Dorte Koefoed Holm

A genome-wide polyketide synthase deletion library uncovers novel genetic links to polyketides and meroterpenoids in Aspergillus nidulans

Fungi possess an advanced secondary metabolism that is regulated and coordinated in a complex manner depending on environmental challenges. To understand this complexity, a holistic approach is necessary. We initiated such an analysis in the important model fungus Aspergillus nidulans by systematically deleting all 32 individual genes encoding polyketide synthases. Wild-type and all mutant strains were challenged on different complex media to provoke induction of the secondary metabolism. Screening of the mutant library revealed direct genetic links to two austinol meroterpenoids and expanded the current understanding of the biosynthetic pathways leading to arugosins and violaceols. We expect that the library will be an important resource towards a systemic understanding of polyketide production in A. nidulans.

Heterologous Reconstitution of the Intact Geodin Gene Cluster in Aspergillus nidulans through a Simple and Versatile PCR Based Approach

Fungal natural products are a rich resource for bioactive molecules. To fully exploit this potential it is necessary to link genes to metabolites. Genetic information for numerous putative biosynthetic pathways has become available in recent years through genome sequencing. However, the lack of solid methodology for genetic manipulation of most species severely hampers pathway characterization. Here we present a simple PCR based approach for heterologous reconstitution of intact gene clusters. Specifically, the putative gene cluster responsible for geodin production from Aspergillus terreus was transferred in a two step procedure to an expression platform in A. nidulans. The individual cluster fragments were generated by PCR and assembled via efficient USER fusion prior to transformation and integration via re-iterative gene targeting. A total of 13 open reading frames contained in 25 kb of DNA were successfully transferred between the two species enabling geodin synthesis in A. nidulans. Subsequently, functions of three genes in the cluster were validated by genetic and chemical analyses. Specifically, ATEG_08451 (gedC) encodes a polyketide synthase, ATEG_08453 (gedR) encodes a transcription factor responsible for activation of the geodin gene cluster and ATEG_08460 (gedL) encodes a halogenase that catalyzes conversion of sulochrin to dihydrogeodin. We expect that our approach for transferring intact biosynthetic pathways to a fungus with a well developed genetic toolbox will be instrumental in characterizing the many exciting pathways for secondary metabolite production that are currently being uncovered by the fungal genome sequencing projects.

Molecular and Chemical Characterization of the Biosynthesis of the 6-MSA-Derived Meroterpenoid Yanuthone D in Aspergillus niger

Secondary metabolites in filamentous fungi constitute a rich source of bioactive molecules. We have deduced the genetic and biosynthetic pathway of the antibiotic yanuthone D from Aspergillus niger. Our analyses show that yanuthone D is a meroterpenoid derived from the polyketide 6-methylsalicylic acid (6-MSA). Yanuthone D formation depends on a cluster composed of ten genes including yanA and yanI, which encode a 6-MSA polyketide synthase and a previously undescribed O-mevalon transferase, respectively. In addition, several branching points in the pathway were discovered, revealing five yanuthones (F, G, H, I, and J). Furthermore, we have identified another compound (yanuthone X1) that defines a class of yanuthones that depend on several enzymatic activities encoded by genes in the yan cluster but that are not derived from 6-MSA.

PHUSER (Primer Help for USER): a novel tool for USER fusion primer design

Uracil-Specific Exision Reagent (USER) fusion is a recently developed technique that allows for assembly of multiple DNA fragments in a few simple steps. However, designing primers for USER fusion is both tedious and time consuming. Here, we present the Primer Help for USER (PHUSER) software, a novel tool for designing primers specifically for USER fusion and USER cloning applications. We also present proof-of-concept experimental validation of its functionality. PHUSER offers quick and easy design of PCR optimized primers ensuring directionally correct fusion of fragments into a plasmid containing a customizable USER cassette. Designing primers using PHUSER ensures that the primers have similar annealing temperature (Tm), which is essential for efficient PCR. PHUSER also avoids identical overhangs, thereby ensuring correct order of assembly of DNA fragments. All possible primers are individually analysed in terms of GC content, presence of GC clamp at 3′-end, the risk of primer dimer formation, the risk of intra-primer complementarity (secondary structures) and the presence of polyN stretches. Furthermore, PHUSER offers the option to insert linkers between DNA fragments, as well as highly flexible cassette options. PHUSER is publicly available at http://www.cbs.dtu.dk/services/phuser/.

Genetics of Polyketide Metabolism in Aspergillus nidulans

Secondary metabolites are small molecules that show large structural diversity and a broad range of bioactivities. Some metabolites are attractive as drugs or pigments while others act as harmful mycotoxins. Filamentous fungi have the capacity to produce a wide array of secondary metabolites including polyketides. The majority of genes required for production of these metabolites are mostly organized in gene clusters, which often are silent or barely expressed under laboratory conditions, making discovery and analysis difficult. Fortunately, the genome sequences of several filamentous fungi are publicly available, greatly facilitating the establishment of links between genes and metabolites. This review covers the attempts being made to trigger the activation of polyketide metabolism in the fungal model organism Aspergillus nidulans. Moreover, it will provide an overview of the pathways where ten polyketide synthase genes have been coupled to polyketide products. Therefore, the proposed biosynthesis of the following metabolites will be presented; naphthopyrone, sterigmatocystin, aspyridones, emericellamides, asperthecin, asperfuranone, monodictyphenone/emodin, orsellinic acid, and the austinols.

Characterization of four new antifungal yanuthones from Aspergillus niger.

Four new yanuthone analogs (1–4) were isolated from the filamentous fungus Aspergillus niger. The structures of the new compounds were elucidated on the basis of UHPLC-DAD-HRMS data and one-dimensional and two-dimensional NMR spectroscopy. Labeling studies with 13C8-6-methylsalicylic acid identified three class I yanuthones originating from the polyketide 6-methylsalicylic acid (yanuthone K, L and M (1–3)) and a class II yanuthone, which was named yanuthone X2 (4). The four new compounds were tested toward the pathogenic yeast Candida albicans and all displayed antifungal activity. Yanuthone X2 represents the first example of a bioactive class II yanuthone, demonstrating the pharmaceutical potential of this class.

Investigation of a 6-MSA Synthase Gene Cluster in Aspergillus aculeatus Reveals 6-MSA-derived Aculinic Acid, Aculins A-B and Epi-Aculin A.

Aspergillus aculeatus, a filamentous fungus belonging to the Aspergillus clade Nigri, is an industrial workhorse in enzyme production. Recently we reported a number of secondary metabolites from this fungus; however, its genetic potential for the production of secondary metabolites is vast. In this study we identified a 6‐methylsalicylic acid (6‐MSA) synthase from A. aculeatus, and verified its functionality by episomal expression in A. aculeatus and heterologous expression in A. nidulans. Feeding studies with fully 13C‐labeled 6‐MSA revealed that 6‐MSA is incorporated into aculinic acid, which further incorporates into three compounds that we name aculins A and B, and epi‐aculin A, described here for the first time. Based on NMR data and bioinformatic studies we propose the structures of the compounds as well as a biosynthetic pathway leading to formation of aculins from 6‐MSA.

Combining UHPLC-High Resolution MS and Feeding of Stable Isotope Labeled Polyketide Intermediates for Linking Precursors to End Products

We present the results from stable isotope labeled precursor feeding studies combined with ultrahigh performance liquid chromatography-high resolution mass spectrometry for the identification of labeled polyketide (PK) end-products. Feeding experiments were performed with (13)C8-6-methylsalicylic acid (6-MSA) and (13)C14-YWA1, both produced in-house, as well as commercial (13)C7-benzoic acid and (2)H7-cinnamic acid, in species of Fusarium, Byssochlamys, Aspergillus, and Penicillium. Incorporation of 6-MSA into terreic acid or patulin was not observed in any of six evaluated species covering three genera, because the 6-MSA was shunted into (2Z,4E)-2-methyl-2,4-hexadienedioic acid. This indicates that patulin and terreic acid may be produced in a closed compartment of the cell and that (2Z,4E)-2-methyl-2,4-hexadienedioic acid is a detoxification product toward terreic acid and patulin. In Fusarium spp., YWA1 was shown to be incorporated into aurofusarin, rubrofusarin, and antibiotic Y. In A. niger, benzoic acid was shown to be incorporated into asperrubrol. Incorporation levels of 0.7-20% into the end-products were detected in wild-type strains. Thus, stable isotope labeling is a promising technique for investigation of polyketide biosynthesis and possible compartmentalization of toxic metabolites.