*2.2. 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 H2O, and cultivated at 28 ◦C for 7 days for the production of sorbicillinoids. A total of 10 kg fermentation sample was harvested.

## *2.3. Extraction and Isolation*

The rice solid fermentation of *A. chrysogenum* was extracted with EtOAc (3 × 5 L) under the ultrasonication processing. The organic solvents were filtered and evaporated by the vaccum to get the crude extracts (25 g). Extracts were fractionated by ODS reverse silica gel using the gradient MeOH/H2O (*v*/*v*, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%) to afford 15 fractions (Fr.1–Fr.15). Fr.8 (MeOH/H2O (*v*/*v*, 65%)) (150 mg) was further subjected to the SephadexTM LH-20 and eluted with MeOH to give 30 subfractions. Fr.8–24 (30 mg) was purified by semi-preparative RP-HPLC using 50% acetonitrile in acidic water (0.1% formic acid) to give compounds **2** (5.0 mg, *t*<sup>R</sup> = 23 min), **4** (4.0 mg, *t*<sup>R</sup> = 24.1 min) and **8** (2.0 mg, *t*<sup>R</sup> = 22.2 min). Fr.10 (MeOH/H2O (*v*/*v*, 75%)) (133 mg) was subjected to the SephadexTM LH-20 and eluted with MeOH to give 30 subfractions. Fr.10–12 (56 mg) was purified by semi-preparative RP-HPLC using 58% acetonitrile in acidic water (0.1% formic acid) to yield compounds **1** (3.5 mg, *t*<sup>R</sup> = 27.2 min) and **6** (2.5 mg, *t*<sup>R</sup> = 26.4 min). Fr.11 (MeOH/H2O (*v*/*v*, 80%)) (220 mg) was subjected to the SephadexTM LH-20 and eluted with MeOH to give 30 subfractions. Fr.11–24 (60 mg) was purified by semi-preparative RP-HPLC using 60% acetonitrile in acidic water (0.1% formic acid) to yield compounds **3** (10.0 mg, *t*<sup>R</sup> = 28.5 min) and **5** (2.9 mg, *t*<sup>R</sup> = 28.3 min). Fr.5 (MeOH/H2O (*v*/*v*, 50%)) (96 mg) was subjected to the SephadexTM LH-20 and eluted with MeOH to give 25 subfractions. Fr.5-16 (10 mg) was purified by semi-preparative RP-HPLC using 37% acetonitrile in acidic water (0.1% formic acid) to yield compound **7** (2.0 mg, *t*<sup>R</sup> = 17.5 min).

Acresorbicillinol A (**1**): pale yellow solid; [*α*] 25 *D* +81 (*c* 0.1, MeOH); UV (MeOH) *λ*max (log *<sup>ε</sup>*) 223 (3.65), 367 (1.72) nm; ECD (*<sup>c</sup>* 3.0 <sup>×</sup> <sup>10</sup>–3 M, MeOH) *<sup>λ</sup>*max (∆*ε*) 200 (+4.51), 228 (–8.29), 270 (–12.89), 315 (–34.60), 352 (+24.06) nm; IR (neat) *ν*max 3399, 2956, 2872, 1722, 1601, 1446, 1381, 1258 cm−<sup>1</sup> ; <sup>1</sup>H and <sup>13</sup>C NMR, Table 1; HRESIMS at *m*/*z* 501.2850 [M + H]<sup>+</sup> (calcd for C29H41O7, 501.2847).


**Table 1.** <sup>1</sup>H NMR (500 MHz) and <sup>13</sup>C NMR data (125 MHz) for **1** and **2**.

<sup>a</sup> Recorded in CD3OD.

Acresorbicillinol B (**2**): pale yellow solid; [*α*] 25 *D* +5 (*c* 0.1, MeOH); UV (MeOH) *λ*max (log *<sup>ε</sup>*) 221 (2.72), 322 (0.68), 351 (0.54) nm; ECD (*<sup>c</sup>* 3.0 <sup>×</sup> <sup>10</sup>–3 M, MeOH) *<sup>λ</sup>*max (∆*ε*) 215 (–14.07), 245 (+36.00), 315 (–75.16) nm, 360 (+13.47) nm; IR (neat) *ν*max 3413, 1724, 1624, 1440, 1378, 1243 cm−<sup>1</sup> ; <sup>1</sup>H and <sup>13</sup>C NMR, Table 1; HRESIMS at *m*/*z* 369.1696 [M + H]<sup>+</sup> (calcd for C22H25O5, 369.1697).

Acresorbicillinol C (**3**): bright yellow solid; [*α*] 25 *<sup>D</sup>* −1048 (*c* 0.1, MeOH); UV (MeOH) *<sup>λ</sup>*max (log *<sup>ε</sup>*) 207 (1.82), 37 (2.12), 278 (2.55), 375 (3.23) nm; ECD (*<sup>c</sup>* 3.0 <sup>×</sup> <sup>10</sup>–3 M, MeOH) *<sup>λ</sup>*max (∆*ε*) 221 (–22.24), 275 (+38.06), 345 (+51.25) nm, 405 (–88.67) nm; IR (neat) *ν*max 3420, 1664, 1606, 1556, 1412, 1347, 1209 cm−<sup>1</sup> ; <sup>1</sup>H and <sup>13</sup>C NMR, Table 2; HRESIMS at *m*/*z* 513.2116 [M + H]<sup>+</sup> (calcd for C28H33O9, 513.2119).


**Table 2.** <sup>1</sup>H NMR (500 MHz) and <sup>13</sup>C NMR data (125 MHz) for **3**.

<sup>b</sup> Recorded in DMSO:CDCl<sup>3</sup> = 3:1.

#### *2.4. 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].

#### *2.5. 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 <sup>×</sup> <sup>10</sup><sup>5</sup> 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<sup>50</sup> 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.

#### *2.6. 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.

#### *2.7. 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.

#### **3. Results and Discussion**

#### *3.1. Isolation and Structure Elucidation*

Acresorbicillinol A (**1**) was obtained as a pale yellow solid, and its molecular formula was established as C29H40O<sup>7</sup> based on HRESIMS data at 501.2850 [M + H]<sup>+</sup> (calcd for C29H41O7, 501.2847), indicating 10 degrees of unsaturation. The IR spectrum indicated the presence of hydroxy (3399 cm−<sup>1</sup> ) and ketone (1722 cm−<sup>1</sup> ) groups. The <sup>1</sup>H NMR data (Table 1 and Figure S1) of **1** showed signals for six methyl signals [*δ*<sup>H</sup> 2.16 (s, H3-24), 1.89 (d, *J* = 7.0 Hz, H3-14), 1.16 (s, H3-28), 1.12 (s, H3-29), 0.86 (d, *J* = 7.0 Hz, H3-26), and 0.81 (d, *J* = 7.0 Hz, H3-27)], five methylene protons [*δ*<sup>H</sup> 2.42 (m, H-22a), 2.38 (m, H-8a), 2.30 (m, H-22b), 2.16 (m, H2-16), 1.97 (dd, *J* = 13.3, 2.8 Hz, H-8b), 1.81 (td, *J* = 13.2, 4.8 Hz, H-15a), 1.64 (m, H-21a), 1.50 (m, H-15b), and 1.23 (m, H-21b)], three methine protons [*δ*<sup>H</sup> 3.18 (t, *J* = 2.8 Hz, H-4), 1.68 (m, H-20), and 1.54 (m, H-25)], six olefinic protons [*δ*<sup>H</sup> 7.26 (dd, *J* = 14.6, 10.9 Hz, H-11), 6.42 (d, *J* = 14.6 Hz, H-10), 6.39 (dd, *J* = 14.6, 10.9 Hz, H-12), 6.20 (dq, *J* = 14.6, 7.0 Hz, H-13), 5.18 (d, *J* = 15.6 Hz, H-18), and 5.13 (dd, *J* = 15.6, 9.0 Hz, H-19)]. Detailed interpretation of the <sup>13</sup>C NMR and HSQC data (Table 1, Figures S2 and S4) of **1** revealed the presence of 29 carbon resonances corresponding to six methyls, five sp<sup>3</sup> methylenes, three sp<sup>3</sup> methines, six sp<sup>2</sup> methines, three sp<sup>3</sup> quarternary carbons with one oxygenated, two sp<sup>2</sup> non-protonated carbons and four carbonyl carbons (*δ*<sup>C</sup> 212.4, 212.3, 200.3 and 178.3, respectively). These data accounted for all <sup>1</sup>H and <sup>13</sup>C NMR resonances of **1** except for three unobserved exchangeable protons, suggesting that **1** was a bicyclic compound. The planar structure of **1** was assigned through detailed analysis of the <sup>1</sup>H-1H COSY and HMBC correlations (Figures 2, S3 and S5). The <sup>1</sup>H-1H COSY (Figure 2) correlations of H-10/H-11/H-12/H-13/H3-14, combined with the HMBC correlations from H-10 to the

olefinic carbons C-3 (*δ*<sup>C</sup> 112.3) and C-9 (*δ*<sup>C</sup> 167.6) and from H-11 to C-9, suggested the presence of the enolic sorbyl side chain. The HMBC correlations (Figure 2) from H-4 to C-3, the sp<sup>3</sup> quarternary carbon C-5 (*δ*<sup>C</sup> 75.4) and two ketone carbons C-2 (*δ*<sup>C</sup> 200.3) and C-6 (*δ*<sup>C</sup> 212.3), from H3-28 to the sp<sup>3</sup> quarternary carbon C-1 (*δ*<sup>C</sup> 70.3), C-2 and C-6, and from H3-29 to C-4, C-5, and C-6 permitted the completion of the cyclohexandione ring, with the enolic sorbyl unit positioned at C-3 and two methyl groups located at C-1 and C-5, respectively. Meanwhile, the <sup>1</sup>H-1H COSY (Figure 2) correlations of H-18/H-19/H-20/H2-21/H2-22 and of H-20/H-25/H3-26/H3-27, as well as the HMBC correlations from H2-22 to the ketone carbon C-23 (*δ*<sup>C</sup> 212.4) and C-24 (*δ*<sup>C</sup> 30.0), and from H3-24 to C-22 and C-23, established the 3-isopropyl-6-oxohept-1-en-1-yl (C-18–C-27) subunit. Moreover, the <sup>1</sup>H-1H COSY (Figure 2) correlations of H2-15/H2-16, and the HMBC correlations from H2-15 and H2-16 to the carbonyl carbon C-17 (*δ*<sup>C</sup> 178.3), indicated that carbonyl carbon C-17 was attached to C-16 directly. Additional HMBC correlations from H2-15 to the sp<sup>3</sup> quarternary carbon C-7 (*δ*<sup>C</sup> 47.8) and the olefinic carbon C-18 (*δ*<sup>C</sup> 135.4), from H2-16 and H-19 to C-7, and from H-18 to C-7 and C-15, indicated that C-7 was located between C-15 and C-18. Key HMBC correlations from H-4, H2-8 and H3-28 to C-7, and from H2-15 and H-18 to C-1 and C-8, along with the <sup>1</sup>H-1H COSY correlations of H-4/H2-8 implied that C-1 and C-8 were all connected to C-7, permitting the completion of the bridged bicyclo [2.2.2]octane-2,6-dione core structure. By consideration of the molecular formula and the chemical shifts of C-5 (*δ*<sup>C</sup> 75.4) and C-17 (*δ*<sup>C</sup> 178.3), these two carbons should be hydroxylated. Thus, the planar structure of **1** was established as shown (Figure 1). ternary carbon C‐5 (*δ*<sup>C</sup> 75.4) and two ketone carbons C‐2 (*δ*<sup>C</sup> 200.3) and C‐6 (*δ*<sup>C</sup> 212.3), from H3‐28 to the sp3 quarternary carbon C‐1 (*δ*<sup>C</sup> 70.3), C‐2 and C‐6, and from H3‐29 to C‐4, C‐5, and C‐6 permitted the completion of the cyclohexandione ring, with the enolic sorbyl unit positioned at C‐3 and two methyl groups located at C‐1 and C‐5, respectively. Meanwhile, the 1H‐1H COSY (Figure 2) correlations of H‐18/H‐19/H‐20/H2‐21/H2‐22 and of H‐20/H‐ 25/H3‐26/H3‐27, as well as the HMBC correlations from H2‐22 to the ketone carbon C‐23 (*δ*<sup>C</sup> 212.4) and C‐24 (*δ*<sup>C</sup> 30.0), and from H3‐24 to C‐22 and C‐23, established the 3‐isopropyl‐ 6‐oxohept‐1‐en‐1‐yl (C‐18–C‐27) subunit. Moreover, the 1H‐1H COSY (Figure 2) correla‐ tions of H2‐15/H2‐16, and the HMBC correlations from H2‐15 and H2‐16 to the carbonyl carbon C‐17 (*δ*<sup>C</sup> 178.3), indicated that carbonyl carbon C‐17 was attached to C‐16 directly. Additional HMBC correlations from H2‐15 to the sp3 quarternary carbon C‐7 (*δ*<sup>C</sup> 47.8) and the olefinic carbon C‐18 (*δ*<sup>C</sup> 135.4), from H2‐16 and H‐19 to C‐7, and from H‐18 to C‐7 and C‐15, indicated that C‐7 was located between C‐15 and C‐18. Key HMBC correlations from H‐4, H2‐8 and H3‐28 to C‐7, and from H2‐15 and H‐18 to C‐1 and C‐8, along with the 1H‐ 1H COSY correlations of H‐4/H2‐8 implied that C‐1 and C‐8 were all connected to C‐7, permitting the completion of the bridged bicyclo [2.2.2]octane‐2,6‐dione core structure. By consideration of the molecular formula and the chemical shifts of C‐5 (*δ*<sup>C</sup> 75.4) and C‐17 (*δ*<sup>C</sup> 178.3), these two carbons should be hydroxylated. Thus, the planar structure of **1** was established as shown (Figure 1).

*J. Fungi* **2022**, *8*, x FOR PEER REVIEW 7 of 15

H‐13), 5.18 (d, *J* = 15.6 Hz, H‐18), and 5.13 (dd, *J* = 15.6, 9.0 Hz, H‐19)]. Detailed interpreta‐ tion of the 13C NMR and HSQC data (Table 1, Figures S2 and S4) of **1** revealed the presence of 29 carbon resonances corresponding to six methyls, five sp3 methylenes, three sp3 me‐ thines, six sp2 methines, three sp3 quarternary carbons with one oxygenated, two sp2 non‐ protonated carbons and four carbonyl carbons (*δ*<sup>C</sup> 212.4, 212.3, 200.3 and 178.3, respec‐ tively). These data accounted for all 1H and 13C NMR resonances of **1** except for three un‐ observed exchangeable protons, suggesting that **1** was a bicyclic compound. The planar structure of **1** was assigned through detailed analysis of the 1H‐1H COSY and HMBC cor‐ relations (Figure 2, Figures S3 and S5). The 1H‐1H COSY (Figure 2) correlations of H‐10/H‐

enolic sorbyl side chain. The HMBC correlations (Figure 2) from H‐4 to C‐3, the sp3 quar‐

**Figure 2.** Key COSY and HMBC correlations of compounds **1**–**3**. **Figure 2.** Key COSY and HMBC correlations of compounds **1**–**3**.

The relative configuration of **1** was determined by NOESY correlations, coupling con‐ stants and HMBC correlations. The NOESY correlation (Figures 3 and S6) of H‐10 with H‐ 4 assigned the olefin C‐3/C‐9 as *Z* geometry. The geometry of the conjugated diene was assigned as 10*E*, 12*E* by the large coupling constants (*J*H‐10/H‐<sup>11</sup> = 14.6 Hz and *J*H‐12/H‐<sup>13</sup> = 14.6 Hz) along with the NOESY correlations of H3‐14 with H‐12 and of H‐13 with H‐11. The *E* geometry of the C‐18/C‐19 double bond was also deduced by the large coupling constant between H‐18 and H‐19 (15.6 Hz). The NOESY correlations of H‐10 with H‐4 and H3‐29 suggested that these protons were close in space. Moreover, the strong HMBC correlations from H‐8a to C‐3 and C‐15, and from H‐8b to C‐5, and the weak correlation from H‐8a to The relative configuration of **1** was determined by NOESY correlations, coupling constants and HMBC correlations. The NOESY correlation (Figures 3 and S6) of H-10 with H-4 assigned the olefin C-3/C-9 as *Z* geometry. The geometry of the conjugated diene was assigned as 10*E*, 12*E* by the large coupling constants (*J*H-10/H-11 = 14.6 Hz and *J*H-12/H-13 = 14.6 Hz) along with the NOESY correlations of H3-14 with H-12 and of H-13 with H-11. The *E* geometry of the C-18/C-19 double bond was also deduced by the large coupling constant between H-18 and H-19 (15.6 Hz). The NOESY correlations of H-10 with H-4 and H3-29 suggested that these protons were close in space. Moreover, the strong HMBC correlations from H-8a to C-3 and C-15, and from H-8b to C-5, and the weak correlation from H-8a to C-5, as well as the lack of HMBC correlation from H-8b to C-3 and C-15, indicated that H-8a and C-15 were eclipsed and that H-8b and C-3 were gauche [20,29]. Meanwhile, the NOESY correlations of H3-28 with H-15a, and of H-8a with H-15b, assigned the relative configurations of C-1 and C-7. However, the relative configuration for C-20 could not be established by the NOESY data. The absolute configuration for **1** was assigned by a comparison of the experimental and calculated ECD spectra of two pairs of enantiomers, (1*R*,4*S*,5*S*,7*R*,20*S*)-**1** (**1a**), (1*S*,4*R*,5*R*,7*S*,20*R*)-**1** (**1b**), (1*R*,4*S*,5*S*,7*R*,20*R*)-**1** (**1c**), and (1*S*,4*R*,5*R*,7*S*,20*S*)-**1** (**1d**). The ECD calculations were conducted using time-dependent density functional theory (TDDFT) at the CAM-B3LYP/6-

31G(d) level. The overall calculated ECD spectrum of **1a**–**1d** was then generated according to Boltzmann weighting of the conformers (Figure S19). For compound **1** the experimental first positive (200 nm), second negative (228 nm), third negative (270 nm), fourth negative (315 nm) and fifth positive (352 nm) Cotton effects compared well with the calculated ECD curve for (1*R*,4*S*,5*S*,7*R*,20*S*)-**1** (**1a**), which showed five corresponding Cotton effects around 200, 222, 270, 315 and 350 nm (Figure 4). Therefore, qualitative analysis of the result allowed the assignment of the absolute configuration of **1** as 1*R*,4*S*,5*S*,7*R*,20*S*. the conformers (Figure S19). For compound **1** the experimental first positive (200 nm), second negative (228 nm), third negative (270 nm), fourth negative (315 nm) and fifth pos‐ itive (352 nm) Cotton effects compared well with the calculated ECD curve for (1*R*,4*S*,5*S*,7*R*,20*S*)‐**1** (**1a**), which showed five corresponding Cotton effects around 200, 222, 270, 315 and 350 nm (Figure 4). Therefore, qualitative analysis of the result allowed the assignment of the absolute configuration of **1** as 1*R*,4*S*,5*S*,7*R*,20*S*. the conformers (Figure S19). For compound **1** the experimental first positive (200 nm), second negative (228 nm), third negative (270 nm), fourth negative (315 nm) and fifth pos‐ itive (352 nm) Cotton effects compared well with the calculated ECD curve for (1*R*,4*S*,5*S*,7*R*,20*S*)‐**1** (**1a**), which showed five corresponding Cotton effects around 200, 222, 270, 315 and 350 nm (Figure 4). Therefore, qualitative analysis of the result allowed the assignment of the absolute configuration of **1** as 1*R*,4*S*,5*S*,7*R*,20*S*.

C‐5, as well as the lack of HMBC correlation from H‐8b to C‐3 and C‐15, indicated that H‐ 8a and C‐15 were eclipsed and that H‐8b and C‐3 were gauche [20,29]. Meanwhile, the NOESY correlations of H3‐28 with H‐15a, and of H‐8a with H‐15b, assigned the relative configurations of C‐1 and C‐7. However, the relative configuration for C‐20 could not be established by the NOESY data. The absolute configuration for **1** was assigned by a comparison of the experimental and calculated ECD spectra of two pairs of enantiomers, (1*R*,4*S*,5*S*,7*R*,20*S*)‐**1** (**1a**), (1*S*,4*R*,5*R*,7*S*,20*R*)‐**1** (**1b**), (1*R*,4*S*,5*S*,7*R*,20*R*)‐**1** (**1c**), and (1*S*,4*R*,5*R*,7*S*,20*S*)‐**1** (**1d**). The ECD calculations were conducted using time‐dependent density functional theory (TDDFT) at the CAM‐B3LYP/6‐31G(d) level. The overall calcu‐ lated ECD spectrum of **1a**–**1d** was then generated according to Boltzmann weighting of

C‐5, as well as the lack of HMBC correlation from H‐8b to C‐3 and C‐15, indicated that H‐ 8a and C‐15 were eclipsed and that H‐8b and C‐3 were gauche [20,29]. Meanwhile, the NOESY correlations of H3‐28 with H‐15a, and of H‐8a with H‐15b, assigned the relative configurations of C‐1 and C‐7. However, the relative configuration for C‐20 could not be established by the NOESY data. The absolute configuration for **1** was assigned by a comparison of the experimental and calculated ECD spectra of two pairs of enantiomers, (1*R*,4*S*,5*S*,7*R*,20*S*)‐**1** (**1a**), (1*S*,4*R*,5*R*,7*S*,20*R*)‐**1** (**1b**), (1*R*,4*S*,5*S*,7*R*,20*R*)‐**1** (**1c**), and (1*S*,4*R*,5*R*,7*S*,20*S*)‐**1** (**1d**). The ECD calculations were conducted using time‐dependent density functional theory (TDDFT) at the CAM‐B3LYP/6‐31G(d) level. The overall calcu‐ lated ECD spectrum of **1a**–**1d** was then generated according to Boltzmann weighting of

*J. Fungi* **2022**, *8*, x FOR PEER REVIEW 8 of 15

*J. Fungi* **2022**, *8*, x FOR PEER REVIEW 8 of 15

**Figure 3.** Key NOESY correlations of compounds **1**–**3**. **Figure 3.** Key NOESY correlations of compounds **1**–**3**. **Figure 3.** Key NOESY correlations of compounds **1**–**3**.

 **Figure 4.** Calculated and experimental ECD spectra of compounds **1**–**3**.

**Wavelength (nm)**

**Figure 4.** Calculated and experimental ECD spectra of compounds **1**–**3**. **Figure 4.** Calculated and experimental ECD spectra of compounds **1**–**3**. Acresorbicillinol B (**2**) was obtained as a pale yellow solid. The molecular formula of **2** was assigned as C22H24O<sup>5</sup> (11 degrees of unsaturation) based on its HRESIMS data at *m/z* 369.1696 [M + H]<sup>+</sup> (calcd for C22H25O5, 369.1697). The <sup>1</sup>H and <sup>13</sup>C NMR spectroscopic data (Table 1, Figures S7 and S8), in association with the HSQC spectrum (Figure S10), indicated 22 carbon resonances including 3 methyl groups, 1 sp<sup>3</sup> methylenes, 2 sp<sup>3</sup> methines, 2 sp<sup>3</sup> non-protonated carbons with 1 oxygenated, 12 olefinic or aromatic carbons (8 protonated), and 2 carbonyl carbons (*δ*<sup>C</sup> 211.4, and 199.7, respectively), which were similar to those of **1**.

**Wavelength (nm)**

Analysis of the <sup>1</sup>H-1H COSY and HMBC data (Figure 2, Figures S9 and S11) of **2** determined the same bicyclo [2.2.2]octane-2,6-dione moiety with the enolic sorbyl substituted at C-3. However, the substitutes at C-7 of **2** were different from those of **1**. The HMBC correlations from H2-8 to C-15 (*δ*<sup>C</sup> 133.9), from H-7 to C-15, C-16 (*δ*<sup>C</sup> 130.5) and C-20 (*δ*<sup>C</sup> 130.5), from H-16/H-20 to C-7 (*δ*<sup>C</sup> 47.5) and C-18 (*δ*<sup>C</sup> 157.7) and from H-17/H-19 to C-15 completed the *para*-hydroxyphenyl group located at C-7. On the basis of these data, the planar structure of **2** was established as shown (Figure 1).

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) of H3-14 with H-12 and of H-13 with H-11 indicated that the geometry of the conjugated diene was 10*E*, 12*E*. Furthermore, the NOESY correlation (Figure 3) of H-4 with H-10 implied a *Z* geometry of the C3/C9 double bond. Other NOESY correlations of H-10 with H-4 and H3-22, and of H-4 with H3-22, placed these protons on the same side. While NOESY correlations of H-8b (*δ*<sup>H</sup> 1.80, ddd, *J* = 13.6, 6.1, 2.7 Hz) with H-16 (H-20), and of H-7 with H3-21, 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 (1*R*,4*S*,5*S*,7*R*)-**2** (**2a**) and (1*S*,4*R*,5*R*,7*S*)-**2** (**2b**). As shown in Figure 4, the experimental ECD spectrum of **2** showed good agreement with the calculated ECD spectrum of (1*R*,4*S*,5*S*,7*R*)-**2** (**2a**), suggesting the absolute configuration of 1*R*,4*S*,5*S*,7*R* for **2**. Thus, the structure of **2** was defined as shown.

Acresorbicillinol C (**3**) was obtained as a bright yellow solid, and its molecular formula was deduced to be C28H32O<sup>9</sup> (13 degrees of unsaturation) on the basis of the HRESIMS data at *m*/*z* 513.2116 [M + H]<sup>+</sup> (calcd for C28H33O9, 513.2119). The IR absorptions suggested the presence of hydroxy (3420 cm−<sup>1</sup> ) and ketone (1664 cm−<sup>1</sup> ) groups. Its <sup>1</sup>H NMR data (Table 2 and Figure S13) revealed signals of eight olefinic protons [*δ*<sup>H</sup> 6.10–7.49], one methine proton [*δ*<sup>H</sup> 3.71 (s, H-1)] and six methyls [*δ*<sup>H</sup> 1.89 (d, *J* = 6.8 Hz, H3-12'), 1.83 (d, *J* = 6.8 Hz, H3-12), 1.31 (s, H3-14'), 1.30 (s, H3-13), 1.29 (s, H3-14), 1.17 (s, H3-13')]. The <sup>13</sup>C NMR spectrum (Table 2 and Figure S14) and the HSQC data (Figure S16) displayed a total of 28 carbon resonances, which were assignable to 6 methyl groups, 8 sp<sup>2</sup> methines, 1 sp<sup>3</sup> methines, 13 non-protonated carbons containing 2 carbonyls (*δ*<sup>C</sup> 199.3, and 190.9), 4 sp<sup>2</sup> non-protonated with two oxygenated, 7 sp<sup>3</sup> non-protonated carbon with 5 oxygenated. These signals (Table 2 and Figures S13−S16) were very similar to those of trichodimerol (**5**) [31,32], except that the proton at the C-1' position in **5** was changed to a hydroxy moiety in **3**. This was evidenced by the HRESIMS data and HMBC correlations (Figures 2 and S17) from H3-13' and H3-14 to C-1' (*δ*<sup>C</sup> 78.3). Therefore, **3** was 1'-hydroxylated analogue of **5**.

The relative configuration of **3** was confirmed by NOESY correlations and coupling constants. The NOESY correlations (Figures 3 and S18) of H-9/H-11, of H-8/H-10/H-12, of H-9'/H-11' and of H-8'/H-10'/H-12', along with the large coupling constants (*J*H-8/H-9 = *J*H-10/H-11 = *J*H-8'/H-9' = *J*H-10'/H-11' = 14.6 Hz) suggested the 8*E*, 10*E*, 8'*E* and 10'*E* configurations of the conjugated dienes in the sorbyl side chains. Meanwhile, the NOESY correlations of H-1/H-8 and H3-14/H-8' suggested the *Z* geometry of C-6/C-7 and C-6'/C-7' double bonds. Furthermore, the NOESY correlations of H-1/H3-13, of H3-13'/H3-14 and of H3-14'/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 (1*S*,2*S*,3*R*,4*R*,1'*R*,2'*S*,3'*R*,4'*R*)-**3** (**3a**) matched well with the experimental data, suggesting the absolute configuration to be 1*S*,2*S*,3*R*,4*R*,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 [20–22]. The resulting EtOAc extracts of *A. chrysogenum* cultivated on the rice were screened by HPLC analysis (Figure S20).

#### *3.2. 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<sup>50</sup> values of 86.93 ± 1.72 and 69.06 ± 10.50 µM, respectively. However, other compounds did not give IC<sup>50</sup> 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**.

Through the DPPH radical scavenging assay, compound **3** exhibited strong activity with the IC<sup>50</sup> 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<sup>50</sup> 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<sup>50</sup> 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). Compound **8** exhibited the radical scavenging activity with the IC<sup>50</sup> values of 155.40 ± 12.42 and 55.36 ± 14.92 µM for 0.5 and 24 h, respectively. Although the IC<sup>50</sup> values of **4**, **5** and **6** were over 200 µM for 0.5 h, their radical scavenging activity significantly enhanced at 24 h, and the IC<sup>50</sup> values were 151.87 ± 15.63, 116.83 ± 3.93 and 102.48 ± 5.04 µM, respectively (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<sup>50</sup> 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<sup>50</sup> 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. μM, respectively [21,37]. To date, compound **3** displayed the best DPPH radical scaveng‐ ing activity for 24 h among all reported sorbicillinoids.

of sorbicillinoids for radical scavenging was previously reported, including for oxosorb‐ 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 bisorbicil‐ linol, bisvertinolone and bisorbibetanone, which showed ED50values of 31.4, 44.3 and 62.5

*J. Fungi* **2022**, *8*, x FOR PEER REVIEW 11 of 15

**Figure 5.** DPPH radical scavenging activity of compound **3** and ascorbic acid as the positive control at 0.5, 1, 4, 6, 8 and 24 h. **Figure 5.** DPPH radical scavenging activity of compound **3** and ascorbic acid as the positive control at 0.5, 1, 4, 6, 8 and 24 h.


*icillinoid*

**Table 4.** DPPH radical scavenging activities of compounds **1**–**8**. **Table 4.** DPPH radical scavenging activities of compounds**1**–**8**.

#### 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 *3.3. Determination of Acsor Cluster Boundary and Its Proposed Biosynthetic Pathway of Sorbicillinoid*

(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* 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. *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 tran‐ scriptional trend was significantly different from other genes in the *Acsor* cluster. Thus,

*J. Fungi* **2022**, *8*, x FOR PEER REVIEW 12 of 15

**Figure 6.** (**A**) Organization of the sorbicillinoid biosynthetic gene cluster. FMO, FAD‐dependent monooxygenase; PKS, polyketide synthase; TF, transcriptional factor; AM, auxiliary modifier; MFS, major facilitator superfamily transporter. (**B**) Transcriptional profiles of the *Acsor* genes during fer‐ mentation. **Figure 6.**(**A**) Organization of the sorbicillinoid biosynthetic gene cluster. FMO, FAD-dependent monooxygenase; PKS, polyketide synthase; TF, transcriptional factor; AM, auxiliary modifier; MFS, major facilitator superfamily transporter. (**B**) Transcriptional profiles of the *Acsor* genes during fermentation.

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 de‐ rivatives 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. chryso‐ genum*. 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*. *J. Fungi* **2022**, *8*, x FOR PEER REVIEW 13 of 15

In summary, eight sorbicillinoid derivatives including three new ones, acresorbicil‐ linols A–C (**1**–**3**), were isolated from the marine‐derived fungus *A. chrysogenum*. The ab‐

pound **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 sorbicilli‐

**Supplementary Materials:** The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figure S1: 1H NMR spectrum of acresorbicillinol A (**1**; 500 MHz, CD3OD), Figure S2: 13C NMR spectrum of acresorbicillinol A (**1**; 125 MHz, CD3OD), Figure S3: 1H‐1H COSY spectrum of acresorbicillinol A (**1**, CD3OD), Figure S4: HSQC spectrum of acresorbicillinol A (**1**, CD3OD), Figure S5: HMBC spectrum of acresorbicillinol A (**1**, CD3OD), Figure S6: NOESY spectrum of acresorbicillinol A (**1**, CD3OD), Figure S7: 1H NMR spectrum of acresorbicillinol B (**2**; 500 MHz, CD3OD), Figure S8: 13C NMR spectrum of acresorbicillinol B (**2**; 125 MHz, CD3OD), Figure S9: 1H‐ 1H COSY spectrum of acresorbicillinol B (**2**, CD3OD), Figure S10: HSQC spectrum of acresorbicillinol B (**2**, CD3OD), Figure S11: HMBC spectrum of acresorbicillinol B (**2**, CD3OD), Figure S12. NOESY spectrum of acresorbicillinol B (**2**, CD3OD) Figure S13: 1H NMR spectrum of acresorbicillinol C (**3**; 500 MHz, DMSO:CDCl3 = 3:1), Figure S14: 13C NMR spectrum of acresorbicillinol C (**3**; 125 MHz, DMSO:CDCl3 = 3:1), Figure S15: 1H‐1H COSY spectrum of acresorbicillinol C (**3**, DMSO:CDCl3 = 3:1), Figure S16: HSQC spectrum of acresorbicillinol C (**3**, DMSO:CDCl3 = 3:1), Figure S17: HMBC spec‐ trum of acresorbicillinol C (**3**, DMSO:CDCl3 = 3:1), Figure S18: NOESY spectrum of acresorbicillinol C (**3**, DMSO:CDCl3 = 3:1), Figure S19: ECD conformers of acresorbicillinols A–C (**1**–**3**). Figure S20: HPLC profiles of the extracts from the rice solid medium of *A. chrysogenum* after 7 days fermentation.

**Author Contributions:** Conceptualization, C.D. and S.W.; methodology, C.D., S.W. and J.R.; soft‐ ware, R.H.; validation, S.W.; formal analysis, E.L., M.W. and R.H.; investigation, C.D. and S.W.; re‐ sources, G.L.; data curation, C.D.; writing—original draft preparation, C.D., S.W. and R.H.; writ‐ ing—review and editing, Y.P., L.L. and G.L.; visualization, R.H.; supervision, Y.P., L.L. and G.L.; project administration, Y.P., L.L. and G.L.; funding acquisition, Y.P. and L.L. All authors have read

**Figure 7.** Proposed biosynthetic pathway of compounds **1**–**8.** and agreed to the published version of the manuscript. **Figure 7.** Proposed biosynthetic pathway of compounds **1**–**8**.

**4. Conclusions**

noids and radical scavenging agents.

Tale S1: Primers used in this study.

#### **4. 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.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof8050530/s1, Figure S1: <sup>1</sup>H NMR spectrum of acresorbicillinol A (**1**; 500 MHz, CD3OD), Figure S2: <sup>13</sup>C NMR spectrum of acresorbicillinol A (**1**; 125 MHz, CD3OD), Figure S3: <sup>1</sup>H-1H COSY spectrum of acresorbicillinol A (**1**, CD3OD), Figure S4: HSQC spectrum of acresorbicillinol A (**1**, CD3OD), Figure S5: HMBC spectrum of acresorbicillinol A (**1**, CD3OD), Figure S6: NOESY spectrum of acresorbicillinol A (**1**, CD3OD), Figure S7: <sup>1</sup>H NMR spectrum of acresorbicillinol B (**2**; 500 MHz, CD3OD), Figure S8: <sup>13</sup>C NMR spectrum of acresorbicillinol B (**2**; 125 MHz, CD3OD), Figure S9: <sup>1</sup>H-1H COSY spectrum of acresorbicillinol B (**2**, CD3OD), Figure S10: HSQC spectrum of acresorbicillinol B (**2**, CD3OD), Figure S11: HMBC spectrum of acresorbicillinol B (**2**, CD3OD), Figure S12. NOESY spectrum of acresorbicillinol B (**2**, CD3OD) Figure S13: <sup>1</sup>H NMR spectrum of acresorbicillinol C (**3**; 500 MHz, DMSO:CDCl<sup>3</sup> = 3:1), Figure S14: <sup>13</sup>C NMR spectrum of acresorbicillinol C (**3**; 125 MHz, DMSO:CDCl<sup>3</sup> = 3:1), Figure S15: <sup>1</sup>H-1H COSY spectrum of acresorbicillinol C (**3**, DMSO:CDCl<sup>3</sup> = 3:1), Figure S16: HSQC spectrum of acresorbicillinol C (**3**, DMSO:CDCl<sup>3</sup> = 3:1), Figure S17: HMBC spectrum of acresorbicillinol C (**3**, DMSO:CDCl<sup>3</sup> = 3:1), Figure S18: NOESY spectrum of acresorbicillinol C (**3**, DMSO:CDCl<sup>3</sup> = 3:1), Figure S19: ECD conformers of acresorbicillinols A–C (**1**–**3**). Figure S20: HPLC profiles of the extracts from the rice solid medium of *A. chrysogenum* after 7 days fermentation. Tale S1: Primers used in this study.

**Author Contributions:** Conceptualization, C.D. and S.W.; methodology, C.D., S.W. and J.R.; software, R.H.; validation, S.W.; formal analysis, E.L., M.W. and R.H.; investigation, C.D. and S.W.; resources, G.L.; data curation, C.D.; writing—original draft preparation, C.D., S.W. and R.H.; writing—review and editing, Y.P., L.L. and G.L.; visualization, R.H.; supervision, Y.P., L.L. and G.L.; project administration, Y.P., L.L. and G.L.; funding acquisition, Y.P. and L.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the National Key Research and Development Program of China (2021YFC2300400) and the National Natural Science Foundation of China (31770056, 32022002, 21977113 and 32170067).

**Institutional Review Board Statement:** Not applicable.

**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.

**Acknowledgments:** We thank Francisco Fierro (Universidad Autónoma Metropolitana-Unidad Iztapalapa, Mexico) for providing the *A. chrysogenum* C10 and Wenzhao Wang (Institute of Microbiology, CAS) for HRESIMS analysis. We also appreciate Guanghua Huang (Fudan University, Shanghai, China) and Linqi Wang (Institute of Microbiology, CAS) providing the strains of *Candida albicans* SC5314 and *Cryptococcus neoformans* W1585, respectively.

**Conflicts of Interest:** The authors declare no conflict of interest.
