Chun-Jun Guo

Two separate gene clusters encode the biosynthetic pathway for the meroterpenoids austinol and dehydroaustinol in Aspergillus nidulans.

Meroterpenoids are a class of fungal natural products that are produced from polyketide and terpenoid precursors. An understanding of meroterpenoid biosynthesis at the genetic level should facilitate engineering of second-generation molecules and increasing production of first-generation compounds. The filamentous fungus Aspergillus nidulans has previously been found to produce two meroterpenoids, austinol and dehydroaustinol. Using targeted deletions that we created, we have determined that, surprisingly, two separate gene clusters are required for meroterpenoid biosynthesis. One is a cluster of four genes including a polyketide synthase gene, ausA. The second is a cluster of 10 additional genes including a prenyltransferase gene, ausN, located on a separate chromosome. Chemical analysis of mutant extracts enabled us to isolate 3,5-dimethylorsellinic acid and 10 additional meroterpenoids that are either intermediates or shunt products from the biosynthetic pathway. Six of them were identified as novel meroterpenoids in this study. Our data, in aggregate, allow us to propose a complete biosynthetic pathway for the A. nidulans meroterpenoids.

Prototype of an intertwined secondary-metabolite supercluster

Significance Filamentous fungi are well known for their ability to produce a wide range of natural products. Until now, biosynthetic genes contiguously aligned in a cluster have been associated with the production of a distinct natural product. This work demonstrates an additional layer of complexity in fungal secondary-metabolite gene clusters. In contrast to the view of stand-alone secondary-metabolite clusters, our collective data have revealed the existence of superclusters with intertwined biosynthetic genes involved in formation of more than one chemical product. Comparative genomics indicates this supercluster is maintained in a rapidly evolving region of diverse fungal genomes. This intertwined design confounds predictions from established secondary-metabolite cluster search algorithms and provides an expanded view of natural product evolution. The hallmark trait of fungal secondary-metabolite gene clusters is well established, consisting of contiguous enzymatic and often regulatory gene(s) devoted to the production of a metabolite of a specific chemical class. Unexpectedly, we have found a deviation from this motif in a subtelomeric region of Aspergillus fumigatus. This region, under the control of the master regulator of secondary metabolism, LaeA, contains, in its entirety, the genetic machinery for three natural products (fumitremorgin, fumagillin, and pseurotin), where genes for fumagillin and pseurotin are physically intertwined in a single supercluster. Deletions of 29 adjoining genes revealed that fumagillin and pseurotin are coregulated by the supercluster-embedded regulatory gene with biosynthetic genes belonging to one of the two metabolic pathways in a noncontiguous manner. Comparative genomics indicates the fumagillin/pseurotin supercluster is maintained in a rapidly evolving region of diverse fungal genomes. This blended design confounds predictions from established secondary-metabolite cluster search algorithms and provides an expanded view of natural product evolution.

Molecular Genetic Characterization of a Cluster in A. terreus for Biosynthesis of the Meroterpenoid Terretonin

Meroterpenoids are natural products produced from polyketide and terpenoid precursors. A gene targeting system for A. terreus NIH2624 was developed, and a gene cluster for terretonin biosynthesis was characterized. The intermediates and shunt products were isolated from the mutant strains, and a pathway for terretonin biosynthesis is proposed. Analysis of two meroterpenoid pathways corresponding to terretonin in A. terreus and austinol in A. nidulans reveals that they are closely related evolutionarily.

Biosynthetic pathway for the epipolythiodioxopiperazine acetylaranotin in Aspergillus terreus revealed by genome-based deletion analysis.

Epipolythiodioxopiperazines (ETPs) are a class of fungal secondary metabolites derived from diketopiperazines. Acetylaranotin belongs to one structural subgroup of ETPs characterized by the presence of a seven-membered 4,5-dihydrooxepine ring. Defining the genes involved in acetylaranotin biosynthesis should provide a means to increase the production of these compounds and facilitate the engineering of second-generation molecules. The filamentous fungus Aspergillus terreus produces acetylaranotin and related natural products. Using targeted gene deletions, we have identified a cluster of nine genes (including one nonribosomal peptide synthetase gene, ataP) that is required for acetylaranotin biosynthesis. Chemical analysis of the wild-type and mutant strains enabled us to isolate 17 natural products from the acetylaranotin biosynthesis pathway. Nine of the compounds identified in this study are natural products that have not been reported previously. Our data have allowed us to propose a biosynthetic pathway for acetylaranotin and related natural products.

Low-Volume Toolbox for the Discovery of Immunosuppressive Fungal Secondary Metabolites

The secondary metabolome provides pathogenic fungi with a plethoric and versatile panel of molecules that can be deployed during host ingress. While powerful genetic and analytical chemistry methods have been developed to identify fungal secondary metabolites (SMs), discovering the biological activity of SMs remains an elusive yet critical task. Here, we describe a process for identifying the immunosuppressive properties of Aspergillus SMs developed by coupling a cost-effective microfluidic neutrophil chemotaxis assay with an in vivo zebrafish assay. The microfluidic platform allows the identification of metabolites inhibiting neutrophil recruitment with as little as several nano-grams of compound in microliters of fluid. The zebrafish assay demonstrates a simple and accessible approach for performing in vivo studies without requiring any manipulation of the fish. Using this methodology we identify the immunosuppressive properties of a fungal SM, endocrocin. We find that endocrocin is localized in Aspergillus fumigatus spores and its biosynthesis is temperature-dependent. Finally, using the Drosophila toll deficient model, we find that deletion of encA, encoding the polyketide synthase required for endocrocin production, yields a less pathogenic strain of A. fumigatus when spores are harvested from endocrocin permissive but not when harvested from endocrocin restrictive conditions. The tools developed here will open new “function-omic” avenues downstream of the metabolomics, identification, and purification phases.

Microbial metabolomics in open microscale platforms

The microbial secondary metabolome encompasses great synthetic diversity, empowering microbes to tune their chemical responses to changing microenvironments. Traditional metabolomics methods are ill-equipped to probe a wide variety of environments or environmental dynamics. Here we introduce a class of microscale culture platforms to analyse chemical diversity of fungal and bacterial secondary metabolomes. By leveraging stable biphasic interfaces to integrate microculture with small molecule isolation via liquid–liquid extraction, we enable metabolomics-scale analysis using mass spectrometry. This platform facilitates exploration of culture microenvironments (including rare media typically inaccessible using established methods), unusual organic solvents for metabolite isolation and microbial mutants. Utilizing Aspergillus, a fungal genus known for its rich secondary metabolism, we characterize the effects of culture geometry and growth matrix on secondary metabolism, highlighting the potential use of microscale systems to unlock unknown or cryptic secondary metabolites for natural products discovery. Finally, we demonstrate the potential for this class of microfluidic systems to study interkingdom communication between fungi and bacteria.

Application of an Efficient Gene Targeting System Linking Secondary Metabolites to their Biosynthetic Genes in Aspergillus terreus

Nonribosomal peptides (NRPs) are natural products biosynthesized by NRP synthetases. A kusA-, pyrG- mutant strain of Aspergillus terreus NIH 2624 was developed that greatly facilitated the gene targeting efficiency in this organism. Application of this tool allowed us to link four major types of NRP-related secondary metabolites to their responsible genes in A. terreus. In addition, an NRP affecting melanin synthesis was also identified in this species.

Recent advances in genome mining of secondary metabolites in Aspergillus terreus.

Filamentous fungi are rich resources of secondary metabolites (SMs) with a variety of interesting biological activities. Recent advances in genome sequencing and techniques in genetic manipulation have enabled researchers to study the biosynthetic genes of these SMs. Aspergillus terreus is the well-known producer of lovastatin, a cholesterol-lowering drug. This fungus also produces other SMs, including acetylaranotin, butyrolactones, and territram, with interesting bioactivities. This review will cover recent progress in genome mining of SMs identified in this fungus. The identification and characterization of the gene cluster for these SMs, as well as the proposed biosynthetic pathways, will be discussed in depth.

Molecular Genetic Characterization of Terreic Acid Pathway in Aspergillus terreus

Terreic acid is a natural product derived from 6-methylsalicylic acid (6-MSA). A compact gene cluster for its biosynthesis was characterized. Isolation of the intermediates and shunt products from the mutant strains, combined with bioinformatic analyses, allowed for the proposition of a biosynthetic pathway for terreic acid.

Characterization of the product of a nonribosomal peptide synthetase-like (NRPS-like) gene using the doxycycline dependent Tet-on system in Aspergillus terreus

Genome sequencing of the fungus Aspergillus terreus uncovered a number of silent core structural biosynthetic genes encoding enzymes presumed to be involved in the production of cryptic secondary metabolites. There are five nonribosomal peptide synthetase (NRPS)-like genes with the predicted A-T-TE domain architecture within the A. terreus genome. Among the five genes, only the product of pgnA remains unknown. The Tet-on system is an inducible, tunable and metabolism-independent expression system originally developed for Aspergillus niger. Here we report the adoption of the Tet-on system as an effective gene activation tool in A. terreus. Application of this system in A. terreus allowed us to uncover the product of the cryptic NRPS-like gene, pgnA. Furthermore expression of pgnA in the heterologous Aspergillus nidulans host suggested that the pgnA gene alone is necessary for phenguignardic acid (1) biosynthesis.