Strains and plasmids
We based this work on the E. coli strain W3110, which is a derivative of E. coli K-12 [25]. A detailed description of the strains and plasmids used in this work is given in Table 7. Strain W(pheA−)Rg is a p-coumaric acid producer, which was previously reported by our group [21]. The genes corresponding to 4CL from S. coelicolor A2 (NP_628552.1) and STS from A. hypogaea (BAA78617.1) were synthesized by Life Technologies with codon optimization for E. coli.
Table 7 Strains and plasmids used in this work
The DNA sequence of the STS gene from V. vinifera was synthesized by Epoch Life Science Inc. and was codon harmonized for improved protein expression by performing synonymous codon replacement of the original sequence, with the codon having the best frequency match to that in the native organism. This approach was based on a previously published work for codon harmonization [26]. Species-specific codon usage tables were obtained from the Codon Usage Database (http://www.kazusa.or.jp/codon/), and analysis of the codon frequency differences was performed with the website-tool Graphical Codon Usage Analyzer available at http://www.gcua.schoedl.de/index.html [27].
Plasmids construction
The construction of plasmids pTrc-Sc4CL-AhSTS and pTrc-Sc4CL-VvSTS (Table 7), were designed so that both genes are under the control of the Trc promoter in an operon with a gene encoding 4CL placed next to the promoter and followed by the STS gene. The genes encoding the 4CL from S. coelicolor A2 and the STS from A. hypogaea were placed each after a ribosome binding site (RBS) taken from the pTrc vector while the STS gene from V. vinifera (VvSTS) was designed with a customized RBS sequence using RBS calculator v1.1 [28].
The first construction step consisted on the individual cloning of the 4CL and STS synthesized genes into the vector pTrc99A by digestion with restriction enzymes NcoI/KpnI, thus generating the plasmids pTrc-sc4CL and pTrc-ahSTS. The gene AhSTS was then subcloned in the KpnI/HindIII sites of pTrc-Sc4CL, producing the plasmid pTrc-Sc4CL-AhSTS. Each plasmid was confirmed by PCR and sequencing. For the construction of the plasmid pTrc-Sc4CL-VvSTS, we started from pTrc-Sc4CL-AhSTS and replaced the sequence of the AhSTS gene with that of the VvSTS gene using restriction sites KpnI/HindIII.
Culture media and conditions
For plasmids construction experiments, solid and liquid Luria–Bertani (LB) media was used, and the antibiotics carbenicillin (Cb; 100 µg/mL), tetracycline (Tc; 30 µg/mL) and kanamycin (Km; 30 µg/mL) were used when required for strain selection.
Cultures for the characterization of production were performed in M9 mineral medium (Na2HPO4-7H2O 12.8 g/L, KH2PO4 3 g/L, NaCl 0.5 g/L, NH4Cl 1 g/L, MgSO4 249 mg/L, CaCl2 11.1 mg/L, and thiamine 10 μg/L). The carbon source was either glucose or glycerol at 10 g/L for shake-flask experiments and the medium was supplemented with l-phenylalanine 0.3 mM to allow the growth of the pheA
− mutant strain.
Culture conditions for strain characterization
Experiments for growth profile characterization were started with an overnight LB culture followed by an adaptation step carried out at a starting density of 0.1 O.D.600 in a 250 mL baffled shake flasks with 50 mL of M9 medium supplemented with 10 g/L of either glucose or glycerol. Flasks were placed in a shaking incubator at 30 °C and 300 rpm for 12 h. Cells were then harvested and used to inoculate again 250 mL baffled shake flasks with 50 mL of fresh M9 medium at a starting density of 0.1 O.D.600. Cultures of strains carrying 4CL and STS genes were also supplemented with p-coumaric acid at a concentration of 3 mM. Cultures were incubated at 30 °C, 300 rpm and induced with 0.5 mM of IPTG when they reached 0.8 O.D.600. Then, cultures continued for another 20 h after induction. All cultures were performed at least in triplicate.
Coculture fermentations
Batch coculture fermentations were performed in 1 L autoclavable glass bioreactors (Applikon, The Nederlads) connected to an Applikon ADI 1010 BioControlleer and ADI1025 controllers to control pH, temperature, stirrer speed, and aeration rate. The working volume was 750 mL and culture conditions were set at pH 7.0, 30 °C, 600 rpm and an aeration rate of 0.5 vvm of sterile air. pH was controlled automatically by adding a solution of 10 % w/v NH4OH.
LB overnight cultures were used to start the inocula in 250 mL baffled shake flasks with 50 mL of mineral M9 medium without addition of p-coumaric acid and supplemented with 0.3 mM l-phenylalanine and 10 g/L glycerol. Cells were then harvested to inoculate bioreactors. Cultures in bioreactors were started in the same fresh medium as the inoculum at an O.D.600 of 0.05 for each strain, to give a final starting inoculum of 0.1 O.D.600. Growth was monitored, and when reached 0.8 O.D.600, cultures were induced at a final concentration of 0.5 mM of IPTG. Cultures continued for 20 h more after induction. Samples of 500 µL of the culture were collected for resveratrol extraction and 1 mL for O.D.600 and glycerol determination.
Protein expressions assays
Total protein expression was analyzed using 12 % SDS–polyacrylamide gel electrophoresis and Coomassie blue staining. Briefly, 1 mL aliquots of culture were collected at the end of the cultures and O.D.600 was determined. All samples were standardized by O.D.600 (dilution to a volume of 1 mL at 3.0 O.D.600). Cells were then centrifuged, and the pellet resuspended in 100 µL of distilled water. Then 20 µL were mixed with 10 µL of loading buffer with β-mercaptoethanol and boiled in a water bath at 100 °C for 5 min. Samples of 5 µL each were loaded and resolved on a 12 % SDS–polyacrylamide gel, followed by Coomassie blue staining.
p-Coumaric acid and trans-resveratrol extractions
To quantify p-coumaric acid and trans-resveratrol, 500 µL aliquots of complete culture were mixed with an equal volume of ethyl acetate. The mixture was vortexed for 30 s and after centrifugation, 400 µL of the organic layer was separated in a clean microfuge tube and evaporated till dryness in an Eppendorf Concentrator. After that, samples were resolubilized in 400 µL of methanol. Finally, samples were filtered through Whatman Anopore inorganic filters of 0.2 µm before injecting into an HPLC system.
Analytical methods
Cell growth was monitored measuring optical density at 600 nm (O.D.600) in a spectrophotometer (Beckman DU-70; Fullerton, CA, USA). Glucose and glycerol concentrations were determined using an HPLC system (600E quaternary bomb, 717 automatic injector, 2410 refraction index, Waters, Milford, MA, USA) using an Aminex HPX-87H column (Bio-rad, Hercules, CA, USA). 20 µL of a sample was injected using as mobile phase 5 mM H2SO4, with a flow rate of 0.5 mL/min. The column temperature was set at 50 °C. Sugars were detected by their refractive index. Identification and quantitation of compounds was performed by comparison of each peak retention time and interpolation in a standard curve. For analysis of p-coumaric acid and trans-resveratrol, we used an HPLC system (Agilent 1100 System, Agilent Technologies, Palo Alto, CA, USA), with a reverse phase column (Phenomenex Synergi Hydro RP C18, 150 × 4.6 mm, 4 um, Phenomenex, Torrance, CA, USA) and equipped with photodiode array detection. We eluted the sample using an isocratic mobile phase of 0.1 % TFA in water (A) and 0.1 % TFA in methanol (B) in a ratio of 60:40 (A:B v/v). Flow was set at one mL/min, column temperature at 45 °C and the injection volume was 10 µL. Identification of p-CA and trans-resveratrol was performed by comparison of the retention times and UV/Vis spectra of each peak with the standards. For quantification of each component, calibration curves were constructed using component standards and by interpolation of the sample signals within the calibration curves.