Isolation of a Novel Polyketide from Neodidymelliopsis sp.

Fungi have become an invaluable source of bioactive natural products, with more than 5 million species of fungi spanning the globe. Fractionation of crude extract of Neodidymelliopsis sp., led to the isolation of a novel polyketide, (2Z)-cillifuranone (1) and five previously reported natural products, (2E)-cillifuranone (2), taiwapyrone (3), xylariolide D (4), pachybasin (5), and N-(5-hydroxypentyl)acetamide (6). It was discovered that (2Z)-cillifuranone (1) was particularly sensitive to ambient temperature and light resulting in isomerisation to (2E)-cillifuranone (2). Structure elucidation of all the natural products were conducted by NMR spectroscopic techniques. The antimicrobial activity of 2, 3, and 5 were evaluated against a variety of bacterial and fungal pathogens. A sodium [1-13C] acetate labelling study was conducted on Neodidymelliopsis sp. and confirmed that pachybasin is biosynthesised through the acetate polyketide pathway.


Isolation and Structure Elucidation
Freeze-dried plates (dry weight 108.6 g) inoculated with Neodidymelliopsis sp. (ICMP 11463) were extracted with a combination of MeOH and CH2Cl2. Preliminary fractionation of the brown gum was carried out using C8 reversed-phase flash chromatography, eluting with a MeOH/H2O gradient, to generate five fractions (F1-F5). Further purification of F3 and F4 by a combination of Sephadex LH20 and silica gel column chromatography afforded compounds 1-6 ( Figure 3).

Isolation and Structure Elucidation
Freeze-dried plates (dry weight 108.6 g) inoculated with Neodidymelliopsis sp. (ICMP 11463) were extracted with a combination of MeOH and CH2Cl2. Preliminary fractionation of the brown gum was carried out using C8 reversed-phase flash chromatography, eluting with a MeOH/H2O gradient, to generate five fractions (F1-F5). Further purification of F3 and F4 by a combination of Sephadex LH20 and silica gel column chromatography afforded compounds 1-6 ( Figure 3).
An 1 H-NMR time course experiment was used to explore the ease of conversion of (2Z)cillifuranone (1) to (2E)-cillifuranone (2), and to answer the possibility that conversion of 1 to 2 occurred during chromatography and with time. The time course experiment involved dissolving 1 in d 4 -methanol and storing the sample open to light at room temperature and acquiring a 1 H-NMR spectrum every three months (Figure 4). Over a nine-month period, 1 was observed to isomerise to 2. After three months, approximately 70% of (2Z)-cillifuranone (1) had isomerised, while, after nine months, approximately 77% had isomerised.
(2Z)-cillifuranone (1) to (2E)-cillifuranone (2), and to answer the possibility that conversion of 1 to 2 occurred during chromatography and with time. The time course experiment involved dissolving 1 in d4-methanol and storing the sample open to light at room temperature and acquiring a 1 H-NMR spectrum every three months (Figure 4). Over a nine-month period, 1 was observed to isomerise to 2. After three months, approximately 70% of (2Z)-cillifuranone (1) had isomerised, while, after nine months, approximately 77% had isomerised. This time-controlled experiment clearly demonstrated that (2Z)-cillifuranone (1) is ambient light and temperature sensitive, isomerising to (2E)-cillifuranone (2). Since 2 appeared to be the more stable isomer, this could explain some of difficulties that we had in acquiring 1 in amounts required for characterisation. The extraction procedure could have led to 1 isomerising to 2. The standard extraction procedure used does not seek to minimise light exposure and the process of drying initial fractions collected from reversed-phase C8 column chromatography requires removal of water that is achieved under reduced pressure in a 40 °C water bath. Further extractions of Neodidymelliopsis sp. were undertaken in the absence of light and with water baths at room temperature. These This time-controlled experiment clearly demonstrated that (2Z)-cillifuranone (1) is ambient light and temperature sensitive, isomerising to (2E)-cillifuranone (2). Since 2 appeared to be the more stable isomer, this could explain some of difficulties that we had in acquiring 1 in amounts required for characterisation. The extraction procedure could have led to 1 isomerising to 2. The standard extraction procedure used does not seek to minimise light exposure and the process of drying initial fractions collected from reversed-phase C 8 column chromatography requires removal of water that is achieved under reduced pressure in a 40 • C water bath. Further extractions of Neodidymelliopsis sp. were undertaken in the absence of light and with water baths at room temperature. These changes were made to minimise isomerisation of 1. However, no further isolation of pure (2Z)-cillifuranone (1) was achieved.

Evaluation of Bioactivity
During initial inhouse whole cell screening, Neodidymelliopsis sp. (ICMP 11463) showed antibacterial activity against Staphylococcus aureus and Mycobacterium abscessus but not Escherichia coli or Pseudomonas aeruginosa. Three of the six compounds isolated from Neodidymelliopsis sp. were evaluated for their bioactivity against Mycobacterium abscessus and M. marinum using inhouse assays, and against a more extensive panel of microorganisms (Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Acinetobacter baumannii, Methicillin-resistant Staphylococcus aureus, Candida albicans, and Cryptococcus neoformans) by the Community for Open Antimicrobial Drug Discovery (COADD) at The University of Queensland. COADD test compound activity as inhibition of microbial growth at a single concentration of 32 µg/mL and their assays did not identify any significant inhibition of growth for any compound tested (Table 2). Similarly, we found no activity for 2 and 3 against M. abscessus or M. marinum (data not shown). However, we did find some anti-mycobacterial activity for 5 with Minimum Inhibitory Concentrations (MIC) of 32 and 64 µg/mL for M. marinum and M. abscessus, respectively ( Figure 5).

Proposed Biosynthesis
In 2010, three secondary metabolites, sorbifuranone A-C (7-9), were isolated from Penicillium chryogenum [8]. These metabolites contained a (2Z)-cillifuranone fragment incorporated into the larger sorbifuranone structures (Figure 2). The authors postulated a biosynthesis for sorbifuranone A (7) using (2Z)-cillifuranone (1) and sorbicillinol (12) as intermediates ( Figure 6) [8]. Recent studies on the chemistry of Penicillium chryogenum led to the discovery of (2E)-cillifuranone (2) that was found in conjunction with sorbifuranone A-C (7-9) [4]. An unconfirmed signal in their UV-spectrum led the researchers to postulate that (2Z)-cillifuranone (1) must exist and be the unfavoured isomer in the fungus [4]. We speculate that it is possible to achieve the biosynthesis of both (2Z)-cillifuranone and taiwapyrone using the previously proposed intermediate 11 as a common biosynthetic intermediate (Figure 7). The biosynthesis of 1 can begin with putative enzymatic oxidation of 10 to form 11, which can be mediated by dioxygenase followed by reduction of the aldehyde to a secondary hydroxyl to form 13. From 13, it is possible for two different cyclization pathways to lead to two different products. The first cyclization We speculate that it is possible to achieve the biosynthesis of both (2Z)-cillifuranone and taiwapyrone using the previously proposed intermediate 11 as a common biosynthetic intermediate (Figure 7). The biosynthesis of 1 can begin with putative enzymatic oxidation of 10 to form 11, which can be mediated by dioxygenase followed by reduction of the aldehyde to a secondary hydroxyl to form 13. From 13, it is possible for two different cyclization pathways to lead to two different products. The first cyclization (red) begins with attack of the ketone by the allylic alcohol to form the furanone ring followed by expulsion of water, the resulting product is (2Z)-cillifuranone (1). The second possible cyclization (blue) pathway involves the vinyl alcohol attacking the carbonyl of the acid forming the six membered α-pyrone ring with the expulsion of water. Subsequent reduction of the ketone forms taiwapyrone (3).
Molecules 2021, 26, x FOR PEER REVIEW 7 of 1 (red) begins with attack of the ketone by the allylic alcohol to form the furanone rin followed by expulsion of water, the resulting product is (2Z)-cillifuranone (1). The second possible cyclization (blue) pathway involves the vinyl alcohol attacking the carbonyl o the acid forming the six membered α-pyrone ring with the expulsion of water. Subsequen reduction of the ketone forms taiwapyrone (3). Having such commonly related intermediates allows Neodidymelliopsis sp. to hav flexibility in production of these two secondary metabolites. Depending on the condition Neodidymelliopsis sp. is exposed to, the fungus can respond with rapid production of (2Z) cillifuranone (1) or taiwapyrone (3). Given that some investigations of the chemistry o Neodidymelliopsis sp. yielded no (2E/Z)-cillifuranone, it is plausible that the pathway i sensitive to growing conditions.

Sodium [1-13 C] Acetate Incorporation Study
Due to the lack of biosynthetic knowledge for compounds, such as taiwapyrone and (2E)-cillifuranone, a sodium [1-13 C] acetate labelling study was conducted to test th  Having such commonly related intermediates allows Neodidymelliopsis sp. to have flexibility in production of these two secondary metabolites. Depending on the conditions Neodidymelliopsis sp. is exposed to, the fungus can respond with rapid production of (2Z)-cillifuranone (1) or taiwapyrone (3). Given that some investigations of the chemistry of Neodidymelliopsis sp. yielded no (2E/Z)-cillifuranone, it is plausible that the pathway is sensitive to growing conditions.

Sodium [1-13 C] Acetate Incorporation Study
Due to the lack of biosynthetic knowledge for compounds, such as taiwapyrone and (2E)-cillifuranone, a sodium [1-13 C] acetate labelling study was conducted to test the hypothesis that these compounds were produced through the acetate pathway. The organism was regrown in solid culture with 1 mg/mL sodium [1-13 C] acetate incorporated in the agar. Standard extraction and fractionation unfortunately only yielded pachybasin (5), with no chromatographic evidence observed for 1-4 and 6.
Further examination of the enriched carbon chemical shifts and determination of pairs of chemical shifts led to the reassignment of three quaternary carbons (C-3, C-4a, and C-9a) previously assigned incorrectly in isolation reports of pachybasin (5) ( Table 3).    To determine the exact polyketide folding mode that leads to pachybasin, a second experiment would need to be devised using 1,2-13 C sodium acetate. In the established F folding mode, the octaketide cyclization point lies between C-2 and C-3 [11,13]. The use of double labelled acetate would identify intact acetate units, with F-mode leaving C-2 as a single labelled position (Figure 9). In contrast, the proposed F -mode folding leaves C-11 as a single labelled position [11,13]. Both cyclization modes preserve the F-mode labelling pattern. To generate the evidence required to prove the F folding mode, a 13 C-COSY experiment would be acquired, with the 13 C-13 C connectivity being used to definitively establish the cyclization mode that leads to pachybasin.
Molecules 2021, 26, x FOR PEER REVIEW 9 of 14 COSY experiment would be acquired, with the 13 C-13 C connectivity being used to definitively establish the cyclization mode that leads to pachybasin.

Fungal Material
Fungal material was provided by Manaaki Whenua-Landcare Research, a New Zealand Crown Research Institute (Auckland, New Zealand) responsible for the curation of the International Collection of Microorganisms from Plants (ICMP). The ascomycete fungus Neodidymelliopsis was described as a new genus in 2015 as a group of plant-associated Phoma-like fungi [14]. Culture ICMP 11463 was isolated in October 1991 from a leaf spot on New Zealand native Pittosporum. As the ITS sequence does not closely match any known Neodidymelliopsis species, it may be a novel species in this genus [14]. Freezer stocks were made by growing the fungus on 1.5% potato dextrose agar (PDA) plate and excising small cubes of agar (5-6 mm in length) from the fungus' growing edge. These cubes were placed within a cryovial containing 1 mL of 10% glycerol. The cryovials were rested for 1 h, after which the remaining liquid glycerol was removed, and the tubes were stored at −80 • C.
For the antifungal assay, fungi strains were cultured for 3 days on YPD agar at 30 • C. A yeast suspension of 1 × 10 6 to 5 × 10 6 CFU/mL was prepared from five colonies. These stock suspensions were diluted with yeast nitrogen base (YNB) (Becton Dickinson, 233520, New South Wales, Australia) broth to a final concentration of 2.5 × 10 3 CFU/mL. The compounds were added in duplicate to the wells of a 96-well plate (Corning 3641, nonbinding surface) at a concentration of 64 µg/mL and a final volume of 50 µL. Then, 50 µL of the fungi suspension that was previously prepared in YNB broth to the final concentration of 2.5 × 10 3 CFU/mL were added to each well of the compound-containing plates, giving a final compound concentration of 32 µg/mL. Plates were covered and incubated at 35 • C for 36 h without shaking. C. albicans MICs were determined by measuring the absorbance at OD 530 . For C. neoformans, resazurin was added at 0.006% final concentration to each well and incubated for a further 3 h before MICs were determined by measuring the absorbance at OD 570−600 .
Colistin and vancomycin were used as positive bacterial inhibitor standards for Gramnegative and Gram-positive bacteria, respectively. Fluconazole was used as a positive fungal inhibitor standard for C. albicans and C. neoformans. The antibiotics were provided in 4 concentrations, with 2 above and 2 below its MIC value, and plated into the first 8 wells of Column 23 of the 384-well NBS plates. The quality control (QC) of the assays was determined by the antimicrobial controls and the Z'-factor (using positive and negative controls). Each plate was deemed to fulfil the quality criteria (pass QC), if the Z'-factor was above 0.4, and the antimicrobial standards showed full range of activity, with full growth inhibition at their highest concentration, and no growth inhibition at their lowest concentration.

Conclusions
A study of Neodidymelliopsis sp. led to the isolation of a novel polyketide, (2Z)cillifuranone (1), and five known natural products: (2E)-cillifuranone (2), taiwapyrone (3), xylariolide D (4), pachybasin (5), and N-(5-hydroxypentyl)acetamide (6). Three of the six natural products were tested against a range of bacterial and fungal pathogens; however, none of the natural products showed any significant antimicrobial activity, with the exception of 5, which showed some activity against M. abscessus and M. marinum with MIC of 64 and 32 µg/mL, respectively. A sodium [1-13 C] acetate incorporation experiment was performed to determine the biosynthesises of these natural products. Only pachybasin (5) was isolated from the labelled extract of Neodidymelliopsis sp., with the pattern of label incorporation being consistent with biosynthesis via cyclization of an acetate polyketide pathway-derived octaketide.
Supplementary Materials: The following are available online: the 1 H-, 13 C-, COSY, HSQC, HMBC, and NOESY NMR spectra for compound 1, 1 H-and 13 C-NMR spectra of 4 and 6, 13 C-NMR spectra of 5. The raw data for the antimicrobial activity of 5 against M. abscessus and M. marinum are also available online.