Sesquiterpenoids From the Antarctic Fungus Pseudogymnoascus sp. HSX2#-11

The fungal strains Pseudogymnoascus are a kind of psychrophilic pathogenic fungi that are ubiquitously distributed in Antarctica, while the studies of their secondary metabolites are infrequent. Systematic research of the metabolites of the fungus Pseudogymnoascus sp. HSX2#-11 led to the isolation of six new tremulane sesquiterpenoids pseudotremulanes A–F (1–6), combined with one known analog 11,12-epoxy-12β-hydroxy-1-tremulen-5-one (7), and five known steroids (8–12). The absolute configurations of the new compounds (1–6) were elucidated by their ECD spectra and ECD calculations. Compounds 1–7 were proved to be isomeride structures with the same chemical formula. Compounds 1/2, 3/4, 1/4, and 2/3 were identified as four pairs of epimerides at the locations of C-3, C-3, C-9, and C-9, respectively. Compounds 8 and 9 exhibited cytotoxic activities against human breast cancer (MDA-MB-231), colorectal cancer (HCT116), and hepatoma (HepG2) cell lines. Compounds 9 and 10 also showed antibacterial activities against marine fouling bacteria Aeromonas salmonicida. This is the first time to find terpenoids and steroids in the fungal genus Pseudogymnoascus.

The extreme environments of Antarctica, including cold, dry climate and intense solar radiations, have nurtured a number of unique microbial resources (Cong et al., 2020). It has been proved that Antarctic microorganisms, especially fungi, have the potential capacity to produce novel secondary metabolites to adapt to the harsh environments (Kwon et al., 2017;Rusman et al., 2018;Yu et al., 2019;. Pseudogymnoascus are known as a kind of psychrophilic pathogenic fungi with ubiquitous distribution in Antarctica (Rosa et al., 2020;Santos et al., 2020;Martorell et al., 2021). These fungal strains have been proved to have the abilities to produce cold-adapted enzymes to adapt severe cold Antarctic environment (Loperena et al., 2012;Poveda et al., 2018). Pseudogymnoascus can be antagonistic fungi against potato scab pathogens from potato field soils (Tagawa et al., 2010) and have been certified to be one of the predominant microbial colonizers in the root endosphere and rhizosphere of turfgrass systems (Xia et al., 2021). The extracts of some of Pseudogymnoascus strains exhibit potent bioactivities, such as antimicrobial, herbicidal, and antitumoral activities (Henríquez et al., 2014;Gonçalves et al., 2015;Gomes et al., 2018;Ferrarezi et al., 2019). However, only four studies have been done on the secondary metabolites of the genus Pseudogymnoascus until now, as far as we know, and most of the obtained structures focus on polyketides, showing antimicrobial activities (Figueroa et al., 2015;Guo et al., 2019;Fujita et al., 2021;Shi et al., 2021). Rare studies about the secondary metabolites of these fungi enlighten that there is latent space for searching novel compounds. Pseudogymnoascus sp. HSX2#-11 was an Antarctic fungus isolated from a soil sample of the Fields Peninsula, which can produce abundant and various secondary metabolites, according to our previous research on the fingerprint spectrum and molecular network of its ethyl acetate extract of the fermentation broth . Further chemical investigation resulted in the isolation and identification of six new tremulane sesquiterpenoids, pseudotremulanes A-F (1-6), together with one known analog 11,12-epoxy-12β-hydroxy-1-tremulen-5-one (7; Zhou et al., 2008), and five known steroids, ganodermasides A (8), B (9), and D (10; Weng et al., 2010Weng et al., , 2011Feng et al., 2010), and dankasterone B (12;Amagata et al., 2007; Figure 1). Compounds 8 and 9 exhibited cytotoxicities against human breast cancer cell line MDA-MB-231, colorectal cancer cell line HCT116, and hepatoma cell line HepG2 (Table 3). Compounds 9 and 10 showed antibacterial activity against marine fouling bacteria Aeromonas salmonicida. Here, we address the isolation, structure elucidation, and biological activity evaluation of the isolated compounds.

Fungal Materials
The soil samples were collected in ice-free areas (about 10 cm underground) of the Fields Peninsula using sterile spatulas and sterilized WhirlPak bags (Sigma-Aldrich, United States), and were transported to the lab in sealed foam package with dry ice added by airplane, at the Chinese 35th Antarctic expedition in 2019. The fungus Pseudogymnoascus sp. HSX2#-11 was isolated from a soil sample from Fields Peninsula. The strain was deposited at −80 • C in the State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong University, Qingdao, China.
Steps 2-4 were repeated 30 times. The PCR products were then submitted for sequencing (BGI, China) with the primers ITS1 and ITS4. The sequence of HSX2#-11 was searched in the NCBI nucleotide collection database through the BLAST program. The phylogenetic tree of the top 20 most similar to this fungal sequence identified the strain HSX2#-11 as
Adriamycin was used as a positive control. The cell lines of MDA-MB-231, HCT116, A549, PANC-1, and HepG2 in the logarithmic growth phase were seeded into 96-well plates with 5,000 cells/well (100 µl/well), respectively. After 24 h of culture, the isolated compounds to be tested were added (the final concentration was shown in Supplementary Table 1), and three replicates were set for each concentration. The dosage of DMSO in the solvent control group was 0.1% of the maximum dose used in the test group. After 72 h of drug treatment, 10% (m/v) of cold trichloroacetic acid was added to each well to fix the cells. After SRB staining, 150 µl/well Tris solution was added to determine the optic density (OD) values at 515 nm on a microplate reader (TriStar 2 S LB 942 Multimode Reader, Berthold Technologies, Germany). The inhibition rates of the tumor cell growth were calculated by the following formula: The IC 50 values were calculated using the method of log (inhibitor) vs. normalized response in the software package GraphPad Prism 5.

Antibacterial Activity Assays
The antibacterial activities were evaluated by the conventional broth dilution assay (Appendino et al., 2008). Nine marine fouling bacteria, Pseudomonas fulva, Aeromonas hydrophila, A. salmonicida, Vibrio anguillarum, V. harveyi, Photobacterium halotolerans, P. angustum, Enterobacter cloacae, and E. hormaechei, were used, and cipofloxacin was used as a positive control. The initial screening of antibacterial activity assays was tested in a 96-well plate. Each well contained 198 µl tested bacterial suspension (2-5 × 10 5 CFU/ml in LB broth) and 2 µl compound (final concentration was 20 µM). Three replicates were performed. The plates were incubated at 37 • C for 24 h, and then the OD values were tested at 600 nm in a microplate reader (TriStar 2 S LB 942 Multimode Reader, Berthold Technologies, Germany). The inhibitory rates were calculated according to the following formula: The MIC values of some active target compounds were evaluated using the twofold serial dilution method. The concentrations of the compounds ranged from 100 to 6.25 µM. The other steps were the same as in the primary screening. The MIC values were calculated using the method of log (inhibitor) vs. normalized response in the software package GraphPad Prism 5.

Structure Elucidations of Isolated Compounds
Pseudotremulane A (1) was obtained as a colorless oil. Its molecular formula, C 15 H 22 O 3 , was determined by the Frontiers in Microbiology | www.frontiersin.org  HR-APCI-MS spectrum (Supplementary Figure 7), with five degrees of unsaturation. The analysis of 1 H NMR and 13 C NMR spectra (Supplementary Figures 1, 2) combined with the HSQC spectrum (Supplementary Figure 3)  , δ C 69.20, three methines, and four quaternary carbon signals, including two olefinic carbons at δ C 125.0 and δ C 138.5, and one ester group at δ C 177.8, which represented two degrees of unsaturation (Tables 1, 2). The other degrees of unsaturation revealed that there had been three rings in the structure of 1. These data suggested that 1 was tremulane-type sesquiterpenoid similar to 11,12-epoxy-12βhydroxy-1-tremulen-5-one (7; Zhou et al., 2008). There had been three obvious differences between 1 and 7. The disappeared ketone carbonyl in 7 was replaced by the arisen methylene at C-5 in 1 (Tables 1, 2); this was further confirmed by the key HMBC correlation from H-13 to C-5 (Figure 3). The HMBC correlations from H-11 to C-12, and H-4 to C-12 indicated the  ester group carbon at C-12 (Figure 3). The lower field shift of C-15 data (Tables 1, 2) compared with those of 7, combined with the HMBC correlations from H-15 to C-8, and H-14 to C-15 elucidated the oxidation of C-15 (Figure 3). Thus, the planer structure of 1 was unambiguously confirmed. The relative configurations of 1 were determined by NOESY spectra analysis (Supplementary Figure 6). The NOESY correlations between H-14 and H-8b, H-8b and H-13, and H-13 and H-3 indicated that H-14, H-13, and H-3 were in the same orientation (Figure 4). The other orientation of H-6, H-7, and H-15 was suggested by the NOESY cross-peaks of H-6/H-8a and H-7/H-15 (Figure 4). Therefore, the relative configurations of 1 were assigned as 3R * ,6R * ,7R * ,9S * . Pseudotremulane B (2) was gained as a colorless oil, with the molecular formula of C 15 H 22 O 3 determined by HR-APCI-MS indicating five degrees of unsaturation and had the same molecular formula as 1 (Supplementary Figure 14). The 1 H and 13 C NMR data of 2 were very similar to those of 1 (Tables 1, 2). The downfield shift of C-2, C-3, C-5, C-7, C-9, C-11, and C-12 and the high-field shift of C-4, C-6, C-8, C-13, and C-14 in 13 C NMR suggested the difference configurations between 1 and 2. The NOESY cross-peaks of H-15/H-3 and H-3/H-7 declared that H-3, H-7, and H-15 were in the same face (Figure 4). The NOESY correlation of H-13 and H-14 indicated that H-13 and H-14 were in another face. Therefore, the relative configurations of 2 were assigned as 3S * ,6R * ,7R * ,9S * . Pseudotremulane C (3) was acquired as a colorless oil. The HR-APCI-MS of 3 exhibited the same molecular formula with 1 and 2 (Supplementary Figure 21). The strong similar 1 H and 13 C NMR data between 2 and 3 (Tables 1, 2) suggested that they shared the same planer structures. The high-field shift of C-14 and the downfield shift of C-15 (Table 2) revealed the difference configurations of C-9 of 2 and 3. The α-orientation of H-3, H-7, and H-14 was determined by the NOESY correlations of H-3/H-7 and H-7/H-14 (Figure 4). The β-orientation of H-13 and H-15 was determined by the NOESY cross-peaks of H-13/H-8a and H-8a/H-15 (Figure 4). Compounds 2 and 3 were a pair of epimeride at the location of C-9.
Pseudotremulane D (4) was obtained as a colorless oil, with the same molecular formula with 1-3, according the analysis of its HR-APCI-MS spectrum (Supplementary Figure 28). Careful analysis of the 1 H and 13 C NMR data of 1 and 4 indicated that they had the same planer structures. The difference configurations of C-9 of 1 and 4 were determined by the highfield shift of C-14 and the downfield shift of C-15 ( Table 2) (Figure 4). Compounds 1 and 4 were a pair of epimeride at the location of C-9.
Pseudotremulane F (6) was isolated as a colorless oil. The same molecular formula of C 15 H 22 O 3 was determined by the HR-APCI-MS spectrum (Supplementary Figure 42). The three methyls, four methylenes (one oxygenated), four methines (one oxygenated), and four quaternary carbons (one ester group carbon, two olefinic, and one sp 3 quaternary carbon) exhibited in the NMR spectra (Supplementary Figures 29-31), indicating the similar structures of 6 and 5. The most obvious differences of 13 C NMR data between 6 and 5 were the downfield shift of C-1 (δ C 140.8 in 6 vs δ C 45.0 in 5) and the high-field shift of C-2 (δ C 132.6 in 6 vs δ C 162.9 in 5) and C-3 (δ C 44.7 in 6 vs δ C 128.8 in 5; Table 2), elucidating that the olefinic bond location was changed from C-2/C-3 in 5 into C-1/C-2 in 6. This was further confirmed by the HMBC correlations from H-11 to C-2, H-4 to C-2, and H-8 to C-1 (Figure 3). The β-orientation of H-13 and H-14 was revealed by the NOESY cross-peaks of H-13/H-4b and H-4b/H-14, and the α-orientation of H-3, H-7, H-15, and H-10 was suggested by the NOESY correlations of H-3/H-7, H-7/H-15, and H-15/H-10 (Figure 4).
To further conform these results, the theoretical ECDs of compounds 1-6 ( Figure 6) were calculated to compare with their experimental ECD spectra (Mazzeo et al., 2013;Cao et al., 2020). The MMFF94S method was used to conformational searches of 1a-6a to obtain the lowest energy conformers with relative energies between 0 and 10 kcal/mol. Gaussian 09 package was used to optimize the searched conformations. The first optimization was set at the gas-phase RB3LYP/6-31G(d) level to  get preferential conformations with the relative energies less than 2.5 kcal/mol. Then the conformers were optimized again at the set of gas-phase B3LYP/6-311 + G(d). The total 60 electronic excited states were calculated at the set of gas-phase RB3LYP/6-311 + + G(2d,p). Boltzmann statistics were used to simulate ECD with a standard deviation of σ 0.4 eV. The theoretical ECD spectra of 1b-6b were obtained by directly reversing the spectra of 1a-6a, respectively. The results exhibited that the experimental ECDs of 1-6 were matched well with the calculated ECDs of 1a-6a, respectively, which further verified the absolute structures of 1-6 ( Figure 6). Interestingly, compounds 1/2, 3/4, 1/4, and 2/3 were identified as four pairs of epimeride at the locations of C-3, C-3, C-9, and C-9, respectively.

DISCUSSION
The genus Pseudogymnoascus as a kind of psychrophilic pathogenic fungi is widely distributed in Antarctica (Rosa et al., 2020;Santos et al., 2020;Martorell et al., 2021). Pseudogymnoascus can be one of the antagonistic fungi against potato scab pathogens from potato field soils, which could be used as potential agents to control potato scab disease (Tagawa et al., 2010). Pseudogymnoascus spp. has been certified to be one of the predominant microbial colonizers in the root endosphere and rhizosphere of turfgrass systems (Xia et al., 2021). The extracts of some Pseudogymnoascus strains exhibit potent bioactivities, such as antimicrobial, herbicidal, and antitumoral activities (Henríquez et al., 2014;Gonçalves et al., 2015;Gomes et al., 2018;Ferrarezi et al., 2019). To the best of our knowledge, only 22 natural products, including 6 new compounds, were discovered from Pseudogymnoascus up to now (Figueroa et al., 2015;Guo et al., 2019;Fujita et al., 2021;Shi et al., 2021). More than 70% of the previously isolated structures belong to polyketides; others are alkaloids (13.6%), benzene derivative (9.1%), and fatty acid (4.5%). Our research isolated 12 natural products (1-12), including 6 new compounds (1-6), from the fungus Pseudogymnoascus 2#-11. All of the isolated compounds are first obtained from the genus Pseudogymnoascus. This is the first time to discover terpenoids and steroids from the genus Pseudogymnoascus. The whole number of the fungal strain secondary metabolites increased by 35%, and the number of their new compounds is doubled. This greatly enriches the number and diversity of natural products of the genus Pseudogymnoascus. Except for antimicrobial activities of some of the previously obtained polyketides (Figueroa et al., 2015;Fujita et al., 2021;Shi et al., 2021), no other activities were found in Pseudogymnoascus in previous studies. This study is the first to identify secondary metabolites with cytotoxic activities (8 and 9) in Pseudogymnoascus.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.

AUTHOR CONTRIBUTIONS
TS contributed to experimental design and operation, data analysis, and manuscript preparation. X-QL contributed to manuscript revision. LZ supported the sample of the Antarctic soil. Y-HZ contributed to ECD calculations. J-JD contributed to activity evaluations. E-LS contributed to software drawing guidance. Y-YY, Y-TZ, and W-PH contributed to activity evaluations. D-YS was the project leader organizing and guiding the experiments. All authors contributed to the article and approved the submitted version.

ACKNOWLEDGMENTS
We would like to thank Antarctic Great Wall National Observation and Research Station of Polar Ecosystem for sample collection; Jing-Yao Qu, Jing Zhu, and Zhi-Feng Li in MS, and Hai-Yan Sui in NMR for help and guidance from State Key Laboratory of Microbial Technology of Shandong University.