Chermebilaenes A and B, New Bioactive Meroterpenoids from Co-Cultures of Marine-Derived Isolates of Penicillium bilaiae MA-267 and Penicillium chermesinum EN-480

The co-cultivation of two or more different microbial strains in one culture vessel was supposed to be a viable experimental approach for enhancing the diversity of the compounds produced. Two new meroterpenoid derivatives, chermebilaenes A (1) and B (2), together with three known sesquiterpenoids, sesquicaranoic acid B (3), cyclonerodiol (4) and bisabol-l-on-13-säuremethylester (5), were characterized from a co-culture of the marine-derived fungal isolates of Penicillium bilaiae MA-267 and Penicillium chermesinum EN-480. Neither fungus produced these compounds when cultured alone under the same conditions. Compound 1 represents an unprecedented acorane-type sesquiterpene hybridized with an octadecadienoic acid skeleton. The structures were elucidated on the basis of spectroscopic analysis, and the absolute configurations were assumed on the basis of acidic hydrolysis combined with modified Mosher’s method and electronic circular dichroism (ECD) calculations. Compound 1 showed potent inhibitory activities against Ceratobasidium cornigerum and Edwardsiella tarda.


Introduction
Marine fungi are able to synthesize a wide range of structurally unique secondary metabolites endowed with numerous biological activities [1][2][3]. However, mining the full-genome sequences of fungi demonstrates their potential to produce many more compounds than previously expected. It is well recognized that most of the microbial biosynthesis gene clusters remain silent and are apparently not transcribed under conventional cultivation conditions [4,5]. The co-cultivation of two or more different microbial strains in one culture vessel was supposed to be a viable experimental approach for enhancing the diversity of the compounds produced [6][7][8]. In our efforts to identify new bioactive secondary metabolites from marine-derived fungi, we previously investigated the secondary metabolites of two strains of the fungal genus Penicillium, namely P. bilaiae MA-267 and P. chermesinum EN-480, from which several sesquiterpenes with a tricyclo (6.3.1.0 1,5 )dodecane skeleton [9], spiromeroterpenoids containing a drimane-type sesquiterpene skeleton [10], and sesquiterpenoids [11] have been isolated, respectively. In order to investigate the chemical potential of P. bilaiae MA-267 and P. chermesinum of P. bilaiae MA-267 and P. chermesinum EN-480, we initiated the co-culture fermentation of these two fungal strains, which showed the production of several metabolites that were not produced when the two fungi were cultured alone ( Figure S23, Supplementary Material). As a result, chermebilaene A (1), the first natural sesquiterpene hybridized with octadecadienoic acid, together with a new orthoester meroterpenoid, chermebilaene B (2), as well as three known sesquiterpenoids, sesquicaranoic acid B (3), cyclonerodiol (4) and bisabol-l-on-13-säuremethylester (5) (Figure 1), were isolated from the co-culture extract of P. bilaiae MA-267 and P. chermesinum EN-480. Herein, details of the isolation, structure determination, and biological activities of these compounds are described.
Mar. Drugs 2020, 18, x FOR PEER REVIEW 3 of 10 respectively. This deduction was further supported by the COSY and HMBC correlations ( Figure 2). This is the first time that this type of meroterpenoid, which contains an unprecedented acorane-type sesquiterpene hybridized with octadecadienoic acid skeleton, has been described. The relative configuration of 1 was determined by the analysis of the J-values and NOESY (nuclear overhauser effect spectroscopy) data ( Figure 3). The coupling constants between H-9'/13' and H-10'/12' (10.9 Hz) suggested the geometry of the C=C bond at C-9' and C-13' to be Z. Moreover, NOE correlations from H3-14 to H-1, H-6β, and H-7, and from H-7 to H-1, suggested the same orientation of these groups, while the correlations from H-2 to H-4 placed these protons on the opposite face. To confirm the structure and absolute configuration of 1, we pursued a strategy consisting of the acidic hydrolysis of 1 to yield the sesquiterpene diol (6) and 9,12-octadecadienoic acid (7), followed by modified Mosher's method for the obtained 6, and NMR analysis of the resulting Mosher esters allowed the assignment of the 2R and 7S absolute configuration in compound 6 ( Figure 4) and, consequently, the absolute configuration of compound 1 was deduced to be 1R, 2R, 4S, 5S, and 7S.  The relative configuration of 1 was determined by the analysis of the J-values and NOESY (nuclear overhauser effect spectroscopy) data ( Figure 3). The coupling constants between H-9'/13' and H-10'/12' (10.9 Hz) suggested the geometry of the C=C bond at C-9' and C-13' to be Z. Moreover, NOE correlations from H 3 -14 to H-1, H-6β, and H-7, and from H-7 to H-1, suggested the same orientation of these groups, while the correlations from H-2 to H-4 placed these protons on the opposite face.
Mar. Drugs 2020, 18, x FOR PEER REVIEW 3 of 10 respectively. This deduction was further supported by the COSY and HMBC correlations ( Figure 2). This is the first time that this type of meroterpenoid, which contains an unprecedented acorane-type sesquiterpene hybridized with octadecadienoic acid skeleton, has been described. The relative configuration of 1 was determined by the analysis of the J-values and NOESY (nuclear overhauser effect spectroscopy) data ( Figure 3). The coupling constants between H-9'/13' and H-10'/12' (10.9 Hz) suggested the geometry of the C=C bond at C-9' and C-13' to be Z. Moreover, NOE correlations from H3-14 to H-1, H-6β, and H-7, and from H-7 to H-1, suggested the same orientation of these groups, while the correlations from H-2 to H-4 placed these protons on the opposite face.  To confirm the structure and absolute configuration of 1, we pursued a strategy consisting of the acidic hydrolysis of 1 to yield the sesquiterpene diol (6) and 9,12-octadecadienoic acid (7), followed by modified Mosher's method for the obtained 6, and NMR analysis of the resulting Mosher esters allowed the assignment of the 2R and 7S absolute configuration in compound 6 ( Figure 4) and, consequently, the absolute configuration of compound 1 was deduced to be 1R, 2R, 4S, 5S, and 7S. To confirm the structure and absolute configuration of 1, we pursued a strategy consisting of the acidic hydrolysis of 1 to yield the sesquiterpene diol (6) and 9,12-octadecadienoic acid (7), followed by modified Mosher's method for the obtained 6, and NMR analysis of the resulting Mosher esters allowed the assignment of the 2R and 7S absolute configuration in compound 6 ( Figure 4) and, consequently, the absolute configuration of compound 1 was deduced to be 1R, 2R, 4S, 5S, and 7S.
respectively. This deduction was further supported by the COSY and HMBC correlations ( Figure 2). This is the first time that this type of meroterpenoid, which contains an unprecedented acorane-type sesquiterpene hybridized with octadecadienoic acid skeleton, has been described. The relative configuration of 1 was determined by the analysis of the J-values and NOESY (nuclear overhauser effect spectroscopy) data ( Figure 3). The coupling constants between H-9'/13' and H-10'/12' (10.9 Hz) suggested the geometry of the C=C bond at C-9' and C-13' to be Z. Moreover, NOE correlations from H3-14 to H-1, H-6β, and H-7, and from H-7 to H-1, suggested the same orientation of these groups, while the correlations from H-2 to H-4 placed these protons on the opposite face. To confirm the structure and absolute configuration of 1, we pursued a strategy consisting of the acidic hydrolysis of 1 to yield the sesquiterpene diol (6) and 9,12-octadecadienoic acid (7), followed by modified Mosher's method for the obtained 6, and NMR analysis of the resulting Mosher esters allowed the assignment of the 2R and 7S absolute configuration in compound 6 ( Figure 4) and, consequently, the absolute configuration of compound 1 was deduced to be 1R, 2R, 4S, 5S, and 7S.  Chermebilaene B (2) was isolated as a colorless, amorphous solid with a determined molecular formula of C 25 H 36 O 9 (eight degrees of unsaturation) on the basis of (+)-HRESIMS (high resolution electrospray ionization mass spectroscopy) data. The 1 H and 13 C NMR spectroscopic data (Table 1) contained 25 carbon signals, which were sorted by DEPT and HSQC experiments into the following categories: six methyls, five methylenes (with one oxygenated), six methines (with two oxygenated), and eight quaternary carbons (with two carbonyl and four oxygenated), as well as two exchangeable protons. Detailed analysis of the 1 H and 13 C NMR spectroscopic data revealed that 2 was a spiromeroterpenoid derivative, similar to asnovolin G isolated from the fungus Aspergillus novofumigatus [14]. However, one of the methylenes resonating at δ C/H 28.4/1.61 (CH 2 -7) in asnovolin G was replaced by an oxymethine unit in 2, as shown by the HRESIMS data as well as by the observation of additional resonance signals at δ C 67.0/δ H 3.71 (CH-7) and δ H 4.54 (7-OH) in the NMR spectra of 2 ( Table 2). The deduction was supported by the COSY and HMBC correlations (Figure 2).
The relative configuration was determined by the detailed analysis of the NOESY data ( Figure 3). Key NOESY correlations from H-13α to H 3 -15 and H 3 -12, and from the protons of 7-OH to H 3 -12, revealed the cofacial orientation of these groups, while the NOE cross-peaks from H-5 to H-1α and H-7 placed these groups on the opposite face, which determined the relative configuration of the A/B rings. Furthermore, the NOE correlations from H-3' to H 3 -8' and H-4', and from H-5' to H-4' and H-11β, indicated the cofacial orientation of these groups, while correlations from H 3 -9' to H 3 -10' placed the two methyls on the opposite face. The (5R*, 7S*, 8S*, 9S*, 10S*, 1'S*, 2'S*, 3'S*, 4'R*, 5'S*, 6'R*) relative configuration could thus be deduced for 2.
Mar. Drugs 2020, 18, x FOR PEER REVIEW 4 of 10 Chermebilaene B (2) was isolated as a colorless, amorphous solid with a determined molecular formula of C25H36O9 (eight degrees of unsaturation) on the basis of (+)-HRESIMS (high resolution electrospray ionization mass spectroscopy) data. The 1 H and 13 C NMR spectroscopic data (Table 1) contained 25 carbon signals, which were sorted by DEPT and HSQC experiments into the following categories: six methyls, five methylenes (with one oxygenated), six methines (with two oxygenated), and eight quaternary carbons (with two carbonyl and four oxygenated), as well as two exchangeable protons. Detailed analysis of the 1 H and 13 C NMR spectroscopic data revealed that 2 was a spiromeroterpenoid derivative, similar to asnovolin G isolated from the fungus Aspergillus novofumigatus [14]. However, one of the methylenes resonating at δC/H 28.4/1.61 (CH2-7) in asnovolin G was replaced by an oxymethine unit in 2, as shown by the HRESIMS data as well as by the observation of additional resonance signals at δC 67.0/δH 3.71 (CH-7) and δH 4.54 (7-OH) in the NMR spectra of 2 ( Table 2). The deduction was supported by the COSY and HMBC correlations (Figure 2).

Biological Activities of the Isolated Compounds
The isolated compounds, as well as the hydrolysis products 6 and 7, were evaluated for antimicrobial activity [19] against nine human-and aqua-pathogenic bacteria, Aeromonas hydrophilia, Edwardsiella ictarda, E. tarda, Escherichia coli, Staphylococcus aureus, Vibrio alginolyticus, V. anguillarum, V. harveyi, and V. parahemolyticus, as well as four plant-pathogenic fungi, Alternaria solani, Ceratobasidium cornigerum, Colletotrichum glecosporioides, and Fusarium graminearum ( Table 2). Compound 1 exhibited significant activity against Edwardsiella tarda and Ceratobasidium cornigerum, with MIC values of 0.25 and 0.5 µg/mL, respectively, whereas one of its hydrolysis product 9,12-octadecadienoic acid (7) demonstrated activities against each of the tested pathogens, with MIC (minimum inhibitory Mar. Drugs 2020, 18, 339 6 of 10 concentration) values ranging from 1.0 to 8.0 µg/mL. In contrast, compounds 2-6 were inactive toward all pathogens in our experiments. These data indicated that the incorporation of a fatty acid into the acorane-type sesquiterpenoid derivative likely significantly increases the antimicrobial activity. The addition of a fatty acid could improve the penetration into the cell membrane, which may cause the antimicrobial activity [20]. Table 2. Antimicrobial activities of compounds 1-7 (MIC, µg/mL) a .

Fungal Material
The fungus Penicillium bilaiae MA-267 was isolated from the rhizosphere of the marine mangrove plant Lumnitzera racemosa that was collected at Hainan Island, P. R. China, in March 2013. P. chermesinum EN-480 was isolated from the fresh tissue of marine red algal Pterocladiella tenuis, collected from Shandong province, P. R. China, in July 2014. The fungi were identified as Penicillium bilaiae and Penicillium chermesinum, respectively, by sequence analysis of the ITS (internal transcribed spacer) regions of their 18S rDNA, as described previously [21]. The resulting sequence data obtained were deposited in GenBank (accession nos. KP096311 for P. bilaiae MA-267 and KT119566 for P. chermesinum EN-480). Both strains EN-480 and MA-267 are preserved at the Key Laboratory of Experimental Marine Biology, Institute of Oceanology of the Chinese Academy of Sciences.

Fermentation
P. bilaiae MA-267 and P. chermesinum EN-480 were each grown on PDA (potato dextrose agar) medium at 28 • C for four days, and P. chermesinum EN-480 was then inoculated into 1 L conical flasks (100 × 100 mL, a total of 10 L), each containing 100 mL of rice medium (70 g rice, 0.3 g peptone, 0.1 g corn syrup, and 100 mL naturally sourced and filtered seawater that was obtained from the Huiquan Gulf of the Yellow Sea near the campus of IOCAS) at room temperature. After 3 days, a full loop of P. bilaiae MA-267 was transferred aseptically to each flask culture of P. chermesinum EN-480 and re-incubated at room temperature for 28 days.
Mar. Drugs 2020, 18, x FOR PEER REVIEW 7 of 10 P. bilaiae MA-267 was transferred aseptically to each flask culture of P. chermesinum EN-480 and reincubated at room temperature for 28 days.

Hydrolysis of Compound 1
Compound 1 (10.0 mg, 18.5 µM) was dissolved in 5 mL of THF/MeOH/H 2 O (2/2/1) mixed solvent, to which an excess amount of lithium hydroxide (10.5 mg, 43.7 µM) was added. The reaction mixture was allowed to stir at room temperature for 36 h and was then evaporated to dryness under reduced pressure. The progress of the reaction was monitored by TLC analysis. The resulting reaction mixture was then dissolved in 2 mL of 10wt% NaH 2 PO 4 , extracted with dichloromethane (3 × 4 mL), dried with Na 2 SO 4 , and then concentrated in vacuo to obtain the product.

Conclusions
In summary, we isolated and identified two new meroterpenoid derivatives (1 and 2), as well as three known sesquiterpenoids (3−5), from the co-culture broth of two marine-derived fungi, P. bilaiae MA-267 and P. chermesinum EN-480, whereas neither fungus could produce these compounds when cultured alone. It is noteworthy that compound 1 represents an unprecedented acorane-type sesquiterpene hybridized with an octadecadienoic acid skeleton, and it may prove useful as an antibiotic agent against aquatic or plant pathogens. The fact that co-cultivation in the present study induced the production of new fungal metabolites with improved antimicrobial activity demonstrates the general value of such co-cultivation experiments and encourages future studies.