reconstituted in vivo and in vitro, unequivocally showing that this FADH2 -dependent enzyme is uniquely capable of the stepwise gem-dichlorination of a non-activated carbon atom on a freestanding substrate. Genome mining indicated that related hybrid enzymes are encoded in cryptic gene clusters in numerous ecologically relevant fungi., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aspirochlorine (1) is an epidithiodiketopiperazine (ETP) toxin produced from koji mold (Aspergillus oryzae), which has been used in the oriental cuisine for over two millennia. Considering its potential risk for food safety, we have elucidated the molecular basis of aspirochlorine biosynthesis. By a combination of genetic and chemical analyses we found the acl gene locus and identified the key role of AclH as a chlorinase. Stable isotope labeling, biotransformation, and mutational experiments, analysis of intermediates and an in vitro adenylation domain assay gave totally unexpected insights into the acl pathway: Instead of one Phe and one Gly, two Phe units are assembled by an iterative non-ribosomal peptide synthetase (NRPS, AclP), followed by halogenation and an unprecedented Phe to Gly amino acid conversion. Biological assays showed that both amino acid transformations are required to confer cytotoxicity and antifungal activity to the mycotoxin., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Enzyme quartet: Isolation of the first sulfur-bearing intermediate of the gliotoxin pathway in Aspergillus fumigatus and successful in vitro conversion of the bisglutathione adduct into an intact epidithiodiketopiperazine by a four-enzyme cascade (including glutamyltransferase GliK and dipeptidase GliJ) revealed an outstanding adaptation of a primary metabolic pathway into natural product biosynthesis that is widespread in fungi., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The genome of the fungal model organism Aspergillus nidulans harbors nearly 30 polyketide synthase genes, yet the majority of these genes remain silent in the absence of particular stimuli. In this study, environmental conditions such as low specific microbial growth rate as well as nitrate, orthophosphate and glucose limitations were simulated under a continuous cultivation regime to induce the expression of silent polyketide synthase genes. In addition to offline and online bioprocess parameters, the physiological equilibrium was defined at the transcript level in terms of indicator gene expression. The different cultivation parameters resulted in a differential expression of two polyketide synthase genes coding for the biosynthesis of a variety of phenolic compounds, such as orsellinic acid, lecanoric acid, emodins, chrysophanol, shamixanthone, and sanghaspirodin. Further investigation of the metabolome revealed the formation of a novel prenylated benzophenone derivative designated as pre-shamixanthone. Our data indicate that employing chemostat fermentations in combination with genome mining, transcriptome analysis and metabolic profiling represents a valuable approach for triggering cryptic biosynthetic pathways., (Copyright © 2012 Elsevier B.V. All rights reserved.)