Total Heterologous Biosynthesis of Fungal Natural Products in <i>Aspergillus nidulans</i>

Fungal natural products comprise a wide range of bioactive compounds including important drugs and agrochemicals. Intriguingly, bioinformatic analyses of fungal genomes have revealed that fungi have the potential to produce significantly more natural products than what have been discovered so far. It has thus become widely accepted that most biosynthesis pathways of fungal natural products are silent or expressed at very low levels under laboratory cultivation conditions. To tap into this vast chemical reservoir, the reconstitution of entire biosynthetic pathways in genetically tractable fungal hosts (total heterologous biosynthesis) has become increasingly employed in recent years. This review summarizes total heterologous biosynthesis of fungal natural products accomplished before 2020 using Aspergillus nidulans as heterologous hosts. We review here Aspergillus transformation, A. nidulans hosts, shuttle vectors for episomal expression, and chromosomal integration expression. These tools, collectively, not only facilitate the discovery of cryptic natural products but can also be used to generate high-yield strains with clean metabolite backgrounds. In comparison with total synthesis, total heterologous biosynthesis offers a simplified strategy to construct complex molecules and holds potential for commercial application

Rational Domain Swaps Reveal Insights about Chain Length Control by Ketosynthase Domains in Fungal Nonreducing Polyketide Synthases

A facile genetic methodology in the filamentous fungus <i>Aspergillus nidulans</i> allowed exchange of the starter unit ACP transacylase (SAT) domain in the nonreduced polyketide synthase (NR-PKS) AfoE of the asperfuranone pathway with the SAT domains from 10 other NR-PKSs. The newly created hybrid with the NR-PKS AN3386 is able to accept a longer starter unit in place of the native substrate to create a novel aromatic polyketide <i>in vivo</i>

Reengineering an Azaphilone Biosynthesis Pathway in <i>Aspergillus nidulans</i> To Create Lipoxygenase Inhibitors

Sclerotiorin, an azaphilone polyketide, is a bioactive natural product known to inhibit 15-lipoxygenase and many other biological targets. To readily access sclerotiorin and analogs, we developed a 2–3 step semisynthetic route to produce a variety of azaphilones starting from an advanced, putative azaphilone intermediate (<b>5</b>) overproduced by an engineered strain of <i>Aspergillus nidulans</i>. The inhibitory activities of the semisynthetic azaphilones against 15-lipoxygenase were evaluated with several compounds displaying low micromolar potency

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

Nonribosomal peptides (NRPs) are natural products biosynthesized by NRP synthetases. A <i>kusA-</i>, <i>pyrG-</i> mutant strain of <i>Aspergillus terreus</i> 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 <i>A. terreus</i>. In addition, an NRP affecting melanin synthesis was also identified in this species

Engineering Fungal Nonreducing Polyketide Synthase by Heterologous Expression and Domain Swapping

We reannotated the <i>A</i>. <i>niger</i> NR-PKS gene, e_gw1_19.204, and its downstream R domain gene, est_GWPlus_C_190476, as a single gene which we named <i>dtbA</i>. Heterologous expression of <i>dtbA</i> in <i>A</i>. <i>nidulans</i> demonstrated that DtbA protein produces two polyketides, 2,4-dihydroxy-3,5,6-trimethylbenzaldehyde (<b>1</b>) and 6-ethyl-2,4-dihydroxy-3,5-dimethylbenzaldehyde (<b>2</b>). Generation of DtbAΔR+TE chimeric PKSs by swapping the DtbA R domain with the AusA (austinol biosynthesis) or ANID_06448 TE domain enabled the production of two metabolites with carboxylic acids replacing the corresponding aldehydes

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, <i>ataP</i>) 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

Molecular Genetic Characterization of the Biosynthesis Cluster of a Prenylated Isoindolinone Alkaloid Aspernidine A in <i>Aspergillus nidulans</i>

Aspernidine A is a prenylated isoindolinone alkaloid isolated from the model fungus <i>Aspergillus nidulans</i>. A genome-wide kinase knockout library of <i>A. nidulans</i> was examined, and it was found that a mitogen-activated protein kinase gene, <i>mpkA</i>, deletion strain produces aspernidine A. Targeted gene deletions were performed in the kinase deletion background to identify the gene cluster for aspernidine A biosynthesis. Intermediates were isolated from mutant strains which provided information about the aspernidine A biosynthesis pathway

Illuminating the Diversity of Aromatic Polyketide Synthases in <i>Aspergillus nidulans</i>

Genome sequencing has revealed that fungi have the ability to synthesize many more natural products (NPs) than are currently known, but methods for obtaining suitable expression of NPs have been inadequate. We have developed a successful strategy that bypasses normal regulatory mechanisms. By efficient gene targeting, we have replaced, <i>en masse</i>, the promoters of nonreducing polyketide synthase (NR-PKS) genes, key genes in NP biosynthetic pathways, and other genes necessary for NR-PKS product formation or release. This has allowed us to determine the products of eight NR-PKSs of <i>Aspergillus nidulans</i>, including seven novel compounds, as well as the NR-PKS genes required for the synthesis of the toxins alternariol (<b>8</b>) and cichorine (<b>19</b>)