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. 2023 Feb 10;86(2):398–405. doi: 10.1021/acs.jnatprod.2c01027

Identification and Functional Characterization of Fungal Chalcone Synthase and Chalcone Isomerase

Sho Furumura 1, Taro Ozaki 1, Akihiro Sugawara 1, Yohei Morishita 1, Kento Tsukada 1, Tatsuya Ikuta 1, Asuka Inoue 1, Teigo Asai 1,*
PMCID: PMC9972472  PMID: 36762727

Abstract

graphic file with name np2c01027_0005.jpg

By mining fungal genomic information, a noncanonical iterative type I PKS fused with an N-terminal adenylation–thiolation didomain, which catalyzes the formation of naringenin chalcone, was found. Structural prediction and molecular docking analysis indicated that a C-terminal thioesterase domain was involved in the Claisen-type cyclization. An enzyme responsible for formation of (2S)-flavanone in the biosynthesis of fungal flavonoids was also identified. Collectively, these findings demonstrate unprecedented fungal biosynthetic machinery leading to plant-like metabolites.

Naringenin chalcone (1) is a common precursor of flavonoids that are ubiquitously distributed among the plant kingdom as essential and biologically active secondary metabolites.1 The biosynthesis of chalcone is mediated by chalcone synthase (CHS), which is a single-domain enzyme consisting of a ketosynthase (KS) domain, a representative enzyme of so-called type III polyketide synthases (PKSs).2,3 CHS adopts p-coumaroyl CoA as a starting material and extends it to a tetraketide intermediate by using three molecules of malonyl CoA. Finally, 1 is released via a Claisen-type cyclization.4 Since the cloning of the corresponding gene in 1983,5 CHS has been extensively studied.

Fungal genomes contain a huge number of untapped biosynthetic gene clusters (BGCs) that are responsible for the biosynthesis of cryptic natural products, making fungi attractive targets for genome mining of novel natural products and biosynthetic machineries.6,7 Among fungal PKSs, there is a group composed of PKSs with an A-T didomain of NRPS at its N-terminus. In this article, this type of PKS is defined as AT-PKS. The same type of PKS was also defined as NRPS-PKS in previous studies.8−10 Only five fungal AT-PKSs have been identified and characterized.8−12 An N-terminal A-T didomain incorporates various starting materials to PKS assembly lines, thereby enabling AT-PKSs to produce unique polyketides distinct from the products of canonical PKSs. During genome mining focusing on noncanonical PKSs of fungal origins,13−16 we identified DiapA, which has a unique domain architecture, A-T-KS-acyltransferase (AT)-dehydratase (DH)-ketoreductase (KR)-acyl carrier protein (ACP)-thioesterase (TE), in a Discosia fungus from our own culture collection (Figures 1A, S1). A similar chimeric AT-PKS, P168DRAFT_323099 (CfvA in this study), was proposed to be involved in the biosynthesis of chlorflavonin,17 which is the first example of a fungal flavonoid, the producer of which was confirmed to be Aspergillus candidus (Figure 1B).18 More recently, Zhang et al. demonstrated that this type of AT-PKS catalyzes the formation of 1 and naringenin (2).19 Herein, we report the functional characterization of DiapA, showing that this enzyme also catalyzes the formation of 1 and that its TE domain is responsible for the characteristic Claisen-type cyclization. In addition to its natural substrate, DiapA accepted benzoic acid analogues and gave unnatural flavanones as products. We also identified a chalcone isomerase (CHI) gene involved in the biosynthesis of chlorflavonin and demonstrated that this CHI catalyzes the conversion of chalcones to corresponding (2S)-flavanones. Collectively, our findings demonstrated a unique fungal strategy for the biosynthesis of plant-like metabolites.

Figure 1.

Figure 1

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Comparison of fungal AT-PKSs. (A) Representative examples of fungal AT-PKSs. The chemical structures of the starter units are shown in magenta. (B) Comparison of diapA and a putative BGC for chlorflavonin (cfv).

Results and Discussion

To identify the function of DiapA, we expressed this gene in Aspergillus oryzae to construct a transformant AO-diapA (Tables S1–S4). As a result, naringenin (2), a well-known precursor of plant flavonoids, was observed as a racemic mixture in the culture medium of AO-diapA, although in a very small amount (Figure 2A,B,E, Table S5). A feeding experiment of [1,2-13C2]-labeled sodium acetate to AO-diapA indicated incorporation of four acetate units from C-3 to C-8a of 2, suggesting that the starter substrate of DiapA was 4-hydroxybenzoic acid (3) (Figure S2, Table S6). Consistently, addition of 3 to the culture medium of AO-diapA increased the production of 2 (Figure 2B). In this experiment, phloretin (4) was also observed as a minor product with negligible levels of bis-noryangonin (BNY) produced (Figures 2B, S3, Table S7). The lack of the ER domain in DiapA indicated that endogenous enzyme(s) in A. oryzae reduces the double bond of 1 to yield 4. This assumption was further supported by the observation that isoliquiritigenin, a deoxy analogue of 1, was readily converted to davidigenin during the cultivation of A. oryzae (Figure S4). Co-production of phloretin 4 and racemic 2 indicated that DiapA synthesizes 1.

Figure 2.

Figure 2

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Heterologous production of chalcone and flavanones in Aspergillus oryzae. (A) Proposed biosynthetic pathway for the observed metabolites. (B) HPLC profiles of the metabolites extracted from A. oryzae transformants. (C) Stereoselective conversion of chalcones 1 and 8 to corresponding (2S)-flavanones 2 and 7 by LjCHI or CfvF. (D) Conversion of l-tyrosine to 3. (E, F) Chiral HPLC analysis of 2 (E) and 7 (F) isolated from the transformants. Chromatograms were monitored at 280 nm.

In vitro adenylation of 3 with the recombinant A-T didomain resulted in 4-hydroxybenzoyl-AMP as a product (Figures S5 and S6). Because a conserved domain search of the NCBI database suggested the presence of a putative CoA binding site, CoA was added to the reaction mixture. However, even in the presence of CoA, 4-hydroxybenzoyl-CoA was not detected. This result indicated the DiapA-A domain catalyzed adenylation as observed for the known NRPS-A domain. The substrate specificity of the A domain was then examined by detecting concomitant formation of pyrophosphate using a pyrophosphatase and a malachite green phosphate exchange assay kit. The DiapA-A domain exhibited substrate tolerance against a commercially available benzoic acid analogue (Figure S7). Although the production level was much lower than that of 2, AO-diapA could produce unnatural flavanone 6 when 4-aminobenzoic acid was added to the culture broth (Figures 2A, S8, Table S8).

To construct a fungal platform able to produce plant flavonoids, de novo biosynthesis of (2S)-2 by co-expressing plant CHI with diapA was investigated (Figure 2C). Because several Aspergilli are known to degrade trans-p-coumaric acid (5) to 3,20,21 it was assumed that the introduction of tyrosine ammonia lyase (TAL) into A. oryzae would result in 3 being produced from l-tyrosine via 5 (Figure 2D). After confirming that A. oryzae converted 5 to 3 (Figure S9), the metabolic pathway from l-tyrosine to (2S)-2 in A. oryzae was constructed. For endogenous conversion of l-tyrosine to 5 and stereoselective conversion of 1 to (2S)-2, the TAL gene derived from Rhodotorula glutinis (RgTAL)22 and CHI derived from Lotus japonicus (LjCHI)23 were selected, respectively. Both RgTAL and LjCHI were co-expressed with DiapA in A. oryzae. The resulting transformant AO-diapA/TAL/CHI successfully produced 2 with concomitant formation of 3 (Figure 2B). Efficient conversion of 5 to 3 by endogenous enzyme(s) of A. oryzae was confirmed by feeding of [1,2-13C2] sodium acetate to AO-diapA/TAL/CHI, which clearly showed incorporation of four acetate units into 2 (Table S6), although the in vitro adenylation assay indicated that DiapA could also accept 5 as shown for FnsA (Figure S7).19 Based on these feeding experiments, we concluded that 3 would be a preferred substrate of DiapA at least in our experimental conditions employing the A. oryzae expression system. Subsequent chiral HPLC analysis readily confirmed the selective production of (2S)-2 (2S/2R = 7:3 with LjCHI and 1:1 without LjCHI, Figure 2E). The activity of LjCHI in A. oryzae seemed to be low when the chiral HPLC result was compared to that in the literature.23 In plants, CHS and CHI were known to interact with each other.24 Therefore, this low activity of LjCHI in A. oryzae might be derived from the absence of the correct protein–protein interactions between DiapA and LjCHI. This CHI-dependent formation of (2S)-2 supports the formation of free 1 as a product of DiapA reaction.

As observed for AO-diapA, the transformants harboring diapA homologues, pfiapA (fnsA gene cloned from Pestalotiopsis fici MAFF 237190) and pmiapA, also produced 2 as a racemic mixture (Figure S10). Despite these observations, fungi may possess enzyme(s) for stereospecific conversion of 1 to 2 because enzymes generally recognize only one stereoisomer and do not accept the other. Interestingly, the putative BGC for chlorflavonin contains an enzyme, P168DRAFT_323099 (CfvF), which shows weak homology to bacterial CHI (Figures 1B, S11).25,26 Given this observation, it was suspected that CfvF catalyzes the stereospecific conversion of chalcone(s) to the corresponding flavanone(s) in the biosynthesis of chlorflavonin. To address this hypothesis, co-expression of cfvA and cfvF in A. oryzae was examined. When cfvF was co-expressed with cfvA, (2S)-pinocembrin (7) was selectively produced (Figures 2C,F, S12). On the other hand, racemic 7 was produced without cfvF. These observations indicated that CfvF catalyzes the stereospecific cyclization of pinocembrin chalcone (8). Recombinant CfvF was prepared, and in vitro cyclization of 1 and 8 as substrates was investigated (Figure S5). As a result, CfvF-dependent conversion of both substrates to the corresponding flavanones in (2S)-forms was observed (Figure S12), further confirming the function of CfvF as CHI.

Considering that 1 was produced as a product of DiapA, the TE domain likely catalyzes Claisen-type cyclization rather than the canonical hydrolytic release of an ACP-tethered polyketide chain. A similar TE-catalyzed cyclization was proposed for the fungal NR-PKSs, such as PksA for aflatoxin biosynthesis27 and PKS1 for 1,3,6,8-THN.28 While DiapA-TE and its orthologs fell into a distinct clade from other TE domains (Figure 3A), amino acid sequence alignment revealed that the catalytic triads, S2550, D2579, and H2690, are conserved in DiapA (Figure S13). Intriguingly, DiapA-TE shows a closer relationship to bacterial TE domains than fungal counterparts. To investigate the function of catalytic triads, the A. oryzae transformants expressing DiapA (S2550A) and DiapA (H2690A) were constructed. Both mutants abolished the production of 2 and BNY (Figure 3B). The production of other derailment compounds was not observed in either case. This observation indicated that the TE domain was involved in DiapA catalysis and responsible for the formation of both 2 and BNY. This hypothesis was further confirmed by gene complementation analysis. Both reverse mutation to WT sequence and in trans complementation of the ACP-TE didomain recovered the production of 2 (Figure S14).

Figure 3.

Figure 3

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Functional analysis of the DiapA-TE domain. (A) Phylogenetic tree of TE domains of DiapA and other related enzymes. (B) HPLC-MS traces of the metabolites extracted from A. oryzae transformants expressing diapA and its mutants. (C) Molecular docking analysis of the DiapA-TE domain. See Figure S16 for the results using all possible diastereomers. (D) Amino acid sequence alignment of the TE domains (Pfam ID: PF00975). See Figure S9 for the full alignment.

To explore the other amino acid residues related to the function of the TE domain, structural prediction utilizing AlphaFold v2.2 was employed.29 Protein folding of the obtained 3D model structure was consistent with that of the TE domain of deoxyerythronolide B synthase (DEBS) (PDBID: 5D3K(30)). A molecular docking analysis using this model structure and putative intermediate of Claisen-type cyclization identified the three aromatic residues, F2482, Y2581, and F2635, that likely interact with the intermediate (Figures 3C, S15). These residues are conserved among DiapA homologues that are classified into the same clade in the phylogenetic tree, but not in the other TE domains (Figure 3D). The three DiapA mutants, F2482A, Y2581A, and F2635A, were constructed and expressed in A. oryzae to investigate whether these residues are involved in the DiapA catalysis. Although the resulting transformants still produced 2 and BNY, the production was dramatically decreased in all cases, without increasing the production of any other compounds (Figure 3B). Considering that Y171 of DEBS-TE, which corresponds to Y2581 of DiapA, was proposed to be important for forming the active site and correct positioning of catalytic histidine residue,30,31 Y2581 might also have a similar function to Y171 of DEBS-TE, while F2482 and F2635 characteristic of DiapA-TE possibly contribute to chalcone formation.

In the present study, we identified and functionally characterized fungal AT-PKS DiapA. The DiapA-catalyzed chalcone-synthesizing pathway is proposed as shown in Figure 4A. The DiapA-A domain adenylates 3 and loads it to the T domain. Using T domain-tethered p-hydroxybenzoate as a starter, downstream PKS iteratively catalyzes chain elongation with four molecules of malonyl CoAs to yield a pentaketide intermediate. During four rounds of chain elongation, KR and DH domains only function in the first round. The pentaketide is finally transferred to the terminal TE domain, which catalyzes the Claisen-type cyclization to furnish 1. Unlike plant CHSs, which synthesize tri- and tetraketide pyrones as byproducts (Figure 4B),32 only a negligible amount of BNY was produced by AO-diapA. Therefore, it was concluded that DiapA is a dedicated enzyme that selectively synthesizes 1.

Figure 4.

Figure 4

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Comparison of the chalcone-synthesizing reactions identified in this study and those in plants. (A) Proposed model for the DiapA-catalyzed reaction. After starter unit 3 is adenylated and loaded onto the T domain by the action of the A domain, downstream PKS domains iteratively catalyze chain elongation and Claisen-type cyclization to yield 1. In the biosynthesis of chlorflavonin, (2S)-7, which is generated by CfvF, might serve as a biosynthetic intermediate. (B) Plant CHS-catalyzed formation of 1. CHS catalyzes three rounds of chain elongation using p-coumaroyl-CoA as a starter unit. Note that tetraketide pyrone CTAL is concomitantly generated. X = CoA or catalytic Cys residue of CHS.

We also compared the nonribosomal code of the DiapA-A domain with those of FnsA-A and CfvA-A domains to obtain insights into their substrate specificities. The nonribosomal codes of DiapA and FnsA show close similarity (Figure S16). This observation is consistent with the observation that both DiapA and FnsA synthesized 1 derived from 3. In contrast, the nonribosomal code of CfvA, which likely utilizes benzoic acid as a starter, differs from those of DiapA and FnsA (Figure S16). This subtle change may reflect the difference in the substrate specificities of these enzymes. The production level of 2 in AO-diapA was low without addition of 3 or co-expression of RgTAL, suggesting that the fungal strains that originally possess diapA or its homologues may have the biosynthetic pathway that provides the starter unit for these AT-PKSs.

While the detailed reaction mechanism remains to be elucidated, the catalytic triads of canonical TE domains are conserved in the DiapA-TE and essential for its function. As no other candidate residues are found around the putative active sites, it was speculated that conserved H2690 not only deprotonates S2550 with the assistance of D2579 for initial nucleophilic attack to the mature polyketide chain but also functions as a catalytic base during the cyclization as proposed for the TE domain of PksA.27

In summary, we conducted genome mining of fungal genomic information, which is regarded as a rich source of novel biosynthetic machineries, and identified an unprecedented fungal AT-PKS that synthesizes 1. Heterologous expression in A. oryzae, isolation and structural analysis of the metabolites, and structural prediction of the terminal TE domain and molecular docking analysis clearly indicated that DiapA is a dedicated chalcone synthase distinct from plant type III PKS. The previous study also identified FnsA, a DiapA homologue, synthesized 1 and 2.19 However, it remains unclear whether the cyclization of 1 to 2 is an enzymatic process or not. We identified a fungal CHI that is also distinct from plant CHI, demonstrating that fungi adopt unique biosynthetic machineries for the synthesis of plant-like metabolites. This study paves the way for further genome mining of related pathways and sets the stage for biosynthetic engineering of plant flavonoids using fungal expression systems.

Experimental Section

General Experimental Procedures

TLC was performed on silica gel 60 F254 (Merck) and RP-18 F254 (Merck). Column chromatography was carried out on silica gel 60 (40–50 mesh) and silica gel 120 (spherical) RP-18 (Kanto Chemical Co., Inc.). The 600 MHz NMR spectra were recorded on a JEOL ECA-600 spectrometer (1H NMR, 600 MHz; 13C NMR, 150 MHz). Chemical shifts for 1H and 13C NMR are given in parts per million (δ) relative to residual solvent signals (δH 3.30, δC 49.0) for CD3OD or (δH 2.05) for acetone-d6 as internal standards. Mass spectra were measured on an Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific). HPLC analysis was performed on a JASCO AS-1555-10 intelligent sampler, JASCO PU-4180 RHPLC pump, and JASCO MD-4017 photo diode array detector (JASCO), which was equipped with COSMOSIL packed column 5C18-MSII (φ 4.6 mm × 150 mm) (nacalai tesque). LC-MS analysis was performed on an Acquity H-Class PLUS system (Waters) with a PDA eλ detector (Waters) and SQ detector 2 (Waters), which was equipped with an acquity UPLC BEH C18 column (130 Å, 1.7 μm, 2.1 mm × 50 mm; Waters). For the analysis of adenylated products, a Dionex Ultimate3000 (Thermo Scientific) with an Exactive Orbitrap mass spectrometer was used. In this case, L-column3 C8 3 μm (120 Å, 3.0 μm, 2.0 mm × 100 mm; CERI) was used for analysis. For malachite green phosphate assays, Absorbance 96 (Byonoy) was used to measure the absorption at 620 nm. NanoPad DS-11+ (DeNovix) was used to measure absorption at 395 and 360 nm.

Strains

Escherichia coli DH5α was used for cloning following standard recombinant DNA techniques. E. coli BL21(DE3) was used for expression of protein. Aspergillus oryzae NSAR1 (niaD–, sC–, ΔargB, adeA–) and A. oryzae NSPlD1 (niaD–, sC–, ΔpryG, ΔligD) were used as the host for fungal expression. Discosia sp. batta1 was used for genomic DNA preparation and draft genome sequencing. Pestalotiopsis fici MAFF 237190, P. microspora NBRC 30316, and Aspergillus candidus NBRC 8816 were used for genomic DNA preparation.

Genome Sequencing and Accession Numbers

Genome sequencing of Discosia sp. batta1 was conducted by Macrogen Japan Corp. using an Illumina Hiseq system (Illumina). Assembly of obtained reads was conducted by Research Institute of Bio-System Informatics (Tohoku Chemical Co., Ltd.) using Velvet 1.2.10.33 The sequence of diapA gene has been deposited in the DNA Data Bank of Japan (DDBJ) with the accession number LC733667. The primers used for the cloning of pfiapA and pmiapA were designed based on the nucleotide sequences deposited in the database with the following accession numbers: ETS82484.1 for pfiapA and APX43991.1 for pmiapA. The nucleotide sequences of RgTAL (AAA33883.1) and LjCHI (Q8H0G2.1) were optimized for expression in A. oryzae and synthesized by Integrated DNA Technologies.

Gene Cloning and Construction of A. oryzae Transformants

Each gene or donor cassette was amplified by PrimeSTAR MAX DNA polymerase (Takara) with primers listed in Table S1 and cloned into appropriate vectors or inserted into the targeted chromosomal region (HS201, HS401, HS601, identified in the previous study34) by homologous recombination. The constructed plasmids are listed in Table S2. Transformation of A. oryzae was performed using the protoplast-polyethylene glycol method as previously described.7,34 The transformants created in this study and the plasmids used for the transformation are shown in Tables S3 and S4. The details for the construction of each transformant are described in the Supporting Information.

Cultivation of Transformants and HPLC Analysis

The obtained transformants were cultivated on a selection agar plate at 30 °C, and its mycelia were inoculated in 1/2 CPS medium (1.75% Czapek-Dox broth, 0.17% meat peptone, 0.17% soy peptone, 0.17% casein peptone, 1.0% soluble starch, 0.5% maltose monohydrate, 0.2% sodium acetate (NaOAc)) and incubated at 30 °C for 5 days. The culture media were extracted as previously reported7 and analyzed by reversed-phase HPLC and LC-MS in the following conditions. HPLC analysis was performed on COSMOSIL packed column 5C18-MSII (φ 4.6 mm × 150 mm) with acetonitrile and water containing 0.01% trifluoroacetic acid (0–2 min: 20:80, 2–12 min: 20:80 to 100:0, 12–24 min: 100:0) at a flow rate of 1 mL min–1 using a photodiode array detector. LC-MS analysis was performed on an Acquity UPLC BEH C18 column with acetonitrile and water containing 0.1% formic acid (0–0.2 min: 20:80, 0.2–3.3 min: 20:80 to 100:0, 3.3–4.3 min: 100:0) at a flow rate of 0.5 mL min–1 in the ESI negative mode.

Isolation and Structure Determination of Metabolites

For isolation of compounds 2, 4, 6, and BNY, each A. oryzae transformant was cultivated on a larger scale (0.3–3.6 L) at 30 °C and 150 rpm for 3 or 5 days, and organic extracts of the culture supernatants or mycelia were fractionated by consecutive column chromatography using silica gel and/or reversed-phase column chromatography as stationary phase and mixtures of n-hexane–EtOAc and CHCl3–MeOH as well as water–acetonitrile as mobile phase. Schemes for isolation and structure determination and NMR and HR-MS spectra for each compound are summarized in Tables S5–S8 and the Supporting Information.

Preparation of 1 and 8 from 2 and 7

7 was prepared from chrysin by means of the reported procedure.35 A 20 mg amount of 2 and 7 was dissolved into 600 μL of 10 M KOH. After incubation for 1 h on ice, 6 M HCl was added and adjusted to pH 4–6. The precipitate was collected and purified by silica gel column chromatography (1: hexane–EtOAc = 2:1 to 1:1, 8: hexane–EtOAc = 3:1 to 2:1) to give 1 (2.2 mg) and 8 (1.6 mg).

Expression and Purification of Recombinant Proteins

The DiapA-AT didomain, CfvF, and LjCHI were expressed in E. coli BL21(DE3) as recombinant proteins fused with a N- or C-terminal histidine tag and/or maltose binding protein (MBP). The expression and purification followed standard molecular biology techniques. The detailed procedures for each protein are described in the Supporting Information.

In Vitro Assay of the DiapA-A-T Domain

Purified DiapA-AT (7 μM) was incubated with 2 mM 3 (dissolved in DMSO) in 200 μL of buffer, containing 28.8 mM hydroxylamine, 1 mM dithiothreitol, 3 mM ATP, 2 mM CoA trilithium salt, 10 mM MgCl2, 10% glycerol, 300 mM NaCl, and 50 mM Tris-HCl buffer (pH 7.5). The reaction was incubated for 60 min at 30 °C, and then 1 μL of this mixture was LC-MS analyzed. LC-MS analysis was performed on L-column3 C8 3 μm with acetonitrile and 10 mM NH4HCO3 (0–10 min: 2:98 to 30:70, 10–15 min: 30:70 to 95:5) and monitored in ESI negative mode. Substrate scope of the A domain was investigated by the following procedures. Purified DiapA-A-T (5 μM) was incubated with 2 mM substrates (dissolved in DMSO) in 50 μL of buffer, containing 28.8 mM hydroxylamine, 1 mM dithiothreitol, 0.4 U/mL pyrophosphatase (Sigma-Aldrich), 2 mM ATP, 10 mM MgCl2, 10% glycerol, 300 mM NaCl, and 50 mM Tris-HCl buffer (pH 7.5). The reaction was incubated for 30 min at 30 °C and then quenched by adding 50 μL of the working reagent from the malachite green phosphate assay kit (Enzo).36 After a 30 min incubation at 30 °C, the absorption at 620 nm (A620) was measured. The control A620 value was subtracted from the A620 value of the reaction mixture without substrate, and then the relative adenylation activity was calculated.

In Vitro Assay of CHI and CfvF

One milliliter of buffer C (50 mM potassium phosphate buffer (pH 7.5), 150 mM NaCl, 10% glycerol) was heated at 30 °C and transferred to a cuvette. After measuring the blank, 40 μg of 1 or 8 dissolved in 40 μL of EtOH was added. After measuring A395 (1) or A360 (8) for 60 s, purified protein was added to the reaction mixture (final concentration: 100 nM). Then A395 or A360 was measured for 120 s. The absorption was measured every 10 s, and residual ratio was calculated against absorbance at 0 s. The reaction mixture was extracted with EtOAc and concentrated in vacuo. The extract was dissolved in MeOH and used for chiral HPLC analysis (Figure S12). Chiral HPLC analyses were conducted with Chiralpak AD-H (150 mm × 4.6 mm i.d., dp = 5 μm, D) with reported conditions.37 Absolute configurations of 2 were determined by the elution order of two peaks. The absolute configuration of 7 was determined by comparing the specific rotation of 7 eluted around 6.7 min with that in the literature ([α]24D −44 (c 0.002, MeOH) in this study; [α]32D −52 (c 0.188, MeOH) in the literature38).

Phylogenetic Analysis of Fungal AT-PKSs and TE Domains

Amino acid sequences of the fungal AT-PKSs and their TE domains were aligned with MUSCLE, and phylogenetic analyses were conducted using MEGA version 10.1.839 by means of the bootstrap neighbor joining method. Accession numbers of each sequence used for phylogenetic analysis of fungal TE domains and AT-PKSs are listed in Tables S9 and S10, respectively. The results were visualized and analyzed using MEGA or FigTree v1.4.4.

Molecular Docking

The prediction model of the TE domain (residues 2496–2709) was prepared using AlphaFold 2.229 provided at the NIG supercomputer as a singularity image. The model was manually inspected, and the substrate binding pocket was determined by comparison with the closely related homologues. Two of the catalytic triad30 (D2579, H2690) and cavity-forming residues were defined as flexible side chains, and the other residue in the triad, S2550, was defined to form a covalent bond with the intermediate products. Since covalently bound intermediates have two chiral centers, all four enantiomers were prepared with seven active torsions. Covalent docking was performed using the program adfr40 using 50 GA evolutions with 2,500,000 maxEvals each. The resultant poses were aggregated with the cutoff of 2.0 Å, and the most dominant and lowest energy cluster was picked for each ligand. The results were visualized and analyzed using PyMOL (Schrödinger).

Acknowledgments

We thank Prof. Katsuya Gomi (Tohoku University) and Prof. Katsuhiko Kitamoto (The University of Tokyo) for providing the expression vectors and the fungal strain, Prof. Hideaki Oikawa (Hokkaido University), Prof. Atsushi Minami (Hokkaido University), and Prof. Jun-ichi Maruyama (The University of Tokyo) for the genome editing system of A. oryzae, Prof. Ikuro Abe (The University of Tokyo) and Prof. Takayoshi Awakawa (The University of Tokyo) for providing the protein expression vector, and Prof. Tomoyoshi Akashi (Nihon University) for the CHI expression plasmid. We also thank Yuto Homma (Tohoku University) for his help in bioinformatic analysis of A domains. Molecular docking was performed on the NIG supercomputer at ROIS National Institute of Genetics. This work was supported by JSPS KAKENHI (22H02775 (T.A.), 22K19095 (T.O.), 21H04791 (A.I.), and 21H05113 (A.I.), AMED (20wm0325003 (T.A.) and 22gm1610007 (T.A.)), JST FOREST Program (JPMJFR205W (T.A.) and JPMJFR215T (A.I.)), and Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED (JP22am121038 (T.A.) and JP22ama121040 (T.O.)).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jnatprod.2c01027.

  • Experimental details, figures, tables and additional information, and NMR and HR-MS spectra of the isolated compounds (PDF)

Author Contributions

S.F. and T.O. contributed equally.

The authors declare no competing financial interest.

Supplementary Material

np2c01027_si_001.pdf (3.8MB, pdf)

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