Complexity generation in fungal peptidyl alkaloid biosynthesis: a two-enzyme pathway to the hexacyclic MDR export pump inhibitor ardeemin

Abstract

Ardeemins are hexacyclic peptidyl alkaloids isolated from Aspergillus fischeri as agents that block efflux of anticancer drugs by MultiDrug Resistance (MDR) export pumps. To evaluate the biosynthetic logic and enzymatic machinery for ardeemin framework assembly, we sequenced the A. fischeri genome and identified the ardABC gene cluster. Through both genetic deletions and biochemical characterizations of purified ArdA and ArdB we show this ArdAB enzyme pair is sufficient to convert anthranilate (Ant), L-Ala, and L-Trp to ardeemin. ArdA is a 430 kDa trimodular nonribosomal peptide synthase (NRPS) that converts the three building blocks into a fumiquinazoline (FQ) regioisomer termed ardeemin FQ. ArdB is a prenyltransferase that takes tricyclic ardeemin FQ and dimethylallyl diphosphate to the hexacyclic ardeemin scaffold via prenylation at C2 of the Trp-derived indole moiety with intramolecular capture by an amide NH of the fumiquinazoline ring. The two-enzyme ArdAB pathway reveals remarkable efficiency in construction of the hexacyclic peptidyl alkaloid scaffold.

Figure 1

Structures of ardeemins

Figure 2

Synthetic route to ardeemin FQ and illustration of isomers observed

Figure 3

Identification of the ardeemin (ard) gene cluster. (a) Organization of the ard cluster; (b) HPLC analysis (λ = 292 nm) of 3-day metabolites produced by A. fischeri strains (1) WT A. fischeri; (2) synthetic ardeemin FQ standard; (3) A. fischeri with ardA KO (4) A. fischeri with ardB KO.

Figure 4

Characterization of ArdA. a) HPLC analyses (at λ = 292 nm) of in vivo and in vitro reconstitution of ArdA. (1) 2 mM of the amino acid building blocks and no enzyme, (2) 2 mM of the amino acid building blocks and 10 µM ArdA, (3) metabolites extracted from 3-day culture of BJ5464-NpgA expressing ArdA, (4) synthetic ArdFQ synthetic standard. b) SDS-PAGE of the heterologously expressed ArdA protein purified by FLAG-tag affinity chromatography (432 kDa). c) HPLC-based time course study of the turnover of ArdFQ by ArdA.

Figure 5

HR-LCMS characterisation of the transformation of synthetic ardeemin FQ to ardeemin by the action of ArdB and comparison to authentic ardeemin from A. fischeri. (1) EIC at m/z 359.1503 (2) EIC at m/z 427.2129 (3) EIC at m/z 427.2129.

Scheme 1

Differentiation in NRPS mediated biosynthesis of FQF and Ardeemin FQ: formation of regioisomeric quinazolinones by swapping the order of A domains

Scheme 2

Comparison of post-NRPS tailoring of FQ scaffolds in fumiquinazoline and ardeemin pathways: capture of C-β of a Trp-derived indole moiety by oxygen (fumiquinazoline) vs carbon (ardeemin) electrophiles.