A Macrosphelide as the Unexpected Product of a Pleurotus ostreatus Strain-Mediated Biotransformation of Halolactones Containing the gem-Dimethylcyclohexane Ring. Part 1

The aim of the study was to obtain new compounds during biotransformation of two halocompounds, the δ-bromo and δ-iodo-γ-bicyclolactones 1 and 2. Unexpectedly Pleurotus ostreatus produced together with the hydroxylactone, 2-hydroxy-4,4-dimethyl-9-oxabicyclo[4.3.0]nonane-8-one (3), its own metabolite (3S,9S,15S)-(6E,12E)-3,9,15-trimethyl-4,10,16-trioxacyclohexa-deca-6,12-diene-1,5,8,11,14-pentaone (4). The method presented here, in which this macrosphelide 4 was obtained by biotransformation, has not been previously described in the literature. To the best of our knowledge, this compound has been prepared only by chemical synthesis to date. This is the first report on the possibility of the biosynthesis of this compound by the Pleurotus ostreatus strain. The conditions and factors, like temperature, salts, organic solvents, affecting the production of this macrosphelide by Pleurotus ostreatus strain were examined. The highest yield of macroshphelide production was noticed for halolactones, as well with iodide, bromide, iron and copper (2+) ions as inductors.


Introduction
Fungi are remarkable organisms that easily produce a wide range of secondary metabolites with broad spectrum biological activity [1]. Forty-two percent of about 23,000 active compounds from microorganisms are made by fungi and thirty two percent by actinomycete-filamentous bacteria. The most important group of biologically active compounds produced by microorganisms are antibiotics, of which about 100,000 tons are produced worldwide annually. Over half of these antibiotics are produced by actinomycetes, 10%-15% by non-filamentous bacteria and about 20% from filamentous fungi [2].

Results and Discussion
Bromo-and iodolactone 1 and 2 with the gem-dimethylcyclohexane ring were selected as substrates for the biotransformations. The method of preparation was described in our earlier paper [23]. Strains from the Department of Chemistry's collection, which were used in order to obtain new derivatives, had not been tested previously in the biotransformation of halolactones.

Results and Discussion
Bromo-and iodolactone 1 and 2 with the gem-dimethylcyclohexane ring were selected as substrates for the biotransformations. The method of preparation was described in our earlier paper [23]. Strains from the Department of Chemistry's collection, which were used in order to obtain new derivatives, had not been tested previously in the biotransformation of halolactones.
The following strains were used as bioreagents: Yarrowia lipolytica AM71, Rhodotorula marina AM77, Rhodotorula rubra AM82, Penicillium vermiculatum AM30, Absidia glauca AM254, Absidia cylindrospora AM336, Penicillium frequentans AM351, Aspergillus ochraceus AM456, and Pleurotus ostreatus AM482. It turned out that some of these microorganisms were able to perform a hydrolytic dehalogenation reaction, by a mechanism similar to SN2, producing hydroxylactone (3)  The results of the screening biotransformations are presented in Table 1. As shown in Table 1, the selected yeast strains were not able to transform substrates 1 and 2 (entries 1-3). The same situation was observed in the case of Aspergillus ochraceus AM456 strain (entry 8). The next four strains transformed substrates 1 and 2 to the product 3, to an extent not exceeding 50% (entries 4-7). Only the Pleurotus ostreatus AM482 strain was able to perform the complete conversion of both substrates, giving hydroxylactone 3 as the only product (entry 9, Scheme 1).

Scheme 1. Biotransformatic hydrolytic dehalogenation of halolactones (1) and (2).
The results of the screening biotransformations are presented in Table 1. As shown in Table 1, the selected yeast strains were not able to transform substrates 1 and 2 (entries 1-3). The same situation was observed in the case of Aspergillus ochraceus AM456 strain (entry 8). The next four strains transformed substrates 1 and 2 to the product 3, to an extent not exceeding 50% (entries 4-7). Only the Pleurotus ostreatus AM482 strain was able to perform the complete conversion of both substrates, giving hydroxylactone 3 as the only product (entry 9, Scheme 1).
In the next step, the Pleurotus ostreatus AM482 strain was used to carry out the preparative biotransformation of both substrates 1 and 2. During the extraction of the preparative biotransformation products, chloroform was used instead of methylene chloride, which is commonly used by us for standard extractions [23][24][25]. In the GC chromatograms, in addition to the signals coming from the substrates 1, 2 and the product 3, a signal from another product 4 was present. The results of the preparative biotransformation are shown in Figure 1. In the next step, the Pleurotus ostreatus AM482 strain was used to carry out the preparative biotransformation of both substrates 1 and 2. During the extraction of the preparative biotransformation products, chloroform was used instead of methylene chloride, which is commonly used by us for standard extractions [23][24][25]. In the GC chromatograms, in addition to the signals coming from the substrates 1, 2 and the product 3, a signal from another product 4 was present. The results of the preparative biotransformation are shown in Figure 1. The preparative biotransformation carried out on bromolactone 1 gave 12.3 mg (16.5%) of hydroxylactone 3 and 7.1 mg of the additional product 4. In turn, the use of iodolactone 2 as a substrate allowed for the production of 10.5 mg (16.8%) of hydroxylactone 3 and 12.8 mg of product 4. 1 H-NMR analysis of hydroxylactone 3 confirmed that this compound was analogous to the hydroxylactone obtained in previous studies conducted by our team [23]. Analysis of the NMR spectra of the unexpected product 4 allowed us to determine that it was a metabolite of the fungus Pleurotus ostreatus. Compound 4 was identified as macrosphelide (6E,12E)-3,9,15-trimethyl-4,10,16trioxacyclohexa-deca-6,12-diene-1,5,8,11,14-pentaone. It turned out that macrosphelide 4, obtained during the biotransformation of halolactones 1 and 2, was identical to the one described in an article by Sunazuka et al. [27]. Comparative analysis of the 1 H-NMR and 13 C-NMR spectra of the compound described in literature [28] with the biotransformation product obtained by us (Tables S1 and S2  The chemical synthesis of the macrosphelide, and the absolute configurations of three chiral centres present in the molecule, was presented in the paper mentioned earlier [27]. Therefore, a collaboration with Yuji Matsuya and Kenji Sugimoto was established, who also received this The preparative biotransformation carried out on bromolactone 1 gave 12.3 mg (16.5%) of hydroxylactone 3 and 7.1 mg of the additional product 4. In turn, the use of iodolactone 2 as a substrate allowed for the production of 10.5 mg (16.8%) of hydroxylactone 3 and 12.8 mg of product 4. 1 H-NMR analysis of hydroxylactone 3 confirmed that this compound was analogous to the hydroxylactone obtained in previous studies conducted by our team [23]. Analysis of the NMR spectra of the unexpected product 4 allowed us to determine that it was a metabolite of the fungus Pleurotus ostreatus. Compound 4 was identified as macrosphelide (6E,12E)-3,9,15-trimethyl-4,10,16trioxacyclohexa-deca-6,12-diene-1,5,8,11,14-pentaone. It turned out that macrosphelide 4, obtained during the biotransformation of halolactones 1 and 2, was identical to the one described in an article by Sunazuka et al. [27]. Comparative analysis of the 1 H-NMR and 13 C-NMR spectra of the compound described in literature [28] with the biotransformation product obtained by us (Tables S1 and S2 in Supplementary Materials) confirmed that these were the same compound (Scheme 2).
Molecules 2016, 21, 859 4 of 11 In the next step, the Pleurotus ostreatus AM482 strain was used to carry out the preparative biotransformation of both substrates 1 and 2. During the extraction of the preparative biotransformation products, chloroform was used instead of methylene chloride, which is commonly used by us for standard extractions [23][24][25]. In the GC chromatograms, in addition to the signals coming from the substrates 1, 2 and the product 3, a signal from another product 4 was present. The results of the preparative biotransformation are shown in Figure 1. The preparative biotransformation carried out on bromolactone 1 gave 12.3 mg (16.5%) of hydroxylactone 3 and 7.1 mg of the additional product 4. In turn, the use of iodolactone 2 as a substrate allowed for the production of 10.5 mg (16.8%) of hydroxylactone 3 and 12.8 mg of product 4. 1 H-NMR analysis of hydroxylactone 3 confirmed that this compound was analogous to the hydroxylactone obtained in previous studies conducted by our team [23]. Analysis of the NMR spectra of the unexpected product 4 allowed us to determine that it was a metabolite of the fungus Pleurotus ostreatus. Compound 4 was identified as macrosphelide (6E,12E)-3,9,15-trimethyl-4,10,16trioxacyclohexa-deca-6,12-diene-1,5,8,11,14-pentaone. It turned out that macrosphelide 4, obtained during the biotransformation of halolactones 1 and 2, was identical to the one described in an article by Sunazuka et al. [27]. Comparative analysis of the 1 H-NMR and 13 C-NMR spectra of the compound described in literature [28] with the biotransformation product obtained by us (Tables S1 and S2 in Supplementary Materials) confirmed that these were the same compound (Scheme 2).  The chemical synthesis of the macrosphelide, and the absolute configurations of three chiral centres present in the molecule, was presented in the paper mentioned earlier [27]. Therefore, a collaboration with Yuji Matsuya and Kenji Sugimoto was established, who also received this The chemical synthesis of the macrosphelide, and the absolute configurations of three chiral centres present in the molecule, was presented in the paper mentioned earlier [27]. Therefore, a collaboration with Yuji Matsuya and Kenji Sugimoto was established, who also received this macrosphelide [28]. The results of the rotation determination of the synthesised compound and macrosphelide obtained by biotransformation proved to be very close to the same value, and identical to the mark. On this basis, we assumed that macrosphelide 4 isolated from oyster mushroom has the absolute configuration 3S, 9S, 15S. It is worth noticing that compound 4 was previously obtained exclusively by chemical synthesis and so far there is no information in the literature about the possibility of its biosynthesis by fungi or yeast.
Because we used chloroform instead of methylene chloride for the extraction of the preparative biotransformation, we discovered that the Pleurotus ostreatus AM482 strain was able to produce macrosphelide 4, a compound obtained only by organic synthesis previously. In order to determine the effect of the extraction solvent, the experiment was repeated, this time extracting the product with a portion of methylene chloride and a second portion with chloroform. This experiment revealed that the methylene chloride extraction showed only the signal derived from the hydroxylactone 3 in the GC-FID chromatogram. When chloroform was used, two signals were observed in the GC-FID chromatograms: the first coming from the hydroxylactone 3 and the second from the macrosphelide 4. The control experiment (without added substrate) conducted in parallel demonstrated that macrosphelide 4 was produced only in the presence of iodolactone 2.
Continuing our research we decided to find out the effect of temperature on the behaviour of the Pleurotus ostreatus strain. It was found that the optimum temperature for growth and biotransformation were in the range of 25˝C to 26˝C. At the lower temperature (21˝C-22˝C) the increase in biomass was very slow, so the substrate did not undergo any transformation. In turn, higher temperatures (28˝C-30˝C) resulted in dieback of the culture, and thereby inhibition of the biotransformation. The optimum temperature for the growth of the culture in the present work was consistent with the literature data [29].
Iodolactone 2 was transformed into hydroxylactone 3, and simultaneously induced the formation of macrosphelide 4, providing that the biotransformation was carried out at a temperature of 25˝C-26˝C. Lowering or raising the temperature by a few degrees dramatically reduced the substrate conversion and thus none of these products were formed.
In another experiment, the effect of substrate (iodolactone 2) addition and a potential inducer (KI) was tested. This time, both compounds (10 mg) were added to the cultures, which were either in the log growth phase (three days) or in the stationary phase (six days). The extractions were carried out three, five, seven and nine days after the addition of substrate. The results are presented in Tables 2 and 3. Table 2. The results obtained with iodolactone 2 in the log growth phase (according to GC-FID).  Cultures to which the substrate was added after three days showed the presence of macrosphelide 4 after an additional three days. Interestingly, this compound was also found in the cultures to which potassium iodide was added, which suggested that the production of macrosphelide 4 was induced by iodolactone 2. It is worth noting that P. ostreatus is one of the few fungal species resistant to potassium iodide that maintains the ability to accumulate iodine [30]. However, there are no literature data about the effects of iodine on the metabolism of Pleurotus.

Time of Biotransformation (Days) Iodolactone (2) (%) Hydroxylactone (3) (%) Macrosphelide (4) (%)
When substrate (2) or KI was added to six day cultures in both cases macrosphelide was also present, but in a smaller amount. After nine days of biotransformation, macrosphelide 4 completely disappeared, probably due to further metabolism of this compound by the microorganism.
Another experiment was designed to test whether macrosphelide 4 was produced only under the influence of halolactone and potassium iodide, or also under the influence of other compounds that might induce secondary metabolites in fungal cultures. The putative inducers were added to flasks containing 100 mL of three day old cultures of Pleurotus ostreatus AM482 strain. The results of this phase of the study are presented in Table 4. Table 4. Potential impact of inducers on macrosphelide 4 production after 7 days (according to GC-FID).
Because halolactones 1 and 2 and potassium iodide induced the synthesis of macrosphelide 4, we evaluated whether or not potassium bromide or sodium chloride could also induce the production of this molecule. Neither of these salts was effective. It is known from the literature [32] that stress conditions can promote the production of antibiotics; it is also known that Pleurotus ostreatus is sensitive to salinity [29]. Because of these facts, NaCl was added to the culture (final concentration of 0.1 M) to induce osmotic stress. It appeared that this strain behaved differently, since a high concentration of salt increased the amount of biomass without inducing the production of macrosphelide 4. Organic compounds such as ethanol, dimethylsulphoxide (DMSO), and hydrogen peroxide have been used previously to elicit secondary metabolite biosynthesis [31]. These data encouraged us to use these compounds as inducers of macrosphelide 4 production, but these experiments have proven unsuccessful.
The Tomprefa group [8] postulated that macrosphelides were synthesized by the polyketide pathway. Since it is known that the biosynthesis of other macrolide antibiotics, erythromycin for example, are stimulated by propionic acid and propanol [33], we decided to use these compounds in our research. Unfortunately, they also proved to be ineffective. Propanol was toxic to microorganisms in concentration similar to ethanol. When propanol was added in a lower concentration, only traces of compound 4 were observed. It is possible that macrosphelide 4 is synthesized by the Pleurotus ostreatus strain by another route or that the strain does not have the enzyme linking alcohol and propionic acid with coenzyme A.
In a review on the effect of heavy metals on secondary metabolite production/cellular differentiation, Weinberg [34] summarizes evidence that regulation primarily occurs at the level of transcription. Furthermore, three heavy metal ions, manganese, ferric and zinc, were identified as having a key role in the secondary metabolism of a wide variety of microorganisms [31]. In addition, Pleurotus ostreatus has the ability to accumulate metals such as iron, zinc, manganese, copper, and nickel [35,36].
In our case, the presence of iron, copper, manganese and cobalt induced the biosynthesis of the macrosphelide. Surprisingly, of the selected metals, only zinc ions proved to be completely inactive, even though it is known from the literature that in Aspergillus parasiticus, zinc overload suppressed the formation of the polyketide, versicolorin A [34]. Such an effect may be due to the fact that zinc is the only one of these metal ions which is not involved in redox processes. Alternatively, the ineffectiveness of zinc could be caused by problems with the transport of this metal into the cell and thus its bioavailability.
The best effect brought the addition of EDTA chelated iron salts, wherein the degree of oxidation of the iron influenced to a small extent the production of macrosphelide 4. It is known from the literature [37] that Pleurotus actively transports iron into the cell by redox processes. It is worth noting that iron plays many functions in cells, it is a component of haem among others. Haem is a cofactor for many enzymes, including oxygenase, which is involved in the biosynthesis of polyketide antibiotics. Enzymes participating in the biosynthesis of the polyketide antibiotic amphotericin by Streptomyces nodosus cultures contain a cysteine residue that coordinates with the haem iron [38].
Because both iron and manganese ions induced the formation of macrosphelide 4, it is feasible that the limiting step in the biosynthesis of this compound are the reactions catalysed by enzymes associated with cytochrome P450; P450 monooxygenase or manganese-dependent peroxidase. Further research on the biosynthesis of macrosphelide 4 is in progress.

Microorganisms
The yeast and fungal strains were obtained from a collection of the Institute of Biology and Botany, Medical University, Wroclaw, Poland: Yarrowia lipolytica AM71, Rhodotorula marina AM77, Rhodotorula rubra AM82, Penicillium vermiculatum AM30, Absidia glauca AM254, Absidia cylindrospora AM336, Penicillium frequentans AM351, Aspergillus ochraceus AM456, Pleurotus ostreatus AM482. All these strains are available in the Department of Chemistry, Wroclaw University of Environmental and Life Sciences. The strains were cultivated on Sabouraud's agar containing 0.5% of aminobac, 0.5% of peptone, 4% of glucose and 1.5% of agar dissolved in distilled water at 28˝C and stored in a refrigerator at 4˝C.

Screening Procedure of Substrates Biotransformation
The yeast and fungal strains which were used for biotransformation were cultivated at 25˝C in 300 mL Erlenmayer flasks, containing 100 mL of the medium consisting of glucose (3 g) and peptobac (1 g). After three days, 10 mg of the substrate 1 or 2 dissolved in 1 mL of acetone was added to each flask. The shaken cultures were incubated with the substrate for nine days. After three, five, seven and nine days of incubation, the mixture of unreacted substrate, products and mycelium was extracted with 15 mL of dichloromethane or chloroform. These solutions were dried over anhydrous magnesium sulphate and then filtered. After evaporation of the solvent, the residue was dissolved in 2 mL of acetone and analyzed by TLC (hexane-acetone, 3:1) and GC (DB-17 column).

Preparative Biotransformations
Ten Erlenmeyer flasks with the three-day cultures of Pleurotus ostreatus were prepared as described in the screening procedure. Then 100 mg of the halolactones 1 or 2 were dissolved in 10 mL of acetone and distributed between these flasks. After nine days the products were extracted with chloroform (3ˆ40 mL). The combined organic solutions were dried over anhydrous magnesium sulphate and the solvent was evaporated in vacuo. The mixture containing the products, the unreacted substrate and the fungal metabolites was separated by column chromatography (silica gel, hexane:acetone, 3:1) to obtain the pure products-hydroxylactone 3 and macrocyclic lactone 4.

The Influence of Various Factors on the Production of Macrosphelide 4
Temperature Six Erlenmeyer flasks with the three-day cultures of P. ostreatus were prepared as described in the screening procedure. The culture was grown at three different temperatures: 21˝C-22˝C, 25˝C-26˝C and 28˝C-30˝C. After three, five, seven and nine days of biotransformation, one-fourth the volume of the medium with overgrown mycelium was taken from each flask. Such samples were extracted with chloroform, dried and analysed by means of GC.
Growth Phase of the Microorganism Substrate 2 was added to a culture of P. ostreatus after three or six days. After three, five, seven and nine days of biotransformation the culture was extracted with chloroform and analysed by means of GC. As a control, in one flask the microorganism was grown without substrate 2.

Conclusions
From the nine tested microorganism strains, four were able to biotransform bromolactone 1, whereas iodolactone 2 was transformed by five strains. A complete conversion of the substrate was observed only in the Pleurotus ostreatus culture. Both substrates were converted into hydroxylactone 3 by hydrolytic dehalogenation. When chloroform was used for the extraction instead of methylene chloride, the presence of a fungal metabolite, macrosphelide 4, was observed in the extract in addition to hydroxylactone 3. Further studies have demonstrated that the biosynthesis of macrosphelide 4 was induced not only by substrates 1 and 2, but also by iodide, bromide, iron, copper, manganese and cobalt ions. The present work is the first to discover the production of macrosphelide 4 in Pleurotus ostreatus culture and to investigate the induction of its biosynthesis.