Bacteria strains, plasmids, and oligonucleotides
The bacterial strains and plasmids used in this study are listed in Additional file 1. The specific oligonucleotides used for PCR amplification were synthesized by Integrated DNA Technologies (IDT) and listed in Additional file 3. We constructed Modules 4–7 (p15A-amp-\(\Delta\)N50LsLCYe-OfCCD1-trxA (TM1) with three mutations of OfCCD1 active site loop, adding katG) and 4–8 (p15A-amp-\(\Delta\)N50LsLCYe-OfCCD1-trxA (TM1) with three mutations of OfCCD1 active site loop, adding ahpC/F). The backbone was amplified with Module 4–1 as a template and the genes of the katG and ahpC/F were amplified from E. coli BL21. Cloning was performed using the iProof ™ High-Fidelity DNA Polymerase (BIO-RAD).
Media and culture conditions
All the cells were grown in LB media . For the production test, a chemically defined auto-induction medium was used, which contained 2 g/L glucose and 8 g/L glycerol, 2 g/L ammonium sulfate, 4.2 g/L KH2PO4, 11.24 g/L K2HPO4, 1.7 g/L citric acid, 0.5 g/L MgSO4, and 10 mL/L trace element solution. The trace element solution (100 x) contained 0.25 g/L CoCl2·6H2O, 1.5 g/L MnSO4·4H2O, 0.15 g/L CuSO4·2H2O, 0.3 g/L H3BO3, 0.25 g/L Na2MoO4·2H2O, 0.8 g/ L Zn(CH3COO)2, 5 g/L Fe(III) citrate, and 0.84 g/L ethylenediaminetetraacetic acid (EDTA) at pH 8.0. The auto-induction medium was supplemented with the appropriate antibiotics (100 mg/L ampicillin, 34 mg/L chloramphenicol, 50 mg/L kanamycin, and 50 mg/L spectinomycin) to maintain corresponding plasmids. Cells were induced by 15 mM lactose . 1% fresh cell culture was inoculated into 1 ml of auto-induction medium in 14 ml snap cap tubes and 10 ml of auto-induction medium in 100 ml flasks. After induction, dodecane (200 μL for the 1 ml culture and 10 ml for the 10 ml culture) was added to the culture to extract ionone, and the cells were incubated at 28 °C for 72 h with a shaking speed of 100 rpm or 300 rpm before harvest.
Quantification of α-ionone and 6-Methyl-5-hepten-2-one (MHO)
The α-ionone, or MHO samples were prepared by diluting 10–50 times of organic layer into 1000 μL hexane. Gas chromatography–mass spectrometry (GC–MS) analyses of the samples were performed on an Intuvo 9000 GC system attached with a 5977B MS detector (Agilent Technologies, USA). The system was equipped with a polar DB wax column (polyethylene glycol (PEG); 30 m × 0.25 mm I.D. × 0.25 µm: Agilent Technologies, USA) and a split injector (split ratio 1:10). The oven program started at 80 °C for 1 min, with temperature increased by 20 °C/min until 130 °C, held for 1.5 min, before being increased by 40 °C/min until 200 °C. This was held for 2 min, before being finally increased by 80 °C/min and maintained at 230 °C for another 2 min. Helium was used as the carrier gas at a constant flow rate of 1.0 mL/min. The Agilent 5977B mass spectrometer was operated in the electron ionization mode at 70 eV with a source temperature of 230 °C, transfer line temperature set at 250 °C, and a scan range of m/z 50–500 in the full scan mode at an acquisition rate of 3.6 scans/s. Methanol was the solvent for the column wash and hexane for the needle wash. The injection volume was 1 µL. The ionone concentrations were calculated by interpolating with a standard curve prepared by commercial standards (Sigma-Aldrich Pte Ltd, Singapore). The mass spectrometer was operated in EI mode with a full scan analysis.
ROS-Glo™ H2O2 Assay
ROS-Glo (Promega, Madison, WI, USA) assay was used as specified to quantify H2O2 production [18, 19]. E. coli strains AI_2211 and AI_2218 were inoculated into 1 ml auto-induced R-medium by RTS-1C (Personal Bioreactor, Biosan). Cells were grown to an OD600 of 1.5–2.0 and then the H2O2 substrate was added. The culture was subsequently incubated for 1.5 h, the cells harvested, and the amount of luciferin precursor produced measured. The H2O2 substrate reacts directly with H2O2 to generate a luciferin precursor. Upon addition of ROS-Glo™ Detection Reagent containing Ultra-Glo™ Recombinant Luciferase and d-Cysteine, the precursor is converted to luciferin by the d-Cysteine, and the luciferin produced reacts with Ultra-Glo™ Recombinant Luciferase to generate a luminescent signal that is proportional to H2O2 concentration (ROS-Glo™ H2O2 Assay, Promega). 800 µL of cells were incubated with 200 µL of H2O2 substrate followed by the addition of the ROS-Glo detection reagent. Luminescence corresponding to H2O2 levels was measured using a micro plate reader (Molecular Devices, San Jose, CA, USA).
Quantification of carotenoids
Intracellular carotenoids were extracted from cellular pellets according to the previous method . Briefly, 10–50 μL bacterial culture was collected and centrifuged. Cell pellets were washed with PBS and were resuspended in 20 μl of water, followed by addition of 180 μl of acetone. The high performance liquid chromatography (HPLC) method was modified as previously described. Briefly, the analysis employed an Agilent 1260 Infinity LC System equipped with a ZORBAX, Eclipse Plus C18, 4.6 × 250 mm, 5 μm column and diode array detector (DAD). Isocratic condition (50% methanol, 48% ethyl acetate, and 2% water) was maintained at 1.5 mL/min for 5 min. The carotenoids were detected at wavelength of 450 nm. Standard curves were generated using commercial standards of ε-carotene (CaroteNature, Switzerland) and lycopene (Santa Cruz Biotechnology, Dallas, TX).
The starting medium was a chemically defined medium modified from previous studies , which contained 5 g/L of glucose transferred into a 5L bioreactor with an initial working volume of 1.8L. The E. coli strain AI_2218 was inoculated into the sterile defined medium to obtain an initial optical density at 600 nm (or OD600) of 0.1. The fermentation was first carried out under the controlled set points of pH, temperature, and dissolved oxygen at 7.0, 37 °C, and 30%, respectively. After inoculation, peristatic pumping of the feedstock solution (containing 750 g/L glucose and 7.5 g/L MgSO4) at 1.62 mL/h flow rate was carried out overnight (~ 13 h). The pH of the culture was controlled at 7.0 using an alkaline solution (a mixture of 28% ammonium hydroxide and 1 M sodium hydroxide solution; in ratio 1:1 by volume) throughout the experiments. The flow rate of feedstock was changed to an exponential feeding rate after 13 h, cells were induced by 0.1 mM Isopropyl β-d-1-thiogalactopyranoside (IPTG) when OD600 reached about 40.0 and 700 mL of sunflower oil as an extractant was then added to the bioreactor. Dissolved oxygen was subsequently lowered to 15% and the flow rate of the feedstock was kept constant at 15.6 mL/h after induction.