Plasmid construction
The plasmids used in this study are listed in Supplementary Table S1. Codon-optimization of the CYP71D18 gene was performed for E. coli using the GeneArt Strings DNA Fragments Service by Life Technologies Corporation (Carlsbad, CA, USA). Codon-optimization of the remaining genes, except ATR2, was performed for E. coli using the OptimumGene algorithm by GenScript Biotech Corporation (Piscataway, NJ, USA). Detailed sequences are shown in Supplementary Note. The hypothetical membrane-anchoring region was removed from Arabidopsis thaliana-derived cytochrome P450 reductase (ATR2, Accession number NM_119167). The DNA sample of ATR2 was obtained by reverse transcription from Arabidopsis thaliana mRNA and subsequent PCR amplification. The use of plant parts in the present study complies with international, national and/or institutional guidelines. The tag sequence for soluble expression was inserted at multiple cloning site 2 (MCS2) of the pCDFDuet-1 vector to construct the pCDF-ATR2 plasmid. The hypothetical membrane-anchoring region was removed from Mentha spicata-derived cytochrome P450 (CYP71D18, Accession number AF124815) and was codon-optimized for E. coli and artificially synthesized. The tag sequence for soluble expression was inserted at multiple cloning site 1 (MCS1) of the pCDF-ATR2 vector to construct pCDF-CYP71D18-ATR2. The CDH (ISPD, Accession number AY641428) gene from Mentha piperita was codon-optimized for E. coli expression, artificially synthesized, and inserted at the SalI site (between P450 and ATR2 genes) of the pCDF-CYP71D18-ATR2 plasmid with the Shine-Dalgarno sequence to construct the pCDF-CYP71D18-ATR2-ISPD plasmid. The ISPD gene was also inserted at the NdeI-BamHI site of the pET-3a plasmid to construct the pET-ISPD plasmid. Similarly, ISPD was inserted at the KpnI-SalI site of pMW218 to construct pMW-ISPD. The CDH (limC, Accession number AJ006869) gene from Rhodococcus erythropolis DCL14 was codon-optimized for E. coli expression, artificially synthesized, and inserted at the NdeI-BamHI site of the pET-3a plasmid to construct the pET-limC plasmid. The QconCAT1 gene (design details are described in the QconCAT standard protein preparation) was codon-optimized for E. coli expression and inserted at the BamHI-XhoI site of the pET-28a plasmid to construct the pET-QconCAT1 plasmid. The QconCAT2 gene (design details are described in the QconCAT standard protein preparation) was codon-optimized for E. coli expression and inserted at the BamHI-XhoI site of the pET-28a plasmid to construct the pET-QconCAT2 plasmid.
Strains
The strains used in this study are listed in Supplementary Table S2. E. coli BL21(DE3) was used as the host strain for protein expression and biocatalysis.
Culture method and biocatalysis condition
The recombinant strain BL21(DE3) harboring expression plasmids was grown at 37 °C in LB medium as the seed culture. The seed culture was inoculated into 20 mL of terrific broth (TB) medium (12 g Bacto tryptone, 24 g Bacto yeast extract, 4 mL glycerol, 2.31 g KH2PO4, 12.54 g/L K2HPO4) at a ratio of 1% and incubated at 37 °C, using BR-300LF (Taitec Corporation, Saitama, Japan). For P450 expression, 80 mg/L 5-amino levulinic acid and 100 μM Fe(NH4)(SO4)2 were added to TB medium to facilitate heme biosynthesis27. The antibiotic spectinomycin (100 mg/L), carbenicillin (100 mg/L), and kanamycin (50 mg/L) were used to maintain the plasmid. All chemicals used in this study were purchased from Sigma Aldrich (St. Louis, Mo, USA), Nacalai Tesque (Kyoto, Japan), and Tokyo Chemical Industry (Tokyo, Japan). When the optical density of the culture at 600 nm reached 0.8, isopropyl β-d-1-thiogalactopyranoside (IPTG) was added at a final concentration of 50 μM and incubated at 20 °C for 16 h.
Following induction, cells were harvested by centrifugation at 20,000×g for 2 min at 4 °C using himac CF15RN (Eppendorf Himac Technologies, Ibaraki, Japan) and resuspended in 50 mM potassium phosphate buffer (pH 7.2) containing 5% (v/v) glycerol. The cell suspension was diluted to achieve a final concentration of OD600 = 20, and mixed with the substrate. Biocatalysis was performed at 14 °C for 16 h or otherwise indicated. Reactions were carried out in a gas chromatography (GC) vial or headspace vial with tightly closed lids. The vial and its contents were cooled by ice to reduce substrate volatilization, especially that of (−)-limonene.
Analytical method
Following the conversion reaction, vials were cooled with ice, and ethyl-acetate extraction was conducted. E. coli BL21(DE3) with an empty plasmid were included as negative controls in these experiments. Obtained extracts were analyzed by GC. Gas chromatography-flame ionization detector (GC-FID) analysis was performed on a GC-2010Plus gas chromatograph (Shimadzu, Kyoto, Japan) equipped with an FID (at 300 °C) and DB-1 column (30 m length, 0.25 mm internal diameter, 0.25 μm film thickness, Agilent Technologies, Santa Clara, CA, USA). The analysis was carried out with a temperature program as follows: 65 °C for 5 min, 5 °C/min to 145 °C, 25 °C/min to 250 °C, and then held at 300 °C for 3 min. The carrier gas was helium (120.7 kPa, 19.6 mL/min). The injection conditions were split-flow 1:10 and 250 °C, with a linear velocity of 35.0 cm/s.
GC mass spectrometry (GC–MS) analysis was performed on a GC–MS-QP2010 system (Shimadzu) using an Rt-DEX column (30 m length, 0.25 mm internal diameter, 0.25 μm film thickness, RESTEK, Bellefonte, PA, USA). The analysis was carried out with a temperature program as follows: 50 °C for 5 min, 5 °C/min to 230 °C. The carrier gas was helium (0.7 mL/min). The injection conditions were split-flow 1:10, 220 °C, and linear velocity of 30.4 cm/s. The interface temperature was 220 °C. The detector operated in scan mode, and detection was performed in the range of m/z 40–400.
QconCAT standard protein preparation
Two tryptic peptides were selected based on a preliminary study to represent each protein. Peptide sequences were concatenated in two different orders to produce QconCAT1 and QconCAT2 (Fig. 5A). This artificial gene was synthesized and cloned into the BamHI-XhoI site of the pET-28a expression vector by GenScript Biotech Corporation (Piscataway, NJ, USA). Internal BamHI and XhoI sites were removed by substitution with a synonymous codon. Detailed sequences are shown in Supplementary Note. Resulting QconCAT expression plasmids were transformed into E. coli BL21(DE3) and maintained in LB medium containing kanamycin (50 mg/L). A single colony was inoculated into 2 mL 13C-M9 medium (6.78 g Na2HPO4, 3 g KH2PO4, 1 g NH4Cl, 0.5 g NaCl, 0.24 g MgSO4, 11 mg CaCl2, 10 mg thiamine, and 10 g/1 L [U-13C6] glucose) and incubated overnight at 37 °C. This seed culture was inoculated into 40 mL 13C-M9 medium at a ratio of 1% and incubated at 37 °C. When the optical density of the culture at 600 nm reached 0.6, IPTG was added at a final concentration of 1 mM and incubated at 30 °C for 46 h. Cells were harvested by centrifugation at 3500×g for 10 min at 4 °C and resuspended in 10 mL of xTractor Buffer (Clontech Laboratories, Inc., Mountain View, CA, USA). The crude lysate was obtained according to the manufacturer’s protocol for extracting proteins from bacterial cell culture. The lysate was His-tag purified using the Capturem Maxiprep Kit (Clontech Laboratories, Inc.) according to the manufacturer’s protocol. The purified protein concentration was measured using the Bradford method. Sample purity was confirmed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
Proteome analysis
Total protein was extracted as described previously with minor modifications28. E. coli cells were harvested by centrifugation at 3500×g for 10 min at 4 °C such that the OD600 × volume (mL) = 50, washed once with M9 medium, and subsequently frozen at − 80 °C until analysis. Cell pellets were resuspended in 1 mL lysis buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at pH 7.5, 5% (v/v) glycerol, 15 mM dithiothreitol, 100 mM KCl, and 5 mM EDTA). Resuspended cells were disrupted using a multi-bead shocker (Yasui Kikai Corporation, Osaka, Japan) with glass beads YGB01 (diameter 0.1 mm, Yasui Kikai Corporation) at 10 cycles of 2500 rpm for 30 s at 30 s intervals, and subsequently centrifuged at 3500×g for 10 min at 4 °C to collect the supernatant. The supernatants were used for protein quantitation via the Bradford method.
Thereafter, 50 μg of total protein and 2 μg (100.9 pmol) of QconCAT protein were supplemented with denaturing buffer (500 mM Tris–HCl at pH 8.5, 10 mM EDTA, 7 M guanidine HCl) to a total volume of 220 μL. One microliter of 50 mg/mL dithiothreitol was added and mixed by vortexing using vortex-genie 2 Mixer (Scientific Industries, Bohemia, NY, USA) at 25 °C for 1 h. Protein was subsequently alkylated with 2.5 mL of 50 mg/mL iodoacetamide (IAA) by vortexing in the dark at 25 °C for 1 h. Next, 600 μL of ice-cold methanol, 150 μL of chloroform, and 450 μL of cold water were consecutively added to lysates and gently mixed. Following centrifugation at 20,000×g for 5 min at 4 °C, the upper phase was discarded. Subsequently, 450 μL of methanol was added to the bottom phase and the interphase. Proteins were precipitated by centrifugation under the same conditions. Trypsin/Lys-C digestion was performed as described previously29. Proteins were dissolved in 9 μL of 6 M urea for 10 min by vortexing. Thereafter, 36 μL of 0.1 M Tris–HCl (pH 8.5) was added to the protein solution and mixed via sonication using Bransonic 3510 J-DTH (Emerson Japan, Kanagawa, Japan). Proteolytic digestion into peptides was performed using 1 μL of 0.5 mg/mL lysyl endopeptidase (Lys-C; Wako Pure Chemical Industries, Osaka, Japan) at a final concentration of 1% (w/w) Lys-C per sample protein and 2.5 μL of 1% w/v ProteaseMax Surfactant Trypsin Enhancer (Promega, Madison, WI, USA) at 25 °C for 3 h, followed by 1 μL of 0.5 mg/mL l-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-trypsin (Promega) at a final concentration of 1% (w/w) trypsin per sample protein at 37 °C for 16 h. Following trypsin digestion, 7.5 μL water and 3 mL of 50% (v/v) formic acid were added to the protein sample, which was subsequently centrifuged at 20,000×g for 5 min. Finally, 12 μL of the sample was mixed with 36 μL of 5% formic acid, and the mixtures were desalted using C18-StageTips30,31,32 or MonoSpin C18 column (GL sciences) with acetonitrile as wash solution and 0.1% formic acid, 95% acetonitrile as elution solution.
Samples were analyzed by nano-liquid chromatography-mass spectrometry (nano-LC–MS/MS). The nano-LC–MS/MS system comprised an LC-20Adnano and an LC–MS-8060 triple-quadrupole mass spectrometer with an electrospray ionization ion source (Shimadzu). Sample separation was performed using nano-LC (LC-20Adnano), and electrospray ionization was performed using LC–MS-8060. All analytical methods were performed as described previously33,34,35. The multiple reaction monitoring (MRM) method used to quantify five proteins was created using the open software Skyline version 4.136, and is shown in Supplementary Table S3 online. Peptides were quantified by the peak area ratio of the 12C sample to the 13C sample derived from the QconCAT1 protein. Absolute quantification values were calculated using the known concentration of internal stable labeled QconCAT1 and number of unlabeled cells. The method for data-dependent acquisition mode LC–MS/MS analysis was shown in supplementary method.
