Aromatic Polyketides from the Deep-Sea Cold-Seep Mussel Associated Endozoic Fungus Talaromyces minioluteus CS-138

Five new aromatic polyketides, including a unique benzofuran derivative, talarominine A (1), and four chromone analogs talamins A–D (2–5), along with one known related metabolite, 5-hydroxy-7-methoxy-2,3-dimethylchromone (6), were isolated and identified from the Talaromyces minioluteus CS-138, an endozoic fungus obtained from the deep-sea cold seep mussel Gigantidas platifrons. Their chemical structures were elucidated by detailed analysis of their NMR spectra, HRESIMS and X-ray crystallographic data, and by comparison with literature data as well. The antibacterial and DPPH scavenging activities of compounds 1–6 were evaluated. Compounds 1–3 showed inhibitory activity against some of the tested bacteria whereas compounds 2 and 5 showed potent DPPH radical scavenging activities, which were better than that of the positive control butylated hydroxytoluene (BHT). This work is likely the first report on marine natural products of mussel-derived fungus living in cold seep environments.


Introduction
As most of the marine invertebrates are sessile, soft-bodied, and slow to move, they might need complex secondary metabolites that might be produced by interactions with their symbiotic microorganisms to establish a unique chemical defense system against potential parasite predation and/or harmful microbial colonization. It has been proved that the real producer of a lot of marine natural products isolated from macroorganisms seems to be symbiotic microorganisms, instead of the macrobiota themselves [1]. Gigantidas platifrons (deep-sea mussel) belongs to the subfamily Bathymodiolinae (Bivalvia: Mytilidae). It is the most typical macroinvertebrate in the global deep-sea cold seep environments [2]. Deepsea cold seep is a unique marine environment with a high concentration of methane and low-temperature [3]. In order to survive in this extreme environment, the microorganisms from deep-sea mussel might evolve a more specific metabolic mechanism to produce unique secondary metabolites, which will greatly enrich the research of marine natural products [4].
In our continuing research on secondary metabolites of deep-sea-derived fungi [5][6][7][8], an endozoic fungus Talaromyces minioluteus CS-138 was isolated from the inner fresh tissue of Gigantidas platifrons, a deep-sea mussel collected from the cold seep area in the South China Sea. The fungal species T. minioluteus is widely distributed in various environments and has been reported to produce prolific bioactive metabolites, such as antifungal tetracyclic diterpenes, polyketide-terpenoid hybrids, and hydrazide derivatives [9]. In the present work, the HPLC analysis of the EtOAc extract of T. minioluteus CS-138 showed a series of peaks of typical aromatic polyketides with similar UV absorptions which were not found in our HPLC-UV database. We thus carried out a larger-scale fermentation of T. minioluteus CS-138 for chemical investigation. As a result, six secondary metabolites (Figure 1), including a unique benzofuran derivative talarominine A (1) and four chromone analogs talamins A-D (2)(3)(4)(5), together with a known related compound, 5-hydroxy-7methoxy-2,3-dimethylchromone (6) [10], were isolated and identified. Chemical structures of compounds 1-6 were elucidated by detailed analysis of the spectroscopic data, and the structures of compounds 2, 4, and 5 were further confirmed by single-crystal X-ray diffraction analysis. In this article, the isolation, structure identification, antimicrobial activities, and DPPH scavenging activities of compounds 1-6 were elaborated. The work described in the manuscript appears to be the first report on marine natural products from deep-sea cold seep Gigantidas platifrons-derived fungus.
South China Sea. The fungal species T. minioluteus is widely distributed in various environments and has been reported to produce prolific bioactive metabolites, such as antifungal tetracyclic diterpenes, polyketide-terpenoid hybrids, and hydrazide derivatives [9]. In the present work, the HPLC analysis of the EtOAc extract of T. minioluteus CS-138 showed a series of peaks of typical aromatic polyketides with similar UV absorptions which were not found in our HPLC-UV database. We thus carried out a larger-scale fermentation of T. minioluteus CS-138 for chemical investigation. As a result, six secondary metabolites (Figure 1), including a unique benzofuran derivative talarominine A (1) and four chromone analogs talamins A-D (2)(3)(4)(5), together with a known related compound, 5-hydroxy-7-methoxy-2,3-dimethylchromone (6) [10], were isolated and identified. Chemical structures of compounds 1-6 were elucidated by detailed analysis of the spectroscopic data, and the structures of compounds 2, 4, and 5 were further confirmed by single-crystal X-ray diffraction analysis. In this article, the isolation, structure identification, antimicrobial activities, and DPPH scavenging activities of compounds 1-6 were elaborated. The work described in the manuscript appears to be the first report on marine natural products from deep-sea cold seep Gigantidas platifrons-derived fungus.

Structure Elucidation of the New Compounds
Compound 1 was obtained as a yellow amorphous solid, and its molecular formula was determined as C21H22O6 by analyzing the HRESIMS data at m/z 369.1341 [M − H] − (calculated for C21H21O6, 369.1344, Figure S1 in the Supplementary Materials), indicating 11 degrees of unsaturation. The 1 H NMR data of 1 (DMSO-d6, Table 1 and Figure S2) displayed signals for four methyls (including one methoxyl), three aromatic protons, and three exchangeable protons. The 13 C NMR and DEPT data (DMSO-d6, Table 1 and Figure S3) revealed the presence of 21 carbons, including four methyls (with one oxygenated), two sp 3 -hybridized methylenes, three sp 2 -hybridized methines, and 12 non-protonated carbons (including five O-bearing carbons and one ester carbonyl carbon).
The structure of compound 1 was further identified by detailed analysis of 1 H-1 H COSY and HMBC data ( Figure 2). HMBC correlations from H-8 to C-6 and C-10 and from H-10 to C-6, C-8, and C-9 indicated a 1,2,3,5-tetrasubstituted benzene ring in compound 1. Furthermore, a methyl group at C-7 of the benzene ring was confirmed by the HMBC correlations from CH3-7 (δH 1.86) to C-6, C-7, and C-8, whereas a hydroxy group at C-9 was established by the HMBC correlations from the proton of OH-9 to C-8 ( Figure  2). In addition, a pentasubstituted benzene ring in 1 was confirmed by the HMBC correlations from H-6′ to C-2′, C-4′, and C-5′. Two methyl groups were attached to C-2′ and C-4′ of the pentasubstituted benzene ring as evidenced by the HMBC correlations from CH3-2′ (δH 1.79) to C-1′, C-2′, and C-3′ and from CH3-4′ (δH 2.04) to C-3′, C-4′, and C-5′ ( Figure 2). Supported by the HMBC correlations from OH-5′ to C-4′ and the chemical shift of δC 153.7 (C-3′), the locations of two hydroxy groups were designated at C-5′ and C-3′, respectively. The side chain was determined by the HMBC cross peaks from the    Table 1 and Figure S2) displayed signals for four methyls (including one methoxyl), three aromatic protons, and three exchangeable protons. The 13 C NMR and DEPT data (DMSO-d 6 , Table 1 and Figure S3) revealed the presence of 21 carbons, including four methyls (with one oxygenated), two sp 3hybridized methylenes, three sp 2 -hybridized methines, and 12 non-protonated carbons (including five O-bearing carbons and one ester carbonyl carbon).
The structure of compound 1 was further identified by detailed analysis of 1 H-1 H COSY and HMBC data ( Figure 2). HMBC correlations from H-8 to C-6 and C-10 and from H-10 to C-6, C-8, and C-9 indicated a 1,2,3,5-tetrasubstituted benzene ring in compound 1. Furthermore, a methyl group at C-7 of the benzene ring was confirmed by the HMBC correlations from CH 3 -7 (δ H 1.86) to C-6, C-7, and C-8, whereas a hydroxy group at C-9 was established by the HMBC correlations from the proton of OH-9 to C-8 ( Figure 2). In addition, a pentasubstituted benzene ring in 1 was confirmed by the HMBC correlations from H-6 to C-2 , C-4 , and C-5 . Two methyl groups were attached to C-2 and C-4 of the pentasubstituted benzene ring as evidenced by the HMBC correlations from CH 3 -2 (δ H 1.79) to C-1 , C-2 , and C-3 and from CH 3 -4 (δ H 2.04) to C-3 , C-4 , and C-5 ( Figure 2). Supported by the HMBC correlations from OH-5 to C-4 and the chemical shift of δ C 153.7 (C-3 ), the locations of two hydroxy groups were designated at C-5 and C-3 , respectively. The side chain was determined by the HMBC cross peaks from the proton of a methoxy group at δ H 3.55 (3H, s) to C-1, from H 2 -3 and H 2 -2 to C-1, and by the COSY correlations from H 2 -3 to H 2 -2 ( Figure 2). proton of a methoxy group at δH 3.55 (3H, s) to C-1, from H2-3 and H2-2 to C-1, and by the COSY correlations from H2-3 to H2-2 ( Figure 2). The chemical shifts of δC 154.7 (C-11), 149.8 (C-4), 116.9 (C-5), and 119.5 (C-6) and the HMBC cross peaks from H-2 to C-4 and from H-6′ and H-3 to C-5 determined that the 1,2,3,5-tetrasubstituted benzene ring and the side chain were connected to a furan ring through C-5 and C-4, respectively ( Figure 2). Thus, the structure of compound 1 was determined as a unique benzofuran derivative and was given the trivial name talarominine A. A literature survey revealed that talarominine A is a new naturally occurring benzofuran derivative with unique substitution patterns. Compound 2 was obtained as yellow crystals and its molecular formula was determined to be C12H12O5 on the basis of HRESIMS data at m/z 237.0762 [M + H] + (calculated for C12H13O5, 237.0757, Figure S7), requiring 7 degrees of unsaturation. The 1 H (DMSO-d6, Table 2 and Figure S8) and 13 C (DMSO-d6, Table 3 and Figure S9) NMR data revealed the presence of three methyls (one oxygenated), one methine, and eight non-protonated carbons, which were quite similar to those of previously reported 5,7-dihydroxy-6-methoxy-2-methylchromone [11]. Discreet analysis and comparison of the NMR data disclosed that compound 2 and The chemical shifts of δ C 154.7 (C-11), 149.8 (C-4), 116.9 (C-5), and 119.5 (C-6) and the HMBC cross peaks from H-2 to C-4 and from H-6 and H-3 to C-5 determined that the 1,2,3,5-tetrasubstituted benzene ring and the side chain were connected to a furan ring through C-5 and C-4, respectively ( Figure 2). Thus, the structure of compound 1 was determined as a unique benzofuran derivative and was given the trivial name talarominine A. A literature survey revealed that talarominine A is a new naturally occurring benzofuran derivative with unique substitution patterns.
Compound 2 was obtained as yellow crystals and its molecular formula was determined to be C 12 H 12 O 5 on the basis of HRESIMS data at m/z 237.0762 [M + H] + (calculated for C 12 H 13 O 5 , 237.0757, Figure S7), requiring 7 degrees of unsaturation. The 1 H (DMSO-d 6 , Table 2 and Figure S8) and 13 C (DMSO-d 6 , Table 3 and Figure S9) NMR data revealed the presence of three methyls (one oxygenated), one methine, and eight non-protonated carbons, which were quite similar to those of previously reported 5,7dihydroxy-6-methoxy-2-methylchromone [11]. Discreet analysis and comparison of the NMR data disclosed that compound 2 and 5,7-dihydroxy-6-methoxy-2-methylchromone shared the same benzopyran-4-one ring but with different substituents. HMBC correlations from the proton at δ H 6.49 (1H, s, H-6) to C-5, C-7, C-8, and C-10 led to the designation of this proton as H-6 ( Figure 3), whereas the methyl groups were assigned at C-2 and C-3 by the HMBC cross peaks from CH 3 -2 to C-2 and C-3 and from CH 3 -3 to C-2, C-3, and C-4. Moreover, HMBC correlations from OCH 3 -7 to C-7, from OH-8 to C-7, C-8, and C-9, and from OH-5 to C-5, C-6, and C-10 designated the locations of the methoxy and two hydroxy groups ( Figure 3). Furthermore, the structure of compound 2 was confirmed by single-crystal X-ray diffraction experiment ( Figure 4). Thus, compound 2 was determined as 5,8-dihydroxy-7-methoxy-2,3-dimethylchromone and was named as talamin A.  3 3.79, s 7-OCH 3 3.87, s 3.88, s 3.92, s 8-OCH 3 3.74, s 3.74, s fraction experiment ( Figure 4). Thus, compound 2 was determined as 5,8-dihydroxy-7-methoxy-2,3-dimethylchromone and was named as talamin A.   Figure S13), with 14 units more than that of 2 but having same degrees of unsaturation with 2. The 1 H NMR data (DMSO-d6, Table 2 and Figure S14) was similar to that of compound 2, except that the hydroxy group at C-8 of 2 was replaced by a methoxy group in 3, and the 13 C NMR data (DMSO-d6, Table 3 and Figure S15) and and C-10 designated this proton as H-3. In addition, chemical shifts of the carbonyl and several aromatic carbon atoms changed significantly, which indicated that the substituent on benzene ring has changed. HMBC correlations from OCH3-7 (δH 3.92) to C-7 and from OCH3-5 (δH 3.79) to C-5 verified the locations of the methoxy groups. The single-crystal X-ray diffraction experiment ( Figure 4) further verified the locations of the hydroxy and methoxy groups. Thus, compound 5 was identified as 8-hydroxy-5,7-dimethoxy-2-methylchromone and was named as talamin D.

Antibacterial Assays
All of the isolated compounds were tested for antibacterial activities against three humanic and 9 aquatic pathogenic bacteria. As shown in Table 4  Compound 3 was also obtained as yellow powder, and its molecular formula was determined to be C 13 Figure S13), with 14 units more than that of 2 but having same degrees of unsaturation with 2. The 1 H NMR data (DMSO-d 6 , Table 2 and Figure S14) was similar to that of compound 2, except that the hydroxy group at C-8 of 2 was replaced by a methoxy group in 3, and the 13 C NMR data (DMSO-d 6 , Table 3 and Figure S15) and HMBC correlations ( Figure 3) confirmed this designation. Thus, compound 3 was determined as 5-hydroxy-7,8-dimethoxy-2,3-dimethylchromone and gave the trivial name talamin B.
Compound 4 was also isolated as yellow crystals. Its molecular formula, C 12 H 12 O 6 , was determined by HRESIMS at m/z 275.0520 [M + Na] + (calculated for C 12 H 12 O 6 Na, 275.0526, Figure S18). Compared to 2, compound 4 has one less methyl signal and one more oxygenated methylene signal from the 1 H and 13 C NMR data (DMSO-d 6 , Tables 2 and 3 and Figures S19 and S20). HMBC correlations (Figure 3) from the methylene proton of 3-CH 2 OH (δ H 4.35) to C-2, C-3, and C-4 designated the location of the oxygenated methylene group at C-3. In addition, the chemical shifts of the carbon atoms of 4 at benzene ring have changed greatly when compared with that of compound 2, which was speculated to be caused by the different substitution patterns of the methoxy and hydroxy groups on the benzene ring. HMBC correlations from the proton of OCH 3 -8 (δ H 3.74) to C-8 and the chemical shifts of δ C 159.7 (C-7) verified the locations of the hydroxy and methoxyl groups. After the NMR data collection, we set 4 to single-crystal X-ray diffraction experiment and confirmed our deduction ( Figure 4). Thus, compound 4 was assigned as a new chromone derivative and was named as talamin C.
Compound 5 was obtained as light yellow crystals and its molecular formula was assigned as C 12 Figure S24), the same as that of compound 2. The 1 H NMR data (DMSO-d 6 , Table 2 and Figure S25) of 5 was similar to that of 2. However, the olefinic-methyl group at C-3 and the OH group at C-5 in 2 were replaced by an olefinic proton and a methoxy group in 5, respectively. HMBC correlations (Figure 3) from δ H 5.90 (1H, s, H-3) to C-2 and C-10 designated this proton as H-3. In addition, chemical shifts of the carbonyl and several aromatic carbon atoms changed significantly, which indicated that the substituent on benzene ring has changed. HMBC correlations from OCH 3 -7 (δ H 3.92) to C-7 and from OCH 3 -5 (δ H 3.79) to C-5 verified the locations of the methoxy groups. The singlecrystal X-ray diffraction experiment (Figure 4) further verified the locations of the hydroxy and methoxy groups. Thus, compound 5 was identified as 8-hydroxy-5,7-dimethoxy-2methylchromone and was named as talamin D.

Antibacterial Assays
All of the isolated compounds were tested for antibacterial activities against three humanic and 9 aquatic pathogenic bacteria. As shown in Table 4, compound 1 exhibited inhibitory activities against methicillin-resistant Staphylococcus aureus (MRSA), Micrococcus luteus, Pseudomonas aeruginosa, Vibrio harveyi, and Vibrio vulnificus, with MIC values ranging from 32 to 64 µg/mL. Compound 2 showed certain activity against V. vulnificus with an MIC value of 32 µg/mL, whereas compound 3 exhibited inhibitory activities against MRSA and V. vulnificus. For both, the MIC values were 64 µg/mL. Compounds 4-6 showed no or weak activity to the tested strains (MIC > 64 µg/mL).

General Experimental Procedures
One-dimensional and 2D NMR date was acquired on a Bruker Avance 500 spectrometer (Bruker Biospin Group, Karlsruhe, Germany). UV spectra were read from a PuXi TU-1810 UV-visible spectrophotometer (Shanghai Lengguang Technology Co., Ltd., Shanghai, China). Mass spectra were recorded on an API QSTAR Pulsar 1 mass spectrometer (Applied Biosystems, Foster City, CA, USA). Analytical HPLC was performed using a SHI-MADZU prominence HPLC system equipped with LC-20AT pump, SIL-20A automated sample injector, CTO-20AC colomn oven, and SPD-M20A diode array detector controlled by LCSolution software.

Fungal Material
The fungal strain Talaromyces minioluteus CS-138 was isolated from the inner fresh tissue of the Gigantidas platifrons, which is a deep-sea mussel collected from the cold seep area of the south Sea of China in July 2018. After strain identification with the morphological character and ITS region sequence [12], it was found that the fungal strain was the same (100%) as that of Talaromyces minioluteus (JX091487). The strain (sequence in GenBank with acccession No. OM670209) is preserved at the Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences (IOCAS).

Fermentation, Extraction, and Isolation
For chemical investigations, fresh mycelia of T. minioluteus CS-138 were grown on PDA medium with seawater at 28 • C for five days. Then, the strain was inoculated on rice medium for large-scale fermentation in 130 × 1 L Erlenmeyer flasks (70 g rice, 0.1 g corn syrup, 0.3 g peptone, 0.1 g methionine, and 100 mL naturally sourced and filtered seawater) and statically cultured for 30 days at room temperature.
Crystal data for compound 2:

DPPH Radical Scavenging Assay
The scavenging activity against DPPH radicals was carried out according to the method of Sharma with some modifications [18,19]. Certain amounts of compounds 1-6 were individually dissolved in methanol and diluted into six gradients. Then, 100 mL aliquot of samples was added to 100 mL of 0.16 mM DPPH methanolic solution in 96-well plates. After mixing evenly, the mixtures were left to stand at room temperature for 30 min in the dark, and the absorbance was read at 517 nm (A sample ). Butylated hydroxytoluene (BHT) was used as positive control. All the measurements were performed in triplicate and each value was presented as the mean ± standard deviation. The ability to scavenge the DPPH was calculated according to the equation: Scavenging effect (%) = 100 − (A sample − A sample blank ) × 100/(A control − A blank ) A sample blank: : the absorbance of the test sample without DPPH solution; A control : the absorbance of the DPPH solution; A blank : the absorbance of methanol.

Conclusions
In summary, the secondary metabolites of T. minioluteus CS-138, which was isolated from the fresh tissues of Gigantidas platifrons in the cold seep area of South China Sea, were chemically studied. Six aromatic polyketides, including five new compounds (1-5), were isolated and identified. Among them, compounds 2, 4, and 5 were further confirmed by single-crystal X-ray diffraction analysis. Compounds 1, 2, and 3 exhibited inhibitory activities against some human pathogenic and aquatic bacteria, with MIC values ranging from 32 to 64 µg/mL. Moreover, compounds 1, 2, and 5 exhibited potent DPPH radical scavenging activities, significantly better than that of the positive control BHT, possessing the potential to be developed as antioxidants.