Strains and culture conditions
Metabolic engineering for natural compound production can be enhanced through gene modifications that increase enzyme activity. All experimental materials used in this study are listed in Table 1. E.coli were grown in LB (tryptone 10 g/L, yeast extract 5 g/L and NaCl 10 g/L). During the production of (R)-(+)-limonene and (R)-(+)-perillyl alcohol, strains were cultivated in shake-flasks in medium containing glucose 10 g/L, MgSO4 1 mM and riboflavin 0.05 mM during fed-batch fermentation in glucose 20 g/L, K2HPO4 9.8 g/L, ferric ammonium citrate 0.3 g/L, citric acidmonohydrate 2.1 g/L, MgSO4 1 mM，riboflavin 0.05 mM and 1 mL trace element solution, including (NH4)6Mo7O24·4H2O 0.37 g/L, ZnSO4·7H2O 0.29 g/L, H3BO4 2.47 g/L, CuSO4·5H2O 0.25 g/L, and MnCl2·4H2O 1.58 g/L. As required, ampicillin (100 μg/ml) and kanamycin (50 μg/ml) were added for selection.
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 on (www.jcat.de), 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 amplified and ligated into pUC57 using Genewiz Biotech Co., Ltd. (Suzhou, China), producing pUC57-ClLS&pUC57-GPPS. MvaE-mvaS was then excised from pYJM20  and ligated into pET-28a (+) to create pET28a-mvaE-mvaS. GPPS and ClLS were cloned and assembled into pET28a-mvaE-mvaS at the SacI/AatII sites to generate pSC02 (Table 1). CymAa-cymAb fragments were obtained through Pseudomonas putida using AatII and PacI and ligated into pSC02 to create pSC03 (Table 1). 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 1).
pYJM14 was constructed from pTrcHis2B through the introduction of ERG8, ERG12, ERG19 and IDI from S. cerevisiae [39,60]. All plasmids and primers are shown in Table S1.
Total RNA was isolated from 24 h cultures using commercially available SPAKeasy RNA kits. RNA was reverse transcribed using TaKaRa Primer Script RT reagent Kit and RT-PCRs were performed. Each reaction contained 1 μL cDNA, 5μL TB green Premix Ex TaqII, 0.2μL 50×ROX Reference Dye, 0.2 μM for/rev primer, and ddH2O up to 10 µl. RT-PCR conditions were as follows: 30 s at 95 °C, 40 cycles of 95 °C for 5 s, 60 °C for 30 s. Gene expression was normalized to the absolute transcript levels of rpoD. qRT-PCRs were performed on a Primer 5.0 program (Table S2). Relative gene expression was calculated using the 2ΔΔCt method for each treatment. Reactions were repeated a minimum of 3 times.
Cultures were produced in in 25 ml of LB. E. coli strains with each recombinant plasmid inoculated in a gyratory shaker at 37 °C and 180 rpm. IPTG (0.2 mM) was added to induce recombinant protein expression upon an OD600 of 0.6. Cultures were incubated at 30°C with 2-phase fermentation  used for limonene and perillyl alcohol extraction from the aqueous broth due to the toxicity of terpenes. We added 10% (v/v) n-dodecane or DINP (disononyl phthalate) following IPTG induction and cultures were incubated for 72 h. Cell densities, glucose levels, limonene levels and perillyl alcohol production were then assessed.
For (R)-(+)-perillyl alcohol production on a larger scale, fed-batch cultivations were performed in a 5 L bioreactor system (Biostat B plus MO 5L) using 2 L of fermentation fluid. Seed cultures (100 ml, 10 g NaCl, 5 g yeast extract, and 10 g of tryptone per 1 L) were added to shake flasks overnight at 37 °C and Sparger aeration was performed to maintain high dissolved oxygen (DO) levels. Post-fermentation, the pH of the broth was maintained at 7.0 through ammonia addition. Fermentation was performed during the growth stages under the following conditions: 37 °C, agitation 400 rpm and airflow at 1 L/min. Antifoam 204 was added as required. DO was maintained at 20% saturation through the control of air flow and stirrer speed (1-2 L/min and 400-900 rpm, respectively). When cells reached an OD OD600 of ~20, the temperature was switched to 30 °C and 0.2 mM IPTG and 0.05 mM riboflavin were added. DINP (10%) was added after 4 h when the initial glucose levels were exhausted, as indicated by the increase in DO. The fed batch mode was initiated through the feeding of 60% glucose at appropriate rates. Residual glucose levels were maintained to low levels through the addition of acetic acid. Samples were periodically collected and OD600 values were determined prior to centrifugation for the separation of the organic and aqueous phases. Organic layers were removed for all GC–MS analysis.
E. coli growth was determined through OD600 measurements on a spectrophotometer (Cary 50 UV-vis, Varian). The Shimadzu GC-MS system (TQ8050) was used for Limonene and perillyl alcohol identification. GC-MS conditions were as follows: 30 m DB-5MS column (internal diameter 0.32 mm, film thickness 0.25 μm); temperature: 50 °C hold, ramped up 10 °C/min to 250 °C with a final hold at 250 °C for 10 min. Highly pure helium was used as a carrier at a linear velocity of 1 ml/min; an injector temperature of 250 °C; a split ratio of 1:10; an ion source temperature of 230 °C and mass range of m/z 40–500. Limonene and perillyl alcohol peaks were identified through the retention times of external standards and MS comparisons via the National Institute of Standards and Technology (NIST) database. Fermentation broths were mixed, centrifuged, and the organic layer was taken for GC-MS analysis.
The enantiomeric distribution of limonene was analyzed using the Agilent Technologies 7890B GC System on a Cyclodex B column (30m×0.25mm internal diameter; film thickness=0.25μm). GC conditions were as follows: 50 °C hold, 2°C/min ramping to 160 °C; carrier: high-purity helium, linear velocity: 1 ml/min; temperature of the injector: 250 °C; split ratio: 1:20. Compounds of interest: (S)-(-)-limonene at 21.2 min and (R)-(+)-limonene at 22.02 min. The fermentation broth was mixed and centrifuged, and the organic layer was analyzed.