Genome Mining of α-Pyrone Natural Products from Ascidian-Derived Fungus Amphichorda felina SYSU-MS7908

Culturing ascidian-derived fungus Amphichorda felina SYSU-MS7908 under standard laboratory conditions mainly yielded meroterpenoid, and nonribosomal peptide-type natural products. We sequenced the genome of Amphichorda felina SYSU-MS7908 and found 56 biosynthetic gene clusters (BGCs) after bioinformatics analysis, suggesting that the majority of those BGCSs are silent. Here we report our genome mining effort on one cryptic BGC by heterologous expression in Aspergillus oryzae NSAR1, and the identification of two new α-pyrone derivatives, amphichopyrone A (1) and B (2), along with a known compound, udagawanone A (3). Anti-inflammatory activities were performed, and amphichopyrone A (1) and B (2) displayed potent anti-inflammatory activity by inhibiting nitric oxide (NO) production in RAW264.7 cells with IC50 values 18.09 ± 4.83 and 7.18 ± 0.93 μM, respectively.


Introduction
Fungal natural products are an indispensable source for drug development [1,2]. However, under typical laboratory culture conditions, most of the biosynthetic gene clusters (BGCs) from fungi are "silent"; thus, it becomes increasingly difficult to discover novel natural products. Currently, several genome mining strategies have been reported for activating silent BGCs [3], and heterologous expression of target BGCs in a suitable host is one effective approach. Zhu et al. [4] expressed a cryptic BGC from Trichoderma harzianum t-22 in Aspergillus nidulans A1145 and successfully isolated several tetronate natural products, such as trihazone A-F. Similarly, Li et al. [5] characterized a cryptic BGC from A. hancockii in A. nidulans LO8030 and discovered a metabolite with a unique prenylated 6/6/6/5 tetracarbocyclic skeleton. The quadruple auxotroph A. oryzae NSAR1 is also a frequently-used heterologous host for genome mining of fungal natural products. Recently, Jiang et al. [6] used A. oryzae as a heterologous host to express two fungal bifunctional terpene synthases and obtained four terpenes featuring 5-6-7-3-5 ring systems; Yan et al. [7] genome mined four new meroterpenoids, funiculolides A−D, by heterologous expression of a cryptic BGC from A. funiculosus CBS 116.56.
Discovering novel secondary metabolites from marine-derived fungi has been our group's long-term research interest [8][9][10][11]. The fungus Amphichorda felina SYSU-MS7908 was isolated from a marine ascidian Styelaplicata and fermentation under standard laboratory conditions mainly gave meroterpenoids and nonribosomal peptides [12,13]. To probe the biosynthetic potential of A. felina SYSU-MS7908, the genome was sequenced by Illumina second-generation sequencing, and the assembled genome was analyzed by the antibiotics and secondary metabolite analysis shell (antiSMASH) [14]. Fifty-six secondary metabolites BGCs were found ( Figure S1), suggesting that the fungus has great potential to produce structurally diverse natural products.
Pyrones are six-membered cyclic unsaturated esters exhibiting a broad range of activities, such as antifungal, antibiotic, cytotoxic, immunosuppressive, and phytotoxic ( Figure 1) [15][16][17]. One gene cluster, the amp cluster discovered from A. felina SYSU-MS7908, shares similarities with the sol cluster that is responsible for the biosynthesis of solanapyrone D (Figure 2), an α-pyrone containing natural product [18]. However, we had not observed the production of such metabolites from the wild type of A. felina SYSU-MS7908, suggesting that this BGC might be silent. We decided to activate this BGC by heterologous expression in A. oryzae NSAR1 as it is a robust host to mine fungal natural products [19][20][21].
Mar. Drugs 2022, 20, x FOR PEER REVIEW 2 of 1 was isolated from a marine ascidian Styelaplicata and fermentation under standard labor atory conditions mainly gave meroterpenoids and nonribosomal peptides [12,13]. To probe the biosynthetic potential of A. felina SYSU-MS7908, the genome was sequenced by Illumina second-generation sequencing, and the assembled genome was analyzed by the antibiotics and secondary metabolite analysis shell (antiSMASH) [14]. Fifty-six secondary metabolites BGCs were found ( Figure S1), suggesting that the fungus has great potentia to produce structurally diverse natural products. Pyrones are six-membered cyclic unsaturated esters exhibiting a broad range of ac tivities, such as antifungal, antibiotic, cytotoxic, immunosuppressive, and phytotoxic (Fig  ure 1) [15][16][17]. One gene cluster, the amp cluster discovered from A. felina SYSU-MS7908 shares similarities with the sol cluster that is responsible for the biosynthesis of solanapy rone D (Figure 2), an α-pyrone containing natural product [18]. However, we had no observed the production of such metabolites from the wild type of A. felina SYSU-MS7908 suggesting that this BGC might be silent. We decided to activate this BGC by heterologou expression in A. oryzae NSAR1 as it is a robust host to mine fungal natural products [19 21].  Comparison of the amp cluster with the sol cluster was performed using the Clinker too [22].  Figure 1. Representative natural products containing α-pyrone motif. atory conditions mainly gave meroterpenoids and nonribosomal peptides [12,13]. probe the biosynthetic potential of A. felina SYSU-MS7908, the genome was sequenced Illumina second-generation sequencing, and the assembled genome was analyzed by t antibiotics and secondary metabolite analysis shell (antiSMASH) [14]. Fifty-six seconda metabolites BGCs were found ( Figure S1), suggesting that the fungus has great potent to produce structurally diverse natural products.
Pyrones are six-membered cyclic unsaturated esters exhibiting a broad range of tivities, such as antifungal, antibiotic, cytotoxic, immunosuppressive, and phytotoxic (F ure 1) [15][16][17]. One gene cluster, the amp cluster discovered from A. felina SYSU-MS79 shares similarities with the sol cluster that is responsible for the biosynthesis of solanap rone D (Figure 2), an α-pyrone containing natural product [18]. However, we had n observed the production of such metabolites from the wild type of A. felina SYSU-MS79 suggesting that this BGC might be silent. We decided to activate this BGC by heterologo expression in A. oryzae NSAR1 as it is a robust host to mine fungal natural products [1 21].

Bioinformatic Analysis of the Amp Cluster
The amp cluster contains 10 potential biosynthetic genes (transcription factor and transport excluded, Table 1). AmpB is a polyketide synthase (PKS) that shares 52% protein identity with Sol1 ( Figure S2), the PKS involved in solanapyrone D biosynthesis [18,23,24]. AmpC, a putative methyltransferase, shares 49.5% protein identity with Sol2 ( Figure S3). Interestingly, no other genes share significant sequence homology between those two clusters ( Figure 2), suggesting that the amp cluster might produce structurally divergent natural products from solanapyrone D.

Heterologous Expression of the Amp Cluster in A. oryzae
Previous studies suggested that the PKS, Sol1, could produce an advanced biosynthetic intermediate; thus, only the ampB gene was introduced into the A. oryzae NSAR1 host strain. As expected, the production of amphichopyrone A (1) was detected from the expression of the AO-ampB construct ( Figure 3). With amphichopyrone A (1) determined, we next focused on the tailoring genes of the amp cluster. AmpC, a putative O-methyltransferase, was then introduced into AO-ampB to give the construct AO-ampBC. We found that two additional metabolites were produced by AO-ampBC. Large-scale fermentation and spectroscopic analyses determined the structures as amphichopyrone B (2) and udagawanone A (3) [15][16][17]. To investigate the final metabolites produced by the amp cluster, the remaining eight genes, ampADEFGHIJ, were included in the construct; unfortunately, the AO-ampA-BCDEFGHIJ construct generated the same product profile as AO-ampBC (Figure 3), suggesting amphichopyrone (2) and udagawanone A (3) might be the final products catalyzed by the amp cluster ( Figure 4). These results suggest that AmpC catalyzed the meth- To investigate the final metabolites produced by the amp cluster, the remaining eight genes, ampADEFGHIJ, were included in the construct; unfortunately, the AO-ampABCDEFGHIJ construct generated the same product profile as AO-ampBC ( Figure 3), suggesting amphichopyrone (2) and udagawanone A (3) might be the final products catalyzed by the amp cluster ( Figure 4). These results suggest that AmpC catalyzed the methylation reaction at C-4 hydroxyl of amphichopyrone A (1) to give amphichopyrone B (2). Hydroxylation of amphichopyrone B (2) to udagawanone A (3) might be catalyzed by endogenous enzymes from A. oryzae NSAR1 host ( Figure 5). To investigate the final metabolites produced by the amp cluster, the re genes, ampADEFGHIJ, were included in the construct; unfortunately, t BCDEFGHIJ construct generated the same product profile as AO-ampBC ( gesting amphichopyrone (2) and udagawanone A (3) might be the final lyzed by the amp cluster ( Figure 4). These results suggest that AmpC cataly ylation reaction at C-4 hydroxyl of amphichopyrone A (1) to give amphich Hydroxylation of amphichopyrone B (2) to udagawanone A (3) might be endogenous enzymes from A. oryzae NSAR1 host ( Figure 5).    To investigate the final metabolites produced by the amp cluster, the remaining eight genes, ampADEFGHIJ, were included in the construct; unfortunately, the AO-ampA-BCDEFGHIJ construct generated the same product profile as AO-ampBC (Figure 3), suggesting amphichopyrone (2) and udagawanone A (3) might be the final products catalyzed by the amp cluster ( Figure 4). These results suggest that AmpC catalyzed the methylation reaction at C-4 hydroxyl of amphichopyrone A (1) to give amphichopyrone B (2). Hydroxylation of amphichopyrone B (2) to udagawanone A (3) might be catalyzed by endogenous enzymes from A. oryzae NSAR1 host ( Figure 5).

Strains and Media
The strain, Amphichorda felina SYSU-MS7908, was isolated from a marine ascidian Styelaplicata collected from the north atoll of the Xisha Islands, South China Sea, China, in 2018, isolated using the standard protocol [29] and identified by the morphological and the internal transcribed spacer (ITS) of the nuclear ribosomal DNA data analysis (Accession number MT786206). The whole genome sequences of A. felina SYSU-MS7908 have been deposited in the GenBank database with an accession number JAEMHR000000000. The sequences of amp A-J have been submitted to GenBank (OL906410-OL906419). The proposed functions of ampA-J in Table 1 are based on the protein BLAST results. The gene cluster comparison figure between the amp and sol clusters was generated with the Clinker tools with the corresponding annotated sequence files using default parameters [22]. Primer sequences are listed in Table S1.

Construction of Recombinant Plasmids
To construct the expression plasmids for A. oryzae, the genes (ampA-ampJ) of the amp cluster were first amplified using the genomic DNA of Amphichorda felina SYSU-MS7908 as a template. Each amplified DNA fragment was then introduced into the pTAex3 vector [31], and the gene expression cassette, amyB promoter, the target gene, and the amyB terminator were amplified from the pTAex3-based plasmid. These gene expression cassettes were then inserted into the HindIII-linearlized pPTRI [32], pBARI [33], SpeI-linearized pAdeA [34], and XbaI-linearized pUNA [35]. Plasmids constructed in this study are listed in Table S2.

Transformation of A. oryzae NSAR1
The transformation of A. oryzae NSAR1 was conducted via the protoplast−polyethylene glycol method. Mycelia of the parent strain from the solid culture in potato dextrose agar were inoculated in 10 mL DPY medium and cultured at 28 • C and 220 rpm for 2 days, which were then transferred to 100 mL DPY medium and cultured for 24 h. The mycelia were collected by filtration and digested by 1% Yatalase in 0.6 M (NH 4 ) 2 SO 4 , 50mM maleic acid, pH 5.5 at 30 • C for 4 h to remove cell walls. The resulting protoplasts were collected by centrifugation at 1500 rpm for 10 min and washed once with Solution 2 (1.2 M sorbitol, 50 mM CaCl 2 .2H 2 O, 35 mM NaCl, 10M Tris-HCl, pH 7.5). Then, 200 µL protoplast suspension (1 × 10 7 cells/mL) and about 10 µg plasmids were gently mixed and incubated on ice for 30 min, followed by the addition of 1.3 mL Solution 3 (60% PEG4000, 50 mM CaCl 2 .2H 2 O, 10 M Tris-HCl, pH 7.5) at three times. After the mixture was placed at room temperature for 20 min, 5 mL Solution 2 was added. Following centrifugation at 1500 rpm for 10 min, the precipitates were suspended in 200 µL of Solution 2 and spread on the bottom selective medium with 0.8% agar, which was then covered with the selective overlay medium containing 1.5% agar. The selective medium was composed of 0.2% NH 4 Cl, 0.1% (NH 4 ) 2 SO 4 , 0.05% KCl, 0.05% NaCl, 0.1% KH 2 PO 4 , 0.05% MgSO 4 .7H 2 O, 0.002% FeSO 4 .7H 2 O, 2% glucose, 1.2 M sorbitol supplemented with 0.15% methionine, 0.1% arginine, 0.01% adenine, 0.1 µg/mL pyrithiamine hydrobromide and 35 µL/mL glufosinate-ammonium based on the plasmids used. The A. oryzae strain containing the amp cluster was verified by checking the relevant exogenous target genes using PCR analysis ( Figure S10). A. oryzae transformants constructed in this study are listed in Table S3.

Anti-Inflammatory Activity
The RAW264.7 cells were used to evaluate the anti-inflammatory activity of compounds 1-3 following a literature procedure [36]. The cells were seeded in 96-well plates at a density of 5 × 10 5 cells/mL. After 12 h, LPS (1 µg/mL) and samples were added to the cells and then incubated for 24 h at 37 • C. The quantity of nitrite accumulated in the culture medium was measured as an indicator of NO production. Then, 50 µL of cell culture medium with 100 µL Griess reagent were mixed and incubated for 10 min at room temperature. The absorbance was determined at 540 nm wavelength with a microplate reader.

Conclusions
Many genome mining strategies have been reported for activating silent BGCs to produce novel natural products. In this study, we heterologously expressed a cryptic gene cluster from Amphichorda felina SYSU-MS7908 in A. oryzae NSAR1 and two new metabolites, amphichopyrone A (1) and amphichopyrone B (2), along with a known compound, udagawanone A (3), were isolated. Amphichopyrone A (1) and amphichopyrone B (2) show potent anti-inflammatory activity by inhibiting LPS-induced NO production with IC 50 values 18.09 ± 4.83 and 7.18 ± 0.93 µM, respectively.