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. 2026 Feb 11;13:240–250. doi: 10.1016/j.synbio.2026.01.032

Biosynthesis of ent-acu-dioxomorpholine A and its conversion to prenyl-rearranged and rare ketooxadiazepane products in heterologous host

Qiaoqiao Tao 1, Yi Zou 1,⁎
PMCID: PMC12914822  PMID: 41717496

Abstract

Diketomorpholines (DKMs) represent an intriguing class of biologically active heterocycles. To identify unusual DKM natural products, we conducted a comprehensive analysis of fungal genomes from NCBI and our laboratory databases. A non-ribosomal peptide synthetase (NRPS) biosynthetic gene cluster (BGC) was identified in Aspergillus aculeatus CRI323-04, which also harbors a dimethylallyl tryptophan synthase (DMATS) gene and an NAD(P)H-dependent reductase gene. Heterologous expression of this BGC in chassis organism A. nidulans resulted in the production of prenylated DKM (3), seco-diketomorpholines (seco-DKMs) with regular (6–7) or reverse prenyl group (8), as well as rare ketooxadiazepane alkaloids (4–5). Through integrated in vivo and in vitro experiments, we elucidated the biosynthetic pathway of the prenylated DKM (3). Moreover, the feeding experiment showed that the complex conversion of prenylated DKM (3) to seco-DKMs (6–8) and ketooxadiazepane (4–5) alkaloids may be mediated by heterologous host A. nidulans. This study elucidates the biosynthetic pathway of 3 and further facilitates the exploration of structurally diverse prenylated DKMs and ketooxadiazepane alkaloids from fungal resources.

Keywords: Diketomorpholines (DKMs), Ketooxadiazepane alkaloid, Non-ribosomal peptide synthetase (NRPS), Dimethylallyl tryptophan synthase (DMATS), Biosynthetic pathway

Graphical abstract

Image 1

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1. Introduction

Nitrogen- and oxygen-containing heterocyclic scaffolds are widely distributed in both synthetic and natural products, and have long been favored by biochemists due to their structural diversity and pharmaceutical potential [1,2]. Diketomorpholines (DKMs) featuring the morpholine-2,5-dione scaffold, which belong to the cyclodepsipeptide family alongside well-known diketopiperazines (DKPs), possess an N–O heterocyclic ring containing lactone and lactam moieties [3,4] (Fig. 1a). Based on the biosynthetic mechanisms, DKPs in fungi are typically assembled by non-ribosomal peptide synthetases (NRPSs) utilizing free amino acids as substrates [5]. Nevertheless, the biosynthesis of DKMs involves both an NRPS and a reductase. The reductase first reduces an α-keto acid to an α-hydroxy acid, which is then condensed with an amino acid by the NRPS to form characteristic morpholine-2,5-dione scaffold [6]. At present, more than 1000 diketopiperazine products have been identified from natural sources. In contrast to the structural diversity of DKPs, only 22 natural DKMs have been reported to date, and the majority (21 out of 22) are derived from fungi [[7], [8], [9], [10], [11], [12], [13], [14]]. Although DKM scaffolds appear less frequently in natural products, they exhibit prominent bioactivity against various biomolecular targets [15]. For instance, bassiatin from Fusarium oxysporum J8-1-2 suppressed oestrogen-dependent cell proliferation by down-regulating the mRNA and protein levels of ERα and the oestrogen-responsive gene cyclin D1, with enhanced phospho-cyclin D1 (Thr286) levels in MCF-7 ERα-positive breast cancer cells [16]. Furthermore, shornephine A, isolated from a marine-derived Aspergillus sp. CMB-M081F, acted as a noncytotoxic inhibitor of P-glycoprotein-mediated drug efflux, thereby overcoming multidrug resistance in human colon cancer cells [14]. Additionally, mollenines A and B produced by Eupenicillium molle NRRL 13062 exhibited moderate cytotoxicity and antibacterial activity [17].

Fig. 1.

Fig. 1

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Representative natural products of DKP, DKM, and indole alkaloids. (a) Typical DKP natural products biosynthesized by an NRPS, and DKM natural products biosynthesized by an NRPS and a reductase. (b) Acu-dioxomorpholine A in previous work and its enantiomer (ent-acu-dioxomorpholine A, 3) in this work. (c) The only two indole alkaloids (versiquinazolines A–B) featuring a seven-membered N–O heterocycle in previous work, and compounds 4–5 possessing rare ketooxadiazepane scaffold with C-13 ketone in this work.

Among these DKMs, only the biosynthetic pathway of acu-dioxomorpholine A (Fig. 1b) was first identified using stable isotope labeling, which was derived from tryptophan and phenylalanine and synthesized by the adx gene cluster [6]. In order to discover complex DKMs from fungi, we used the reductase (AdxB) involved in the acu-dioxomorpholine A biosynthesis as a probe to search for its homologue proteins in the National Center for Biotechnology Information (NCBI) and our laboratory database. Fortunately, a homologous reductase (here designated AauB) was mined from Aspergillus aculeatus CRI323-04. Apart from AauB, the aau gene cluster contains an NRPS gene (aauA) and a dimethylallyl tryptophan synthase (DMATS) gene (aauC). This result implies that the aau cluster is possibly responsible for the biosynthesis of DKMs.

In order to investigate the products of the aau gene cluster, it was heterologously expressed in the filamentous fungus A. nidulans LO8030 (AN-WT), a well-established host for heterologous expression. Consequently, eight products (1–8) classified into DKMs, seco-DKMs, prenylated DKMs and fascinating ketooxadiazepanes were identified from the A. nidulans mutants with different gene combinations of the aau cluster. Four of these products (3–5 and 7) were new to science. In vitro enzymatic assay disclosed that the DMATS AauC catalyzed C-3 reverse prenylation of 1 to form 3, namely ent-acu-dioxomorpholine A. In comparison to acu-dioxomorpholine A, 3 is its enantiomer, possessing an entirely opposite configuration. Compound 3 was fed to the host A. nidulans, leading to its biotransformation into 4–8. To the best of our knowledge, only two indole alkaloids with a seven-membered N–O heterocycle, versiquinazoline A-B from A. versicolor, were reported up to date [18] (Fig. 1c). Compound 4 belongs to this class of indole alkaloids and represents the first natural product featuring a ketooxadiazepane (1,3,5-oxadiazepan-6-one) scaffold. We proposed that ketooxadiazepane alkaloids were derived from the seco-DKMs, which were modified by the host A. nidulans. Moreover, the ketooxadiazepane group represents a promising pharmacophore. Chemosynthetic compounds with this moiety exhibit multiple bioactivities, including antiviral replication [19,20], β-lactamase inhibition [21], and phospholipase A2 inhibition [22].

2. Materials and methods

2.1. Strains and culture conditions

A. culeatus CRI323-04 was cultured under two conditions: 1) in solid CD medium (10 g/L glucose, 50 mL/L 20 × nitrate, 1 mL/L trace elements, 20 g/L agar) at 30 °C, 5 days to induce sporulation; 2) in liquid potato dextrose broth (PDB, 26 g/L potato dextrose powder) at 30 °C, 220 rpm, 5 days for genomic DNA extraction. A. nidulans LO8030 was used as chassis strain for heterologous expression and cultivated with four conditions: 1) in solid CD medium (compositions as above) at 37 °C, 3 days for sporulation; 2) in solid CDSD medium (CD medium supplemented with 218.6 g/L d-sorbitol) at 37 °C, 2–3 days for protoplast regeneration; 3) in solid CDST medium (pH 7.0, 20 g/L starch, 10 g/L casein hydrolysate, 50 mL/L 20 × nitrate salt, 1 mL/L trace elements, 20 g/L agar) at 25 °C, 3.5 days for producing heterologous products; 4) in liquid CDST medium (pH 7.0, compositions as above) at 25 °C, 220 rpm, 2.5 days for RNA extraction. Saccharomyces cerevisiae BJ5464-NpgA was used for plasmid construction, which was cultivated at 28 °C for 2–3 days. Escherichia coli XL-1 was used for general cloning. E. coli BL-21 was used for protein expression of AauC. Both E. coli were incubated in Luria-Bertani broth (25 g/L Luria-Bertani powder) or solid Luria-Bertani medium (25 g/L Luria-Bertani powder, 20 g/L agar) supplemented with corresponding antibiotics at 37 °C for cloning or 16 °C for protein expression. All strains and shuttle plasmids were listed in Table S1.

2.2. General analysis methods

All the regents were purchased from commercial suppliers, including Sigma-Aldrich, TCI, Cytiva, Adamas, Adamas Life, Takara, Yeasen, and New England Biolabs. Primers were synthesized by Sangon Biotech. Medium-pressure liquid chromatography (MPLC) was performed on a Buchi Pure C-805 preparative liquid chromatography system equipped with a Buchi FlashPure EcoFlex C18 column (80 g, 50 μm). Semi-preparative HPLC was conducted on a Shimadzu prominence liquid chromatograph with the COSMOSIL Packed Column (5C18-MS-II 10 × 250 mm). LC-MS data were recorded on a Waters Acquity H-class Plus UPLC system coupled with a PDA eλ detector and an SQ2 mass spectrometer (electrospray ionization source, ESI). Sample separation was achieved using an Acquity UPLC® BEH C18 column (1.7 μm, 2.1 × 50 mm). The elution program consisted of a linear gradient from 5% to 99% acetonitrile in water over 10 min, followed by 100% acetonitrile within 3 min at a flow rate of 0.4 mL/min. Nuclear magnetic resonance (NMR) data were acquired on a Bruker AVANCE III 400 MHz spectrometer. Chemical shifts were reported relative to tetramethylsilane (TMS) as the internal standard. High-resolution mass spectrum (HRMS) data were obtained from a Waters Acquity UPLC/Xexo G3 QTOF mass spectrometer equipped with an ESI source.

2.3. Bioinformatic analysis

Bioinformatic analysis of the A. culeatus CRI323-04 genome was performed using antiSMASH fungal version to identify putative biosynthetic gene clusters (BGCs) for secondary metabolites. The identified BGCs were annotated for open reading frames and non-coding regions using the 2ndFind tool. Gene functions were determined by the BlastP search in the NCBI database. The domains within NRPS were analyzed by the InterPro website. The substrate specificity of the NRPS adenylation (A) domain was predicted with the PKS/NRPS Analysis Website and PARAS.

2.4. Plasmid construction

Primers used in the study were summarized in Table S2. The recombinant plasmids utilized for heterologous expression in A. nidulans LO8030 or protein expression in E. coli BL21 were listed in Table S3. In order to construct the recombinant plasmids for heterologous expression, the aau A–C genes and their corresponding terminator were amplified from A. culeatus genomic DNA. These genes were subsequently assembled into linear vectors (i.e., pANR, pANU, and pANP), which contained constitutive promoter gpdA, inducible promoters glaA and amyB, and selectable markers. All assemblies were performed via homologous recombination in S. cerevisiae BJ5464-NpgA. The aau A (6462 bp) and its terminator (500 bp) were divided into three overlapping fragments and amplified by PCR with the primer pairs pANR-Aau-NRPS-F1/pANR-Aau-NRPS-R1, pANR-Aau-NRPS-F2/pANR-Aau-NRPS-R2, and pANR-Aau-NRPS-F3/pANR-Aau-NRPS-R3. The shuttle plasmid pANR with the gpdA promoter was digested by the restriction enzyme BamHI. The aforementioned three fragments and linearized pANR were simultaneously transformed into BJ5464-NpgA for in vivo recombination, resulting in plasmid pIM5578.

To construct plasmid pIM5585, aauB and its terminator were obtained by PCR using primer pairs pANP-Aau-KR-F/pANP-Aau-KR-R. The shuttle plasmid pANP with the promoter amyB was linearized with BamHI and assembled with the aauB gene by homologous recombination. The aauC gene was amplified by primer pairs pANU-Aau-DMAT-F/pANU-Aau-DMAT-R. Then the DMATS gene was cloned into the linearized vector pANU, which was digested with PacI, obtaining recombinant plasmid pIM5586. In order to gain plasmid pIM5611 for protein expression, the aauC (DMATS) gene was amplified from the cDNA using primers pET28a-Aau-DMAT-F1/pET28a-Aau-DMAT-R1, and then inserted into pET28a(+), which was digested with BamHI and HindIII. All the recombinant plasmids were further cloned into E. coli XL-1 and extracted using the alkaline lysis method. Each plasmid was confirmed by restriction digestion analysis, and the inserted aau A–C genes were sequenced.

2.5. Heterologous expression of the aau cluster in A. nidulans

In order to heterologously express the aau cluster in chassis fungus A. nidulans, the protoplast was prepared and transformed as follows. First, A. nidulans spores were inoculated into 50 mL CD liquid medium supplemented with uracil (0.5 mM), uridine (10 mM), VB2 (0.125 μg/mL), and VB6 (0.5 μg/mL), and cultivated at 37 °C, 220 rpm for 9–10 h. Germinated mycelium was then collected by centrifugation at 3750 rpm for 10 min, washed twice with osmotic buffer (1.2 M MgSO4, 10 mM sodium phosphate, pH 5.8), and resuspended in 10 mL osmotic buffer containing 20 mg Yatalase and 30 mg lysing enzyme from Trichoderma harzianum. The mycelium was digested overnight with the two aforementioned enzymes at 28 °C with gentle shaking (80 rpm) for 12–14 h. After digestion overnight, the mixture was transferred into a 50 mL sterile tube, and 10 mL ice-cold trapping buffer (0.6 M sorbitol, 0.1 M Tris-HCl, pH 7.0) was slowly layered on top. The tube was centrifuged at 4 °C, 3750 rpm for 15 min. The protoplasts, which accumulated at the interface between the two layers, were carefully collected and transferred into a 15 mL sterile tube. The protoplasts were then diluted with 2 vol of STC buffer (1.2 M sorbitol, 10 mM CaCl2, 10 mM Tris-HCl, pH 7.5) and pelleted by centrifugation at 4 °C, 3750 rpm for 10 min. The resulting pellets were resuspended in an appropriate volume of STC buffer and aliquoted into 1.5 mL Eppendorf tubes. For transformation, 100 μL protoplasts were mixed with 2 μL recombinant plasmids using a PEG-mediated method. The transformed protoplasts were regenerated on solid CDSD plates at 37 °C for 2–3 days. Positive transformants were selected and cultured on CD plates for 3 days to induce sporulation. Subsequently, a small number of spores were spread on CDST (pH 7.0) plates and fermented at 25 °C for 3.5 days to facilitate heterologous production. The metabolites were extracted from two fungal plugs (0.8 cm in diameter) using ethyl acetate/acetone (3:1, v/v) and analyzed by LC-MS with an injection volume of 1 μL.

2.6. Fermentation and isolation of compounds 1–8

Upon detection of heterologous products in the A. nidulans transformants, large-scale fermentations of these strains were carried out on CDST (pH 7.0) plates to isolate the target compounds. The cultures were extracted with ethyl acetate/acetone (3:1, v/v), and the extracts were concentrated under reduced pressure using a rotary evaporator. These crude extracts were preliminarily separated by MPLC and further purified by semi-preparative HPLC to gain the desired compounds.

Compounds 1 and 2 were isolated from the crude extract of the AN-aauAB strain with heterologously expressed aauA and aauB. The crude extraction was subjected to MPLC chromatography and eluted as follows: 25% ACN for 20 min, a linear gradient from 25% to 100% ACN over 50 min, and 100% ACN for 20 min at a flow rate of 25 mL/min. Fraction 2 (eluted with 44% ACN–55% ACN in H2O) was further purified by HPLC with isocratic elution of 35% ACN in H2O at a flow rate of 2.5 mL/min to obtain compound 2 (25.2 mg, tR = 29.0 min). Fraction 3 (eluted with 58%–65% ACN in H2O) was similarly separated by HPLC with 40% ACN in H2O to yield compound 1 (18.0 mg, tR = 27.0 min).

Compounds 3–6 were obtained from the concentrated extract of the AN-aauABC transformant with heterologously expressed aauA, aauB, and aauC. The extract was initially fractionated by MPLC chromatography and eluted according to the following procedure: 40% MeOH for 20 min, a linear gradient of 40%–100% MeOH within 50 min, and 100% MeOH for 20 min at a flow rate of 25 mL/min. Fraction 3 (eluted by 88%–91% MeOH) was separated by HPLC with 60% ACN in H2O to afford compounds 3 (50.0 mg, tR = 37.0 min), 4 (25.2 mg, tR = 25.0 min), and 6 (14.9 mg, tR = 17.0 min). Fraction 4 (eluted by 91%–96% MeOH) was also subjected to HPLC with isocratic elution of 60% ACN in H2O to obtain compounds 5 (15.0 mg, tR = 53.0 min).

A larger-scale fermentation of the AN-aauABC strain resulted in the isolation of compounds 7–8. The concentrated crude extract was preliminarily separated by MPLC using an ACN-H2O gradient at 2.5 mL/min: 35% ACN for 20 min, 35%–100% ACN over 50 min, and 100% ACN for 20 min. Fraction 2 (eluted by 58%–62% ACN) was purified by HPLC with 42% ACN in H2O to give compound 8 (11.0 mg, tR = 36.0 min). Fraction 3 (eluted by 62%–71% ACN) was purified by HPLC using 43% ACN in H2O to obtain compound 7 (8.1 mg, tR = 47.0 min).

2.7. Protein expression and purification of AauC in E. coli

For the expression of protein AauC, E. coli BL21 was transformed with plasmid pIM5611 and cultured in Luria-Bertani broth with 50 μg/mL kanamycin. When the OD600 reached 0.4–0.6, protein expression was induced by adding 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 16 °C for 20 h. The cell pellets were harvested by centrifugation, resuspended in buffer A (50 mM Tris-HCl, 500 mM NaCl, 10% glycerol, pH 7.5), and then disrupted by sonication on ice. The lysate was centrifuged at 4 °C, 14000 rpm for 30 min, and the supernatant was filtrated through a 0.22 μm membrane. Continuously, the resulting supernatant was loaded onto a Ni-affinity column three times for binding proteins. The column was eluted stepwise with buffer A containing 50 mM, 100 mM, 150 mM, and 250 mM imidazole until the Bradford dye reagent no longer turned blue. All eluted fractions were analyzed by 10% SDS-PAGE. The 10 mL of 250 mM imidazole eluent was concentrated to approximately 1 mL using an Amicon® Ultra-15 centrifugal filter (30 kDa) at 4 °C and 4000 rpm. The concentrated sample was subsequently desalted using a PD-10 column equilibrated with buffer C (50 mM Tris-HCl, 50 mM NaCl, 5% glycerol, pH 7.5) and eluted with 5 mL of the same buffer. The desalted protein was further concentrated to a final volume of 250 μL under the same conditions (4 °C, 4000 rpm). The purified AauC protein solution was aliquoted (40 μL per tube) into 1.5 mL Eppendorf tubes and stored at −80 °C.

2.8. In vitro assay of AauC

In order to characterize the function of AauC, the in vitro assay was performed as follows: 10 μM AauC, 10 mM MgCl2, 0.2 mM DMAPP, 0.1 mM compound 1 or 2 were mixed with buffer C to a volume of 50 μL and incubated at 25 °C for 2 h. Simultaneously, a negative control was set up in parallel under the same conditions without adding AauC. The reaction mixture was quenched by adding 50 μL of acetonitrile and centrifuged at 13300 rpm for 5 min. The resulting supernatants were analyzed by LC-MS with an injection volume of 2 μL.

2.9. Feeding assays of compound 3 in host A. nidulans

The host A. nidulans was cultured on 4 mL CDST solid medium (pH 7.0) at 25 °C for 2.5 days. Compound 3 (200 μM) dissolved in dimethyl sulfoxide was spread on the surface of the mycelia. Meanwhile, compound 3 was added to the medium as a blank control with the same concentration. After an additional 1 days of culture at 25 °C, the mycelia and medium were extracted with ethyl acetate/acetone (3:1, v/v), and the mixture was centrifuged at 12000 rpm for 3 min. 250 μL of supernatant was dried in vacuo and dissolved in methanol for LC-MS analysis.

2.10. Marfey's method

The amino acid configuration in compound 1 was determined by Marfey's method [23]. Compound 1 (0.6 mg) in ACN (200 μL) was mixed with 12 N HCl (500 μL) and ddH2O (300 μL). The mixture was heated at 115 °C for 24 h, dried in vacuo, and redissolved in ddH2O (50 μL). Subsequently, 10 μL of 1 M NaHCO3 was added, followed by 50 μL of Nα-(5-fluoro-2,4-dinitrophenyl)-l-leucinamide (l-FDLA, 10 mg/mL in ACN). The reaction mixture was incubated at 45 °C for 3 h and quenched with 5 μL 2 N HCl. And then the reaction mixture was dried and dissolved in 200 μL ACN. The resulting derivatives were subjected to LC-MS analysis with an injection of 1 μL. Standard amino acids (l-Trp and d-Trp) were also derivatized with the above method. The retention times of l-Trp-l-FDLA (m/z 499) and d-Trp-l-FDLA (m/z 499) were 7.3 min and 7.6 min, respectively. The retention time of the l-FDLA derivative of acid hydrolysate of 1 was 7.3 min.

3. Results and discussion

3.1. Identification biosynthetic gene cluster (BGC) for the diketomorpholines

Based on careful comparative analysis utilizing AdxB as a probe, the aau gene cluster was identified from A. aculeatus CRI323-04 (Fig. 2a). Synteny analysis between this DKM-associated BGC and homologous BGCs in other Aspergillus species revealed that the aau cluster harbored three conserved genes: an NRPS gene (aauA), an NAD(P)H-dependent reductase gene (aauB), and a DMATS gene (aauC). Protein BLAST analysis revealed that AauA, AauB, and AauC resemble AdxA, AdxB, and AdxC, which are involved in the biosynthesis of acu-dioxomorpholine A with 90%, 85%, and 94% sequence identity, respectively (Table S5) [6,24]. Consequently, we reasoned that this BGC might create natural products related to acu-dioxomorpholine A. This hypothesis is supported by the presence of a two-module NRPS (A1-T1-C1-A2-T2-CT domain) and a reductase in the aau BGC. Furthermore, this NRPS putatively activates two molecules of l-amino acid derivatives as substrates due to the absence of the epimerase domain.

Fig. 2.

Fig. 2

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The aau gene cluster and functional confirmation of AauA and AauB in vivo. (a) The aau cluster contained an NRPS (aauA, A1-T1-C1-A2-T2-CT) gene, a reductase gene (aauB), and a DMATS gene (aauC). (b) LC-MS analysis showed that heterologous expression of aauAB in A. nidulans produced compounds 1–2. (c) Structural elucidation of compounds 1–2.

3.2. Heterologous expression of the aau gene cluster and products interconversion

To clarify the products of the aau cluster, the aauA–aauC was amplified from A. aculeatus genomic DNA and then refactored into shuttle plasmids with constitutive gpdA promoter, inducible glaA and amyB promoters, and selectable markers (Fig. S1). These recombinant plasmids were transformed into the host A. nidulans and carried out heterogeneous expression. The aauA gene was initially expressed in A. nidulans. Unexpectedly, no products were detected from the metabolites of A. nidulans transformants. The aauA and aauB genes were next co-expressed in A. nidulans owing to the proposed biosynthetic pathway for acu-dioxomorpholine A. In contrast to the AN-WT control, compounds 1 (m/z 335 [M + H]+) and 2 (m/z 353 [M + H]+) were detected from the transformants (Fig. 2b). Subsequently, large-scale fermentation resulted in the isolation of 1 and 2, and their structures were identified by HRMS and NMR (Fig. 2c).

Compound 1 possessed a molecular formula of C20H18N2O3 in accordance with the ion mass at m/z 335.1402 [M + H]+ (calcd for 335.1396, Table S6 and Fig. S2), indicating thirteen degrees of unsaturation. The 1H, 13C, DEPT-135, and HSQC NMR data (Table S7 and Fig. S10–S16) indicate that 1 had a total of twenty carbons that were assigned as two carbonyls (δC 167.7 and 166.0), twelve aromatic carbons (nine protonated, δC 136.5, 136.0, 129.2, 129.2, 128.0, 128.0, 127.2, 126.4, 121.0, 118.7, 118.6, and 111.4), one sp2-hybridized quaternary carbon (δC 108.2), one olefinic methine (δC 124.7), an oxygenated-methine carbon (δC 78.3), one methine carbon (δC 53.8) attached to nitrogen, and two methylene carbons (δC 37.1 and 28.0). The tryptophan moiety was deduced by the COSY spin systems of H-1/H-2, H-4/H-5/H-6/H-7 and H2-8/H-9/H-14, and the HMBC correlations from H-1, H-2, and H-6 to C-7a, from H-2 and H2-8 to C-3a, from H-4 and H-9 to C-3, from H-5 and H-7 to C-3a, from H2-8 to C-2, from H-8 and H-14 to the carbonyl C-10. The phenyllactic acid unit was determined by COSY correlations along with HMBC correlations of H2-15 with C-13, C-17, and C-21, H-19 with C-17 and C-21, and H-20 with C-16 and C-18. This unit represents a phenylalanine-like moiety, where the α-amine group in phenylalanine is replaced with a hydroxyl. Moreover, the two above moieties were connected via an ester bond and an amide bond that was supported by HMBC correlations of H-14 with C-10 and C-12, H-9 with C-13. The planar structure of 1 was indeed confirmed and satisfied the unsaturated degree. The relative configurations of the two stereocenters (C-9 and C-12) were established according to NOESY data. The NOESY correlation of H-9 with H-12 indicated that H-9 and H-12 faced the same direction. To further confirm the configuration of 1, we performed the acid hydrolysis and chemical derivatization using Marfey's reagent (l-FDLA). These reactions elucidated that 1 contains one l-(S)-tryptophan (Fig. S17). Owing to the aforementioned analysis and the NOESY correlations, AauA specifically recognizedl-(S)-tryptophan and l-(S)-phenyllactic acid to assemble the diketomorpholine core. Compound 1 was designated as cyclo-(l-tryptophan-l-phenyllactic acid), which was reported as a natural product for the first time [14].

Compound 2 had the molecular formula of C20H20N2O4 (twelve degrees of unsaturation) based on HRMS at m/z 353.1512 [M + H]+ (calcd for 353.1501, Fig. S3). The similarities observed in the 1H and 13C NMR spectra of compounds 1 and 2 suggested they are structurally related. Compound 2 had a mass 18 units larger than compound 1. In addition, it had an additional hydroxyl proton signal (δH 5.71), and both carbonyls shifted downfield. Detailed analysis of NMR data (Table S8 and Fig. S18–24) revealed that compound 2 was the ester hydrolysis product of compound 1. Therefore, 2 was assigned as seco-(l-tryptophan-l-phenyllactic acid) with the same absolute configuration due to the biogenesis [25]. Hence, AauA and AauB were regarded as the key roles for the biosynthesis of the DKM scaffold. The reductase AauB first reduces phenylpyruvic acid (PPA) to l-(S)-phenyllactic acid (l-PLA), which is then condensed with l-(S)-tryptophan by AauA to generate the DKM scaffold.

In order to investigate the following biosynthetic steps, we focused on the putative DMATS gene (aauC), as prenyl groups appeared in some natural products with the DKM scaffold. Based on this idea, we continued to characterize the function of AauC in vivo. We constructed the recombinant strain AN-aauABC with co-expression of aauA, aauB, and aauC. As a result, compounds 3–8 were detected from this strain, clearly indicating that AauC catalyzed the tailoring step following the AauA-mediated condensation (Fig. 3a). Subsequent large-scale fermentation led to the isolation of 3–8. Their structures were determined by HRMS and NMR analysis (Fig. 3b). Among them, compounds 3–5 and 7 were firstly reported, especially 4–5 possessing unique ketooxadiazepane scaffold with C-13 ketone. Retro-biosynthetic analysis suggested that 4–5 may derive from 3 as a common precursor.

Fig. 3.

Fig. 3

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Confirmation of the function of AauC in vivo and in vitro. (a) LC-MS analysis showed that heterologous expression of aauABC in A. nidulans produced compounds 3–8. (b) Structural elucidation of compounds 3 (ent-acu-dioxomorpholine A) and compounds 4–8. (c) HPLC analysis (λ = 210 nm) demonstrated that AauC converted 1 to 3in vitro. (d) HPLC analysis (λ = 210 nm) showed that AauC could not recognize 2 as the substrate in vitro.

The molecular formula of ent-acu-dioxomorpholine A (3), C25H26N2O3, was determined from HRMS data (m/z 403.2030 [M + H]+, calcd for 403.2022, Fig. S4). Compared to the NMR data of 1, compound 3 has an additional prenyl group (Table S9 and Fig. S25–S31). Furthermore, the COSY correlation of H-2′ and H-3′ in combination with HMBC correlations from H-4′ to C-2′, from H-5′ to C-4′ and C-3, and from H2-8 to C-1′ confirmed the location of a prenyl group at C-3. The relative configuration of 3 was determined as shown by NOESY correlations of H-2 with H-8β (δH 2.31), H-4′ and H-5′, H-8α (δH 2.47) with H-4 and H-9, as well as H-9 with H-12. Additionally, the larger coupling constant of 10.7 Hz between H-8β (δH 2.31) and H-9 supported the relative configuration of compound 3. Collectively, 3 was derived from 1 by means of reverse prenylation modification catalyzed by DMATS AauC, which possessed the same absolute configuration assigned as 2S,3R,9S,12S. Notably, compound 3 exhibits configurations at C-2/C-3/C-9/C-12 that are completely opposite to those of acu-dioxomorpholine A.

The molecular formula of 4 was assigned as C26H28N2O4 (fourteen degrees of unsaturation) according to HRMS m/z 455.1967 [M + Na]+ (calcd for 455.1947, Fig. S5). Analysis of its 1D NMR and HSQC data (Table S10, and Fig. S39–S42) revealed the presence of two carbonyls (δC 174.2 and 171.8), fourteen olefinic carbons (eleven protonated, δC 148.0, 144.9, 139.5, 132.0, 130.5, 130.5, 129.8, 129.1, 129.1, 127.2, 126.5, 120.4, 114.5, and 108.2), two quaternary carbons (δC 62.9 and 41.6), three methines (δC 82.5, 81.3, and 60.6), three methylene (δC 78.4, 37.7, and 34.9), two methyls (δC 23.2 and 22.7). Further analysis of 2D NMR spectra (Fig. S43–S45) suggested that 4 was a seco-DKM derivative of 3. Similarly, the tryptophan moiety was deduced from the COSY correlations of H-4/H-5/H-6/H-7 together with HMBC correlations from H-2 to C-3, C-3a, C-7a, and C-9, from H-4 and H-6 to C-7a, from H-5 and H-7 to C-3a, from H-8 to C-3, C-3a and C-10. The phenyllactic acid unit was confirmed from the COSY correlation of H-12 and H-15 along with HMBC correlations from H-12 to C-13 and C-16, from H-15 to C-13, C-17 and C-21, from H-20 to C-16 and C-18, and from H-19 to C-17 and C-21. Unexpectedly, the two aforementioned segments were assembled by a ketooxadiazepane scaffold firstly reported from natural products, which was determined from HMBC correlations from H-9 to C-13, from H-12 to C-22, and from H-22 to C-2 and C-7a. In addition, the substitution of a prenyl group at C-3 was supported by the COSY correlation of H-2′ and H-3′ in combination with HMBC correlations from H-4′ to C-2′ and H-5′ to C-4′ and C-3. NOESY correlations from H-2 to H-4′, H-8β (δH 2.33), H-12 and H-22β (δH 4.77), and from H-8α (δH 2.62) to H-4 and H-9 in combination with a large coupling constant (J = 10.0 Hz) between H-8β (δH 2.33) and H-9 demonstrated that H-2, H-12 and the prenyl group faced the same direction. Otherwise, H-9 orientated the opposite side. Given that l-(S)-tryptophan was involved in the biosynthetic process of 4, similar to that of 3, the absolute configuration was finally determined to be 2S,3R,9S,12S.

The HRMS data of 5 indicated a molecular formula of C27H30N2O4 (fourteen degrees of unsaturation) based on the HRMS ion at m/z 447.2304 for [M + H]+ (calcd for 447.2284, Fig. S6). The 1H and 13C NMR data (Table S11, Fig. S46–S47) closely resembled those of 4 except for an extra methoxy group (δH/δC 3.62/52.6). The HMBC correlation from H-23 to C-10 confirmed the location of the methoxy group at C-10. The relative configuration of 5 was illustrated by NOE correlations similar to those of 4, along with a large coupling constant (J = 10.0 Hz) between H-8β (δH 2.30) and H-9. Compared with the two compounds, it was not difficult to find that 5 was derived from 4 via methyl esterification, likely mediated by host A. nidulans. Thus, the absolute configuration of 5 was the same as that of 4 as shown in Fig. 3b.

The molecular formula of compound 6 was deduced as C25H28N2O4 on the basis of HRMS data (Fig. S7). The NMR data of 6 (Table S12, Fig. S53–S59) were similar to those of 2 except for an additional prenyl group. The prenyl group attached to N-1 was supported by the COSY correlation of H-1′/H-2′ together with HMBC correlations from H-2 to C-1′, from H-4′ to C-2′, C-3′ and C-5′, and from H-5′ to C-2′ and C-3′. In addition, two seco-diketomorpholine derivatives 7–8 with the same molecular formula C25H28N2O4 were identified according to HRMS (Fig. S8–S9). Detailed interpretation of NMR data for 7–8 (Table S13–S14, and Fig. S60–S73) assigned their structures with a regular prenyl group or a reverse prenyl group substituted on the tryptophan unit at C-2, respectively. The co-expression of aauC with aauAB in A. nidulans yielded DKM derivatives with reverse prenyl group (3–5, 8) or regular prenyl group (6–7) at different positions of the tryptophan moiety. Based on the structures of compounds 3–8, we hypothesized that AauC might catalyze regular or reverse prenylation reactions on the tryptophan moiety. This hypothesis required validation through in vitro experiments with AauC.

3.3. Characterization of AauC by in vitro assay

To validate the function of AauC in vitro, the aauC gene was cloned from the cDNA and expressed in E. coli BL21. Ni-NTA chromatography was utilized for the purification of His-tagged AauC (Fig. S74), and the purified protein AauC was sequentially used for the in vitro biochemical assay. In order to verify whether 1 or 2 can be used as the substate, each compound was incubated with AauC at 25 °C for 2 h. AauC efficiently converted 1 to 3 by prenylation rather than recognized 2 as the substrate (Fig. 3c and d). Hence, these in vitro experiments clearly demonstrated that AauC specifically catalyzed the reverse prenylation of 1 at C-3 on the tryptophan unit, giving rise to 3. Moreover, AauC was unable to catalyze prenylation at other positions of 1 or 2 to generate 4–8.

3.4. Chemical feeding of compound 3 in host A. nidulans

We initially presumed that compounds 6–8 were derived from 2 prenylated by AauC. However, in vitro assays revealed that 2 was not the substrate of AauC. In addition, no enzymes in the aau cluster are responsible for the post-modifications to produce 4–8. We supposed that the formation of 4–8 was likely attributed to tailoring steps catalyzed by the chassis organism A. nidulans. Thus, to identify the biosynthetic origin of 4–8, we conducted feeding experiments using 3 as substrates. Simultaneously, 3 was added to the medium as a blank control. Interestingly, when 3 was fed into the host A. nidulans, it was simultaneously converted into 4–8 (Fig. 4a). These findings collectively demonstrate that A. nidulans may mediate the conversion of 3 into 4–8 in vivo. It was suggested that compounds 4–8 were biosynthesized from 3 as the precursor. Hence, we proposed the biosynthetic pathway for 4–8 (Fig. 4b).

Fig. 4.

Fig. 4

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Chemical feeding of compound 3 as substrate in A. nidulans. (a) LC-MS analysis demonstrated that compounds 4–8 were produced when 3 was fed into A. nidulans. (b) The proposed pathway of the conversion of 3 to 4–8.

The DKM (morpholine-2,5-dione) scaffold is inherently acid-labile and unstable in the solvent due to the ester bond; it undergoes facile (auto) acid-mediated ring opening rearrangement to yield the corresponding acid [14]. Due to this instability, we infer that the DKM scaffold of 3 tends to undergo hydrolytic ring-opening to generate seco-DKM (seco-3), subsequent incorporation of one-carbon unit, and eventual formation of seven-membered N–O heterocycle to yield the unique ketooxadiazepane scaffold of 4 and 5. Furthermore, the cleavage of the ester bond of 3 prompts the protonation at N-14, the cleavage of the C2–N14 bond, and sigmatropic rearrangements of the prenyl group to produce 6–8. Compounds 4 was further transformed to 5 through O-methylation.

3.5. Proposed biosynthetic pathway of 1–8

According to heterologous expression of the aau cluster in A. nidulans and in vitro assays, we confirmed that the biosynthesis of 3 comprises three enzymes: AauA, AauB, and AauC, which sequentially mediate the reduction of PPA, condensation and cyclization of l-tryptophan and l-PLA, and C-3 reverse prenylation (Fig. 5).

Fig. 5.

Fig. 5

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Whole biosynthetic pathway of the aau gene cluster which was responsible for the biosynthesis of DKMs 1–3, and the formation of seco-DKMs (6–8) and ketooxadiazepane alkaloids (4–5) in A. nidulans.

AauB shows 42% sequence identity and 56% similarity with NAD(P)H-dependent ketoreductase AdrE (accession number B6HV33), which reduces the carbonyl group to alcohol during andrastin A biosynthesis [26]. Similarly, AauB is predicted to bind NAD(P)H and catalyze the reduction of phenylpyruvic acid (PPA) to phenyllactic acid (PLA). In general, PLA biosynthesis contains two steps: phenylalanine (Phe) is first transaminated to generate PPA, and PPA is further reduced to PLA by NAD-dependent hydroxyl acid dehydrogenases [27]. PLA is a conventional weapon used by lactic acid bacteria (LAB) and some other Gram-positive bacteria [28], yeast Wickerhamia fluorescens [29], and yeast-like Geotrichum candidum in their ecological niches [30], which is not reported in other fungi to the best of our knowledge. Therefore, AauB is the essential enzyme in the biosynthesis of PLA as a precursor participating in the subsequent reactions.

AauA is a two-module NRPS proposed to assemble the DKM core with l-tryptophan and l-phenyllactic acid (l-PLA) as substrates. AauA contains two adenylation (A) domains, and its specificity code in the substrate-binding pocket was used to predict which substrate will be recognized [31]. In view of the specificity code, the first A domain of AauA (AauA-A1) is inferred to recognize hydrophobic amino acids, such as tryptophan (Fig. S75). Nevertheless, the highly conserved Asp (D) in the second A domain (AauA-A2) is replaced by a noncanonical Glycine (G), implicating that Aau-A2 activates an amino acid analogue. The specificity code of Aau-A2 exhibits similarity to those of A domains from other fungi which load α-hydroxy acid, such as PF1022 synthetase that loads phenyllactic acid [32]. Then the two condensation (C) domains of AauA (AauA-C1 and AauA-C2) catalyze the stepwise formation of the ester and amide bond within the DKM ring. Significantly, the canonical histidine (H) of AauA-C1 is replaced by an atypical arginine (R) (Fig. S76), implying that the arginine plays an essential role in the formation of a unique ester bond instead of a conventional amide bond.

AauC has 52% sequence identity and 68% similarity to prenyltransferase RoqD (UniProt B6HJU1) in the roquefortine biosynthesis that catalyzes the reversed prenylation of the indole moiety at the C-3 position as well as ring closure between C-2 and the DKP nitrogen (N-14) [33]. This protein homology demonstrates that AauC catalyzes similar reactions, which is consistent with the in vitro biochemical assay.

Aside from 3, co-expression of aauABC in A. nidulans also resulted in the production of 4–8. The conversion from 3 to 4–8 was mediated by the host A. nidulans. The lactone hydrolysis of 3 to generate seco-3, which underwent incorporation of one-carbon unit, and putative cyclization to yield 4 and methyl easter 5. The intermediate seco-3 mainly underwent the cleavage of the C–N bond and sigmatropic rearrangements to afford 4–8.

4. Conclusion

In this study, we elucidated the biosynthesis of prenylated DKM (3) based on heterologous expression of the aau gene cluster and enzymatic experiments in vitro. The assembly line of DKMs mainly includes: 1) a ketoreductase (AauB) reducing PPA to l-PLA through carbonyl reduction; 2) a two-module NRPS (AauA) accomplishing condensation and cyclization between l-tryptophan and l-PLA; 3) a dimethylallyl tryptophan synthase DMATS (AauC) catalyzing C-3 reverse prenylation on the tryptophan moiety. Furthermore, feeding experiments demonstrated that 3 was biotransformed to ketooxadiazepanes 4–5 and seco-DKM derivatives 6–8 in A. nidulans. Notably, we identified the first natural product featured with a ketooxadiazepane scaffold (4) derived from the seco-DKM precursor. Although the detailed mechanism underlying the formation of the ketooxadiazepane backbone remains to be fully clarified, its biosynthesis likely involves endogenous enzymes from the chassis strain A. nidulans. Such enzymes responsible for constructing this scaffold in A. nidulans remain to be elucidated. Our work exemplifies the successful discovery of structurally diverse DKM and ketooxadiazepane alkaloids from fungal resources.

CRediT authorship contribution statement

Qiaoqiao Tao: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Funding acquisition, Formal analysis. Yi Zou: Writing – review & editing, Supervision, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization.

Data availability statement

The data that support this study are available in the supplementary materials of this article.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank Prof. Prasat Kittakoop from Chulabhorn Graduate Institute and the Center for Environmental Health, Thailand, for kindly providing the fungus A. aculeatus CRI323-04. This work is supported by National Natural Science Foundation of China (32400059), Chongqing Postdoctoral Science Foundation Special Funded Project (2412013581196684) and Special Fund for Youth Team of Southwest University (SWU-XJLJ202306).

Footnotes

Peer review under the responsibility of Editorial Board of Synthetic and Systems Biotechnology.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.synbio.2026.01.032.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

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Data Availability Statement

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