Kenneth D. Clevenger

A scalable platform to identify fungal secondary metabolites and their gene clusters

The genomes of filamentous fungi contain up to ~90 biosynthetic gene clusters (BGCs), encoding diverse secondary metabolites, an enormous reservoir of untapped chemical potential. However, recalcitrant genetics, cryptic expression, and unculturability prevent the systematic exploitation of these gene clusters and harvesting of their products. With heterologous expression of fungal BGCs largely limited to expression of single or partial clusters, we established a scalable process for expression of large numbers of full-length gene clusters, called FAC-MS. Using Fungal Artificial Chromosomes (FACs) with Metabolomic Scoring (MS) we screened 56 secondary metabolite BGCs from diverse fungal species for expression in A. nidulans. Fifteen new metabolites were discovered and confidently assigned to their BGCs. A new macrolactone, valactamide A, and its hybrid PKS-NRPS gene cluster were characterized extensively using this integrated platform. Regularizing access to fungal secondary metabolites at an unprecedented scale stands to revitalize drug discovery platforms with renewable sources of natural products.

Rational Design of a Transition State Analogue with Picomolar Affinity for Pseudomonas aeruginosa PvdQ, a Siderophore Biosynthetic Enzyme

The Pseudomonas aeruginosa enzyme PvdQ can process different substrates involved in quorum-sensing or in siderophore biosynthesis. Substrate selectivity was evaluated using steady-state kinetic constants for hydrolysis of N-acyl-homoserine lactones (HSLs) and p-nitrophenyl fatty acid esters. PvdQ prefers substrates with alkyl chains between 12 and 14 carbons long that do not bear a 3-oxo substitution and is revealed here to have a relatively high specificity constant for selected N-acyl-HSLs (kcat/KM = 10(5) to 10(6) M(-1) s(-1)). However, endogenous P. aeruginosa N-acyl-HSLs are ≥100-fold disfavored, supporting the conclusion that PvdQ was not primarily evolved to regulate endogenous quorum-sensing. PvdQ plays an essential biosynthetic role for the siderophore pyoverdine, on which P. aeruginosa depends for growth in iron-limited environments. A series of alkylboronate inhibitors was found to be reversible, competitive, and extremely potent (Ki ≥ 190 pM). A 1.8 Å X-ray structure shows that 1-tridecylboronic acid forms a monocovalent bond with the N-terminal β-chain Ser residue in the PvdQ heterodimer, mimicking a reaction transition state. This boronic acid inhibits growth of P. aeruginosa in iron-limited media, reproducing the phenotype of a genetic pvdQ disruption, although co-administration of an efflux pump inhibitor is required to maintain growth inhibition. These findings support the strategy of designing boron-based inhibitors of siderophore biosynthetic enzymes to control P. aeruginosa infections.

n-Alkylboronic acid inhibitors reveal determinants of ligand specificity in the quorum-quenching and siderophore biosynthetic enzyme PvdQ.

The enzyme PvdQ (E.C. 3.5.1.97) from Pseudomonas aeruginosa is an N-terminal nucleophile hydrolase that catalyzes the removal of an N-myristyl substituent from a biosynthetic precursor of the iron-chelating siderophore pyoverdine. Inhibitors of pyoverdine biosynthesis are potential antibiotics since iron is essential for growth and scarce in most infections. PvdQ also catalyzes hydrolytic amide bond cleavage of selected N-acyl-l-homoserine lactone quorum-sensing signals used by some Gram-negative pathogens to coordinate the transcription of virulence factors. The resulting quorum-quenching activity of PvdQ has potential applications in antivirulence therapies. To inform both inhibitor design and enzyme engineering efforts, a series of n-alkylboronic acid inhibitors of PvdQ was characterized to reveal determinants of ligand selectivity. A simple homologation series results in compounds with Ki values that span from 4.7 mM to 190 pM, with a dependence of ΔGbind values on chain length of -1.0 kcal/mol/CH2. X-ray crystal structures are determined for the PvdQ complexes with 1-ethyl-, 1-butyl-, 1-hexyl-, and 1-octylboronic acids at 1.6, 1.8, 2.0, and 2.1 Å resolution, respectively. The 1-hexyl- and 1-octylboronic acids form tetrahedral adducts with the active-site N-terminal Ser217 in the β-subunit of PvdQ, and the n-alkyl substituents are bound in the acyl-group binding site. The 1-ethyl- and 1-butylboronic acids also form adducts with Ser217 but instead form trigonal planar adducts and extend their n-alkyl substituents into an alternative binding site. These results are interpreted to propose a ligand discrimination model for PvdQ that informs the development of PvdQ-related tools and therapeutics.

Substrate Trapping in the Siderophore Tailoring Enzyme PvdQ

Siderophore biosynthesis by Pseudomonas aeruginosa enhances virulence and represents an attractive drug target. PvdQ functions in the type-1 pyoverdine biosynthetic pathway by removing a myristoyl anchor from a pyoverdine precursor, allowing eventual release from the periplasm. A circularly permuted version of PvdQ bypasses the self-processing step of this Ntn-hydrolase and retains the activity, selectivity, and structure of wild-type PvdQ, as revealed by a 1.8 Å resolution X-ray crystal structure. A 2.55 Å resolution structure of the inactive S1A/N269D-cpPvdQ mutant in complex with the pyoverdine precursor PVDIq reveals a specific binding pocket for the d-Tyr of this modified peptide substrate. To our knowledge, this structure is the first of a pyoverdine precursor peptide bound to a biosynthetic enzyme. Details of the observed binding interactions have implications for control of pyoverdine biosynthesis and inform future drug design efforts.

Interrogation of Benzomalvin Biosynthesis Using Fungal Artificial Chromosomes with Metabolomic Scoring (FAC-MS): Discovery of a Benzodiazepine Synthase Activity

The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-CT and the internal C-domains as BenZ-C1 and BenZ-C2. Unexpectedly, we also uncovered evidence suggesting BenY-CT or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal CT domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities.

Clicking on the lights to reveal bacterial social networking.

“No man is an island.” 1 With apologies to John Donne, the same could be said for a bacterium. The discovery of bacterial quorum sensing and its relevance to microbial ecology and pathogenesis have fueled the increasing scrutiny of the molecular mechanisms responsible for the apparent group behavior of microbes. 2 A number of chemically diverse small molecules act as diffusible signaling molecules that regulate gene expression in a population‐dependent manner. Some of these signals, such as the N‐acyl‐L‐homoserine lactones, are produced and sensed by others in the same or closely related species, and other chemical classes of signals are used more broadly for interspecies and even interkingdom communication. 3 As a field, the study of these microbial social networks has been termed “sociomicrobiology.” 4

Identification of the First Diketomorpholine Biosynthetic Pathway Using FAC-MS Technology

Filamentous fungi are prolific producers of secondary metabolites with drug-like properties, and their genome sequences have revealed an untapped wealth of potential therapeutic leads. To better access these secondary metabolites and characterize their biosynthetic gene clusters, we applied a new platform for screening and heterologous expression of intact gene clusters that uses fungal artificial chromosomes and metabolomic scoring (FAC-MS). We leverage FAC-MS technology to identify the biosynthetic machinery responsible for production of acu-dioxomorpholine, a metabolite produced by the fungus, Aspergilllus aculeatus. The acu-dioxomorpholine nonribosomal peptide synthetase features a new type of condensation domain (designated CR) proposed to use a noncanonical arginine active site for ester bond formation. Using stable isotope labeling and MS, we determine that a phenyllactate monomer deriving from phenylalanine is incorporated into the diketomorpholine scaffold. Acu-dioxomorpholine is highly related to orphan inhibitors of P-glycoprotein targets in multidrug-resistant cancers, and identification of the biosynthetic pathway for this compound class enables genome mining for additional derivatives.