Native Promoter Strategy for High-Yielding Synthesis and Engineering of Fungal Secondary Metabolites

Strategies are needed for the robust production of cryptic, silenced, or engineered secondary metabolites in fungi. The filamentous fungus <i>Fusarium heterosporum</i> natively synthesizes the polyketide equisetin at >2 g L^–1 in a controllable manner. We hypothesized that this production level was achieved by regulatory elements in the equisetin pathway, leading to the prediction that the same regulatory elements would be useful in producing other secondary metabolites. This was tested by using the native <i>eqxS</i> promoter and <i>eqxR</i> regulator in <i>F. heterosporum</i>, synthesizing heterologous natural products in yields of ∼1 g L^–1. As proof of concept for the practical application, we resurrected an extinct pathway from an endophytic fungus with an initial yield of >800 mg L^–1, leading to the practical synthesis of a selective antituberculosis agent. Finally, the method enabled new insights into the function of polyketide synthases in filamentous fungi. These results demonstrate a strategy for optimally employing native regulators for the robust synthesis of secondary metabolites

Thiazoline Peptides and a Tris-Phenethyl Urea from <i>Didemnum molle</i> with Anti-HIV Activity

As part of our screening for anti-HIV agents from marine invertebrates, the MeOH extract of <i>Didemnum molle</i> was tested and showed moderate <i>in vitro</i> anti-HIV activity. Bioassay-guided fractionation of a large-scale extract allowed the identification of two new cyclopeptides, mollamides E and F (<b>1</b> and <b>2</b>), and one new tris-phenethyl urea, molleurea A (<b>3</b>). The absolute configurations were established using the advanced Marfey’s method. The three compounds were evaluated for anti-HIV activity in both an HIV integrase inhibition assay and a cytoprotective cell-based assay. Compound <b>2</b> was active in both assays with IC₅₀ values of 39 and 78 μM, respectively. Compound <b>3</b> was active only in the cytoprotective cell-based assay, with an IC₅₀ value of 60 μM

Mollecarbamates, Molleureas, and Molledihydroisoquinolone, <i>o</i>‑Carboxyphenethylamide Metabolites of the Ascidian <i>Didemnum molle</i> Collected in Madagascar

The extract of a sample of the tunicate <i>Didemnum molle</i> (MAY13-117) collected in Mayotte afforded eight new metabolites, mollecarbamates A–D (<b>1</b>–<b>4</b>) and molleureas B–E (<b>5</b>–<b>8</b>), along with the two known natural products, <i>N,N</i>′-diphenylethyl urea (<b>10</b>) and molleurea A (<b>11</b>). Another sample of <i>D. molle</i> (MAD11-BA065) collected in Baie des Assassins, Madagascar, afforded molledihydroisoquinolone (<b>9</b>). Mollecarbamates <b>1</b>–<b>4</b> are a family of compounds that possess repeating <i>o</i>-carboxyphenethylamide units and a carbamate moiety, while the molleureas <b>5</b>–<b>8</b> contain tetra- and penta-repeating carboxyphenethylamide units and a urea bridge in different positions. Molledihydroisoquinolone (<b>9</b>) is a cyclic form of <i>o</i>-carboxyphenethylamide. We propose that these unique natural products are most probably produced by an unprecedented biosynthetic pathway that contains a yet unknown chorismate mutase variant. The structures of the compounds were elucidated by interpretation of the data from 1D and 2D NMR, HRESIMS, and MS/MS analyses of the positive ESIMS experiments. Compounds <b>1</b>–<b>8</b> were tested against pathogenic bacteria and in a cytoprotective HIV cell based assay but did not show any significant effects in these assays

Plakinamine M, a Steroidal Alkaloid from the Marine Sponge Corticium sp.

By means of bioassay-guided fractionation, a new steroidal alkaloid, plakinamine M (<b>1</b>), and the known compound, plakinamine L (<b>2</b>), with a unique acyclic side chain, were isolated from the marine sponge Corticium sp. collected from New Britain, Papua New Guinea. The structures were determined on the basis of extensive 1D and 2D NMR and HRESIMS. The two compounds showed inhibition of Mycobacterium tuberculosis with MIC values of 15.8 and 3.6 μg/mL, respectively

Reactivation of HIV by H37Ra, PIM6, and H37Rv lysate in a primary T_CM model of latency.

<p>Cultured T_CM cells following 72-hour incubation with test conditions or co-stimulation with αCD3/αCD28. (A) Levels of intracellular p24 Gag were measured by flow cytometry. The horizontal line within the box represents the median, the boundaries of the box represent the 25^th- and 75^th-percentile, and the whiskers represent the maximum and minimum values. Significance for intracellular p24 Gag was determined using a 2-tailed, paired Student’s t-test versus PBS (*p<0.05). (B) Relative luminescence was measured from supernatant of cultured T_CM cells following 72-hour incubation with conditions or co-stimulation with αCD3/αCD28. The horizontal line within the box represents the median, the boundaries of the box represent the 25^th- and 75^th-percentile, and the whiskers represent the maximum and minimum values. Significance was determined using a 2-tailed, paired Student’s t-test versus PBS (*p<0.05). Significance of individual test conditions are as follows: αCD3/αCD28 (p≤0.01), PIM6 (p<0.05), H37Ra (p≤0.01), and <i>M</i>. <i>smegmatis</i> (p≤0.01).</p

PIM6 and H37Rv lysate induce GFP expression through TLR-2.

<p>A) JLAT cells were incubated for 16 hours and GFP expression was measured by flow cytometry. Data represent mean ± SD of four independent experiments run in triplicate. *p<0.05 compared to PBS control. B) JLAT-TLR2 cells were incubated for 16 hours and GFP expression was measured by flow cytometry. Data represent mean ± SD of ten independent experiments run in triplicate. *p<0.05 compared to PBS control. <b>C</b>) JLAT-TLR2 cells were pre-incubated with the TLR-2 neutralizing antibody, PAb-hTLR2, for 30 minutes prior to addition of test conditions. Cells were subsequently incubated for 16 hours and GFP expression was measured by flow cytometry. Data represent mean ± SD. *p<0.05 test condition in the absence of TLR-2 neutralizing antibody compared to test condition in the presence of TLR-2 neutralizing antibody. D) JLAT-TLR2 cells pre-incubated with BAY 11–7082 for 30 minutes prior to addition of conditions. Cells were subsequently incubated for 16 hours and GFP expression was measured by flow cytometry. Data represent mean ± SD. *p<0.05 test condition in the absence of BAY 11–7082 compared to test condition in the presence of BAY 11–7082.</p

4‑Quinolone Alkaloids from <i>Melochia odorata</i>

The methanol extract of <i>Melochia odorata</i> yielded three 4-quinolone alkaloids including waltherione A (<b>1</b>) and two new alkaloids, waltherione C (<b>2</b>) and waltherione D (<b>3</b>). Waltheriones A and C showed significant activities in an in vitro anti-HIV cytoprotection assay at concentrations of 56.2 and 0.84 μM and inhibition of HIV P24 formation of more than 50% at 1.7 and 0.95 μM, respectively. The structures of the alkaloids were established by spectroscopic data interpretation

Oxazinin A, a Pseudodimeric Natural Product of Mixed Biosynthetic Origin from a Filamentous Fungus

A racemic, prenylated polyketide dimer, oxazinin A (<b>1</b>), was isolated from a novel filamentous fungus in the class Eurotiomycetes, and its structure was elucidated spectroscopically. The pentacyclic structure of oxazinin A (<b>1</b>) is a unique combination of benzoxazine, isoquinoline, and a pyran ring. Oxazinin A (<b>1</b>) exhibited antimycobacterial activity and modestly antagonized transient receptor potential (TRP) channels

Myristicyclins A and B: Antimalarial Procyanidins from <i>Horsfieldia spicata</i> from Papua New Guinea

An antimalarial screen for plants collected from Papua New Guinea identified an extract of <i>Horsfieldia spicata</i> as having activity. Isolation of the active constituents led to the identification of two new compounds: myristicyclins A (<b>1</b>) and B (<b>2</b>). Both compounds are procyanidin-like congeners of myristinins lacking a pendant aromatic ring. Myristicyclin A was found to inhibit the ring, trophozoite, and schizont stages of <i>Plasmodium falciparum</i> at similar concentrations in the mid-μM range