Biotransformation of eugenol by an endophytic fungus Daldinia sp. IIIMF4010 isolated from Rosmarinus officinalis

Abstract Natural value-added compounds produced from biological sources have attained immense significance in medicinal, food, flavourings, and agrochemical industries. Further, biotransformation is a powerful tool used to produce value-added compounds cost-effectively and selectively. In the present study, biotransformation of eugenol using an endophytic fungus Daldinia sp. IIIMF4010 isolated from the fresh leaves of the plant Rosmarinus officinalis leads to the production of two known value-added compounds. The biotransformation reaction of eugenol (50 mM) resulted in the production of eugenol-β-D-glucopyranoside (6.2%) and vanillin (21.8%). These biotransformed products were further characterized by liquid chromatography-mass spectroscopy (LC-MS) and nuclear magnetic resonance (NMR). Graphical Abstract


Introduction
Recently, the demand for natural phytochemicals for pharmaceuticals and cosmeceuticals applications has increased. For the production of these compounds, there is a need of safe, eco-friendly and effective production processes (Singh et al. 2019) as their biosynthetic pathway is complicated and hence their proper synthesis is not achieved. In the existing literature, a wide range of compounds such as aromatics, alkaloids, coumarins, steroids, and terpenoids are synthesized through biotransformation, as it results in regiospecific and stereospecific products (Suga and Hirata 1990;Giri et al. 2001;Kim et al. 2012). These biotransformation reactions involve oxidation, reduction, hydroxylation, hydrolysis, esterification, methylation, isomerization and glycosylation (Ishihara et al. 2003). Further, microbial whole/immobilized cells and their enzymatic preparations are considered as effective and environment friendly bioconversion tools in such reactions (Ashengroph et al. 2012).
A diversified family of organic compounds, phenylpropanoids derived from plants, are the potential precursor for microbial biotransformation reactions (Giedraityte and Kal _ edien _ e 2014). Various naturally available substrates such as eugenol, iso-eugenol and ferulic acid can possibly be biotransformed into value-added products. Among these, eugenol is found to be apromising substrate for biotransformation reactions as it is economic and readily available (Priefert et al. 2001;Ulanowska and Olas 2021).
Eugenol is an amphipathic hydroxyphenyl propene which is present in a diverse number of plants, including Syzygium aromaticum (Myrtaceae), Ocimum basilicum, O. gratissimum (Lamiaceae), Myristica fragrans (Myristicaceae), and Cinnamomum (Lauraceae) (Kamatou et al. 2012;Marchese et al. 2017). It is commonly used as a flavoring agent in cosmetic and food products. It has also been used as a dental material for temporary filling and root canal sealer as zinc oxide eugenol cement (Kim et al. 2012). Being lipophilic in nature and insoluble in water, eugenol has limited pharmacological applications. Therefore, attaching a glycosidic moiety to it will not only increase its hydrophillicity but also enhance its physicochemical and pharmacokinetic properties (Kren, 1999) which may be useful for its application in cosmetics and food additives. For instance, it is used as a component of hair restorers since eugenol glucoside is gradually degraded into eugenol by the indigenous microorganisms of human skin therefore, it acts as a prodrug of a hair restorer ('NCATS Inxight Drugs -EUGENYL GLUCOSIDE', 2022). Apart from that, eugenol glucoside, also known as citrusin C has also been identified in foods, such as common sages (Salvia officinalis), herbs and spices, and lemons (Citrus limon), thus making it a potential biomarker for the consumption of these foods ('Showing Compound Citrusin C (FDB018116) -FooDB. Foodb.ca ', 2022). It also has tyrosinase inhibitory, superoxide scavenging, and ACE inhibitory activity (Divya et al. 2019;Alvarenga et al. 2020).Recently, several attempts have been made to produce these glucosides, such as b-glucosides by chemical glycosylation (Mastelic et al. 2004). However, not much attention has been paid to the microbial glycosylation of eugenol.
In the present research, biotransformation of eugenol into its respective glucoside using an endophytic fungus Daldinia sp. IIIMF4010 has been carried out. The biotransformation of eugenol also resulted in the formation of vanillin ( Figure 1). Vanillin, a characteristic aroma constituent of the vanilla pods, is used as a flavouring agent in foods, confectionery, and beverages, as a fragrance component in perfumes & cosmetics, and pharmaceuticals (Priefert et al. 2001;Ma et al. 2022).

Results and discussion
Fifteen microbial cultures (12 fungal and 3 bacterial cultures) were screened for eugenol biotransformation and among them, only one strain (RL1) could bio-transform eugenol ( Figure S1). The isolated strain was identified by ITS-based rDNA sequencing and phylogenetic tree was constructed by the neighbor-joining (N-J) method using MEGA 10.1.8 software. Its partial sequence exhibited 85% similarity ( Figure S2) with Daldinia eschscholtzii (MT507840.1), and thus, the isolated strain was named as Daldinia sp. IIIMF4010.
Amongst the existing reports, eugenol has been used as a substrate for biotransformation reactions that resulted in the synthesis of various commercially important products. For instance, bis-eugenol with a yield of 16.3 mg/L after 18 h was synthesised from eugenol using Kalopanax pictus callus culture. (Kim et al. 2012). Further, Chen et al. (2019) screened a novel strain Gibberella fujikuroi ZH-34, however, it was used for the biotransformation of eugenol to coniferyl aldehyde with the yield of 57.3% after 6 h. Besides this, a couple of bacterial strains, including the recombinant strain of E. coli XL1-Blue (pSKvaomPcalAmcalB) and Bacillus safensis SMS1003 have also been exploited for their potential to convert eugenol into ferulic acid (8.6 g/L in 18 h) and vanillin (0.12 g/L in 96 h) respectively (Overhage et al. 2003;Singh et al. 2019). However, as per literature, few monoterpene glycosides have been isolated from natural sources, and even less have been produced using the microbial and plant wholecell/enzymatic biotransformation process. A research group reported the presence of a gene UGT78A15 from Camellia sinensis which was involved in regulating plant lowtemperature tolerance. It efficiently catalyzed the glucosylation of eugenol to eugenol glucoside in vitro and in vivo (Zhao et al. 2020). Recently, Bashyal et al. (2019) also explored the catalytic promiscuity of glycosyltransferases (YjiC) from Bacillus licheniformis DSM 13 for the synthesis of diverse sugar-conjugated natural products. In this context, based on the successful conversion, we established a biotransformation process for the production of eugenol-b-D-glucopyranoside (6.2%), and vanillin (21.8%) in 48 h using Daldinia sp. IIIMF4010. The proposed biotransformation process using microbial cells may be sustainable, robust and selective, and hence, it may be beneficial in the green synthesis of commercially important glucosides.

Reagents
Eugenol was purchased from Sigma Aldrich (St. Louis, MO, United States), Silica gel (F 254 ) for TLC was obtained from Merck KGaA, 64271 Darmstadt Germany. HPLC purification was done on a 1260 Infinity II Agilent system, equipped with a photodiode array (PDA) detector. HPLC solvents were purchased from Merck (Mumbai). Water used for HPLC analysis was obtained from the A10 Milli-Q Advantage water system (Millipore, France). Column chromatography was done using silica gel (100-200 mesh). NMR solvents were obtained from Aldrich Chemicals (Milwaukee, WI, U.S.A). All other solvents used were of analytical grade and were obtained from commercial sources. TLC was developed using iodine and anisaldehyde as staining reagents.

Endophyte isolation
Fresh leaves of the plant Rosmarinus officinalis were collected in sterile polythene bags from the experimental fields of CSIR-IIIM, India, and were processed immediately. For the isolation of endophytes, leaves were first washed under running tap water in order to remove dirt. Surface sterilization of the leaves was done by immersing them in 70% alcohol for 30 s, followed by 2% sodium hypochlorite for 2 min. The leaves were then cut into small segments with and without midrib using sterile forceps and blades. These segments were then placed on sabouraud dextrose agar (SDA) plates and incubated at 28 C for 5-6 days. Microbial cultures were purified, sub-cultured, and finally preserved on SDA slants at 4 C.

Screening and biotransformation procedure
For the biotransformation of eugenol, isolated cultures were inoculated into 100 mL sterile sabouraud dextrose broth (SDB) medium supplemented with 10 mM eugenol and incubated at 28 C for 4 days at 180 rpm. After 4 days of incubation, a reaction mixture for the biotransformation of eugenol was prepared. For reaction mixture, cell biomass (500 mg wet cell weight) was suspended in 5 ml of 100 mM potassium phosphate buffer (pH 7.0) containing 50 mM eugenol and incubated for 48 h at 28 C in a shaker incubator (180 rpm). The biotransformed reactions were analysed on TLC ( Figure S1)

Extraction and purification
After incubation, reactions were extracted by ethyl acetate twice, and the organic phase containing compound was filtered and dried under the reduced vacuum pressure at 40 C. The small amount of crude extract was reconstituted in 1 mL of methanol and was spotted on TLC Silica gel 60 F 254 plates for product analysis using the solvent system hexane/ethyl acetate (60:30). Extract exhibiting biotransformed products was then subjected to column chromatography for purification on silica gel (100-200 mesh, 60 g) using a gradient of ethyl acetate-hexane (0:100-100:0). Total twentyfive fractions (50 mL each) were collected, and analyzed by TLC, and the fractions showing same TLC profile were grouped together into five fractions (F1-F5). Compound 1 (5 mg), eugenol-b-D-glucopyranoside, was purified from the fraction F4 using semi-preparative reverse phase HPLC (column Merck RP-18 5 lm; 10 Â 250 mm; 1.5 ml/min; 60-40% over 40 min). Thereafter, fraction F2 was further purified using semi-preparative RP-HPLC (column Merck RP-18 5 lm; 10 Â 250 mm; 1.5 ml/min; 60-40% over 40 min) to give compound 2 (7 mg), vanillin.

Disclosure statement
No potential conflict of interest was reported by the authors.