The FPS1 gene was tagged at its 3' end replacing the carboxy-terminal threonine residue with a sequence encoding three HA epitopes to permit immunodetection: SGRIFYPYDVPDYAGYPYDVPDYAGYPYDVPDYAAQCGR. The HA sequences are underlined. The construct was expressed from the TPI promoter in the 2 μpYX212 and pYX222 vectors (Novagen; now discontinued) which contain the URA3 and HIS3 selection markers respectively. The gene was cloned into the BamH1 and HindIII sites and the vectors transformed into S. cerevisiae using the lithium acetate method. The GFP (GenBank U62636) and hA2aR  genes were amplified by PCR and cloned into the HindIII and XmaI sites and the HindIII and SalI sites of pYX212 and pYX222, respectively. The GFP gene was cloned with an α-secretion factor to facilitate secretion into the culture medium.
Yeast strains and culturing conditions
S. cerevisiae BY4741 is the parental strain for the deletion mutants spt3Δ, srb5Δ and gcn5Δ from the EUROSCARF collection http://web.uni-frankfurt.de/fb15/mikro/euroscarf and the yTHCBMS1 strain (Open Biosystems) used in this study, and as such provided the wild-type control. Yeast cells were cultured in 2.5 L bioreactors containing 2 L of either CSM ± myo-inositol or 2 × CBS. CSM was composed of 1.7 g/L yeast nitrogen base (YNB) without amino acids, 5 g/L ammonium sulphate supplemented with 2% glucose ± 10 μg/mL additional myo-inositol, 2 × DO solution minus histidine or minus uracil (Clontech Yeast Protocols Handbook Version PR13103) and 10 mM MES pH 6. 2 × CBS was composed of 10 g/L ammonium sulphate, 6 g/L potassium dihydrogen phosphate, 1 g/L magnesium sulphate supplemented with 2% glucose, 2 mL/L each of trace element solution and vitamin stock solution (recipes shown below) and 2 × DO solution minus histidine or minus uracil. The pH was adjusted to, and maintained at, 6 via the online addition of 0.5 M NaOH. The agitation, aeration and temperature of the cultures were maintained at 700 rpm, 1 L per min and 30°C respectively. 1L trace element solution was composed of the following: 15 g EDTA, 4.5 g ZnSO4·7H20, 1 g MnCl2·4H2O, 0.3 g CoCl2·6H2O, 0.3 g CuSO4·5H2O, 0.4 g Na2MoO4·2H2O, 4.5 g CaCl2·2H2O, 3 g FeSO4·7H2O, 1 g H3BO3 and 0.1 g KI. The pH was maintained at 6.0 with 1 M NaOH throughout the addition and finally adjusted to pH 4 with 1 M HCl prior to autoclave sterilisation and storage at 4°C. 1 L vitamin solution was composed of the following: 0.05 g D-biotin, 1 g Ca D (+) panthothenate, 1 g nicotinic acid, 25 g myo-inositol, 1 g thiamine hydrochloride, 1 g pyridoxol hydrochloride and 0.2 g D-amino benzoic acid. pH maintained at 6.5 with 1 M HCl. The vitamin solution was filter sterilized and stored as 20 mL aliquots at 4°C. Plasmid retention was verified by the plating of the cells onto CSM + inositol agar in the absence of histidine or uracil and incubation at 30°C for 4 days. To initiate each experiment, 50 mL of a given medium were inoculated with fresh yeast cells in a baffled shake flask and cultured for up to 72 h in a shaking incubator at 30°C, 220 rpm. This pre-culture was subsequently used to inoculate 200 mL of the same medium in a baffled shake flask and cultured under the conditions outlined above to an OD600 of 1. This was then used to inoculate the bioreactors to initial OD600 = 0.05. For the production of hA2aR, the strains BY4714 and yTHCBMS1 were transformed with hA2aR vectors and cultured in 1.75 L 2 × CBS supplemented with 10 mM theophylline and 0.5, 1.0 or 10.0 μg/mL doxycycline. The cells were harvested by centrifugation once the glucose concentration of the cultures was in the range 5 – 10 mM.
Sampling, extracellular substrate determination and membrane preparation
Samples were withdrawn at various points in both the glucose and ethanol phases. 15 – 100 mL culture were centrifuged at 5000 × g, 4°C for 5 min. 0.5 mL of the supernatant was stored at -20°C for glucose and ethanol analyses. The cell pellets were frozen in liquid nitrogen and stored at -80°C for subsequent membrane preparation. Ethanol analysis (10176290035, R-Biopharm, Germany) was performed according to the manufacturer's instructions. Glucose concentrations were calculated with an Accu-Chek Active glucose analyzer (Roche Diagnostics, UK). Cell pellets were fractionated with glass beads (1:1 ratio) in 2 mL cell breaking buffer (50 mM potassium phosphate pH 7.4, 100 mM NaCl, 0.5 mM EDTA, 5% glycerol, 4 mM PMSF). The cells were agitated in a Fast Prep (Thermo Fisher Scientific, UK) at speed 6.5, employing 6 × 40 s pulses with 2 min incubations on ice between pulses. The samples were clarified at 10,000 × g, 4°C for 30 min and the total membrane pellet recovered from the supernatant at 100,000 × g, 4°C for 60 min. Total membranes were re-suspended in 50 μL of Buffer A (20 mM Hepes pH 8, 50 mM NaCl, 10% glycerol w/v) and the total protein concentration determined using a Bio-Rad (Hemel Hempstead, UK) Bradford-based assay with bovine serum albumin as standard. Dry-weight determinations were performed by collecting two samples of 5 mL by centrifugation for 5 min at 5,000 g. The cells were washed once in 5 mL water, dried for 24 h at 110°C, and stored in a desiccator for 24 h before being weighed. Typical dry weights were 2.1 g/L for low-yielding conditions and 2.0 g/L for high yielding conditions after completion of the glucose growth phase.
Immunoblotting and yield analysis
30 – 75 μg of total membranes were loaded per lane on an 8% polyacrylamide gel and separated by SDS-PAGE at 150 V for 1.25 h. Proteins were subsequently transferred to a nitrocellulose membrane (ProTran; Geneflow, UK) at 100 V for 1 h. The membrane was blocked with phosphate-buffered saline (PBS) containing 5% milk overnight at 4°C before incubating with mouse monoclonal anti-HA (clone 12CA5; Roche Diagnostics, UK) at a 1:5,000 dilution in PBS/5% milk for 1 h at room temperature with gentle agitation. The membrane was subsequently washed twice with PBS/0.2% Tween 20 for 5 min before incubating with goat anti-mouse horseradish peroxidase-conjugated secondary monoclonal antibody (Sigma-Aldrich, UK) at a 1:5,000 dilution in PBS/5% milk for 1 h at room temperature with gentle agitation. The membrane was washed as above and developed using an enhanced chemiluminesence detection kit (Geneflow, UK) following the manufacturer's instructions and visualized with a Chemidoc (UVItech, UK). The signal from each lane was quantified using either UVIband or the ImageGauge programme and was expressed as the factor improvement over our internal control (which is the previously-reported reference yield of Fps1 per μg of total membrane ) and was corrected for the amount of total membranes loaded per lane.
Radioligand binding assay
Harvested cells were re-suspended in 30 mL breaking buffer and disrupted at 30,000 psi for 10 min using an Avestin C3. The samples were clarified by centrifugation at 10,000 × g, 4°C for 30 min and total membranes recovered from the supernatant at 100,000 × g, 4°C for 60 min. Total membranes were re-suspended in 2.5 mL Buffer A, the protein concentration determined using a NanoDrop 1000 (Thermo Fisher Scientific, UK) and 0.5 mL aliquots stored at -80°C. Membrane bound hA2aR was then determined using a radioligand binding assay based on the protocol of Fraser . Membranes at 0.5 mg/ml with 0.1 U of adenosine deaminase were incubated with varying concentrations of 3H ZM241385 for 60 min at 30°C and non-specific binding was defined by including 1 μM ZM241385 in the incubations. Assays were terminated by centrifugation at 14,000 rpm in a bench-top centrifuge for 5 min. The supernatant was discarded, the pellets washed superficially with water and solublilized with Soluene, which was added to scintillation fluid and then counted to determine bound radioactivity. Binding was analyzed using PRISM Graphpad v 4.0 to determine Kd and binding capacity.
GFP fluorescence measurements
50 mL of 2 × CBS medium were inoculated with individual yeast colonies transformed with the GFP vector and cultured for up to 48 h in the presence or absence of various doxycycline concentrations at 220 rpm and 30°C. These cultures were used to inoculate fresh 50 mL 2 × CBS medium to a final OD600 of 0.1 and were cultured as above for 16 – 20 h. 1 mL samples were withdrawn and the cells pelletted at 5,000 × g, 4°C for 5 min and the supernatant collected. 200 μL supernatant were loaded in triplicate in a black Nunc MaxiSorp 96-well plate and the fluorescence recorded on a SpectraMax Gemini XS plate reader (Molecular Devices, Wokingham, UK) with excitation and emission wavelengths of 390 nm and 510 nm respectively, and a cut-off of 495 nm. Doxycycline fluorescence accounted for less than 5% of the signal up to concentrations of 10 μg/mL.
RNA preparation and real time quantitative PCR
Yeast cells (60 mL) from two biological replicates and two technical replicates were harvested and frozen in liquid nitrogen. Total RNA was then prepared using the RNeasy kit from Qiagen with on-column DNAse treatment, following the manufacturer's instructions. Analysis of mRNA was performed using real time quantitative PCR (Q-PCR). 1.1 μg RNA was used in the cDNA reaction using the iScript cDNA Synthesis Kit (Bio-Rad, UK). Each sample was amplified using up to 30 cycles (20 s 94°C; 20 s 60°C; 20 s 72°C) in a Bio-Rad iCycler iQ, and the data were analyzed using iCycler IQ version 3.0. The data were normalized using the reference genes PDA1 and ACT1 and the signal was scaled to mRNA copies/cell according to a SAGE study  in which copies of mRNA/cell of all the reference genes had previously been determined.
On-line flow microcalorimetry
Flow microcalorimetric data were collected for bioreactor-grown cultures as detailed above, in the absence or presence of 0.5 μg/mL doxycycline using a Thermal Activity Monitor 2277 (Thermometric AB, Sweden) with a flow vessel working volume of 0.6 cm3
. The system was sterilized by successively pumping through solutions of sterile deionized water, 70% ethanol (v/v), sodium hydroxide (0.5 M) and finally sterile deionized water. The thermostatic water bath was maintained at 30°C, a sampling interval of 600 s was chosen in the Digitam v4.1 software and the pumping rate was set at 48 mL/h (Watson Marlow 400–403 u/VM2, 30 rpm). The calorimeter was calibrated at the same temperature, flow rate and amplifier setting (1000 μW) to be used during experimental data collection in order to establish a steady baseline deflection. Electrical calibration was done in the culture medium. Once the baseline was established, the inlet and outlet tubes were placed in the bioreactor in the presence of 70% ethanol prior to inoculation in order to maintain sterility. Power-time data were imported into Microcal Origin 7.0 where all subsequent data analysis was performed. Pseudo 1st
order rate constants were extracted from the calorimetric data using the equation below [33
], for a flowing system that conforms to 1st
where Φ is the calorimetric output (Js-1); F is the flow rate (dm3s-1); C is the concentration of reactant (mol dm-3); H is the enthalpy of reaction (kJ mol-1); k is the rate constant (s-1); τ is the residence time of reacting solution (s); t is time (s). By plotting the natural logarithm of power against time, a pseudo 1st order rate constant for exponential growth under the conditions employed was obtained. This was validated against the value calculated from OD600 and dry weight measurements. Peak areas were calculated using MicroCal Origin 7.0 using the calculus function embedded in the software. Integration limits were chosen to omit the initial 10 min of data to minimize errors associated with the initiation of the experiment. The high limit was taken as being the very bottom of the down-curve of the glucose peak.
50 mL of 2 × CBS medium were inoculated with individual yeast colonies and cultured for up to 72 h in the presence or absence of 0.5 μg/mL doxycycline at 220 rpm and 30°C. These cultures were used to inoculate fresh 50 mL 2 × CBS medium to a final OD600 of 0.1 and were cultured as above for 24 h. 50 mL 2 × CBS were inoculated to a final OD600 of 0.1 from these cultures and grown as stated above to a final OD600 of 1. Cycloheximide was then added to the cultures to a final concentration of 10 μg/mL and incubated for 10 min. Cells were recovered at 5,000 × g, 4°C, 5 min, stored on ice and then suspended in 0.4 mL lysis buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 30 mM MgCl2, 200 μg/mL heparin, 50 μg/mL cycloheximide, 1 mM DTT, 1 μl/mL RNase inhibitor and one EDTA-free protease inhibitor cocktail per 10 mL buffer) prepared in fresh DEPC-treated water with an equivalent volume of acid-washed glass beads. The cells were then disrupted in a Precellys 24 (Bertin Technology, France) at a speed of 6,500 rpm for 10 s. Samples were cooled on ice for 2 min followed by 2 additional rounds of disruption. The samples were clarified twice at 5,000 × g, 4°C, 5 min and 10,000 × g, 4°C, 20 min and the supernatant recovered. The RNA was subsequently quantified at 260 nm on a Nanodrop (Thermo Fisher Scientific, UK) and 5 – 10 OD600 units loaded on a freshly-prepared 10 mL 10 – 50% sucrose gradient. Gradients were ultracentrifuged at 37,000 rpm, 4°C in a SW40 Beckman rotor for 2 h 40 min. The OD254 profile of the gradients were recorded by chart recorder (Pharmacia LKB REC 102, GE Healthcare Life Sciences, UK) by passing the gradient (from the bottom of the tube to the top of the tube) through an AmershamPharmacia UV detector (GE Healthcare Life Sciences, UK) at a speed of 1.8 mL/min with the simultaneous collection of 0.7 mL fractions. These fractions were stored at -20°C for later analysis. For EDTA polysome disruption profiling, yeast cultures were prepared as above and the cells recovered in the absence of cycloheximide. Cells were resuspended in 0.4 mL lysis buffer lacking cyclohexamide, and disrupted and clarified as above. The RNA was quantified as previously stated and 10 OD600 units were mixed with 50 mM EDTA and loaded on top of freshly-prepared 10 mL 7.5 – 20% sucrose gradients. The gradients were then ultracentrifuged and processed as described above.
Analysis of ribosomal RNA
50 mL 2 × CBS medium were inoculated with individual yeast colonies transformed with the Fps1-HA3 vector and cultured for up to 48 h in the presence or absence of various doxycycline concentrations at 220 rpm and 30°C. These cultures were used to inoculate fresh 50 mL 2 × CBS medium containing various doxycycline concentrations to a final OD600 of 0.1 and were cultured as above to a final OD600 of 1 – 3. Cells from 0.5 mL culture were collected by centrifugation at 1,000 × g, 4°C for 5 min and the supernatant discarded. The pellet was then processed and the RNA collected with an RNeasy Mini Kit (Qiagen) according to the standard enzymatic lysis protocol for yeast. The RNA was quantified and samples diluted to 150 ng/μL. The RNA samples were heated to 70°C for 3 min prior to loading 150 ng samples in triplicate on an Agilent RNA 6000 Nanochip and running on an Agilent Bioanalyzer 2100 according to the manufacturer's instructions.