Plant metabolite 5-pentadecyl resorcinol is produced by the Amazonian fungus Penicillium sclerotiorum LM 5679

Penicillium species, of which there are more than 350, are found in diverse ecosystems ranging from marine habitats to bioaerosols. Their ecology is complex; individual Penicillium strains can gain sustenance as opportunistic pathogens and saprophytes, and can also have commensal relationships with higher organisms (Park et al., 2019; Abstract Since the classic studies of Alexander Flemming, Penicillium strains have been known as a rich source of antimicrobial substances. Recent studies have identified novel metabolites produced by Penicillium sclerotiorum that have antibacterial, antifouling and pharmaceutical activities. Here, we report the isolation of a P. sclerotiorum (LM 5679) from Amazonian soil and carry out a culture-based study to determine whether it can produce any novel secondary metabolite(s) that are not thus-far reported for this genus. Using a submerged culture system, secondary metabolites were recovered by solvent extract followed by thin-layer chromatography, nuclear magnetic resonance, and mass spectroscopy. One novel secondary metabolite was isolated from P. sclerotiorum (LM 5679); the phenolic compound 5-pentadecyl resorcinol widely known as an antifungal, that is produced by diverse plant species. This metabolite was not reported previously in any Penicillium species and was only found once before in fungi (that time, in a Fusarium). Here, we discuss the known activities of 5-pentadecyl resorcinol in the context of its mode-of-action as a hydrophobic (chaotropicity-mediated) stressor.


Fungal cultivation for secondary metabolite production
A spore suspension of isolate LM 5679 was prepared by adding sterile distilled water (5 mL) to a sporulated culture (120-hours-old; on 5-mL PDA on slants in 30-L tubes). The spores were dislodged using a sterile inoculation loop under the aseptic conditions and the number of spores was determined using a hemocytometer (Neubauer, Germany) under a light microscope. The volume of 1 mL of spore suspension containing 1 x 10 4 spores was used for inoculations.

Secondary metabolite analysis
The entire content of the Erlenmeyer 125 mL from fungal cultivation (containing fungal biomass and culture media) was mixed with ethyl acetate (20 mL) and shaken well in an orbital shaker (TECNAL. Model TE140, São Paulo, Brazil) 100 rpm for 60 min. The culture filtrate and solvent were taken in a separating funnel (1:1, v/v) and mixed well and after separation, the solvent fraction was collected (Santos-Ebinuma, 2013). We repeated this procedure 20 times with 20 fresh cultivations resulting in 200 mL of the ethyl acetate phase got from 1000 mL of fungal cultivation. Hao et al., 2020). Penicillium species are well-known for their ability to compete effectively against other microbes and, since the discovery of penicillin (Fleming, 1929), have been recognized as a rich source of antimicrobials and other secondary metabolites (Kozlovsky et al., 2020).
Recent studies have revealed that P. sclerotiorum, isolated from marine habitats, is a rich source of novel secondary metabolites that have antibacterial, trypanocidal, and pharmaceutical activities, and can be used as dyes and as antifouling agents against molluscs (Notarte et al., 2018;Wang et al., 2018;Xin et al., 2019). Soils are key habitats of Penicillium species, where they are known for their ecological versatility, including their various trophic interactions (Bitas et al., 2013;Ceci et al., 2018;Toju and Sato, 2018). Here, we isolate a P. sclerotiorum (LM 5679) from the humic topsoil of a biodiverse ecosystem, the Amazonian rainforest, and carry out a culture-based study to determine whether it can produce any secondary metabolite(s) hitherto unidentified in the Penicillium genus.

Sampling and isolation of fungi
Twenty samples of humic topsoil (5 g each) were collected beneath the forest canopy, at a depth of 5 cm, within the Reserva Florestal Adolpho Ducke, Manaus, Amazonas, Brazil (Latitude: 02°95'43"N; Longitude: 59°93'39"W. These samples were collected with clean and sterile polythene bags using a sterile spatula. From each collected sample, 1 g soil was transferred to a tube containing 9 mL of sterile distilled water; tubes were then shaken for 5 minutes in a vortex mixer (Model K45-2810, Kasvi Paraná-Brazil). From each tube, four successive dilutions were made from 10 -2 to 10 -5 and 0.1 mL of each suspension was inoculated into Petri dishes containing potato dextrose agar (PDA) (agar, 15 g/L, dextrose, 20 g/L and potato extract, 4 g/L) and then spread over the surface using a sterile Drigalski´s spatula (Clark, 1965).
Plates were incubated at 28 °C for between 24 to 120 hours and then used to inoculate tubes containing PDA using a hook for transferring a small section of the developed fungal culture to tubes containing PDA. Individual fungal strains were isolated by plating individually onto fresh PDA and incubating at 28 °C for 7 days. Each strain was identified to the genus level by assessing the morphological characteristics using microscopic description of conidia, phialides, metule, conidiophore and hyphae (Riddell, 1950). All fungal strains were deposited into the Microorganism Collection of National Institute for Amazon Research (Manaus, Brazil).
Among the fungi that were isolated, one of the isolates exhibited morphology and pigment production characteristic of P. sclerotiorum. We selected this isolate to be investigated for its taxonomy and the production of metabolites due to the biotechnological potential of this species. This selected isolate was investigated by sequencing rDNA ITS region. For this purpose, DNA was extracted from mycelium the phenol:chloroform:isoamyl-alcohol method according to Ferrer et al. (2001). The ITS was amplified The ethyl acetate phase (72 mg) was concentrated in a rotary evaporator (IKA, RV10 digital, Santa Clara, CA, USA) and the subjected to Sephadex LH-20 column chromatography (h = 49 cm, Φ = 2.5 cm) (Sigma-Aldrich, San Luis, Missouri, USA), eluted with methanol, and collected as 18 fractions. Fractions 4-10 were subjected to silica gel column chromatography (70-230 mesh; h = 52 cm, Φ = 2.5 cm) eluted sequentially with hexane, hexane:ethyl acetate (10-55% v/v), ethyl acetate:methanol (25-30% v/v) and methanol and collected as 34 fractions. Fractions 27-29 were combined and subjected to microcrystalline cellulose column chromatography (h = 20 cm, Φ = 3.5 cm) (Merck) sequentially eluted with hexane, hexane:ethyl acetate (5-50% v/v) and ethyl acetate, giving 34 fractions.
All decisions during the compound isolation were taken by analyzing the fractions of the chromatographic fractions by thin-layer chromatography (TLC) using ultraviolet light (254 nm/365 nm) and the spray-reagent sulfuric vanillin aiming to isolate novel metabolites presenting a yellow colouration. The Figure 1 summarizes the chromatographic fractionation to reach the compound 1.

Identity of isolate LM 5679
The isolation of fungi resulted in 200 fungal isolates, belonging to the genera: and Mycelium sterilla (0.5%).
The isolate, identified but the fungal collection as LM 5679 was investigated in detail as to its morphology and sequence of the ITS region of rDNA. We found green sporulating colonies on PDA after 7 d at 25 °C that were 18-40 mm in diameter, with underside of the culture turning orange towards the centre, vivid orange. Microscopy revealed conidiophores monoverticillate with short to long necks, phialides ampulliform to cylindrical and conidia produced in columns, ellipsoidal, finely roughened. This morphology description is consistent with that of Penicillium species as described by Pitt and Hocking (2009). In order to identify the specie of LM 5679, the sequences of the Internal Transcribed Spacer (ITS) region from rDNA were analyzed and compared to those in the GenBank database and exhibited 99% sequence similarity with the Accession EF488396.1 of Penicillium sclerotiorum. The ITS1-5,8s-ITS2 sequence of LM 5679 has now been desposited in NCBI-GeneBank database with accession number of MW058060.

Secondary metabolite production
The ethyl-acetate phase was selected for isolation and chemical characterization experiments. Purification procedures with different elution systems resulted in the isolation of a single compound (2.1 mg) (see Figure 1).
The 1 H NMR spectrum (Table 1) showed signals of three aromatic hydrogens with coupled meta (J = 2.1 Hz) at δ 6.18 (H-2) and 6.20 (H-4, H-6). The hydrogens of methylene groups were verified in the region between δ 2.47-1.30 and methyl group at δ 0.90. The signal at δ 8.02 was attributed to the hydrogen of hydroxyl group because it did not show correction with carbon in the HSQC experiment. 13 C NMR data associated with the DEPT allowed to identify olefinic carbons δ 100.89 (C-2) and 107.66 with greater intensity corresponding to two carbons (C-4 and C-6). The chemical shifts of the aromatic ring were indicative of resorcinol (Barrero et al., 1989) and for the side chain methylenes between δ 36.59-23.33 in addition to methyl at δ 14.30 were verified. In the HMBC experiment (Table 1), the hydrogen at δ 2.47 (H-1') showed corrections at J-3 with the carbons at 107.66 (C-4, C-6), and J-2 with 145.84 (C-5) and 32.10 (C-2 '). The high-resolution mass spectrum (negative ionization mode) showed the peak in m/z to be 319.2631 [M-H], indicating a molecular formula of C 21 H 36 O 2 . Thus, this compound was identified as 5-pentadecyl resorcinol, an alkylresorcinol that is also known as adipostatin A or cardol (see Figure 5).
Alkylresorcinols are a group of secondary metabolites that belong to the phenol family and are also known as resorcinol lipids. These substances are comprised of an aromatic ring with hydroxyl groups at the 1-and 3-ring positions, an alkyl chain at the 5-position, and range between 1 and 29 carbon atoms in length (Żarnowski et al., 2002, 2004). The characteristic structure of this group of compounds originates by condensation of acetyl-CoA subunits, which are synthesized by polyketide synthase enzymes type III (PKSs III) (Staunton and Weissman, 2001).
There are about 100 research articles published about the antimicrobial compound 5-pentadecyl-resorcinol (some using the synonyms adispostatin A or cardol) according to Web-of-Science at October 2020. Surprisingly few of these focus on the mechanism-of-action of this antimicrobial. However, biochemical studies of cellular macromolecules indicate that 5-pentadecyl-resorcinol is inhibitory and/or structurally damaging to diverse macromolecular systems, including the plasma membranes and enzymes (Deszcz and Kozubek, 2000;Murata et al., 2013;Kustiawan et al., 2015;Masuoka et al., 2015). This substance is very hydrophobic, with a log P octanol-water of predicted to be in the range 7-8 (e.g. Kozubek, 1995). Previous studies have shown that compounds with log P > 1.95 partition into the hydrophobic domains of the plasma membrane and proteins (Bhaganna et al., 2010). Hydrophobes thereby act as cellular stressors that entropically disorder biomacromolecules via a chaotropicity-mediated mechanism (Bhaganna et al., 2010;McCammick et al., 2010;Ball and Hallsworth, 2015). A considerable number of secondary metabolites, including those hydrophobes classed as volatile organic compounds, act as cellular stressors via this mechanism (Cray et al., 2013a(Cray et al., , b, 2015Suryawanshi et al., 2017).
Such compounds also induce oxidative stress in the cell, usually because they trigger lipid peroxidation. At very low concentrations, the 5-pentadecyl-resorcinol molecule (that has three double bonds) can act as an antioxidant according to in-vitro studies (Struski et al., 1990;Oliveira et al., 2011). However, this hydrophobe that has been shown to cause oxidative stress in vivo (Murata et al., 2013), and is highly inhibitory to microbial cells (Kubo et al., 2003;Murata et al., 2013;Zhou et al., 2016). Given the  Dry biomass (8.6 g) was extracted twice with acetone. Supernatant was extracted twice with ethyl acetate. 5-pentadecyl resorcinol was detected by using preparative TLC silica gel 60 plates, microcolorimetric method, GC and EI\MS.

Zarnowski et al. (2000)
Penicillium sclerotiorum Defined nutrient medium Czapek-Dox broth The production of 5-pentadecyl resorcinol was revealed using an ethyl acetate extraction which yielded an orange pigment, and then confirmed using NMR and mass spectrometry.

Bacteria: none reported
Archaea: none reported a There are many reports of 5-pentadecyl resorcinol production in diverse plant species (and even sponges); b Only reported thus far in fungi.
Brazilian Journal of Biology, 2022, vol. 82, e241863 7/9 Penicillium sclerotiorum produces 5-pentadecyl resorcinol BALL, P. and HALLSWORTH, J.E., 2015. Water structure and chaotropicity: their uses, abuses and biological implications. Physical Chemistry Chemical Physics,vol. 17,no. 13, non-specific nature of hydrophobe-induced damage that is mediated by chaotropicity and oxidative stress (they can potentially affect all types of biomacromolecule and all types of cell), we believe that no forms of life are immune from adverse effects. The minimum inhibitory concentration of 5-pentadecyl-resorcinol for microbial systems is typically in the low mM range (Kubo et al., 2003), which is consistent with a mechanism-of-action as chaotropicity-mediated hydrophobic stressor (see Figure 1 of Cray et al., 2015). It is intriguing to speculate how P. sclerotiorum can produce 5-pentadecyl-resorcinol without sustaining high levels of collateral damage. However, it is well-established that microbes that produce chaotropic substances in quantity have adaptations to stabilize their macromolecular systems that can include more-polar lipid headgroups, longer-chain and/or more-branched membrane lipids, highly efficient protein-stabilization proteins, and accumulation of polar (kosmotropic) substances that stabilize macromolecular systems (Hallsworth, 1998;Hallsworth et al., 2003;Bhaganna et al., 2010;Cray et al., 2015). More work is needed to establish whether P. sclerotiorum LM5679 also has these kinds of adaptation.
The current study demonstrates the incompleteness of our knowledge about the ecophysiology, and the biotechnological potential, of Amazonian soil fungi as secondary metabolite producers. Fungi, like all living systems, are constantly exposed to mechanistically diverse types of stress, from outside as well as those generated by the cell (Hallsworth, 2018). Alkylresorcinols can increase tolerance to stresses caused by oxidative damage (Agil et al., 2016) and high temperature (Deryabin et al., 2014), so it paradoxical that these substances are also highly toxic/ stressful/inhibitory in some species (Goebel et al., 2019;Li et al., 2020). More work is needed to determine the mechanisms at play that underlie this paradox. Further studies are also needed to understand commercial viability, and other ecophysiological roles, of 5-pentadecyl resorcinol in P. sclerotiorum.