Engineering Fungal Nonribosomal Peptide Synthetase-like Enzymes by Heterologous Expression and Domain Swapping

A facile genetic methodology in the filamentous fungus <i>Aspergillus nidulans</i> allowed the exchange of various domains in nonribosomal peptide synthase (NRPS)-like enzymes from <i>Aspergillus terreus</i>. The newly generated engineered enzymes are capable of producing compounds with different chemical structures than its parent enzyme <i>in vivo</i>. This work provides insight in the programing of nonribosomal peptide biosynthesis in filamentous fungi

Molecular Genetic Characterization of Terreic Acid Pathway in <i>Aspergillus terreus</i>

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

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

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

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

Azaphilones Inhibit Tau Aggregation and Dissolve Tau Aggregates <i>in Vitro</i>

The aggregation of the microtubule-associated protein tau is a seminal event in many neurodegenerative diseases, including Alzheimer’s disease. The inhibition or reversal of tau aggregation is therefore a potential therapeutic strategy for these diseases. Fungal natural products have proven to be a rich source of useful compounds having wide varieties of biological activities. We have previously screened Aspergillus nidulans secondary metabolites for their ability to inhibit tau aggregation <i>in vitro</i> using an arachidonic acid polymerization protocol. One aggregation inhibitor identified was asperbenzaldehyde, an intermediate in azaphilone biosynthesis. We therefore tested 11 azaphilone derivatives to determine their tau assembly inhibition properties <i>in vitro</i>. All compounds tested inhibited tau filament assembly to some extent, and four of the 11 compounds had the advantageous property of disassembling preformed tau aggregates in a dose-dependent fashion. The addition of these compounds to the tau aggregates reduced both the total length and number of tau polymers. The most potent compounds were tested in <i>in vitro</i> reactions to determine whether they interfere with tau’s normal function of stabilizing microtubules (MTs). We found that they did not completely inhibit MT assembly in the presence of tau. These derivatives are very promising lead compounds for tau aggregation inhibitors and, more excitingly, for compounds that can disassemble pre-existing tau filaments. They also represent a new class of anti-tau aggregation compounds with a novel structural scaffold

Elevated Bcl-2 expression in HBV pre-S2Δ cells.

<p>(A) Total RNA was isolated from Huh-7 -V, Huh-7 pre-S2Δ, and Huh-7 pre-S cells and then subjected to RT-PCR analysis with Bcl-2 and G3APDH specific primers. (B) For Huh-7 -V, Huh-7 pre-S2Δ, and Huh-7 pre-S cells, the relative levels of Bcl-2 mRNA were quantified. The data represent the mean of Bcl-2 mRNA expression level from three independent experiments. Columns, mean; bars, SD (n = 3). Significant differences (**, <i>P</i><0.01) between the control and experimental group are marked with an asterisks. (C) The cell lysates collected from three stable clones, and cell lysates were analyzed by Western blotting analysis with antibodies for hemagglutinin (HA) tag, Bcl-2, and β-actin. (D) The levels of Bcl-2 protein in the graphs were representative of three independent experiments (lower panel). Columns, mean; bars, SD (n = 3). Significant differences (**, <i>P</i><0.01) between the control and experimental group are marked with an asterisks. (E) Furthermore, expression level of Bcl-2 was determined by using Western blotting in three individual pre-S2Δ stable cell lines. (F) Expression level of Bcl-2 was enhanced by pre-S2Δ in the immortalized human hepatocyte cell line. Total cell lysate from NeHep and NeHep-pre-S2Δ were determined by Western blotting analysis with Bcl-2, pre-S, and β-actin specific antibosies.</p

The pre-S2Δ large surface protein induced expression of Bcl-2 family proteins.

<p>(A) Total cell lysate was isolated from Huh-7 -V, Huh-7 pre-S2Δ, and Huh-7 pre-S cells and then subjected to Western blotting analysis with Bcl-xL, Mcl-1, Bax, and Bad specific antibodies. (B) For Huh-7 -V, Huh-7 pre-S2Δ, and Huh-7 pre-S cells, the relative levels of Bcl-xL, Mcl-1, Bax, and Bad protein were quantified. The data represent the mean of Bcl-xL, Mcl-1, Bax, and Bad expression level from three independent experiments. Columns, mean; bars, SD (n = 3). Significant differences (*, <i>P</i><0.05) between the control and experimental group are marked with an asterisks.</p