Sorbicillinoid Derivatives with the Radical Scavenging Activities from the Marine-Derived Fungus Acremonium chrysogenum C10

Sorbicillinoids are a class of structurally diverse hexaketide metabolites with good biological activities. To explore new structural sorbicillinoids and their bioactivities, the marine-derived fungus Acremonium chrysogenum C10 was studied. Three new sorbicillinoid derivatives, acresorbicillinols A–C (1–3), along with five known ones, trichotetronine (4), trichodimerol (5), demethyltrichodimerol (6), trichopyrone (7) and oxosorbicillinol (8), were isolated. The structures of new sorbicillinoids were elucidated by analysis of nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy (HRESIMS). The absolute configurations of compounds 1–3 were determined by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. Compound 3 exhibited a strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, with the IC50 value ranging from 11.53 ± 1.53 to 60.29 ± 6.28 μM in 24 h. Additionally, compounds 2 and 3 showed moderate activities against Staphylococcus aureus and Cryptococcus neoformans, with IC50 values of 86.93 ± 1.72 and 69.06 ± 10.50 μM, respectively. The boundary of sorbicillinoid biosynthetic gene cluster in A. chrysogenum was confirmed by transcriptional analysis, and the biosynthetic pathway of compounds 1–8 was also proposed. In summary, our results indicated that A. chrysogenum is an important reservoir of sorbicillinoid derivatives, and compound 3 has the potential for new natural agents in DPPH radical scavenging.


Introduction
Marine-derived fungi can thrive in the extreme environments including salinity, high pressure, low temperature and oligotrophic conditions compared to their terrestrial counterparts, which makes them able to produce structurally diverse bioactive compounds more easily [1][2][3]. Meanwhile, these compounds usually have unique structures that also provide the possibility for structural design and modification of the leading compounds [4]. As one special marine-derived fungus, Acremonium chrysogenum has made irreplaceable contributions to controlling the bacterial infections and saving countless patients for production of the β-lactam antibiotic cephalosporin C (CPC) and its derivatives [5]. The genomic sequences and annotation of A. chrysogenum was first completed in 2014, and a total of 42 secondary metabolite biosynthetic gene clusters, including 14 polyketide synthetase (PKS) clusters, 10 terpene synthase clusters, 8 hybrid clusters, 7 nonribosomal peptide synthetase clusters and 3 non-identified secondary metabolite clusters, were predicted [6].
Sorbicillinoids are a class of structurally diverse hexaketide metabolites with a characteristic sorbyl side chain residue [7][8][9]. They were first isolated from Penicillium notatum in 1948 and structurally elucidated by Cram and Tishler [10,11]. Up until now, more than 159 naturally occurring sorbicillinoids have been isolated and have displayed good biological activities in cytotoxic, antimicrobial and phytotoxic activities [7][8][9]. Because free radicals play an important role in the development of aging and many diseases, including cancer, arthritis and atherosclerosis, exploring the novel radical scavengers is crucial for developing new drugs to slow down the aging process and treat these diseases. Some sorbicillinoid derivatives have shown great antioxidative application prospects, such as bisorbicillinol (ED 50 = 31.4 µM) and bisorbibetanone (ED 50 = 62.5 µM), etc. [8]. Additionally, there is an urgent need to find more novel compounds for the emergence of microbial resistance. Some sorbicillinoids showed significant antimicrobial activity, indicating their potential as candidates [7]. Meanwhile, the sorbicillinoid biosynthetic gene clusters from Penicillium chrysogenum and Trichoderma reesei have been identified and their biosynthetic pathway has been partially revealed [12][13][14]. Generally, two PKSs SorA and SorB are responsible for the formation of sorbicillin and dihydrosorbicillin, which are then oxidative dearomatized to give sorbicillinol and dihydrosorbicillinol by the FAD-dependent monooxygenase SorC, respectively [15]. Sorbicillinol is regarded as the precursor of most sorbicillinoids since it is condensed with its derivatives or other compounds to form the dimeric and hybrid sorbicillinoids by Diels-Alder or Michael-addition-like reactions [16,17]. The sorbicillinoid biosynthetic gene cluster in A. chrysogenum has been regarded as the most ancient, based on evolutionary origin, and carries more modifier than other species [13], and disruption of these two PKS encoding genes results in the abolishment of sorbicillinoids [18]. However, there is lack of a systematic investigation about sorbicillinoids produced by A. chrysogenum.

General Experimental Procedure
Optical rotations, ECD spectra, UV and IR data were measured on the Austria Anton Paar MCP 200 Automatic Polarimeter, the Applied Photophysics Chirascan circular di-

General Experimental Procedure
Optical rotations, ECD spectra, UV and IR data were measured on the Austria Anton Paar MCP 200 Automatic Polarimeter, the Applied Photophysics Chirascan circular dichroism spectrometer, the Thermo Scientific GENESYS 10S UV-Vis and the Thermo Scientific Nicolet IS5 spectrophotometers, respectively. HRESIMS data and MS were obtained using an Agilent 6520B Q-TOF Mass instrument equipped with an ESI source. All MS experiments were performed in positive ion mode. NMR data were acquired with the AVANCE-500 spectrometer (Bruker, Bremen, Germany) using solvent signals (CD 3 OD, δ H 3.30/δ C 49.9, DMSO, δ H 2.50, 3.30/δ C 39.5, and CDCl 3 , δ H 7.26/δ C 77.16) as references. Octadecylsilyl (ODS, 50 µm, YMC Co., Ltd. Japan) and Sephadex TM LH-20 (Cytiva, Uppsala, Sweden) were used for column chromatography. High performance liquid chromatography (HPLC) was performed on the SHIMADZU LC20AT system equipped with UV diode array detector using the Thermo Hypersil Gold-C18 columns (5 µm, 250 mm × 4.6 mm) at a flow rate of 1 mL/min. For semi-preparative HPLC, Waters 1525 system equipped with the UV/Visible detector and the Thermo Hypersil Gold-C18 columns (5 µm, 250 mm × 10 mm) was used and performed at a flow rate of 2 mL/min. Solvents including methanol and ethyl acetate (EtOAc) for extraction and chromatographic separation were analytical grade. HPLCgrade solvents (acetonitrile and formic acid) were used for the HPLC and semi-preparative HPLC analysis.

Fungal Materials and Fermentation
One high CPC-producing strain of A. chrysogenum C10 (ATCC 48272) was released by PanLab. This fungus was inoculated on the rice solid medium in 500 mL Erlenmeyer flasks containing 80 g of rice and 120 mL of H 2 O, and cultivated at 28 • C for 7 days for the production of sorbicillinoids. A total of 10 kg fermentation sample was harvested.

ECD Calculations
Conformational analyses were performed using Maestro 10.2 in the OPLS3 molecular mechanics force-field within an energy window of 5.0 or 3.0 kcal/mol. The conformers were then further optimized with the software package Gaussian 09 at the B3LYP/6-31G(d) level for compounds 1-3, respectively, and the harmonic vibrational frequencies were also calculated to confirm their stability. The TDDFT methods at the CAM-B3LYP/6-31G(d) and B3LYP/6-31G(d) level were applied to calculate the 60 lowest electronic transitions to obtain conformers in a vacuum, respectively. The Gaussian function was applied to simulate the ECD spectrum of the conformers. The calculated ECD spectra were obtained according to the Boltzmann weighting of each conformer's ECD spectrum [23].

Antimicrobial Activity Assay
The bacterial strains (Staphylococcus aureus CGMCC 1.89, Pseudomonas aeruginosa ATCC 15692) and the fungal strains (Cryptococcus neoformans W1585, Candida albicans SC5314) were used in this study. The concentration of 50 mM compounds was prepared using dimethyl sulfoxide (DMSO). The bacterial and fungal strains were streaked onto Mueller-Hinton Agar (MHA) and Potato Dextrose Agar (PDA) for growth at 37 • C and 28 • C, respectively. Single colony was picked and adjusted to 2 × 10 5 CFU/mL by Mueller-Hinton Broth (MHB) or Potato Dextrose Broth (PDB). The stock solutions of compounds were diluted into 500, 250, 125, 62.5 and 31.25 µM by MHB or PDB, successively. Fifty microliters of serial dilutions of each compound and 50 µL of microbial suspension were added to the 96-well plates and incubated at 37 • C or 28 • C for 24 h until the results were recorded. IC 50 was defined as the half maximal inhibitory concentrations of the compounds that inhibited the visible microbial growth after 24 h of incubation. Ampicillin and amphotericin B were used as the positive control for detecting the activities of these compounds against bacteria and fungi, respectively.

DPPH Radical Scavenging Assay
The DPPH radical scavenging activity of the compounds was carried out as previously described [24,25]. The modified parameter was the reaction time from 0.5 h to multiple time-points including 0.5, 1, 4, 6, 8 and 24 h. Ascorbic acid and ethanol were used as the positive and negative control, respectively. All experiments were replicated at least three times.

RNA Isolation and Real-Time RT-PCR Analysis
The mycelia of A. chrysogenum C10 grown on the modified MDFA medium were collected at different time-points [26]. RNA isolation and real-time RT-PCR were performed as described previously [27,28]. All primers used in this study were listed in Table S1.

Analysis of the 1 H-1 H COSY and HMBC data
The relative stereochemistry of 2 was determined by NOESY correlations and coupling constants as well as by comparison with those of 1 and the known compound sorbicatechol C [30]. The large coupling constants (J H-10/H-11 = 14.6 Hz and J H-12/H-13 = 14.6 Hz), along with NOESY correlations (Figures 3 and S12)  , and of H-7 with H 3 -21, combined with the strong HMBC correlations from H-8b to C-5 and C-15, the weak correlation from H-8b to C-3 and lack of HMBC correlation from H-8a to C-5 and C-15 determined the relative stereochemistry of C-7 and C-1 as shown. The absolute configuration of 2 was also determined by a comparison of the experimental and calculated ECD spectra for enantiomers (1R,4S,5S,7R)-2 (2a) and (1S,4R,5R,7S)-2 (2b). As shown in Figure 4, the experimental ECD spectrum of 2 showed good agreement with the calculated ECD spectrum of (1R,4S,5S,7R)-2 (2a), suggesting the absolute configuration of 1R,4S,5S,7R for 2. Thus, the structure of 2 was defined as shown.
The relative configuration of 3 was confirmed by NOESY correlations and coupling constants. The NOESY correlations (Figures 3 and S18)  /H-1 inferred that these protons were in close proximity to their related functional groups, respectively. The similar Cotton effects in the ECD spectra of 3 and 5 deduced the absolute configuration of 3 to be the same as that of 5, which was further verified by ECD calculations (Figure 4). The calculated ECD curve of (1S,2S,3R,4R,1'R,2'S,3'R,4'R)-3 (3a) matched well with the experimental data, suggesting the absolute configuration to be 1S,2S,3R,4R,1'R,2'S,3'R,4'R. Thus, the structure of 3 was defined as depicted.
Except for the new compounds 1-3, the structure of five known sorbicillinoids isolated in this study were confirmed by comparison of the spectroscopic data with those in the literature [20][21][22]. The resulting EtOAc extracts of A. chrysogenum cultivated on the rice were screened by HPLC analysis (Figure S20).

Biological Activities Evaluation
To explore the bioactivities of compounds 1-8, their abilities of anti-microorganisms and DPPH radical scavenging were evaluated. The results showed that compounds 2 and 3 exhibited the moderate activities against S. aureus and C. neoformans with the IC 50 values of 86.93 ± 1.72 and 69.06 ± 10.50 µM, respectively. However, other compounds did not give IC 50 value at a concentration below 100 µM (Table 3). No candidate compounds could significantly inhibit the growth of C. albicans and P. aeruginosa. Compound 3 might function as the β-1,6-glucan inhibitor to inhibit the fungal growth as its structural analogue bisvertinolone [33]. Bisvertinolone also exhibited significant inhibitory activity against S. aureus with the minimal inhibitory concentration (MIC) value of 30 µg/mL [34]. However, only several monomeric sorbicillinoids from Scytalidium album exhibited the weak activity against C. neoformans with the MIC value of over 38 µg/mL [35]. Table 3. Anti-microbial inhibitory activities of compounds 1-8.

Compounds
S Through the DPPH radical scavenging assay, compound 3 exhibited strong activity with the IC 50 value of 60.29 ± 6.28 µM after standing for 0.5 h, and then we continued to record its radical scavenging activity for 24 h (at 1, 4, 6, 8 and 24 h). Compound 3 gave the significant activity with the IC 50 values of 43.52 ± 5.93, 22.57 ± 7.34, 15.85 ± 5.94, 12.30 ± 5.74 and 11.53 ± 1.53 µM, respectively, indicating that 3 displays the time-dependent manner for DPPH radical scavenging. Compared with the IC 50 value of ascorbic acid as the positive control, which was 25.36 ± 3.82 to 28.45 ± 3.04 µM, compound 3 represents one novel DPPH radical scavenging agent ( Figure 5 and Table 4).  (Table 4). Compounds 4, 5, 6 and 8 also displayed the time-dependent manner as compound 3. The time-dependent manner of sorbicillinoids for radical scavenging was previously reported, including for oxosorbicillinol, trichotetronine, bisorbicillinolide and methylbisorbibutenolide [22,36,37]. There was a different scavenging values of 4 and 8 between this study and the reports in Hirota's Lab, and the reaction buffer might be the key determination factor. Additionally, the IC 50 values of compounds 1, 2 and 7 exceeded 200 µM, even standing for 24 h, indicating that they did not have DPPH radical scavenging ability (Table 4). DPPH radical scavenging activity of other representative sorbicillinoids has been reported, including for bisorbicillinol, bisvertinolone and bisorbibetanone, which showed ED 50 values of 31.4, 44.3 and 62.5 µM, respectively [21,37]. To date, compound 3 displayed the best DPPH radical scavenging activity for 24 h among all reported sorbicillinoids.
icillinol, trichotetronine, bisorbicillinolide and methylbisorbibutenolide [22,36,37]. There was a different scavenging values of 4 and 8 between this study and the reports in Hirota's Lab, and the reaction buffer might be the key determination factor. Additionally, the IC50 values of compounds 1, 2 and 7 exceeded 200 μM, even standing for 24 h, indicating that they did not have DPPH radical scavenging ability (Table 4). DPPH radical scavenging activity of other representative sorbicillinoids has been reported, including for bisorbicillinol, bisvertinolone and bisorbibetanone, which showed ED50 values of 31.4, 44.3 and 62.5 μM, respectively [21,37]. To date, compound 3 displayed the best DPPH radical scavenging activity for 24 h among all reported sorbicillinoids.

Determination of Acsor Cluster Boundary and Its Proposed Biosynthetic Pathway of Sorbicillinoid
To confirm the boundary of the sorbicillinoid biosynthetic gene cluster, the total RNA was isolated from A. chrysogenum C10 after incubation in the modified MDFA medium (also producing sorbicillinoids as in the rice solid medium) for 1, 3 and 5 days, and used as a template for real-time RT-PCR, the transcriptions of all 10 genes, including orf2

Determination of Acsor Cluster Boundary and Its Proposed Biosynthetic Pathway of Sorbicillinoid
To confirm the boundary of the sorbicillinoid biosynthetic gene cluster, the total RNA was isolated from A. chrysogenum C10 after incubation in the modified MDFA medium (also producing sorbicillinoids as in the rice solid medium) for 1, 3 and 5 days, and used as a template for real-time RT-PCR, the transcriptions of all 10 genes, including orf2 (ACRE_048080), AcsorD (ACRE_048110), AcsorR2 (ACRE_048120), AcsorT (ACRE_048130), AcsorE (ACRE_048140), AcsorR1 (ACRE_048150), AcsorC (ACRE_048160), AcsorB (ACRE_048170), AcsorA (ACRE_048180) and orf1 (ACRE_048200), were analysed ( Figure 6A). Transcriptional results showed that AcsorA, AcsorB, AcsorC, AcsorD, AcsorE, AcsorT, AcsorR1 and AcsorR2 displayed a similar transcriptional pattern. In other words, the transcriptional level gradually increases during the fermentation. However, orf2 was silent during fermentation. Although orf1 was transcribed, the transcriptional trend was significantly different from other genes in the Acsor cluster. Thus, orf1 and orf2 are considered to be situated outside the Acsor cluster ( Figure 6B). Combining with the results from bioinformatic analysis, a 35.5 kb Acsor cluster was identified that contains eight genes encoding one high-reducing polyketide synthase AcsorA, one non-reducing PKS AcsorB, two FAD-dependent monooxygenases AcsorC and AcsorD, one major facilitator superfamily transporter AcsorT, two putative regulators AcsorR1 and AcsorR2 and one putative serine hydrolase AcsorE.
(ACRE_048160), AcsorB (ACRE_048170), AcsorA (ACRE_048180) and orf1 (ACRE_048200), were analysed ( Figure 6A). Transcriptional results showed that AcsorA, AcsorB, AcsorC, AcsorD, AcsorE, AcsorT, AcsorR1 and AcsorR2 displayed a similar transcriptional pattern. In other words, the transcriptional level gradually increases during the fermentation. However, orf2 was silent during fermentation. Although orf1 was transcribed, the transcriptional trend was significantly different from other genes in the Acsor cluster. Thus, orf1 and orf2 are considered to be situated outside the Acsor cluster ( Figure 6B). Combining with the results from bioinformatic analysis, a 35.5 kb Acsor cluster was identified that contains eight genes encoding one high-reducing polyketide synthase AcsorA, one nonreducing PKS AcsorB, two FAD-dependent monooxygenases AcsorC and AcsorD, one major facilitator superfamily transporter AcsorT, two putative regulators AcsorR1 and AcsorR2 and one putative serine hydrolase AcsorE. Based on the confirmation of Acsor cluster, the biosynthetic pathway of compounds 1-8 was proposed. Sorbicillinoid biosynthesis starts from the formation of the polyketide backbone via condensation of acetate units catalyzed by AcsorA and AcsorB to generate sorbicillin and dihydrosorbicillin, and then they are oxidative dearomatized by AcsorC to form the common precursor-sorbicillinol and dihydrosorbicillinol. Sorbicillinol and its derivatives can be converted to 1, 2 and 4 by a Diels-Alder reaction. Compounds 3, 5 and 6 were biosynthesized by a Michael addition of sorbicillinol. Compounds 7 and 8 could be formed from sorbicillinol by an oxidation reaction (Figure 7). The structure diversification of sorbicillinoid derivatives was likely due to the multi-functions of AcsorD in A. chrysogenum. Based on the confirmation of Acsor cluster, the biosynthetic pathway of compounds 1-8 was proposed. Sorbicillinoid biosynthesis starts from the formation of the polyketide backbone via condensation of acetate units catalyzed by AcsorA and AcsorB to generate sorbicillin and dihydrosorbicillin, and then they are oxidative dearomatized by AcsorC to form the common precursor-sorbicillinol and dihydrosorbicillinol. Sorbicillinol and its derivatives can be converted to 1, 2 and 4 by a Diels-Alder reaction. Compounds 3, 5 and 6 were biosynthesized by a Michael addition of sorbicillinol. Compounds 7 and 8 could be formed from sorbicillinol by an oxidation reaction (Figure 7). The structure diversification of sorbicillinoid derivatives was likely due to the multi-functions of AcsorD in A. chrysogenum.

Conclusions
In summary, eight sorbicillinoid derivatives including three new ones, acresorbicillinols A-C (1-3), were isolated from the marine-derived fungus A. chrysogenum. The absolute configurations of compounds 1-3 were determined by ECD calculations. Compound 3 exhibited strong DPPH radical scavenging, indicating that it can be regarded as

Conclusions
In summary, eight sorbicillinoid derivatives including three new ones, acresorbicillinols A-C (1-3), were isolated from the marine-derived fungus A. chrysogenum. The absolute configurations of compounds 1-3 were determined by ECD calculations. Compound 3 exhibited strong DPPH radical scavenging, indicating that it can be regarded as one novel DPPH radical scavenging agent. Compounds 2 and 3 exhibited the moderate activities against S. aureus and C. neoformans, respectively. Meanwhile, the boundary of the Acsor cluster was confirmed and the biosynthetic pathway of compounds 1-8 was also proposed. This study suggests that A. chrysogenum is a potential pool for novel sorbicillinoids and radical scavenging agents.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analyzed in this study are available within the manuscript and are available from the corresponding authors upon request.