Production of non-natural 5-methylorsellinate-derived meroterpenoids in Aspergillus oryzae

Fungal meroterpenoids are diverse structurally intriguing molecules with various biological properties. One large group within this compound class is derived from the aromatic precursor 3,5-dimethylorsellinic acid (DMOA). In this study, we constructed engineered metabolic pathways in the fungus Aspergillus oryzae to expand the molecular diversity of meroterpenoids. We employed the 5-methylorsellinic acid (5-MOA) synthase FncE and three additional biosynthetic enzymes for the formation of (6R,10′R)-epoxyfarnesyl-5-MOA methyl ester, which served as a non-native substrate for four terpene cyclases from DMOA-derived meroterpenoid pathways. As a result, we successfully generated six unnatural 5-MOA-derived meroterpenoid species, demonstrating the effectiveness of our approach in the generation of structural analogues of meroterpenoids.


General experimental procedures
Organic solvents were purchased from Anaqua (Hong Kong) Co. Ltd., and other chemicals were purchased from Wako Chemicals Ltd., Thermo Fisher Scientific, Sigma-Aldrich, or J&K Scientific Ltd., unless noted otherwise.Oligonucleotide primers (Table S1) were purchased from Beijing Genomics Institute.PCR was performed using a T100™ Thermal Cycler (Bio-Rad Laboratories, Inc.) with Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech Co., Ltd).Analytical HPLC was performed on a Dionex Ultimate 3000 UHPLC system (Thermo Scientific).Semipreparative HPLC was performed on a Waters 1525 Binary HPLC pump with a 2998 photodiode array detector (Waters Corporation), using a COSMOSIL 5C18-AR-II column (10 i.d.× 250 mm, Nacalai Tesque, Inc).Flash chromatography was performed using an Isolera Spektra One flash purification system (Biotage).NMR spectra were obtained at 600 MHz ( 1 H)/150 MHz ( 13 C) with a Bruker Ascend Avance III HD spectrometer and analyzed by TopSpin 4.1.3software.Chemical shifts were recorded with reference to solvent signals ( 1 H NMR: CDCl 3 7.26 ppm, CD 3 OD 3.31 ppm; 13 C NMR: CDCl 3 77.0ppm, CD 3 OD 49.0 ppm).HR-ESI-MS spectra were obtained with a SCIEX X500R Q-TOF mass spectrometer.Optical rotations were measured with a P-2000 Digital Polarimeter (JASCO Corporation).X-ray diffraction data were collected on a Bruker D8 Venture Photon II diffractometer.
The primers used in the study and the detailed methods of the plasmid constructions are provided in Table S1 and Table S2.

HPLC analysis of metabolites derived from A. oryzae transformants
To analyze the metabolites produced by each A. oryzae transformant, the transformants were cultivated on a DPY agar plate [2% dextrin, 1% hipolypepton (Nihon Pharmaceutical Co., Ltd.), 0.5% yeast extract, 0.5% KH 2 PO 4 , 0.05% MgSO 4 •7H 2 O, and 1.5% agar] for seven days at 30 °C.A small piece of fungal mycelia and agar was cut from the plate, soaked in ethyl acetate, and extracted using an ultrasonic bath.The ethyl acetate layer was transferred to a new tube, and the solvent was removed using nitrogen gas flow.
The residue was dissolved in methanol and analyzed by HPLC, with a solvent system of 20 mM formic acid (solvent A) and acetonitrile containing 20 mM formic acid (solvent B), at a flow rate of 0.4 mL/min and a column temperature of 40 °C, using a Kinetex 2.6 µm C 18 100 Å column (2.1 i.d.x 100 mm; Phenomenex).Separation was performed using a linear gradient from 10:90 (solvent B/solvent A) to 100:0 for 10 min, 100:0 for the following 3 min, and a linear gradient from 100:0 to 10:90 within the following 2.0 min, and then 10:90 for 2.5 min of equilibrium.

Isolation of each metabolite from A. oryzae transformants
To isolate each metabolite, A. oryzae transformants were cultivated on DPY agar plates (90 mm diameter; the volume of medium in one plate is approximately 20 mL) for seven days at 30 °C.The resulting fungal cultures, including agar medium, were crushed into small pieces, soaked in ethyl acetate, and extracted twice using an ultrasonic bath.After filtration, ethyl acetate was removed in vacuo.The resultant crude extract was fractionated by flash chromatography, and further purified by semipreparative HPLC.Purification methods for each compound are described in detail below.

X-ray crystallographic analysis
Single crystal of 3 was grown in CH 3 OH, whereas those of 5 and 7 were grown in CH 3 OH/CH 2 Cl 2 (1:2, v/v), by a slow evaporation process at room temperature.Single crystal X-ray diffraction measurements were performed on a Bruker D8 Venture diffractometer using Cu Kα radiation at 213 K (for 3), 218 K (for 5), or 243 K (for 7).The data collection was performed with the APEX3 program, and cell refinement and data reduction were carried out using the SAINT program.The structures of 3, 5, and 7 were solved by direct method with the SHELXT program and refined using the SHELXL program.All non-hydrogen atoms were refined anisotropically, whereas hydrogen atoms were placed by geometrical calculations.The absolute configuration of 3, 5, and 7 was determined by the Flack parameters.

Structural determination of 5′-desmethylpreterretonin A (3):
The molecular formula of 3 was established as C 25 H 36 O 5 by HR-MS analysis.The NMR spectra of 3 are similar to those of preterretonin A [7] and revealed that the A-, B-, and C-rings of 3 are identical to those of preterretonin A. However, signals corresponding to the D-ring are missing in the 1 H and 13 C NMR spectra of 3, hampering the complete structural determination based on the NMR analysis.The structure of 3, including its absolute configuration, was established by X-ray crystallographic analysis, confirming that 3 is the 5′-desmethyl form of preterretonin A.

Structural determination of 5′-desmethylprotoaustinoid A (4):
The molecular formula of 4 was established as C 25 H 36 O 5 by HR-MS analysis.The NMR spectra of 4 are highly similar to those of protoaustinoid A [7], except that the signal corresponding to the methyl group bound to C-5′ is missing in the NMR spectra of 4 and that a methylene carbon (δ C 59.5, δ H 3.83/3.33)was observed in replacement of the C-5′ methine of protoaustinoid A. These observations suggested that 4 is the 5′-desmethyl form of protoaustinoid A, which was confirmed by further analysis of 2D NMR spectra of 4.

Structural determination of compound 5:
The molecular formula of 5 was established as C 25 H 36 O 5 by HR-MS analysis.The NMR spectra of 5 are highly similar to those of the major product from the K187A variant of AdrI [6], except that the signal corresponding to the methyl group bound to C-5′ is missing in the NMR spectra of 5 and that a methine signal (δ H 3.12) was observed in the 1 H NMR spectrum of 5. Finally, X-ray crystallographic analysis confirmed that 5 is the 5′desmethyl form of the aforementioned product from the AdrI variant.

Structural determination of 5′-desmethylinsuetusin B1 (6):
The molecular formula of 6 was established as C 25 H 36 O 5 by HR-MS analysis.The NMR spectra of 6 are highly similar to those of insuetusin B1 [1], except that the signal corresponding to the methyl group bound to C-5′ is missing in the NMR spectra of 6 and that an olefinic proton (δ H 5.78) was observed in the 1 H NMR spectrum of 6.These observations suggested that 6 is the 5′-desmethyl form of protoaustinoid A, which was confirmed by further analysis of 2D NMR spectra of 6.

Structural determination of 5′-desmethylinsuetusin A1 (7):
The molecular formula of 7 was established as C 25 H 36 O 5 by HR-MS analysis.The NMR spectra of 7 are highly similar to those of insuetusin A1 [1], except that the signal corresponding to the methyl group bound to C-5′ is missing in the NMR spectra of 7 and that a methine signal (δ H 2.79) was observed in the 1 H NMR spectrum of 7. Finally, X-ray crystallographic analysis confirmed that 7 is the 5′-desmethyl form of insuetusin A1.

Structural determination of compound 8:
The molecular formula of 8 was established as C 25 H 38 O 5 by HR-MS analysis.The NMR spectra of 8 are similar to those of 7, and it was revealed that 8 possesses an identical terpenoid moiety to 7.However, the signals corresponding to the α,β-unsaturated carbonyl system at C-2′-C-4′ of 7 are missing in the 13 C NMR spectrum of 8, which instead revealed the presence of one methine at 38.9 ppm (C-2′) and two sp 2 quaternary carbons at 105.1 ppm (C-3′) and 168.5 ppm (C-4′).In addition, the 1 H-1 H COSY spectrum revealed the spin systems of H-2′/H-8′.Furthermore, the HMBC correlations of an enolic proton (δ H 12.41) to C-3′ and C-4′ elucidated that the double bond at C-2′/C-3′ of 7 was reduced to a single bond in 8 and that 8 contained the enol functionality at S6 the C-4′ position.Finally, the 2′S configuration was established based on the correlations of H-8′ (δ 1.25) and H-9/H11α.

Feeding experiment
To perform the bioconversion experiment with compound 7, A. oryzae NSARU1 was initially cultivated in 5 mL of DPY medium (supplemented with 0.01% adenine, 0.2% uracil, and 0.5% uridine) at 30 ºC and 160 rpm for three days and then transferred to 25 mL of DPY medium (supplemented with 0.01% adenine, 0.2% uracil, and 0.5% uridine) containing 16.7 mg/L of 7.After cultivation at 30 ºC and 160 rpm for three days, the medium and mycelia were separated by filtration.The medium was extracted with ethyl acetate.Meanwhile, the mycelia were soaked in acetone for 1 hour using an ultrasonic bath, and after filtration, acetone was removed using nitrogen gas flow.The broth and mycelial extracts were individually dissolved in methanol and analyzed by HPLC with 60% aqueous acetonitrile containing 20 mM formic acid at a flow rate of 1.0 mL/min and a column temperature of 40 °C, using an Accucore C18 column (4.6 i.d.x 100 mm; Thermo Scientific).

Evaluation of the antibacterial activities of the obtained compounds
The compounds obtained in this study were initially tested for their antimicrobial activity using the Bauer-Kirby method.Five bacterial strains (Staphylococcus epidermidis ATCC 12228, Staphylococcus aureus ATCC 6538, Bacillus cereus, Streptococcus faecalis, and Escherichia coli ATCC 10536) were cultivated in Luria-Bertani (LB) broth at 37 °C for 12 hours and then diluted to a concentration of ≈5 × 10 5 colony forming unit (CFU)/mL using Mueller Hinton broth (beef infusion solids 2 g/L, starch 1.5 g/L, casein hydrolysate 17.5 g/L).
Subsequently, 180 μL of the prepared microbial-containing medium was spread onto LB plates.After the surfaces of the plates dried, a 5 mm filter paper impregnated with a continuous 2-fold dilution of compounds (from 20 to 0.625 mg/mL) was placed at a specific location on each plate.The LB plates were incubated at 37 °C for 24 hours.Ampicillin was used as a positive control.
For compound 3, which exhibited positive activity against Staphylococcus aureus ATCC 6538 and Bacillus cereus, the broth microdilution method was employed to determine the minimum inhibitory concentration (MIC) using a sterile 96-well plate.The bacteria-containing medium was prepared using the same method described above and added to each well of the 96-well plate (100 μL/well).The microbial strain was then treated with a continuous 2-fold dilution of the compound (from 1,000 to 1.95 μg/mL).The 96-well plate was placed in an incubator at 37 °C for 12 hours.The MIC was defined as the lowest concentration at which no bacterial growth was observed.The antibacterial activity of preterretonin A, which was obtained in our previous study [6], was measured in the same manner as described for 3. 5′-Desmethylinsuetusin A1 (7).White crystal; [α] 21.9 D +64.8 (c 1.00, CHCl 3 ); for NMR data see Figure S32 to

Figure S2 .
Figure S2.Predicted mechanism for the formation of 8.

Figure S3 .
Figure S3.HPLC profile of (i) 7 incubated in DPY medium and the metabolites of A. oryzae NSARU1 cultivated (ii) without and (iii) with 7. The chromatograms were extracted at 254 nm.