2 MATERIALS AND METHODS
2.1 Bacterial strains, plasmid and precultures. E. coliK-12 derivatives W3110recA -, and W3110recA - vgb + were obtained as described elsewhere (Jaén, Velazquez, Delvigne, Sigala, & Lara, 2019). The strains were transformed with plasmid pUC18 and preserved at -70 °C. The strains were precultured in lysogenic broth (LB) supplemented with ampicillin disodium salt (100 µg/ml) for 8-10 hr. An aliquot was taken to inoculate 50 mL of mineral medium. The composition of the mineral medium (in g/L) was as follows: K2HPO4, 17; KH2PO4, 5.3; (NH4)2SO4, 2.5; NH4Cl, 1.0; citrate-Na3·2H2O, 2; MgSO4·7H2O, 1.0; and thiamine-HCl, 0.01. The medium was supplemented with a trace element solution of 2 mL/L and 100 μg/mL ampicillin disodium salt. The trace element solution composition is described elsewhere (Jaén, Velazquez, Delvigne, Sigala, & Lara, 2019). Glucose was added at a final concentration of 2.5 g/L. Precultures were performed in baffled shake flasks incubated for 12-16 hr at 250 rpm in a shaker with an orbital diameter of 50 mm. Then, the broth was collected and centrifuged at 7500 rpm for 10 min, and the cell pellet was washed and resuspended in 3 mL of fresh medium and used to inoculate the main cultures.
2.2 Bioreactor cultures. Bioreactor cultures were carried out in a 1-L Biostat B Plus stirred tank bioreactor (Sartorius BBI, Melsungen, Germany) at 37 °C, pH 7.2, air flow 1 vvm. DOT was measured with an optical sensor (Visiferm 120 Arc, Hamilton, Bonaduz, Switzerland). The batch phase started with 500 mL of medium supplemented with 15 g/L glucose and 100 mg/L ampicillin disodium salt. The DOT was controlled at 30 % air sat. during the first 6 h by a PI controller in the agitation cascade mode (ti = 50 s;xp = 140 %; tD = 0 s; dead band = 0.1 %) using MFCS/DA software (Sartorius BBI, Melsungen, Germany). After 6 h of culture, the DOT level was changed to 2 % air sat. The fed-batch phase was started after initial glucose depletion. A concentrated glucose solution (600 g/L) supplemented with ampicillin disodium salt was added to the medium using a Watson-Marlow 101U/R programmable peristaltic pump (Watson Marlow, MA, USA). The exponential feeding rate was programmed to maintain the specific growth rate at 0.15 h−1. Off-gas composition was monitored using a BlueInOne Ferm (BlueSens, Herten, Germany) gas analyzer. Oxygen and CO2 evolution rates (OTR and CER respectively) were calculated from the proper mass balances using the off-gas composition (Jaén, Velázquez, Sigala, & Lara, 2019).
2.3 Off-line analyses. The biomass concentration was determined as the dry cell weight. Glucose and ethanol were quantified by an enzymatic-electrochemical method in a YSI-2900 Biochemistry Analyzer (Yellow Spring Instruments, USA). Organic acids were quantified by HPLC (ProStar 210 HPLC system, Agilent Technologies, Santa Clara, CA, USA). Cell lysis and pDNA purification and extraction were carried out using a QIAprep Miniprep Kit (Qiagen, Germany) according to the manufacturer’s recommendations. pDNA was quantified by UV spectroscopy in a Nanodrop 2000 spectrophotometer (Thermo Scientific, WI, USA). Monomeric pDNA supercoiled fraction (SCF) was determined by electrophoresis of 100 ng of pDNA in 0.8 % agarose gel in TAE buffer for 1 h at 80 V. Restriction analysis was carried out to confirm the plasmid identity usingHind III enzyme.
2.4 PCN, relative RNAII/RNAI and vgb copy number determinations. The PCN was determined through RT-qPCR absolute quantification. Standard curve experiments were executed in a 7500 Real-Time PCR System (Applied Biosystems, CA, USA) using SYBR Green (Applied Biosystems, CA, USA). Plasmid copy quantification was determined using the bla gene from pUC18 and the chromosomal reference gene ihfB . RNA integrity was evaluated by chip microfluidics electrophoresis in a 2100 Bioanalyzer (Agilent CA, USA). The RNA integrity number (RIN) was automatically calculated by the 2100 Expert Software (Agilent, CA, USA). All the samples had RINs between 8 and 9, which are acceptable for our purposes. RNAI and RNAII were determined by RT-qPCR absolute quantification. Standard curve experiments were executed in a 7500 Real-Time PCR System (Applied Biosystems, CA, USA) using SYBR Green. The sequence of primers and reaction conditions are detailed elsewhere (Jaén, Velazquez, Delvigne, Sigala, & Lara, 2019). The same methods were applied for vgbmRNA quantitation using the primers 5’-ATGTTAGACCAGCAAACCATTAACATC-3’ (forward) and 5’-TTATTCAACCGCTTGAGCGTACAAATC-3’ (reverse).
2.5 Flux Balance Analysis. The flux distribution in the metabolic network was calculated based on a linear programming algorithm, using the specific uptake rates (qO2 ,qS ) and specific production rates (µ ,qCO2 , qby-products ) as inputs for the calculations as detailed previously (Jaén, Olivares, Sigala, & Lara, 2017). The network comprised 103 reactions and 76 metabolites (intracellular and extracellular) covering glycolysis, PPP, tricarboxylic acids cycle, and mixed-acid fermentation pathways. It included reactions for transport and exchange. Maximization of biomass synthesis was the objective function employed for all cases Simulations were run using MatLab.