Bioprospection of Tenellins Produced by the Entomopathogenic Fungus Beauveria neobassiana

Fungi are known as prolific producers of bioactive secondary metabolites with applications across various fields, including infectious diseases, as well as in biological control. However, some of the well-known species are still underexplored. Our current study evaluated the production of secondary metabolites by the entomopathogenic fungus Beauveria neobassiana from Thailand. The fermentation of this fungus in a liquid medium, followed by preparative high-performance liquid chromatography (HPLC) purification, resulted in the isolation of a new tenellin congener, namely pretenellin C (1), together with five known derivatives (2–6). Their chemical structures were elucidated by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy in combination with high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). We evaluated the antimicrobial and cytotoxic activities from all isolated compounds, as well as their inhibitory properties on biofilm formation by Staphylococcus aureus. Generally, tenellins displayed varying antibiofilm and cytotoxic effects, allowing us to propose preliminary structure-activity relationships (SARs). Among the tested compounds, prototenellin D (2) exhibited the most prominent antibiofilm activity, while its 2-pyridone congener, tenellin (4), demonstrated potent cytotoxic activity against all tested cell lines. Given the fact that the biological activities of the tenellins have so far been neglected in the past, our study could provide a good starting point to establish more concise structure-activity relationships in the near future.


Introduction
Fungi, renowned for their valuable contributions across a range of different fields, are also recognized for being creative secondary metabolite producers.These compounds have significantly benefited society, particularly in antibiotic development, as exemplified by the pleuromutilins, one of the most recent antibiotics class introduced to the market [1].However, the value of these organisms extends beyond the control of infectious diseases, as fungi are instrumental in a wealth of applications, such as in agriculture, where they serve as biocontrol agents [2].The interactions within fungal ecosystems have been leveraged to create environmentally friendly alternatives for pest control, as in the case of entomopathogenic fungi, which act against specific pests while sparing other organisms from harm [3].
Species belonging to the genus Beauveria (Cordycipitaceae, Ascomycota) are widely employed in agriculture as biocontrol agents [4].Recent taxonomic revisions of this genus, facilitated by a combination of whole-genome sequencing, morphometric analysis, and chemical studies, have aided in the delimitation of the Beauveria asiatica and Beauveria bassiana (sensu lato) species complexes [4].Other studies illustrated the biosynthetic potential of Beauveria spp. to produce diverse secondary metabolites, encompassing sesquiterpenes, steroids, tricyclic diterpenoids, cyclodepsipeptides, and various alkaloids [5,6].Notably, compounds like beauverolides and beauvericins are known for their multifaceted roles as insecticidal, cytotoxic, and antimicrobial agents [4,7].Similarly, the tenellins, polyketide synthase/nonribosomal-peptide synthetase (PKS-NRPS) products, originally isolated from B. bassiana, have been extensively studied regarding their biosynthesis [8].However, only a limited number of studies have explored the biological functions of these secondary metabolites, and yet the full potential of these fungi remains largely untangled.
The knowledge gap extends to the complex interplay of fungal secondary metabolites and their usage in fighting the rapid increase of microbial resistance.For instance, biofilms formed by microbes often play a key role during chronic infections as they reduce the effectiveness of antibiotics, resulting in the overall increase of resistance.Nonetheless, no effective treatment for biofilm formation has been developed so far [9].
During our ongoing search for novel biofilm inhibitors, we investigated the production of secondary metabolites by the entomopathogenic fungus B. neobassiana from Thailand.The results are reported in the following narrative.
Analytical HPLC chromatograms and electrospray ionization mass spectra (ESI-MS) were acquired using an UltiMate 3000 Series UPLC (Thermo Fischer Scientific ® , Waltham, MA, USA) equipped with a C18 column (Acquity UPLC BEH 2.1 × 50 mm, 1.7 µm; Waters, Milford, MA, USA) and an amaZon speed ESI-Iontrap-MS (Bruker) using a sample injection volume of 2 µL and a flow rate of 0.6 mL/min.A gradient elution was applied using a mobile phase consisting of solvent A (H 2 O + 0.1% formic acid (v/v)) and solvent B (MeCN + 0.1% formic acid (v/v)).The gradient started at 5% B, gradually increasing to 100% B over 20 min, followed by a 10 min hold at 100% B and UV-Vis detection in the range from 190 to 600 nm.

Fungal Material
The strain Beauveria neobassiana Khons., Kobmoo & Luangsa-ard BCC 31604 was found growing on an adult unidentified beetle (Coleoptera) and collected and isolated on 22 July 2008, by a team including A. Khonsanit, J.J. Luangsa-ard, K. Tasanathai, P. Srikitikulchai, and S. Mongkolsamrit.The collection site was in Chiang Rai Province, Chiang Khong District, along the scenic Phlu Kaeng Waterfall Nature Trail, located at coordinates 20.26 • N latitude and 100.39 • E longitude.The herbarium material and the culture of the fungus are located in the collections of BIOTEC National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand, under the designation nos.BBH 23856 (specimen) and BCC 31604 (mycelial culture), respectively.The culture represents paratype material.The complete identification and characterization of the fungus was recently reported in an integrative phylogenomic study [4], in which mostly the molecular phylogenetic data served for segregation of the new species.

Cultivation, Extraction and Isolation
Beauveria neobassiana BCC 31604 was cultured on potato dextrose agar (PDA; Fisher Scientific GmbH, Schwerte, Germany) plates for 7 days.A total of 5 mycelial plugs were carefully removed using a 7 mm diameter cork borer.The plugs were transferred into 500 mL Erlenmeyer flasks, each containing 200 mL of potato dextrose broth medium (PDB; 24 g/L; Fisher Scientific GmbH).For the main cultures, 50 flasks were used in total (10 L).The cultures were then incubated at 23 • C and 140 rpm.The glucose level was routinely monitored using urine glucose test strips (Dirui Industrial Co., Ltd., Changchun, China).The cultures were then harvested three days after the glucose was depleted.The mycelia were separated from the supernatant by vacuum filtration, and extracted separately.
A liquid-liquid extraction was conducted twice for the supernatant using ethyl acetate (1:1).The resulting organic phases were combined, filtered through anhydrous sodium sulfate, and evaporated under vacuum, while the aqueous phase was discarded.The mycelia were extracted with acetone (2 × 1 L) and subjected to two rounds of ultra-sonic bathing for 30 min at 40 • C. The resultant suspensions were filtered, and the acetone phase was collected and evaporated under vacuum.The resulting semi-solid residue from mycelial extract was then dispersed in 100 mL of water and subjected to liquid-liquid extraction against ethyl acetate as previously described.The obtained total extracts from the mycelia (91 mg) and the supernatant (269 mg) were analyzed by LC-MS and thereafter purified by preparative HPLC.

Biological Assays
All isolated compounds were assessed for their antimicrobial activity using a serial dilution assay over a concentration range from 67 to 0.5 µg/mL following the previously described protocols [11,12].In brief, the antimicrobial activity of all isolated metabolites was assessed by determining their minimum inhibitory concentration (MIC) against a range of different pathogens, including five fungal species: Candida albicans In our experiments, gentamicin served as the positive control against most bacteria, while nystatin acted as the positive control against all fungi.For specific microorganisms, namely A. baumannii, B. subtilis, and M. smegmatis, ciprofloxacin, oxytetracycline, and kanamycin were used as positive controls, respectively.
The cytotoxicity of isolated compounds was determined as previously described [11,12] over a concentration range between 1 and 37 µg/mL against two mammalian cell lines: mouse fibroblasts (L-929) and human endocervical adenocarcinoma (KB-3.1).For compounds that exhibited cytotoxic properties against the first two cell lines, we also evaluated their cytotoxicity against breast cancer (MCF-7) and lung cancer (A-549) cell lines.Epothilone B served as the positive control.Following 5 days of incubation, we determined the minimum concentration of the tested compounds required to achieve 50% growth inhibition, expressed as IC 50 values.
The compounds were evaluated for their ability to inhibit S. aureus (DSM 1104) biofilm formation, following the established procedure [13].Briefly, serially diluted compounds (125-7.8µg/mL) were co-incubated with S. aureus in 96-well plates in CASO medium supplemented with 4% of glucose (see Supplementary Materials).Biofilm inhibition was assessed by using Crystal Violet staining after 21 h, with microporenic acid A serving as a positive control and methanol as solvent control [14].

Isolation and Structure Elucidation of Secondary Metabolites
Six secondary metabolites (1-6) were isolated after the scaled-up fermentation of B. neobassiana in PDB medium and subsequent purification of the obtained total extracts.As can be seen in Figure 1, the composition of the mycelial and culture filtrate extracts was similar, but the more lipophilic components 3 and 4 were preferentially located in the mycelial extracts.To establish their chemical structures, each of these compounds was analyzed using HR-ESI-MS, 1D-and 2D-NMR spectroscopy.
compounds that exhibited cytotoxic properties against the first two cell lines, we also evaluated their cytotoxicity against breast cancer (MCF-7) and lung cancer (A-549) cell lines.Epothilone B served as the positive control.Following 5 days of incubation, we determined the minimum concentration of the tested compounds required to achieve 50% growth inhibition, expressed as IC50 values.
The compounds were evaluated for their ability to inhibit S. aureus (DSM 1104) biofilm formation, following the established procedure [13].Briefly, serially diluted compounds (125-7.8µg/mL) were co-incubated with S. aureus in 96-well plates in CASO medium supplemented with 4% of glucose (see Supplementary Materials).Biofilm inhibition was assessed by using Crystal Violet staining after 21 h, with microporenic acid A serving as a positive control and methanol as solvent control [14].

Isolation and Structure Elucidation of Secondary Metabolites
Six secondary metabolites (1-6) were isolated after the scaled-up fermentation of B. neobassiana in PDB medium and subsequent purification of the obtained total extracts.As can be seen in Figure 1, the composition of the mycelial and culture filtrate extracts was similar, but the more lipophilic components 3 and 4 were preferentially located in the mycelial extracts.To establish their chemical structures, each of these compounds was analyzed using HR-ESI-MS, 1D-and 2D-NMR spectroscopy.Compound 1 was obtained as a yellow amorphous solid, and its molecular formula was established as C 12 H 9 NO 6 indicating nine degrees of unsaturation based on the HR-ESI-MS spectrum (Figure S2) revealing a protonated molecule at m/z 264.0499 [M+H] + (calculated 264.0503).The 1 H NMR spectrum of 1 (Table 1, Figure S3) revealed the presence of two proton resonances at δ H 7.42 and δ H 6.92, each with an integration index of 2, and both appeared as a doublet peak with an identical coupling constant (J value) of 8.3 Hz, indicating that compound 1 comprises a para-disubstituted aromatic ring.In addition, 1 also revealed two more deshielded singlet proton peaks at δ H 8.18 directly correlated via the HSQC spectrum (Figure S6) to a deshielded olefinic carbon at δ C 138.7, suggesting its presence in a heterocyclic aromatic ring and a second broad peak at δ H 13.90 suggesting that 1 might comprise in its structure a carboxylic acid moiety.
By comparing the obtained results with the reported literature, compound 1 was tentatively identified as a 2-pyridone derivative [8].To further confirm the depicted structure of 1, its HMBC spectrum (Figures 2 and S5) revealed key correlations from the deshielded aromatic proton at δ H 8.18 (H-6) to four aromatic carbons distinguished into a carbonyl carbon (δ C 161.2), an oxygenated aromatic carbon (δ C 170.5), and two other unprotonated carbons (δ C 123.6 and δ C 115.3) that were assigned to C-2, C-4, C-5, and C-8, respectively.The HMBC spectrum of 1 (Figures 2 and S5) revealed key correlations from two electromagnetically equivalent aromatic protons at δ H 7.42 (d, J = 8.3 Hz, H-9/H-13) to a carbon resonance at δ C 123.6 (C-4), confirming the presence of the phenyl moiety at C-4 of the 2-pyridone ring.By comparing the obtained 1 H and 13 C NMR data of 1 (Table 1) to those obtained and reported for pretenellin B (3) and 15-hydroxytenellin (5) [8], compound 1 was confirmed to feature N-OH and 3-carboxylic acid groups in its structure and hence to be a previously undescribed tenellin derivative that was trivially named as pretenellin C. Compound 1 was obtained as a yellow amorphous solid, and its molecular formula was established as C12H9NO6 indicating nine degrees of unsaturation based on the HR-ESI-MS spectrum (Figure S2) revealing a protonated molecule at m/z 264.0499 [M+H] + (calculated 264.0503).The 1 H NMR spectrum of 1 (Table 1, Figure S3) revealed the presence of two proton resonances at δH 7.42 and δH 6.92, each with an integration index of 2, and both appeared as a doublet peak with an identical coupling constant (J value) of 8.3 Hz, indicating that compound 1 comprises a para-disubstituted aromatic ring.In addition, 1 also revealed two more deshielded singlet proton peaks at δH 8.18 directly correlated via the HSQC spectrum (Figure S6) to a deshielded olefinic carbon at δC 138.7, suggesting its presence in a heterocyclic aromatic ring and a second broad peak at δH 13.90 suggesting that 1 might comprise in its structure a carboxylic acid moiety.
By comparing the obtained results with the reported literature, compound 1 was tentatively identified as a 2-pyridone derivative [8].To further confirm the depicted structure of 1, its HMBC spectrum (Figures 2 and S5) revealed key correlations from the deshielded aromatic proton at δH 8.18 (H-6) to four aromatic carbons distinguished into a carbonyl carbon (δC 161.2), an oxygenated aromatic carbon (δC 170.5), and two other unprotonated carbons (δC 123.6 and δC 115.3) that were assigned to C-2, C-4, C-5, and C-8, respectively.The HMBC spectrum of 1 (Figures 2 and S5) revealed key correlations from two electromagnetically equivalent aromatic protons at δH 7.42 (d, J = 8.3 Hz, H-9/H-13) to a carbon resonance at δC 123.6 (C-4), confirming the presence of the phenyl moiety at C-4 of the 2pyridone ring.By comparing the obtained 1 H and 13 C NMR data of 1 (Table 1) to those obtained and reported for pretenellin B (3) and 15-hydroxytenellin (5) [8], compound 1 was confirmed to feature N-OH and 3-carboxylic acid groups in its structure and hence to be a previously undescribed tenellin derivative that was trivially named as pretenellin C.

Biological Effects of Tenellin-Derived Metabolites
All isolated compounds were examined for their cytotoxic activity against two cell lines, L929 (fibroblast) KB3.1 (ovary).The obtained results (Table 2) revealed that tenellin (4) was an equi-and the most potent against both cell lines with IC 50 values of 0.79 µM.In addition, 15-hydroxytenellin (5) and pyridovericin (6) exhibitied less potent cytotoxic activities against the same cell lines with IC 50 values ranging from 4.9 to 6.8 µM, while pretenellin C (1), prototenellin D (2) and pretenellin B (3) showed weak or no activity.Compounds 4-6 were further assessed against two additional cell lines namely, A-549 (lung) and MCF-7 (breast), where tenellin kept its leading potency with IC 50 values of 0.24 and 2.0 µM, respectively, compared to epothilone B, a microtubule stabilizer first reported from the myxobacterium Sorangium cellulosum [15] and exhibited cytotoxic activity in paclitaxel-resistant tumor models [16].Therefore, tenellin (4) with IC 50 values down to low micromolar or nanomolar ranges could be noted as a promising scaffold to derivatize for developing a hit molecule for a cytotoxic lead compound.All the isolated compounds were assessed for their antimicrobial activity against a panel of twelve microorganisms including Gram positive, Gram negative bacterial and fungal strains.The obtained results (Table 3) revealed that compounds 3-6 featured moderate to potent activities in particular against S. aureus and B. subtilis with tenellin (4) recognized as the most active at MIC values of 16.6 and 8.3 µg/mL, respectively.Furthermore, compounds 1-6 were evaluated for their antibiofilm properties against the pathogenic bacterium S. aureus.All tested compounds exhibited moderate inhibitory activities towards S. aureus (Figure 3, Table S1), with prototenellin D (2) being the most effective one.It inhibited the formation of S. aureus biofilms for more than 50% up to a concentration of 7.8 µg/mL.However, biofilm inhibition of pretenellin B (3) and tenellin (4) were also quite notable with activities of ca.30% lasting till concentration of 7.8 µg/mL.Pretenellin C (1) and pyridovericin (6) were least active with activity loss below concentrations of 31.3 µg/mL and 62.5 µg/mL (Table S1), respectively.

Discussion
Secondary metabolites featuring the 2-pyridone moiety are previously reported from different sources with a wide range of biological activities as exemplified by furanpyridones [17], aspyridones [18] and ricinine [19].Tenellins, 2-pyridone-containing compounds previously reported from Beauveria bassiana [8,10], have served as model compounds for investigating the biosynthesis of fungal metabolites.Exploring their biosynthetic pathway unveiled their respective biosynthetic gene clusters (BGCs) [8].Remarkably, similar metabolites have been shown to exhibit a broad range of bioactivities including prominent neuritogenic and cytotoxic activities [20] in addition to their antimicrobial and antimalarial properties [21].
Herein, six tenellin derivatives were obtained from B. neobassiana distinguished into five known (2-6) alongside with one previously undescribed derivative, pretenellin C (1) whose closely related derivative, lacking the hydroxyl group at C-11, was patented in 2004 as an antiviral agent against hepatitis C virus [22].
In the cytotoxicity assay, tenellin (4) was the most potent suggesting a plausible role for both the 2-pyridone moiety and the aliphatic side chain in comparison to pretenellin C (1) and prototenellin D (3), respectively.The aliphatic side chain may impart higher lipophilicity, thus facilitating cell wall penetration.This notion was further supported by the observed 2-to 10-fold reduction in IC50 values for 5 and 6 compared to 4, both featured an additional hydroxyl group at C-15 that might hinder cell penetration by raising the side chain polarity.On the contrary, pretenellin C (1) and prototenellin D (2), lacking the aliphatic side chain or 2-pyridone moiety, respectively, displayed no cytotoxicity that further supports the proposed structure-activity relationships.Intriguingly, the presence of Nhydroxylation, in 4 and 5 compared to 6, did not reveal a notable influence on their cytotoxic effects.

Discussion
Secondary metabolites featuring the 2-pyridone moiety are previously reported from different sources with a wide range of biological activities as exemplified by furanpyridones [17], aspyridones [18] and ricinine [19].Tenellins, 2-pyridone-containing compounds previously reported from Beauveria bassiana [8,10], have served as model compounds for investigating the biosynthesis of fungal metabolites.Exploring their biosynthetic pathway unveiled their respective biosynthetic gene clusters (BGCs) [8].Remarkably, similar metabolites have been shown to exhibit a broad range of bioactivities including prominent neuritogenic and cytotoxic activities [20] in addition to their antimicrobial and antimalarial properties [21].
Herein, six tenellin derivatives were obtained from B. neobassiana distinguished into five known (2-6) alongside with one previously undescribed derivative, pretenellin C (1) whose closely related derivative, lacking the hydroxyl group at C-11, was patented in 2004 as an antiviral agent against hepatitis C virus [22].
In the cytotoxicity assay, tenellin (4) was the most potent suggesting a plausible role for both the 2-pyridone moiety and the aliphatic side chain in comparison to pretenellin C (1) and prototenellin D (3), respectively.The aliphatic side chain may impart higher lipophilicity, thus facilitating cell wall penetration.This notion was further supported by the observed 2-to 10-fold reduction in IC 50 values for 5 and 6 compared to 4, both featured an additional hydroxyl group at C-15 that might hinder cell penetration by raising the side chain polarity.On the contrary, pretenellin C (1) and prototenellin D (2), lacking the aliphatic side chain or 2-pyridone moiety, respectively, displayed no cytotoxicity that further supports the proposed structure-activity relationships.Intriguingly, the presence of N-hydroxylation, in 4 and 5 compared to 6, did not reveal a notable influence on their cytotoxic effects.

Figure 1 .
Figure 1.(A) HPLC−UV/Vis chromatograms (210 nm) of the crude extracts obtained from the mycelia and supernatant from the scaled-up fermentation of B. neobassiana in PDB medium.(B) Chemical structures of the isolated metabolites 1−6.

Figure 1 .
Figure 1.(A) HPLC−UV/Vis chromatograms (210 nm) of the crude extracts obtained from the mycelia and supernatant from the scaled-up fermentation of B. neobassiana in PDB medium.(B) Chemical structures of the isolated metabolites 1-6.
4, C a Measured in DMSO-d6 at 500 MHz.b Assigned based on HMBC and HSQC spectra.c n.d.:Not determined.

Figure 3 .
Figure 3. Inhibitory activity of compounds 1-6 and microporenic acid (MAA, positive control) on biofilm formation of S. aureus compared to the solvent control (MeOH, 0% biofilm inhibition).Error bars indicate SD of triplicates; p values: ** p < 0.01, *** p < 0.001.Differences between samples and the control group were determined by a two-tailed Student's t-test.Statistical significance was defined as p < 0.01.

Figure 3 .
Figure 3. Inhibitory activity of compounds 1-6 and microporenic acid (MAA, positive control) on biofilm formation of S. aureus compared to the solvent control (MeOH, 0% biofilm inhibition).Error bars indicate SD of triplicates; p values: ** p < 0.01, *** p < 0.001.Differences between samples and the control group were determined by a two-tailed Student's t-test.Statistical significance was defined as p < 0.01.
a Measured in DMSO-d 6 at 500 MHz.b Assigned based on HMBC and HSQC spectra.c n.d.:Not determined.

Table 1 .
1H and 13 C NMR data of 1