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.