All chemicals, standards, solvents, and media components were purchased from Sigma-Aldrich (St. Louis, MO), Fisher Scientific (Fair Lawn, NJ, USA), or VWR (Radnor, PA, USA). Deionised water was filtered by Sartorius Arium Pro VF Type 1 water system (18.2 MΩ, 0.2 μm).
2.1. Strain construction
The engineered wild-type strain producing limonene, EcoCTs03, was produced by transforming pJBEI-6409 plasmid, which was a gift from Taek Soon Lee (Addgene plasmid # 47048 ; http://n2t.net/addgene:47048 ; RRID:Addgene_47048) [3] through heat shock in E. coli K-12 MG1655. pJBEI-6409 is a p15A plasmid expressing limonene from acetyl-CoA via geranyl pyrophosphate (GPP) and was used as-is in this study. CRISPR Cas9-assisted recombineering method as described previously [47] was used to delete ldhA (UniProt P52643) and adhE (UniProt P0A9Q7) genes from the E. coli MG1655 strain, resulting in LDH and ALDH-ADH knockouts, respectively. The ldhA and adhE regions of the successfully knockout strains were PCR amplified and thereafter sequenced to confirm gene deletion. The strains were stored at -80 °C in 40% glycerol prior to further use. To produce limonene, the pJBEI-6409 plasmid was subjected to heat shock in ΔldhA and ΔadhE strains. The Mix & Go! E. coli Transformation Kit (Zymo Chem) was utilised following manufacturer’s instructions to prepare competent cells using the ldhA and adhE deletion strains. The pJBEI-6409 transformed strains (EcoCTs03, LDH KO, and ALDH-ADH KO) were placed onto individual chloramphenicol selective Luria-Bertani agar plates and left overnight at 37 °C.
In this study, the plasmids used for the expression of the glk enzyme were derived from the previously published plasmids pJBEI-6409 and pTrc-trGPPS(CO)-LS (pJEBI-3101), which were obtained from Addgene [3]. While pJEBI-3101 is a ColE1 plasmid expressing limonene from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) via GPP, it was modified to express the selected enzyme of interest. A list of strains and primers can be found in Table 1 and Table 2, respectively. The gene of interest was glk (Uniprot P0A6V8).
EcoCTs-CPMS5 was created by restriction-free cloning [48, 49]. Briefly, plasmid backbone was amplified by PCR using iProof™ High-Fidelity DNA Polymerase (Bio-Rad), in which overlapping overhangs were created by primer design. The resulting DNA was treated with DpnI (New England Labs) overnight at 37 °C. From the DpnI-treated PCR product, 1 μL was transformed by heat shock in E. coli DH5α, plated on an ampicillin selective Luria-Bertani agar plate and incubated overnight at 37 °C. Positive colonies were identified by colony PCR using PCRBIO Taq mix red (PCR Biosystems), and were subsequently inoculated overnight in 2 mL Luria-Bertani supplemented with appropriate antibiotic at 37 °C, 300 rpm. Cultures were spun down, where the plasmid was extracted using E.Z.N.A.® Plasmid Mini Kit I (Omega Bio-tek) and sequenced using Sanger sequencing (Bio Basic) to confirm the presence of the deletion. Finally, plasmids were confirmed by performing restriction enzyme digestion (RE) and compared with in-silico agarose gel simulation on SnapGene 7.0.2.
The gene of interest was cloned in EcoCTs-CPMS5 by PCR-amplifying the backbone of the plasmid without limonene synthase (LS), as well as the genes of interest from E. coli K-12 MG1655 genomic DNA. Primers contained overhangs such that the backbone and inserts shared a 15 bp overlap region at the site of integration. Expected band sizes were confirmed on an agarose gel and subsequently treated with DpnI overnight at 37 °C. DpnI-treated PCR products were purified using the E.Z.N.A.® gel extraction kit (Omega Bio-tek). Each of purified DNA backbone (50-100 ng) and inserts were added to a PCR tube with In-Fusion Snap Assembly Master Mix (Takara Bio). The mixture was incubated at 50 °C for 15 mins, and 1 μL was transformed by heat shock in E. coli DH5α, plated on an ampicillin selective Luria-Bertani agar plate and incubated overnight at 37 °C. Colonies were analyzed by colony PCR, amplified, extracted, sequenced, and RE digested as described above.
For expression, plasmids were transformed by heat shock in E. coli K-12 MG1655. Competent cells were prepared using the Mix & Go! E. coli Transformation Kit (Zymo Chem) following manufacturer’s specifications. For the expression experiment, pJBEI-6409 was co-transformed with in EcoCTs-CPMS5-GOI (gene of interest, glk), plated on ampicillin and chloramphenicol selective LB agar plates and incubated overnight at 37°C.
2.2. Growth conditions
From the prepared E. coli strains which were placed onto plates, one colony from each plate was selected after overnight incubation at 37 oC. Each colony was inoculated into 5 mL Luria-Bertani medium (5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl) with 30 µg/mL chloramphenicol and left overnight in a shaking incubator at 220 rpm and 37 oC. After which, cell pellets were washed and re-suspended in 50 mL M9 medium (12.7 g/L Na2HPO4.7H2O, 3.1 g/L KH2PO4, 1 g/L NH4Cl, 0.5 g/L NaCl, 0.25 g/L MgSO4.7H2O, 15 mg/L CaCl2.2H2O, 8.1 mg/L FeCl3, 0.89 mg/L MnCl2.4H2O, 1.7 mg/L ZnCl2, 0.34 mg/L CuCl2, 0.6 mg/L CoCl2.6H2O, 0.51 mg/L Na2MoO4) adapted from a previous study [50] with 10 g/L glucose and left overnight in a shaking incubator at 30 oC and 220 rpm. Each strain was kept at -80 ◦C in 40% glycerol. For each glycerol-stored strain, 100 µL was added to 50 mL M9 medium in 250 mL flasks forming pre-cultures and left overnight at 220 rpm and 30 oC. For the collection of metabolomics data at various time-points, numerous cell culture flasks were prepared for duplicate biological samples for each time-series collection. Through the addition of 100 µL pre-cultures of each strain to 50 mL M9 medium in 250 mL flasks, cell cultures of each strain were prepared and kept overnight in a shaking incubator at 220 rpm and 30 oC. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added resulting in a final concentration of 25 μM when cell cultures reached an optical density of 1 at 600 nm. To enable trapping of secreted limonene, dodecane overlays of 5 mL were added to cell cultures [3] and kept at 30 oC and 220 rpm in a shaking incubator. To obtain the concentrations of the intracellular and extracellular metabolites, and secreted limonene for dynamic modelling of the EcoCTs03 strain, prepared cell culture flasks were sacrificed in duplicates at time-points 2 h, 3 h, 6 h, 7 h, and 8 h post-IPTG induction.
2.3 Metabolomics extraction and analysis
2.3.1 Secreted Limonene and Extracellular Metabolites
Cell cultures of 50 mL EcoCTs03 with dodecane overlay were sacrificed at time points 0 h, 2 h, 3 h, 6 h, 7 h, and 8 h post IPTG induction in duplicates and centrifuged at 3000 rpm for 10 mins. For the mutant strains (HK overexpressed, ALDH-ADH knockout, and LDH knockout), only secreted limonene concentrations were analysed by sacrificing shake flasks in duplicates at time points 2 h, 3 h, 6 h and 7 h post IPTG induction. The dodecane overlay which trapped the secreted limonene was removed from each sample and stored at -80 oC prior to analysis. Diluted limonene extracts in ethyl acetate were run on an Agilent 7890B gas chromatography mass spectrometry (GC-MS) system equipped with a DB-5ms column. A 10:1 split ratio and 10 mL/min split flow was utilised for each run with 1 µL of sample injected. The GC oven was held at 40 oC for 3 min, with a temperature gradient of 10 oC/min to 100 oC followed by a 60 oC/min ramp to 220 oC, which was held for 2 min. The temperatures of the injector and MS transfer line were set at 250 oC and 280 oC, respectively. Selected ion-monitoring (SIM) mode was utilised for the MS with ions of m/z 136, 68 and 93 monitored, which represented the molecular ion and top two abundant fragmental ions of limonene. Calibration standards were prepared for limonene in ethyl acetate with concentrations ranging from 0.05 µg/mL to 10 µg/mL. Limonene concentrations from cell cultures were determined using Agilent Quantitative software.
After centrifugation, supernatants of the EcoCTs03 cell cultures were kept at -80 oC before subjecting the extracellular metabolites to quantitative analysis. Aliquots of 1 mL from the thawed supernatant samples were filtered using polyamide filters and subjected to an Agilent 1200 high performance liquid chromatography (HPLC) system equipped with a Bio-rad Aminex HPX-87H column (300 x 7.8 mm). The HPLC system was run together with a 1260 Infinity II Refractive Index Detector (RID). An isocratic gradient was run for 28 min with 0.01 N sulphuric acid, 0.6 mL/ min flow rate, and 5 µL of sample injected. The HPLC column was at 35 oC while the RID had positive polarity at 30 oC. Concentrations of extracellular metabolites were determined from calibrations mixtures of the following range: glucose – 0.5 to 80 g/L; acetic acid – 0.125 to 80 g/L; lactic acid – 0.125 to 8 g/L; ethanol – 0.5 to 80 g/L. Cell pellets left after centrifugation were oven dried before weighting.
2.3.2 Intracellular Metabolites
Cell cultures of EcoCTs03 were sacrificed in duplicates at time-points of 2 h, 3 h, 6 h, 7 h, and 8 h after IPTG induction, where 10 mL aliquots of cell cultures were subjected to fast filtration followed by rapid quenching in liquid nitrogen as described previously with modifications [51, 52]. Briefly, for fast filtration, 10 mL aliquots of each cell culture were filtered using 0.2 μm polyamide membrane filters (Sartorius, Goettingen, Germany) followed by washing with 5 mL of wash solution (1 g/L NH4Cl, 0.5 g/L NaCl, 12.7 g/L Na2HPO4.7H2O, 3.1 g/L KH2PO4,). The filter membrane containing the cells was placed onto aluminum foil, folded, and quickly plunged into liquid nitrogen. The membrane filters encased in aluminum foil were stored at -80 oC until the extraction of the intracellular metabolites. The intracellular metabolites were extracted by removing the aluminum foil and placing the membranes in 5 mL of a 4:4:2 mixture of methanol, acetonitrile, and water, and left to stand in an ice bath for 10 mins. After which, membranes were vortexed for 1 min and subjected to sonication for 3 mins thrice, where samples were placed in an ice bath for 1 min between each round of sonication. An internal standard mixture consisting of 10 µg/mL thymolphthalein monophosphate (TMP) and 50 µg/mL mevalonic acid-d3 (MVA-d3) in a solvent mixture of methanol: 10 mM ammonium hydroxide (7:3) was prepared. Extracts were spiked with 20 µL of the internal standard mixture after decanting into glass tubes. This was followed by subjection to a vacuum concentrator and reconstitution with 200 µL methanol: 10 mM ammonium hydroxide mixture (7:3) prior to filtration into glass vials for quantitative liquid chromatography mass spectrometry analysis.
An Agilent 6230 time of flight-mass spectrometer (TOF-MS) coupled with a Dual Agilent Jet Stream (AJS) ion source was used together with an Agilent ultra-performance liquid chromatography (UPLC) 1290 system. A VanGuard pre-column (2.1 x 5 mm) was utilized with a Waters Acquity UPLC BEH C18 column (2.1 x 150 mm, 1.7 µm). The chromatographic method utilized was modified from previous work [51, 52]. Samples of 2 µL were injected into the system with mobile phase A consisting of 5 mM ammonium formate in water (pH 9.5) and mobile phase B consisting of 5 mM ammonium formate (pH 9.5) in acetonitrile: water (9:1). The solvent gradient utilized for each run started with a flow rate of 0.1 mL/min and 100% mobile phase A held from 0 to 3.5 min followed by 100% mobile phase B at 12 min which was then held for 8 min and flow rate increased to 0.5 mL/min. The mobile phase was changed to 100% mobile phase A at 20 min for 5 min and kept for another 5 min. The column temperature was held at 35 oC throughout the analysis. For the TOF, negative electrospray ionisation was utilised with the following conditions: Gas flow, 11 L/min; Gas temperature, 325 °C; Nebuliser pressure, 35 psi; Sheath gas flow, 11 L/min; Sheath gas temperature, 375 °C; Vcap voltage, 3500 V; Nozzle voltage, 500 V; Skimmer, 65; OctopoleRFPeak, 750; Scan rate, 2 spectra/s. The fragmentor voltage was altered during each 35 min sample run: 2 – 7.5 min, 140 V; 7.5 – 15 min, 100 V, 140 V and 150 V. The following flow diversions for the UPLC was used: 0 – 2 min to waste, 2 – 15 min to TOF-MS, and 15 – 35 min to waste. Methanol: 10 mM ammonium hydroxide (7:3) mixture was used for preparing standard mixtures used for intracellular metabolite quantitation. The intracellular metabolites determined were R5P+Ru5P+X5P (pool of ribose-5-phosphate, ribulose-5-phosphate, and xylulose-5-phosphate), F16BP (fructose-1,6-biphosphate), DHAP+GAP (pool of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate), PYR (pyruvate), DXP (1-deoxy-D-xylulose-5-phosphate), MVA (mevalonate), MVAP (5-phosphomevalonate), MVAPP (5-pyrophosphomevalonate), IPP+DMAPP (pool of isopentenyl pyrophosphate and dimethylallyl pyrophosphate), FPP (farnesyl diphosphate), and GPP (geranyl diphosphate). Calibration mixtures were made up to 100 µL with 10 µL of internal standard mixture consisting of 50 µg/mL MVA-d3 and 10 µg/mL TMP. The following concentration range was used for the calibration mixtures: R5P (R5P + Ru5P + X5P pool) – 0.04 to 10 µg/mL; F1,6BP – 0.04 to 6 µg/mL; DHAP (DHAP + GAP pool) – 0.04 to 5 µg/mL; PYR – 0.04 to 1.5 µg/mL; DXP – 0.04 to 10 µg/mL; MVA – 0.04 to 10 µg/mL; MVAP – 0.01 to 0.3 µg/mL; MVAPP – 0.01 to 0.3 µg/mL; IPP (IPP + DMAPP pool) – 0.05 to 1.5 µg/mL; FPP – 0.05 to 1.5 µg/mL; GPP – 0.05 to 1.5 µg/mL. Agilent Masshunter Workstation Quantitative Analysis for TOF was utilised for metabolite quantitation.
G6P (glucose-6-phosphate) and F6P (fructose-6-phosphate) were quantified by injecting 2 µL of samples and running the samples through the LC-TOF-MS system with a ZIC-HILIC column (2.1 x 100 mm, 3.5 µm). The solvent gradient started with a 10% mobile phase A with a flow rate of 0.5 mL/min at 0 min, 25% mobile phase A at 1.5 min, 35% mobile phase A at 1.8 min and held until 6 min, 10% mobile phase A at 6.5 min until 9.5 with flow rate of 0.6 mL/min, and 10% mobile phase with flow rate of 0.5 mL/min held until 10 min. The column temperature was constant at 35 oC. For the TOF, the same conditions were utilised as described for the other intracellular metabolites with exception to the fragmentor voltage which was kept at 140 V from 0.1 – 6 min while the flow diversion for UPLC was 0 – 0.1 min to waste, 0.1 – 6 min to TOF-MS, and 6 – 10 min to waste. The following concentration range was used for the calibration mixtures: G6P – 0.04 to 10 µg/mL and F6P – 0.04 to 6 µg/mL, with TMP as the internal standard. Metabolite quantitation was executed using the Agilent Masshunter Workstation Quantitative Analysis for TOF.
2.3.3 13C tracer experiments
For experiments involving 13C tracers, [1-13C]glucose (99 atom% 13C) and [4-13C]glucose (99 atom% 13C) tracers were used in parallel in independent 50 mL cell cultures of the engineered wild-type strain (EcoCTs03) instead of glucose as described in the previous section. The cell cultures were prepared in duplicates and sacrificed 24 h after IPTG induction for cell cultures containing either [1-13C]glucose or [4-13C]glucose. The dodecane layers were removed after centrifugation and subjected to GC-MS analysis in scan mode for limonene, where the metabolic flux ratios for the EMP and ED pathway based on the [1-13C]glucose and the metabolic flux ratios for DXP and MVA pathway based on the [4-13C]glucose were determined based on previous work [45]. The correction of limonene mass distribution vectors (MDVs) for natural 13C and 2H isotopes incorporation was executed as previously described [53], where the resulting corrected MDV (MDV*) was utilized for the calculation of the metabolic flux ratios.
2.4 Model construction
The dynamic model of limonene production by the E. coli strain EcoCTs03 was developed with the open-source and stand-alone program COPASI (build 260; [46]) and it was modified from our recently published model [54]. The dynamic model describes the carbon and energy metabolism of EcoCTs03 expressing the MVA pathway from the exponential growth phase after IPTG induction under aerobic conditions. The model contains 55 species and 56 reactions that consist of the pathways found in Figure 1, such as EMP pathway, ED pathway, tricarboxylic acid cycle (TCA cycle), pentose phosphate pathway, acetate metabolism, MVA pathway and DXP pathway [55-57]. For model simplicity, the model only comprises of one compartment. Extracellular metabolites were denoted with an “-ex” suffix, for example “Glcex” or “ACEex” which denotes extracellular glucose and acetate respectively. Concentration units of metabolites in the model were in mmol/L/g dry cell weight (g DCW; reflected as mmol/l in the COPASI file), which was the same units utilized in the time series metabolomics data. Normalizing metabolites concentrations to g DCW allowed for comparison across the different time points after IPTG induction. All reactions were described using Michaelis-Menten rate law, except for enzymes PDH, LDH, PoxB and LAC transport reactions which were described by mass action instead. Table S1 details the rate laws, rate law equations and the parameter values of the final fitted model.