Biosynthesis of a Novel Diketopiperazine Aspkyncin Incorporating a Kynurenine Unit from Aspergillus aculeatus

Abstract

The simplest cyclo-peptides, also known as diketopiperazines (DKPs), are widespread in nature. The growing interest in these simplest cyclo-peptides is driven by their significant potential for therapeutic applications. In this study, we identified a biosynthetic gene cluster from Aspergillus aculeatus CRI323-04 through genome mining and heterologous expression in Aspergillus nidulans. The two core genes, aacA and aacB, within the gene cluster were characterized for their role in the biossoynthesis of aspkyncin, a novel DKP compound that incorporates a l-kynurenine (l-Kyn) unit. Furthermore, we successfully reconstituted the activities of the minimal bimodular non-ribosomal peptide synthetase (NRPS) AacA and the methyltransferase AacB both in vivo and in vitro. Our findings demonstrate that AacA catalyzes the condensation and cyclization of two non-proteinogenic amino acids, l-Kyn and N-methyl-l-alanine, to produce aspkyncin without the involvement of any release domain. Notably, the N-methyl-l-alanine is generated by a specialized l-alanine N-methyltransferase AacB prior to NRP assembly. This study reveals an unconventional pathway for the biosynthesis of fungal DKPs.

Keywords: N-methyltransferase; diketopiperazine; fungi; l-kynurenine; non-ribosomal peptide synthetase (NRPS).

Figure 1

The structures and activities of representative 2,5-diketopiperazines.

Figure 2

(A) Schematic representation of the aac cluster and homologous gene clusters in Aspergillus. AacA: NRPS; AacB: methyltransferase (MT); AacC: cytochrome P450, AacD: amino-acid oxidase; AacE: hypothetical protein; AacF: transcription factor; AacG: carboxylesterase; IDO: indoleamine-2,3-dioxygenase. (B) Structure of 1 (aspkyncin). (C) ¹H−¹H COSY and HMBC correlations in aspkyncin. (D) In vivo reconstitution of aac. Shown is HPLC analysis (λ = 366 nm) of metabolites extracted from 3-day cultures of (i) untransformed A. nidulans LO8030, (ii) A. nidulans LO8030 expressing AacA, (iii) A. nidulans LO8030 expressing AacAB, and (iv) A. nidulans LO8030 expressing AacABCDG.

Figure 3

Characterization of AacB: (A) amino acid derivatization diagram; (B) in vitro reconstitution of AacB. Shown are LC-MS analyses of compounds from the extraction of reaction mixtures after Marfey’s method of (i) AacB + l-alanine, (ii) no enzyme control, (iii) N-methyl-l-alanine, and (iv) l-alanine.

Figure 4

Characterization of AacA: (A) SDS-PAGE analyses of AacA (~194.3 kDa) with 8 × His-tag; (B) in vitro reconstitution of AacA. Shown are HPLC analyses (λ = 366 nm) of compounds from the extraction of reaction mixtures of (i) AacA + l-alanine + l-Kyn; (ii) AacA + AacB + l-alanine + l-Kyn; (iii) AacA + N-methyl-l-alanine + l-Kyn; (iv) no enzyme control (only N-methyl-l-alanine + l-Kyn in reaction buffer); and (v) standard of aspkyncin.

Figure 5

The proposed biosynthetic pathway of aspkyncin.