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Article

The Potential of Arctic Pseudogymnoascus Fungi in the Biosynthesis of Natural Products

by
Tatiana V. Antipova
1,*,
Kirill V. Zaitsev
2,*,
Valentina P. Zhelifonova
1,
Sergey V. Tarlachkov
1,
Yuri K. Grishin
2,
Galina A. Kochkina
1 and
Mikhail B. Vainshtein
1
1
G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, FRC Pushchino Scientific Centre of Biological Research, Russian Academy of Sciences, 142290 Pushchino, Russia
2
Department of Chemistry, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia
*
Authors to whom correspondence should be addressed.
Fermentation 2023, 9(8), 702; https://doi.org/10.3390/fermentation9080702
Submission received: 21 June 2023 / Revised: 21 July 2023 / Accepted: 24 July 2023 / Published: 26 July 2023
(This article belongs to the Special Issue Pharmaceutical Fermentation: Antibiotic Production and Processing)
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Abstract

:
Scarce research into the secondary metabolites of the fungi Pseudogymnoascus spp. has shown a hidden biosynthetic potential for biologically active compounds. This work investigated the biosynthesis of secondary metabolites by two Pseudogymnoascus fungal strains, VKM F-4518 and VKM F-4519, isolated from the surface soil layer of the Kolyma Lowland, Russia, in the Arctic. In these strains, 16-membered trilactone macrolides, (+)-macrosphelides A and B, were identified using 1D and 2D NMR, UHRMS, and optical rotation data. In the fungi of this genus, these metabolites were found for the first time. The studied strains are highly active producers of macrosphelide A, which is being considered as a promising agent for the cure of cancer. Using the antiSMASH secondary metabolite analysis tool, we found that the genome of strain VKM F-4518 contained 32 of the biosynthetic clusters of the secondary metabolite genes (BGC) and that of VKM F-4519 had 17 BGCs. Based on the comparison of the cluster of macrotriolide genes from the fungus Paraphaeosphaeria sporulosa, we found the complete supposed cluster BGCs of macrosphelides in the genomes of two Pseudogymnoascus strains using the BLAST+ program.

1. Introduction

Secondary metabolites (SM) are a promising object of research due to the possibility of discovering substances with a new mechanism of medical action and less toxicity. In this regard, the search for biologically active natural compounds is constantly ongoing in order to discover new and/or more effective drugs [1]. Fungi are well known as sources of diverse biologically active compounds [2,3]. Fungal SMs exhibit a variety of biological activities: they are used as antibiotics, fungicides, immunomodulators, antioxidants, and natural colorants [4,5,6,7]. For this reason, fungi are the most interesting object of special and close attention for searching for new biologically active compounds. The role of these microorganisms as the main drug sources for the pharmaceutical industry is known and obvious.
The variety of SMs identified in fungi is significant, and it is assumed that only a minority of all naturally occurring SMs are currently known [8]. The best-known SMs belong to four basic chemical families: polyketides, terpenoids, nonribosomal peptides, and a hybrid of nonribosomal peptides and polyketides [4]. As a rule, SMs are synthesized using primary metabolic substrates, among which, acetyl-CoA stands out as a precursor of polyketides and terpenoids [9]. Each SM biosynthetic pathway begins with a characteristic enzyme type and ends with the activity of other specific enzymes, which introduce additional modifications to molecules. At present, more than 1000 fungal genomes are known and available as a result of achievements in proteomics, genomics, and sequencing methods [8]. Nevertheless, despite several decades of efforts towards discovering new compounds from fungi, the analysis of genomes for the presence of clusters of their biosynthetic genes is still quite limited [10].
The fungi Pseudogymnoascus spp. Railo 1929 with Geomyces anamorphs are widespread in nature and can be found almost everywhere, from the Arctic to the Antarctic [11,12,13,14]. They can exist in the marine environment and be spread by deep-sea currents [15]. Very often, they are isolated from low-temperature ecotopes. These fungi are not truly psychrophiles or halophiles, but are capable of developing in cold environments (even at slightly negative temperatures) with a reduced level of nutrition and increased salt content [16]. These conditions cause not only osmotic, but also oxidative stress. It is, therefore, natural that these fungi are capable of growth in microaerophilic conditions that reduce their consequences [17]. They are capable of changing their metabolism in response to complex abiotic conditions. In particular, they, rather than basidial fungi, were discovered in studies of the degradation of wood in a hut built by the Scott and Shackleton expeditions in 1908 at Cape Royds in Antarctica [18]. It has been proven that Pseudogymnoascus spp. have the ability to produce cold-adapted enzymes to survive in low-temperature habitats [19,20,21,22,23]. In particular, the fungus Pseudogynmoascus pannorum is capable of producing a protease, characterized by a high catalytic efficiency at low temperatures [22,24], which can play a significant role in the ability of this organism to survive in extreme conditions.
The fungi of the genus Pseudogymnoascus can be antagonists of potato scab pathogens in the soils of potato fields [19]. They are noted as some of the predominant microbial colonizers of the root endosphere and rhizosphere of turfgrass systems [23]. There are animal pathogens among the representatives of this genus. For instance, Pseudogymnoascus destructans causes white-nose syndrome in bats [24].
There are several studies on the secondary metabolites of the genus Pseudogymnoascus representatives. Most of the established structures belong to polyketides that exhibit antimicrobial activity [25,26,27,28]. Polyketides, asterric acid derivatives, and geomycins A–C have been isolated from Geomyces sp. (obtained from Antarctic soil) and have shown both antifungal and antibacterial activity [29]. Geomyces pannorum, isolated from leaf litter, produces pannomycin, a cis-decalin secondary metabolite with potential antibacterial activity against Staphylococcus aureus [30]. A Pseudogymnoascus strain isolated from marine sediments in Admiralty Bay (Antarctica) has suppressed the growth of the phytopathogenic bacteria Xanthomonas passiflorae and Xanthomonas euvesicatoria (99% and 98%, respectively) [31]. Red pigment amphiol, which has antifungal activity, has been isolated from strain Pseudogymnoascus sp. PF-1464 [25]. Mycocidal activity, in relation to Cladosporium sphaerospermum, similar to the activity of the drug benomyl, has been found in strain Pseudogymnoascus sp. from Antarctic sediments [32]. Research into the antimicrobial, cytotoxic, and antiprotozoal properties of the metabolites of Antarctic fungal communities isolated from the marine and lake sediments of Deception Island included a representative of the genus Pseudogymnoascus and had high selective activity against the fungus Paracoccidioides brasiliensis [33], which is one of the most hazardous fungal pathogens for humans. Six new tremulane sesquiterpenoids with antitumor activities have been found in Antarctic Pseudogymnoascus fungi [34]. On the whole, the scarce research into the secondary metabolites of Pseudogymnoascus fungi from cold environments has shown a hidden biosynthetic potential for the search for biologically active compounds.
In the present work, we investigated the secondary metabolites of the Pseudogymnoascus sp. strains, VKM F-4518 and VKM F-4519, isolated from the surface layer of sediments in the Kolyma Lowland (the Arctic). We report on the isolation and structure of two compounds, which have been identified as macrolide antibiotics macrosphelides A and B. Given the potential interest of these compounds, a suspected biosynthetic cluster of macrosphelides genes has been found.

2. Materials and Methods

Fungal strains. The strains of Pseudogymnoascus sp., VKM F-4518 and VKM F-4519, were isolated from the surface layer of sediments in the Kolyma Lowland in the Arctic. The complete genome of these strains was investigated earlier [14] and deposited in the GenBank under the numbers JPKC00000000.1 for Pseudogymnoascus sp. VKM F-4518 and JPKD00000000.1 for Pseudogymnoascus sp. VKM F-4519. The strains are stored in the All-Russian Collection of Microorganisms (VKM), G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, FRC Pushchino Scientific Centre of Biological Research, Russian Academy of Sciences.
Growth conditions. The strains of Pseudogymnoascus sp. were grown on malt agar slants for 7 days. Then, an aqueous spore suspension (1–2 × 105 conidia/mL) of each strain was inoculated into 3 × 750 mL Erlenmeyer flasks, each containing 150 mL of medium (g/L of distilled water): mannitol—50.0; succinic acid—5.4; MgSO4 × 7H2O—0.3; and KH2PO4—1.0; with a pH adjusted to 5.4 with 25% NH4OH solution for submerged cultivation on a shaker (220 rpm) at 24 ± 1 °C. The sampling was conducted in 11 days.
Isolation and identification of the metabolites. The grown mycelium was separated from the culture liquid by filtration through a cotton filter. The filtrate was acidified with a 3% solution of tartaric acid to pH 4 and extracted with CHCl3 (v/v) three times at room temperature. Then, all the volatile materials were removed in vacuo. The amounts of the studied extracts of strain Pseudogymnoascus sp. VKM F-4518 and strain Pseudogymnoascus sp. VKM F-4519 obtained were, respectively, 267 mg and 166 mg.
The extracts of the culture liquid were analyzed using TLC on silica gel plates in chloroform–methanol–25% NH4OH (90:10:0.1) (eluent I) and chloroform–acetone (93:7) (eluent II) systems. The metabolites were detected using absorption and fluorescence at 254 and 366 nm. An extract of strain VKM F-4518 was used to isolate the metabolites using column chromatography, since it contained a larger amount of target products. The extract was loaded onto a column (3 × 11 cm) filled with silica gel (Silica gel 60, 0.063–0.1 mm, Merck, Germany). Elution was carried out in a gradient of CHCl3 and CHCl3:MeOH (9:1) to yield six fractions (Fr. 1–Fr. 6). Fr. 4 yielded macrosphelide 1 (125 mg). The final purification of compound 2 was achieved using preparative TLC on a silica gel plates of Fr. 2 using eluent II (50 mg).
1H NMR (400.130 MHz) and 13C NMR (100.613 MHz) spectra were recorded with an Agilent 400MR spectrometer at 298 K. Chemical shifts are given in ppm relative to internal Me4Si. The methanol was purified using reflux and distillation over Mg turnings. Optical rotations were measured with a JASCO DIP-1000 digital polarimeter at ambient temperature using a 100 nm cell with a 2 mL capacity. High-resolution mass spectra (UHRMS) were acquired using a commercial 7 Tesla LTQ FT ultra mass spectrometer equipped with an Ion Max electrospray ion source (Thermo Electron Corp., Bremen, Germany). Chemical ionization mass spectra (CI) were registered on an LCQ Advantage MAX quadrupole mass spectrometer (Thermo Fisher Scientific GmbH, Bremen, Germany), using a single-channel syringe pump for the direct injection of a specimen into the chamber for chemical ionization at atmospheric pressure. Column chromatography was performed on silica gel (Silica gel 60, 0.063–0.1 mm, Merck, Germany). Silica gel plates (Silica gel F254, Merck, Germany) were used for thin-layer chromatography (TLC) assays.
Bioinformatic analysis. The search for the macrosphelide biosynthesis genes was carried out in the genomes of Pseudogymnoascus sp. VKM F-4518 (JPKC00000000.1) and Pseudogymnoascus sp. VKM F-4519 (JPKD00000000.1) using the BLAST+ program [35]. The protein sequences of the macrosphelide biosynthesis genes from strain Paraphaeosphaeria sporulosa AP3s5-JAC2a [36] were used as references. The identities between the protein sequences were calculated using the TaxonDC program [37]. The antiSMASH program (fungal version) was used for a bioinformatic analysis of the biosynthetic clusters of the secondary metabolite genes (BCG) in the genomes of the strains.
Macrosphelide A (1). White powder.
1H NMR (400 MHz, CDCl3, RT): δ 6.84 (dd, 3JH-H 15.7, 9.5 Hz, 1H, H-7), 6.82 (dd, 3JH-H 15.6, 9.2 Hz, 1H, H-13), 5.98 (dd, 3JH-H 15.6, 4JH-H 1.4 Hz, 1H, H-12), 5.95 (dd, 3JH-H 15.7, 4JH-H 1.4 Hz, 1H, H-6), 5.35-5.25 (m, 1H, H-3), 4.94-4.85 (m, 1H, H-9), 4.84-4.76 (m, 1H, H-15), 4.16-4.12 (m, 1H, H-8), 4.08-4.03 (m, 1H, H-14), 3.94 (br s, 1H, OH), 3.80 (br s, 1H, OH), 2.56 (br s, 1H, H-2), 2.55 (br s, 1H, H-2), 1.38 (d, 3JH-H 6.5 Hz, 3H, Me-9), 1.31 (d, 3JH-H 6.5 Hz, 3H, Me-15), and 1.28 (d, 3JH-H 6.5 Hz, 3H, Me-3) ppm.
13C NMR (400 MHz, CDCl3, RT): δ 170.10 (C-1), 165.74 (C-11), 165.05 (C-5), 146.54 (C-13), 145.87 (C-7), 122.43 (C-6), 121.99 (C-12), 74.35 (C-9), 74.09 (C-8), 73.43 (C-15), 72.72 (C-14), 67.75 (C-3), 40.86 (C-2), 19.52 (Me-3), 17.75 (Me-9), and 17.60 (Me-15) ppm.
Macrosphelide B (2). Yellowish powder.
1H NMR (400 MHz, CDCl3, RT): δ 7.01 (d, 3JH-H 15.7 Hz, 1H, H-12), 6.90 (dd, 3JH-H 15.8, 3.8 Hz, 1H, H-7), 6.72 (d, 3JH-H 15.7 Hz, 1H, H-13), 6.07 (dd, 3JH-H 15.8, 4JH-H 2.0 Hz, 1H, H-6), 5.47-5.40 (m, 1H, H-3), 5.08-5.01 (m, 2H, H-9/H-15), 4.32-4.28 (m, 1H, H-8), 2.81 (dd, 2JH-H 16.2, 3JH-H 11.2 Hz, 1H, H-2), 2.61 (dd, 2JH-H 16.2, 3JH-H 2.3 Hz, 1H, H-2), 1.48 (d, 3JH-H 6.8 Hz, 3H, Me-9), 1.42 (d, 3JH-H 7.8 Hz, 3H, Me-15), 1.34 (d, 3JH-H 6.4 Hz, 3H, Me-3), and 1.23 (br s, 1H, OH) ppm.
13C NMR (400 MHz, CDCl3, RT): δ 196.19 (C-14), 170.33 (C-1), 165.30 (C-11), 164.23 (C-5), 144.37 (C-7), 132.54 (C-13), 132.05 (C-12), 122.50 (C-6), 76.71 (C-15), 75.75 (C-9), 74.73 (C-8), 67.70 (C-3), 40.59 (C-2), 19.77 (Me-3), 17.88 (Me-9), and 16.04 (Me-15) ppm.

3. Results

The extracts of the strains Pseudogymnoascus sp. VKM F-4518 and VKM F-4519 culture liquid contained metabolites 1 with Rf 0.41 (eluent I, see Experiment) and 2 with Rf 0.62 (eluent I). Compound 1 was obtained as a white powder, m.p. 146 °C, and compound 2 was isolated as a yellowish powder, m.p. 146–147 °C. The CI mass spectrum of 1 and 2 (positive ions) displayed molecular ions at m/z 343.0 [M + H]+ and at m/z 340.8 [M + H]−, respectively (see Supporting Information Figure S1). The exact molecular formulas of compounds 1 and 2 were determined using UHRMS operating in positive ion mode, giving [M1 + H]+ = C16H22O8 (m/z = 343.13871) and [M2 + H]+ = C16H20O8 (m/z = 341.12424), respectively.
In the 1H NMR spectrum of 1 (Table S1, Figure S2), two sets of olefinic proton signals were observed at δ 5.95 (dd, 3JH-H 15.7, 4JH-H 1.4 Hz, 1H, H-6) and δ 6.84 (dd, 3JH-H 15.7, 9.5 Hz, 1H, H-7) ppm, and δ 5.98 (dd, 3JH-H 15.6, 4JH-H 1.4 Hz, 1H, H-12) and δ 6.82 (dd, 3JH-H 15.6, 9.2 Hz, 1H, H-13) ppm. These coupling constants (3JH-H 15.6, 15.7 Hz) showed a trans-configuration of both CH=CH fragments. In the 13C NMR spectrum of 1 (Table S1, Figures S3 and S4), 16 carbon signals were observed, which were assignable to three carbonyl (δ 165.05, 165.74, and 170.10 ppm); five methine (δ 67.75, 72.72, 73.43, 74.09, and 74.35 ppm); one methylene (δ 40.86 ppm); and three methyl (δ 17.60, 17.75, and 19.52 ppm) carbons. In this spectrum, these corresponding olefinic carbon signals were also observed at δ 122.43 (C-6), δ 145.87 (C-7), δ 121.99 (C-12), and δ 146.54 (C-13) ppm, respectively. In the 1H-1H COSY (Figure S5) and 1H-13C HSQC (Figure S6) and HMQC (Figure S7) spectra of 1, the presence of partial structures (2→ 3 → 3Me), (6 → 7→ 8 → 9 → 9Me), and (12 → 13 → 14 → 15 → 15Me) of 1 was demonstrated (Figure 1). Finally, the structure of 1 was determined using HMBC (8 Hz) experiments, as shown in Figure 1.
Both 1 and 2 have similar NMR spectral characteristics (see Supporting Information; Tables S1 and S2, Figures S2–S14); the difference consists of significant signal shifts in the 13C NMR spectra for atoms C12–15, due to the oxidation of hydroxyl C-14 in 1 to carbonyl at the transition to compound 2. Another characteristic feature of 1 and 2 is the E-, E-configuration of both double bonds (the spin–spin coupling constants 3JH-H are 15.7–15.9 Hz). Interestingly, according to this and additional (for example, Nuclear Overhauser effect; Figure S9, Supporting Information) data, we may establish the relative configurations for metabolites 1 (3S*, 8R*, 9S*, 14R*, and 15S*) and 2 (3S*, 8R*, 9S*, and 15S*). Thus, for 1, the cross-signals in the NOESY spectrum, δ 5.31/1.41, 4.90/1.30, and 4.15/1.31 ppm, may be identified as H3/Me-9, H9/Me-3, and H8/Me-15 interactions, respectively. In general, based on the obtained results and the literature data, the NMR spectra for metabolites 1 and 2 (see Supporting Information) correspond to (+)-macrosphelide A and (+)-macrosphelide B, respectively (Figure 1) [38].
As is evident, both compounds are chiral. For 1, (+)-macrosphelide A, [α]24D +76.5 (c 0.031, MeOH) ([α]24D +84.1 (c 0.59, MeOH) [38]; [α]24D +82.0 (c 0.10, MeOH) [39]; [α]27D +82.0 (c 0.10, MeOH) [40]; [α]D +74.3 (c 0.75, MeOH) [41]; [α]30D +85.0 (c 0.046, MeOH) [42]; and [α]25D +81.0 (c 0.195, MeOH) [43,44]. Its absolute configuration corresponds to (3S, 8R, 9S, 14R, and 15S). For 2, (+)-macrosphelide B, [α]24D +3.8 (c 0.007, MeOH) ([α]20D +4.10 (c 0.99, MeOH) [38]; [α]24D +10.8 (c 0.065, MeOH) [45]; [α]24D +10.0 (c 0.39, MeOH) [40]; [α]26D +9.1 (c 0.154, MeOH) [42]; [α]20D +10.8 (c 0.065, MeOH) [46]; and [α]27D +3.71 (c 0.335, MeOH) [43,44]. Its absolute configuration is similar to 1, i.e., it is (3S, 8R, 9S, and 15S). The absolute configuration of (+)-macrosphelide A had been established earlier using XRD and chemical (Mosher esters) methods and was confirmed by the total synthesis [39]. Thus, the identity of metabolites 1 and 2 is unambiguously established using NMR spectroscopy, optic rotation, and UHRMS as (+)-macrosphelides A and B, respectively.
Earlier, Harvey et al. [36] reported that 41 fungal biosynthetic gene clusters from various fungal species had been heterologously expressed into Saccharomyces cerevisiae. This approach permits the detection of metabolites that are hardly detectable in the initial fungal organisms. The most notable is a cluster from the fungus Paraphaeosphaeria sporulosa, which contains genes of the enzymes hydrolase (SH), monooxygenases cytochrome P450 (p450), dehydratase (DH), short-chain dehydrogenase/reductase (SDR), and type 1 polyketide synthase (T1PKS). As the main product, this cluster produces an asymmetric macrotriolide, which closely resembles the family of macrosphelides. The sequence of this cluster is deposited in the GenBank under the number KV441552.1. Based on this sequence, we searched for the macrosphelide biosynthesis genes in the genomes of strains VKM F-4518 and VKM F-4519 using the BLAST+ program (Table 1). The identity between the protein sequences of the genes in strain P. sporulosa AP3s5-JAC2a [36] and the studied Pseudogymnoascus strains was more than 75%. The organization of the gene cluster Pseudogymnoascus, as compared with that of strain P. sporulosa, is presented in Figure 2.
Using the antiSMASH secondary metabolite analysis tool, we scanned the genome sequences of the strains for the presence of the suspected BGC secondary metabolites. Our study showed that the genome of strain VKM F-4518 contained 32 clusters, 11 of which belonged to type 1 polyketide synthases (T1PKS), 9 to non-ribosomal peptide synthases (NRPS), 1 to type 3 polyketide synthases (T3PRS), 1 to mixed type (T1PKS, NRPS), 7 to ribosomally synthesized and post-translationally modified peptide synthases (fungal RiPP), and 3 to terpene cyclases (T) (Figure S14, Supporting Information). Strain VKM F-4519 encoded 17 clusters, 8 of which were T1PKS; 6, NRPS; 1, T3PKS, NRPS; and 2, fungal RiPP (Figure S15, Supporting Information). Strains VKM F-4518 and VKM F-4519 were found to have six BGCs, which were similar to those of the known secondary metabolites with a similarity of 12–100% (Table 2). The regions in the genomes of strains VKM F-4918–399.1 (from 1 to 39,308) and VKM F-4919–850.1 (from 1 to 20,490)–HEx-pks1 polyketides–corresponded to the biosynthetic gene cluster of a macrotriolide from P. sporulosa.

4. Discussion

Macrosphelides A and B are metabolites of a polyketide nature belonging to 16-membered trilactone macrolides. The key structural motif of these macrolides includes a three lactone bonding, forming macrotriolides.
(+)-Macrosphelides A and B were first discovered in the fungus Microsphaeropsis sp. [38]. Later, 13 more natural macrosphelides were identified in Microsphaeropsis sp. and the fungus Periconia byssoides, isolated from the marine mollusk Aplysia kurodai [47,48,49]. Macrosphelides A and J were found in the fungus Tritirachium sp. isolated from Antarctic lichen [50]. Macrosphelide A was revealed in Coniothyrium minitans (current name Paraphaeosphaeria minitans), a mycoparasite of sclerotia-forming soil pathogens of the plants Sclerotinia sclerotiorum, S. minor, and Sclerotium cepivorum [51]. In the basidiomycete Pleurotus ostreatus, a macrosphelide was an unexpected metabolite whose biosynthesis was induced under certain conditions, namely by the addition of iodolactone, iron, copper, magnesium, and cobalt ions to the culture medium [52]. Thus, macrosphelides occur in various taxa of the fungal kingdom. For Pseudogymnoascus fungi, these metabolites were discovered for the first time. The studied strains are highly active producers of macrosphelides A and B in comparison to those known in the literature.
(+)-Macrosphelides A and B have antimicrobial and antitumor activity. Macrosphelide A dose-dependently inhibited the adhesion of HL-60 cells to an LPS-activated HUVEC monolayer (IC50, 3.5 µM); macrophelide B also inhibited HL-60 adhesion, but to a lesser extent (IC50, 36 µM) [53]. Macrosphelide A inhibited the growth of some ascomycetes, basidiomycetes, oomycetes, and Gram-positive bacteria tested, including the medically important Staphylococcus aureus [54]. Macrosphelide A has been shown to be capable of inhibiting the mycelial growth of Sclerotinia sclerotiorum and Sclerotium sepivorum at low concentrations (46.6 and 2.9 µg/mL, respectively) [53,55]. 3-Phenyl-macrosphelide A with higher cytotoxic properties has been obtained [56]. The apoptosis-inducing ability of hybrid compounds composed of macrosphelides and a thiazole-containing side chain of epothilones has been investigated. Among the tested series of hybrid compounds, the one containing a thiazole side chain at C15 (MSt-2) showed the maximum potency for inducing apoptosis [44,57]. The molecular mechanism of the anticancer action of macrosphelide A has been investigated [58]. This compound inhibits the proliferation of cancer cells and induces apoptosis by inhibiting the critical enzymes involved in the Warburg effect (aerobic glycolysis in cancer cells)—aldolase A, enolase 1, and fumarate hydratase. The active role of fungal macrosphelides A and B in suppressing the adhesion of human leukemia cells to the endothelial cells of the human umbilical vein, or in suppressing the growth of ovarian cancer cells, etc., makes these compounds a promising biotechnological product. Tests using monoclonal antibodies have shown that this inhibitory effect is focused on syalil Lewis, which is responsible for this adhesion [59]. At the same time, macrosphelides do not cause any significant inhibition of the growth of other cells, and thus represent a promising biotechnological product for potential drugs. Since the noteworthy biological activity of macrosphelides became apparent, attempts have been made to complete the synthesis of macrosphelides [39,40,41,42,43,44,45,46,60,61]. However, these methods of synthesis are limited by the low yield of the target products and their difficult isolation. Thus, the fungal production of macrosphelides A and B remains an object of interest.
No BGCs of macrosphelides have been described to date. Based on the comparison of the cluster of macrotriolide genes from the fungus Paraphaeosphaeria sporulosa, we found the complete cluster of macrosphelide biosynthesis genes in the genomes of two Pseudogymnoascus strains, VKM F-4518 and VKM F-4519. However, there were differences in the organization of the gene cluster. Thus, the genes for DH, SDR, and p450 were inverted in the representatives of the genus Pseudogymnoascus, as compared to those of strain P. sporulosa (Figure 2). In future, in order to experimentally prove that the BGC identified is actually responsible for the production of macrophelides, a gene knockout or heterologous expression must be performed.
Recent advances in genome sequencing have shown that there are probably over five million fungal species, with each species encoding up to 80 natural product biosynthetic pathways [36]. However, despite the increased ease of DNA sequencing, fungal cultivation remains a bottleneck, as only very limited number of fungal species are cultivated in the laboratory [62]. Even in the cultivated species, most BGCs presented in the genomes are either transcriptionally silent or expressed at an extremely low level. In our study, 32 and 17 BGCs were identified in the VKM F-4518 and VKM F-4519 strains, respectively. During our cultivation of these strains under laboratory conditions, only the biosynthetic cluster of macrosphelide genes was expressed.

5. Conclusions

(+)-Macrosphelides A and B were found in two Pseudogymnoascus strains, VKM F-4518 and VKM F-4519, isolated from the surface layer of sediments in the Kolyma Lowland (the Arctic). For the Pseudogymnoascus genus, these compounds were found for the first time. The studied strains are highly active producers of macrosphelides A, which may be a promising agent for the treatment of cancer. Based on the comparison of the cluster of macrotriolide genes from the fungus Paraphaeosphaeria sporulosa, we found the complete cluster of macrosphelide biosynthesis genes in the genomes of two Pseudogymnoascus strains, VKM F-4518 and VKM F-4519, using the BLAST+ program. On the whole, the presented study of new active arctic Pseudogymnoascus strains, as natural producers of macrosphelides, provides new information and a promising way of obtaining these products.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation9080702/s1, Figure S1. (a) The positive ions CI mass spectrum of 1; (b) The positive ions CI mass spectrum of 2; Table S1. Data of the NMR spectra for (+)-Macrosphelide A (1); Figure S2. 1H NMR Spectrum of (+)-Macrosphelide A (1) (CDCl3, RT); Figure S3. 13C NMR Spectrum of (+)-Macrosphelide A (1) (CDCl3, RT); Figure S4. 13C APT NMR Spectrum of (+)-Macrosphelide A (1) (CDCl3, RT); Figure S5. 1H-1H COSY NMR Spectrum of (+)-Macrosphelide A (1) (CDCl3, RT); Figure S6. HSQC NMR spectrum of (+)-Macrosphelide A (1) (CDCl3, RT); Figure S7. HMQC NMR Spectrum of (+)-Macrosphelide A (1) (CDCl3, RT); Figure S8. HMBC NMR spectrum of (+)-Macrosphelide A (1) (CDCl3, RT); Figure S9. NOESY NMR spectrum of (+)-Macrosphelide A (1) (CDCl3, RT); Table S2. Data of the NMR spectra for (+)-Macrosphelide B (2); Figure S10. 1H NMR Spectrum of (+)-Macrosphelide B (2) (CDCl3, RT); Figure S11. 13C NMR Spectrum of (+)-Macrosphelide B (2) (CDCl3, RT); Figure S12. 13C APT NMR Spectrum of (+)-Macrosphelide B (2) (CDCl3, RT); Figure S13. HSQC NMR Spectrum of (+)-Macrosphelide B (2) (CDCl3, RT); Figure S14. HMBC NMR Spectrum of (+)-Macrosphelide B (2) (CDCl3, RT); Figure S15. Identified secondary metabolites regions in the genome of the strain F-4518; Figure S16. Identified secondary metabolites regions in the genome of the strain F-4519.

Author Contributions

Investigation: T.V.A., K.V.Z. and V.P.Z.; writing original draft preparation: T.V.A., K.V.Z. and S.V.T.; writing—review and editing: G.A.K. and M.B.V.; software: S.V.T.; methodology: K.V.Z. and Y.K.G., visualization: M.B.V.; funding acquisition: G.A.K. and S.V.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research has received funding from the Ministry of Science and Higher Education of the Russian Federation under grant agreement No. 075-15-2021-1051. The registration of NMR spectra in this work was supported in part by M.V. Lomonosov Moscow State University Program of Development.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors gratefully acknowledge A.Ya. Zherebker (Skolkovo Institute of Science and Technology) for the measurements of UHRMS spectra and B.P. Baskunov (IBPM RAS) for the measurements of CI mass spectra.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Fermentation 09 00702 g001 550
Figure 1. Structures of (+)-macrosphelides A and B.
Figure 1. Structures of (+)-macrosphelides A and B.
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Fermentation 09 00702 g002 550
Figure 2. Organization of macrosphelide biosynthesis gene clusters.
Figure 2. Organization of macrosphelide biosynthesis gene clusters.
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Table
Table 1. Comparison of the macrosphelide synthesis gene clusters.
Table 1. Comparison of the macrosphelide synthesis gene clusters.
Paraphaeosphaeria sporulosa
AP3s5-JAC2a		Pseudogymnoascus sp. VKM F-4518Pseudogymnoascus sp. VKM F-4519
Gene locus tag (protein accession)DescriptionGene locus tag (protein accession)Protein identity with P. sporulosaGene locus tag (protein accession)Protein identity with P. sporulosa
CC84DRAFT_1205319 (OAG05541.1)DHV500_01681 (KFY98390.1)75.60%V501_03182 (KFZ14540.1)75.40%
CC84DRAFT_1216878 (OAG05542.1)SDRV500_01680 (KFY98389.1)79.92%V501_03183 (KFZ14541.1)80.72%
CC84DRAFT_1195956 (OAG05543.1)p450V500_01679 (KFY98388.1)83.91%V501_03184 (KFZ14542.1)83.72%
CC84DRAFT_1091541 (OAG05544.1)SHV500_01682 (KFY98391.1)90.29%V501_03181 (KFZ14539.1)90.29%
CC84DRAFT_1091839 (OAG05545.1)KS-AT-DH-ER-KR-ACPV500_01683 (KFY98392.1)85.45%V501_03180 (KFZ14538.1)85.28%
Table
Table 2. Clusters of the known secondary metabolites in Pseudogymnoascus VKM F-4518 and VKM F-4519 revealed using antiSMASH software.
Table 2. Clusters of the known secondary metabolites in Pseudogymnoascus VKM F-4518 and VKM F-4519 revealed using antiSMASH software.
RegionTypeFromToMost Similar Known ClusterSimilarity, %
VKM F-4518
1.1.T1PKS115,076Ustilaginoidins23
53.1T1PKS688932,830Secalonic acids12
399.1T1PKS139,308HEx-pks1 polyketide100
496.1T1PKS460051,229Orsellinic acid50
714.1T1PKS127,887Monacolin K22
940.1T1PKS125,7391,3,6,8-Tetrahydronaphthalene100
VKM F-4519
183.1T1PKS143,063Azanigerones20
342.1T1PKS118,121Scytalone40
384.1NRPS35,66661,986Choline100
850.1T1PKS120,490HEx-pks1 polyketide100
914.1NRPS, T1PKS132,243Phyllostictines20
960.1NRPS, T1PKS123,427Phomacins D, C22
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Antipova, T.V.; Zaitsev, K.V.; Zhelifonova, V.P.; Tarlachkov, S.V.; Grishin, Y.K.; Kochkina, G.A.; Vainshtein, M.B. The Potential of Arctic Pseudogymnoascus Fungi in the Biosynthesis of Natural Products. Fermentation 2023, 9, 702. https://doi.org/10.3390/fermentation9080702

AMA Style

Antipova TV, Zaitsev KV, Zhelifonova VP, Tarlachkov SV, Grishin YK, Kochkina GA, Vainshtein MB. The Potential of Arctic Pseudogymnoascus Fungi in the Biosynthesis of Natural Products. Fermentation. 2023; 9(8):702. https://doi.org/10.3390/fermentation9080702

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Antipova, Tatiana V., Kirill V. Zaitsev, Valentina P. Zhelifonova, Sergey V. Tarlachkov, Yuri K. Grishin, Galina A. Kochkina, and Mikhail B. Vainshtein. 2023. "The Potential of Arctic Pseudogymnoascus Fungi in the Biosynthesis of Natural Products" Fermentation 9, no. 8: 702. https://doi.org/10.3390/fermentation9080702

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