4-cymene

Protein determination was carried out according to the Lowry method [41 (link)]. Total proteolytic enzyme activity using azocasein was assessed in the 4th midgut instars larvae homogenate of R. ferrugineus according to Olga et al., [42 (link)] with modifications. The 4th midgut larvae homogenates of lab strain (10 larvae) were pulled out gently, excised, and washed using a saline solution (0.9% (w/v) NaCl) repeatedly and then homogenized using 500 µL of an assay buffer. The midgut instar was homogenized in 500 µL of a protease assay buffer [50 mM HEPS (N-2-hydroxyelthyl piperazin-N′-2-ehtanesulphonic acid), pH 8.0, 5 mM dithiothreitol (DTT) and 0.1% (v/v) Triton X-100]. The reserve homogenates that were obtained from a previous step were centrifuged at 5000× g for 30 min using a Sigma 3k30 cooling centrifuge. The supernatants were used for estimation of the total proteolytic enzyme activity and protein concentration. Ten microliters of supernatant per assay was incubated in a total volume of 60 µL of assay buffer (pH 8) for 20 min at 37 °C before the addition of 200 µL of azocasien (2%, w/v in an assay buffer).
In all cases, enzyme samples of 10 μL, hydro distillate extract from the cell suspension after 40 days, and pure compounds (thymol, estragole, p-cymene, γ-terpinene, linalool, β-terpineol, ocimene, eugenol, 1,8-cineole, β-Caryophyllene, and germacrene D) (50, 100, 500, 1000, and 5000 mg/L) were pre-incubated together for 10 min. Substrate was then added to start the reaction (20 min for Leupeptin). The reaction lasted for 180 min at 37 °C and was then stopped using 300 µL of cold 10% (v/v) trichloroacetic acid (TCA). The reaction mixture was centrifuged at 5000× g for 20 min using the Sigma 3k30 cooling centrifuge. Ten microliters of NaOH (10 N) was added to the supernatant, and absorbency at 450 nm was measured using an ELISA plate reader. An assay mixture without an enzyme was used as a blank, the specific activity of total proteolytic enzymes was calculated as OD450. mg⁻¹·protein⁻¹·h⁻¹, and a blank sample was determined without an enzyme solution.

Limonene hydroxylase (pOT435) gene (LHBS, GenBank Accession No. AF039527.1) from Bacillus stearothermophilus BR 388, were codon optimized and cloned into pUC57. LHBS were PCR amplified and cloned into pET28a (+) with EcoI/XhoI restriction sites, creating pSC00 (pET28a-LHBS). P-cymene monoxygenase hydroxylase (cymAa, GenBank Accession No. AAB62299.1) and P-cymene monoxygenase reductase (cymAb, GenBank Accession No.:AAB62300.1) from Pseudomonas putida were codon optimized by BGI, and cloned into pUC57. cymAa and cymAb were PCR amplified and subcloned into pET28a (+) with BamHI/SacI restriction sites, creating pSC01 (pET28a-cymAa-cymAb). ClLS (GenBank Accession No.:AF514287.1) of Citrus limon and GPPS (GenBank Accession No.:AF513112.1) of Abies grandis were optimized by BGI and synthesized by GeneWiz (Suzhou, China), producing pUC57-ClLS&pUC57-GPPS. MvaE-mvaS was then excised from pYJM20 [46 (link)] and ligated into pET-28a (+) to create pET28a-mvaE-mvaS. GPPS and ClLS which were truncated in the N-terminus, were cloned and assembled into pET28a-mvaE-mvaS at the SacI/AatII sites to generate pSC02 (Table S1). CymAa-cymAb fragments were obtained through Pseudomonas putida using AatII and PacI and ligated into pSC02 to create pSC03 (Table S1). MvaE, mvaS, and GPPS were cloned from pSC02 into pCOLADuet-1 at the BamHI/XhoI sites, generating pcolaDuet-mvaE-mvaS-GPPS-ClLS. CymAa, cymAb were cloned from pSC03 into pcolaDuet-mvaE-mvaS-GPPS-ClLS at the XhoI/PacI sites to produce pSC05 (Table S1).
pYJM14 was constructed from pTrcHis2B through the introduction of ERG8, ERG12, ERG19 and IDI from S. cerevisiae [33 (link), 58 (link)]. All plasmids and primers are shown in Table S2.

The following reagents were purchased and
used without further purification: 4,4′-bipyridine (Acros Organics),
benzyl bromide (Alfa), methyl iodide (Sigma-Aldrich), 4-bromobenzenediazonium
tetrafluoroborate (Alfa), Di-μ-chlorobis[(p-cymene)chlororuthenium(II)] (Strem), 2,2′-bipyridine (Sigma-Aldrich),
ammonium hexafluorophosphate (Oakwood). Acetonitrile was distilled
from CaH₂ prior to use. Tetrabutylammonium hexafluorophosphate
(TBAPF₆) (TCI America) was recrystallized from hot ethanol,
filtered, and dried in vacuo prior to use. 4,4′-Bis(dipropylamido)-2,2′-bipyridine
(dpab), 4,4′-dihydroxy-2,2′-bipyridine (4,4′-dhbpy), N-methyl-4,4′-bipyridinium hexafluorophosphate (MQ⁺), and [(bpy)₂Ru(4,4′-dhbpy)](PF₆)₂ were synthesized following previously reported literature
procedures.^{10 (link),13 (link),14 (link)} Deuterated solvents (methanol-d₄ and
acetonitrile-d₃) were purchased from Cambridge
Isotope Laboratories (CIL). CD₃CN was dried over 3 Å
molecular sieves prior to use in kinetic isotope studies.

The above synthesis was repeated using the
aforementioned procedure
for [(dpab)₂Ru(4,4′-dhbpy)](PF₆)₂ using [(p-cymene)Ru(bpy)Cl]Cl (0.152 g,
0.329 mmol) and 4,4′-bis(dipropylamido)-2,2′-bipyridine
(0.214 g, 0.656 mmol). For this complex, no column was used to treat
the product, because the presence of [Ru(dpab)₃](PF₆)₂ in trace amounts had no effect on subsequent
experiments. The yield was 0.091 g (23%). The product was characterized
by ¹H NMR (Figure S1), ¹³C NMR, COSY, and ESI-MS (Figure S2). ¹H NMR (300 MHz, CD₃CN, residual internal
(CD₂H)CN δ 1.94 ppm) δ 8.95 (t, J = 1.9 Hz, 4H), 8.54 (dd, J = 8.1, 1.2 Hz, 2H),
8.12 (td, J = 7.9, 1.5 Hz, 2H), 7.88 (t, J = 5.5 Hz, 4H), 7.72 (ddt, J = 7.9, 4.1,
1.9 Hz, 6H), 7.53 (d, J = 5.1 Hz, 4H), 7.44 (ddd, J = 7.2, 5.6, 1.3 Hz, 2H), 3.40 (dtd, J = 7.7, 6.1, 2.1 Hz, 8H), 1.66 (hd, J = 7.3, 2.2
Hz, 8H), 0.98 (td, J = 7.4, 2.1 Hz, 12H).