Fungal Indole Alkaloid Biosynthesis: Genetic and Biochemical Investigation of the Tryptoquialanine Pathway in Penicillium aethiopicum

Abstract

Tremorgenic mycotoxins are a group of indole alkaloids which include the quinazoline-containing tryptoquivaline 2 that are capable of eliciting intermittent or sustained tremors in vertebrate animals. The biosynthesis of this group of bioactive compounds, which are characterized by an acetylated quinazoline ring connected to a 6-5-5 imidazoindolone ring system via a 5-membered spirolactone, has remained uncharacterized. Here, we report the identification of a gene cluster (tqa) from P. aethiopicum that is involved in the biosynthesis of tryptoquialanine 1, which is structurally similar to 2. The pathway has been confirmed to go through an intermediate common to the fumiquinazoline pathway, fumiquinazoline F, which originates from a fungal trimodular nonribosomal peptide synthetase (NRPS). By systematically inactivating every biosynthetic gene in the cluster, followed by isolation and characterization of the intermediates, we were able to establish the biosynthetic sequence of the pathway. An unusual oxidative opening of the pyrazinone ring by an FAD-dependent berberine bridge enzyme-like oxidoreductase has been proposed based on genetic knockout studies. Notably, a 2-aminoisobutyric acid (AIB)-utilizing NRPS module has been identified and reconstituted in vitro, along with two putative enzymes of unknown functions that are involved in the synthesis of the unnatural amino acid by genetic analysis. This work provides new genetic and biochemical insights into the biosynthesis of this group of fungal alkaloids, including the tremorgens related to 2.

Alkaloids from the Sponge‐Associated Fungus Aspergillus sp.

“…[29] The structurally most similar member and proposed biological precursor of the fumiquinazolines, fumiquinazoline F, is known to be biosynthetically derived from anthranilic acid, tryptophan, and alanine [44] involving a trimodular non-ribosomal peptide synthetase (NRPS). [45] Modification of the indole side chain by oxidative coupling of an additional amino acid molecule, commonly alanine or methylalanine, results in the formation of the imidazoindolone part of the compounds. [44] A plausible scheme for the biosynthesis of compounds 1-7 investigated in this study would include oxidative coupling of 1-aminocyclopropane-1-carboxylic acid to form the imidazoindolone unit of the compounds.…”

“…In contrast, an unusual oxidative opening of the pyrazinone ring of fumiquinazoline F has been suggested as an intermediate step in the biosynthesis of tryptoquivalines. [45] Accordingly, 1 is proposed to be biosynthesized from a derivative of 3 with subsequent acetylation at N-16.…”

“…[26] Based on the fact that tryptoquivalines are biosynthetically derived from fumiquinazolines by oxidative opening of the pyrazinone ring, [45] a similar configuration would be expected at the respective chiral centers. However, both (R) and (S) configurations are reported for C-3, C-17, and C-18 in the literature, [44,45,47] whereas C-14 mostly exists in the (R) configuration.…”

Synthesis, Reactions and Antimicrobial Activity of Some New 3-Substituted Indole Derivatives

“…IR spectra were recorded on Shimadzu FT-IR 8101 PC infrared spectrophotometer (υ max in cm −1 ). The 1H NMR and 13 C NMR spectra were determined in DMSO-d6 at 300 MHz on a Varian Mercury VX 300 NMR spectrometer using TMS as an internal standard. Mass spectra were measured on a GCMS-QP1000 EX spectrometer at 70 Ev.…”

“…Also 3-substituted indole derivatives possess various types of broad spectrum's biological activities such as antimicrobial, antitumor, hypoglycemic, anti-inflammatory, analgesic and antipyretic activities [5] [6]. Moreover the substitution at the 3-position of the indole ring can take place by connecting an additional heterocyclic ring, such as imidazole (topsentins, nortopsentins) [7] [8], dihydroimidazole (discodermindole) [9], oxazole (pimprinols A-C, almazole C) [10] [11], thiazole (bacillamide A) [12], quinazoline (tremorgens) [13], and pyrimidine [14]. Therefore, 3-substituted indoles still represent a significant synthetic challenge.…”