Fermentation of T. discophora and purification of 4-amino-4-deoxyarabinitol oxidase (NecC)
A nectrisine producer, T. discophora SANK 18292 (JCM 30947), (Miyauchi et al. 2015) which was deposited in the Japan Collection of Microorganisms, RIKEN BioResource Center (Tsukuba, Japan), was used in this study. The spore suspension was harvested from the cultures grown on potato dextrose agar (Wako Pure Chemical Industries, Osaka, Japan) slants, and were inoculated into A-1 medium (Miyauchi et al. 2015). The culture was incubated at 23 °C on a rotary shaker at 210 rpm for 5 days. Glucose/potato/yeast extract/calcium carbonate no. 3 (GPYC-3) medium containing 8 % glycerol, 1 % potato granule, 2.6 % yeast extract, 0.2 % CaCO3 was inoculated with the 1 % v/v culture and cultivated at 23 °C on a rotary shaker at 210 rpm for 4 days. The mycelium was harvested by centrifugation, and rinsed with a 0.9 % NaCl aqueous solution. The mycelium was frozen with liquid N2, then grinded with a pestle in a mortar, and resuspended with 50 mM sodium phosphate buffer, pH 7.0. The mycelium and debris were removed by centrifugation and filtration. The supernatant was subjected to ammonium sulfate fractionation at room temperature. The ammonium sulfate fraction precipitating between 0 and 30 % saturation was washed with 50 mM sodium phosphate, 5 mM dithiothreitol (DTT), 30 % saturation of ammonium sulfate buffer, pH 7.0 on ice. Then, the precipitate was dissolved in 50 mM sodium phosphate, 5 mM DTT buffer, pH 7.0 and dialyzed against the buffer with a Slide-A-Lyzer Dialysis Cassette (Life Technologies, Tokyo, Japan). The sample was applied to a 5 mL HiTrap DEAE FF anion exchange column (GE Healthcare, Tokyo, Japan) equilibrated with 50 mM sodium phosphate, 5 mM dithiothreitol buffer, pH 7.0 at a flow rate of 5 mL/min operated with an ACTAprime plus chromatography system (GE Healthcare) and linear gradient elution was performed in a range of 0–50 % of 50 mM sodium phosphate, 5 mM DTT, 1 M NaCl buffer, pH 7.0 for 15 min. Fractions showing 4-amino-4-deoxyarabinitol oxidase activity were recovered and adjusted to 50 % glycerol and stored at −20 °C.
Proteins were quantified with a DC Protein Assay Kit (Bio-Rad, Tokyo, Japan) according to the manufacturer’s instructions. Protein purity was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) with molecular weight markers, Sea Blue Plus 2 Pre-Stained Standards (Life Technologies). The proteins were stained with Coomassie Brilliant Blue R-250 (CBB).
Partial protein identification by liquid chromatography-tandem mass spectrometry (LC–MS/MS)
In-gel tryptic digestion and subsequent LC–MS/MS analysis with slight modifications were conducted to identify the protein as reported previously (Kubota et al. 2003; Ishizuka et al. 2010). The CBB-stained protein bands in SDS–PAGE were excised separately and were incubated in 50 mM NH4HCO3, pH 8.0 in 30 % CH3CN for 15 min at 37 °C to remove the CBB. Then, the gel pieces were dehydrated by soaking in CH3CN and subsequent evaporation. The proteins were reduced with 10 mM dithiothreitol, 20 mM NH4HCO3, pH 8.0 for 30 min at 50 °C and alkylated with 55 mM iodoacetamide, 20 mM NH4HCO3, pH 8.0 for 20 min at 20 °C in the dark. The gel pieces were evaporated thoroughly, and then incubated in digestion-buffer containing 50 μL of 20 mM NH4HCO3, 10 ng/μL trypsin (Sequencing Grade Modified Trypsin; Promega, Tokyo, Japan), and 0.005 % dodecyl-maltoside, pH 8.0 for 12 h at 37 °C. The resulting peptides were extracted once with 0.05 % formic acid in H2O and twice with 0.05 % formic acid in CH3CN. The collected extracts were evaporated to an amount of approximately 20 μL.
LC–MS/MS analyses were conducted using a LTQ-Orbitrap (Thermo Fisher Scientific, Yokohama, Japan) mass spectrometer and a DiNa nano-flow liquid chromatography system (KYA Technologies, Tokyo, Japan) equipped with a homemade BEH C18 (1.7 μm, Waters, Tokyo, Japan) electrospray ionization tip column (0.15 mm internal diameter × 50 mm length) and an Inertsil C18 (3 μm, GL Sciences, Tokyo, Japan) trap column (0.3 mm internal diameter × 1 mm length). The mobile phase consisted of solvent A, 0.1 % formic acid in H2O; and solvent B, 0.1 % formic acid in CH3CN. Elution of peptides was carried out at a flow rate of 150 nL/min with a linear gradient of 5–35 % of solvent B for 60 min at room temperature. The MS/MS spectra were searched in the National Center for Biotechnology Information non-redundant protein database using the Mascot program (Matrix Sciences, Tokyo, Japan). In addition, MS/MS spectra of major precursor ions unidentified by the Mascot search were manually inspected to assign amino acid sequences.
Cloning of necC fragment
The fungal chromosomal DNA was isolated with DNeasy plant kits (Qiagen, Tokyo, Japan) from grinded frozen mycelium prepared as described above. Forward and reverse degenerate primers, AO1L-f: 5′-aaiisiccigaiggiaaigarwsigtitaygaygc-3′ and AO3L-r: 5′-acitaitaiatrtcrtgrtccatiarncc-3′, were designed from amino acid sequencing data of tryptic peptides to amplify an necC DNA fragment. PCR amplification was conducted using an Expand High Fidelity PCR System (Roche Diagnostics, Tokyo, Japan) and 16 μM of each of the primers with the following program: 94 °C for 2 min; 30 cycles of 94 °C for 15 s, 52 °C for 30 s, 72 °C for 75 s; and 72 °C for 7 min. The PCR product (717 bp) was gel-purified using Qiaquick Gel Extraction Kits (Qiagen) and its inner sequence was then amplified using the primers AO2L-f: 5′-taygtiggiggiccigtitaytgygtiggngg-3′ and AO3L-r with the following program: 94 °C for 2 min; 10 cycles of 94 °C for 15 s, 54 °C for 30 s, and 72 °C for 45 s; and 15 cycles of 94 °C for 15 s, 54 °C for 30 s, 72 °C for 45 s + 5 s per cycle; and 72 °C for 7 min. The gel-purified PCR product was cloned using the pDrive Cloning Vector in a Qiagen PCR Cloning Kit (Qiagen) and sequenced using a Dye Terminator Cycle Sequencing System with AmpliTaq DNA Polymerase (Life Technologies) and a 3730xl DNA Analyzer (Life Technologies).
Construction and screening of the T. discophora genomic DNA library
The fungal chromosomal DNA was isolated from the grinded frozen mycelium with DNeasy plant kits (Qiagen) without using QIAshredder (Qiagen) and extracted with phenol–chloroform. The DNA (0.1 mg) was partially digested with two units of MboI at 37 °C for 4 min and dephosphorylated with calf intestinal alkaline phosphatase (CIAP). The genomic DNA fragments were ligated into XbaI-digested, dephosphorylated, and BamHI-digested SuperCos1 cosmid vector (Agilent Technologies, Santa Clara, CA). Gigapack III Gold packaging extract (Agilent Technologies) was used for packaging the DNA according to the manufacturer’s instructions. E. Coli XL1-Blue MR (Agilent Technologies) was transfected by the resulting recombinant phage. Approximately 50,000 colonies from the genomic library blotted on Amersham Hybond-N+ membranes (GE Healthcare) were screened by hybridization with digoxigenin (DIG)-labeled partial necC DNA fragments using Dig Easy Hyb (Roche Diagnostics) at 43 °C for 14–16 h. The membranes were washed with 0.5 × SSC/0.1 % SDS at 55 °C. Detection was performed using CDP-Star (Roche Diagnostics). The positive cosmids were isolated, then digested with EcoRI and BamHI separately and subcloned into pUC118 EcoRI/BAP vector or pUC118 BamHI/BAP vector (Takara Bio, Shiga, Japan). The DNA fragments were sequenced as described above and assembled using Genetyx software (Genetyx, Tokyo, Japan).
Bacterial expression of necC gene and purification of C-terminal His-tagged NecC
The total RNA of T. discophora was extracted and isolated using an RNeasy Plant Mini Kit (Qiagen) with an RNase-Free DNase Set (Qiagen) from the grinded frozen cells prepared as described above. The RNA was reverse transcribed using ReverTraAce-α- and Oligo (dT) 20 primer (Toyobo, Ohsaka, Japan) according to the supplier’s instructions. Then, necC cDNA was amplified by PCR using forward and reverse primers containing the restriction-enzyme site sequences NdeI-AO-f, 5′-agatatacacatatggatcatcttctccatatcgaca, and AO-SalI-r, 5′-atagtcgacttgagcgtcgtttccaatcttc, and the polymerase Phusion High-Fidelity DNA Polymerase (New England Biolabs, Tokyo, Japan) with the following program; 98 °C for 30 s; 30 cycles of 98 °C for 10 s, 60 °C for 20 s, 72 °C for 60 s; and 72 °C for 7 min. The gel-purified PCR product, digested with NdeI and SalI, was introduced into NdeI-SalI-digested pET21b vector (Merck, Tokyo, Japan), generating the vector pET21bnecC. For expression of His-tagged NecC, the plasmid was transformed into E coli BL21 (DE3) (Merck). Cells were grown at 37 °C to an OD600 of 1.0 in shake flasks containing 2-YT medium (Life Technologies) with 100 mg/L ampicillin at 170 rpm, and then protein was expressed by induction with 0.4 mM isopropyl beta-d-thiogalactoside (IPTG), and the cultivation was then continued at 16 °C for 21 h at 170 rpm. Cells were pelleted by centrifugation, and resuspended in 100 mM sodium phosphate, 300 mM NaCl buffer, pH 7.8. The cells were freeze-thawed once, then disrupted with sonication on ice followed by centrifugation. For protein purification, the supernatant supplemented with 5 % glycerol was diluted tenfold with buffer A containing 20 mM sodium phosphate, 500 mM NaCl, 30 mM imidazole, pH 7.4 and applied to an Ni Sepharose column (HisTrap HP, GE Healthcare) at a flow rate of 5 mL/min operated with an AKTAexplorer 10S chromatography system (GE Healthcare). The absorbed protein was eluted with buffer B containing 20 mM sodium phosphate, 500 mM NaCl 500 mM imidazole, pH 7.4 using gradient elution, from 0 to 100 % B for 20 column volumes (CV), and 100 % B in 5 CV. The purified sample was dialyzed against phosphate buffered saline (PBS) (Takara bio).
LC–MS analysis of nectrisine and 4-amino-4-deoxyarabinitol
To detect and efficiently separate nectrisine and 4-amino-4-deoxyarabinitol by HPLC, samples were labeled using 4-fluoro-7-nitrobenzofurazan (NBD-F) (Dojindo Laboratories, Kumamoto, Japan) (Watanabe and Imai 1981; Imai and Watanabe 1981). Cell extracts (20 μL) were reduced with 10 μL of NaBH4 solution (1 mg/mL) to convert nectrisine to 1,4-Dideoxy-1,4-imino-d-arabinitol (DAB). The resulting solutions were heated at 60 °C for 2 min with 60 μL of 2 g/L NBD-F in methanol and 10 μL of 100 mM borate buffer, pH 8.2. The solutions were cooled to room temperature and acidified with 10 μL of 0.5 M HCl aqueous solution. LC–MS analysis was conducted using an Acquity UPLC System and a Synapt G2-S Mass Spectrometer (Waters). NBD-labeled samples were injected into a Unison UK C18 4.6 × 150 mm column (Imtakt, Kyoto, Japan) at a flow rate of 1 mL/min at 30 °C. The mobile phase consisted of solvent A, 10 mM NH4CO2H/2.2 mM HCO2H in H2O; and solvent B, 2.2 mM HCO2H in CH3CN, using the gradient elution of, 10 % B for 8 min, from 10 to 90 % B for 20 min, and 90 % B for 2 min.
Physicochemical analysis of NecC
NecC from T. discophora was used for physicochemical analysis. The absorption spectrum of NecC at a concentration of 1 mg/mL in PBS, pH 7.4 was recorded with a DU-730 Spectrophotometer (Beckman-Courter, Tokyo, Japan). To release the cofactor of the enzyme, 5 μL of trichloroacetic acid (TCA) (Wako Pure Chemical Industries) was added to 100 μL of the NecC solution, and incubated for 1 h at room temperature. Insoluble proteins were precipitated by centrifugation at 16,000g for 5 min. Then, the absorption spectrum of the supernatant was recorded. Flavin adenine dinucleotide (FAD) (Nacalai Tesque, Kyoto, Japan) dissolved into PBS containing 4.8 % TCA was used as a control molecule.
The oligomeric state of the native NecC was analyzed by blue native PAGE (Schägger and von Jagow 1991; Schägger et al. 1994) and size exclusion-high performance liquid chromatography (SE-HPLC). Blue native PAGE was performed on a NativePAGE 3–12 % gel (Life Technologies) with NativeMark Unstained Protein Standards (Life Technologies) as molecular weight markers and stained with CBB. SE-HPLC was performed on an LC2010 CHT HPLC system (Shimadzu, Kyoto, Japan) equipped with an Acquity UPLC BEH200 SEC column, 4.6 × 150 mm, 1.7 μm particle size (Waters). The mobile phase was 20 mM sodium phosphate, 0.2 M KCl buffer, pH 6.8. The flow rate was 0.2 mL/min. The column oven temperature was 40 °C. Absorbance at 280 nm was recorded.
Differential scanning calorimetry (DSC) was performed on a VP-Capillary DSC system (Malvern, Worcestershire, UK). The sample, diluted to 0.45 mg/mL with PBS, pH 7.4, was heated from 20 to 90 °C at a scanning rate of 60 °C/h. PBS, pH 7.4 was used as a reference buffer. A buffer scan was subtracted from the protein scan and normalized for the protein concentration.
Activity of the native NecC was determined by measuring the rate of H2O2 production spectrophotometrically (Klei et al. 1990; Soldevila and Ghabrial 2001). The standard assay mixture consisted of 0.6 mM 2,2′-azino-bis (3-ethylbenzthiazoline 6-sulfonic acid) (ABTS) (Sigma-Aldrich, Tokyo, Japan), 3 units of peroxidase from horseradish, 1 mM of 4-amino-4-deoxyarabinitol, 47.1 mM of potassium phosphate, pH 7.0, and an appropriate amount of sample containing NecC in a final volume of 1 mL. After incubation at 30 °C for 10 min, the reaction was stopped by adding 0.1 mL of a 4 N HCl aqueous solution. Absorbance at 420 nm of the solution was measured with a spectrophotometer. One unit of enzyme activity was defined as the amount of enzyme catalyzing the oxidation of 2 μmol of ABTS per min under the conditions described above. Activities against d-sorbitol, d-arabinitol or xylitol were measured by replacing 4-amino-4-deoxyarabinitol with each sugar alcohol in the standard assay mixture.
Accumulation of 4-amino-4-deoxyarabinitol in T. discophora
A T. discophora culture was filtered with filter aids, Radiolite #2000 (Showa Chemical Industry, Tokyo, Japan) then stored at 4 °C until use. Metabolites were extracted by heat treatment as follows. The cooled broth (100 g) was added to 400 mL of tap water previously maintained at 61 °C in an egg-plant shaped flask, and incubated at 51–54 °C for 70 min. Then, it was cooled immediately in an ice-water bath and filtered. The filtrate was stirred at 4 °C to convert 4-amino-4-deoxyarabinitol to nectrisine. To detect nectrisine and 4-amino-4-deoxyarabinitol directly, HPLC with evaporative light scattering detection (ELSD) was performed using an Agilent 1100 HPLC system (Agilent Technologies) equipped with an evaporative light scattering detector Model 300S (SofTA, Westminster, CO) and an Xbridge BEH HILIC column, 4.6 × 75 mm, 2.5 μm (Waters) (Kimura et al. 2004) under the following conditions: mobile phase, 90 % CH3CN/10 % H2O/20 mM CH3CO2NH4; flow rate, 1 mL/min; column oven temperature, 40 °C; spray chamber, 40 °C; and drift tube, optical cell, and exhaust tube, 60 °C.
Nucleotide and amino acid sequence accession numbers
The nucleotide sequence was deposited in the EMBL/GenBank/DDBJ databases under the accession number LC056029.