Chemicals
Enzymes, deoxynucleotide triphosphates and Phusion™ high-fidelity DNA-polymerase was obtained from ThermoFisher Scientific (Vienna, Austria). 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) and hemin were purchased from Sigma-Aldrich (Vienna, Austria). Difco™ yeast nitrogen base w/o amino acids (YNB), Difco™ yeast nitrogen base w/o amino acids and ammonia sulfate (YNB2), Bacto™ tryptone and Bacto™ yeast extract were purchased from Becton–Dickinson (Vienna, Austria). Zeocin™ was purchased from InvivoGen (Toulouse, France) via Eubio (Vienna, Austria).
Microorganisms
For this study, a HRP C1A gene, codon-optimized for P. pastoris, was ordered from GenScript (Nanjing, China) and cloned into a pPICZαC vector, providing a Zeocin™ (Zeo) resistance gene as well as an α-prepro mating signal sequence from Saccharomyces cerevisiae for product secretion, using standard methods. Correct integration was verified by sequencing. The pPICZαC vector was successfully integrated in a P. pastoris GS115 strain (HIS+, pep4Δ, aox1∆), kindly provided by Biogrammatics, Inc. (California, United States) and should yield mainly Man5GlcNAc2 glycosylated HRP C1A after transformation (SuperMan5) [15]. The Och1 deficiency of the SuperMan5 strain is based on the disruption, but not deletion of the OCH1 gene. The strain CBS 7435 (identical to NRRL Y-11430 or ATCC 76273) was used as a benchmark wild type strain (wt), which yields natively hypermannosylated HRP C1A [12]. As described in our previous study, we used a genetically engineered wt strain harboring a knock-out deletion of the OCH1 gene (∆OCH1) to avoid hypermannosylation that yielded mainly Man8–10GlcNAc2 glycosylated HRP C1A upon transformation [12]. Both, the wt and ∆OCH1 strain contained a pPpT4_S, which contained the codon-optimized HRP C1A gene under equal conditions [12, 40]. Therefore, all resulting strains had a MutS phenotype, expressed and secreted HRP C1A upon induction of the AOX1 promoter with MeOH.
Culture media
The growth medium [buffered medium with glycerol for yeast (BMGY)] for the shake-flask screenings contained: 10 g L−1 yeast extract, 20 g L−1 peptone, 13.4 g L−1 YNB2, 4 mg L−1 d(+)-biotin, 10 g L−1 glycerol and 100 mL of a 1 M potassium phosphate buffer pH 6.0. The induction medium [buffered medium with MeOH for yeast (BMMY)] for the shake-flask screenings contained: 10 g L−1 yeast extract, 20 g L−1 peptone, 13.4 g L−1 YNB2, 4 mg L−1 d(+)-biotin, 5 g L−1 MeOH and 100 mL of a 1 M potassium phosphate buffer pH 6.0. The preculture medium for the bioreactor cultivations [yeast nitrogen base medium (YNBM)] contained: 20 g L−1 α-d(+)-glucose monohydrate, 3.4 g L−1 YNB2, 10 g L−1 (NH4)2SO4, 0.4 g L−1 d(+)-biotin, 0.1 M potassium phosphate buffer, pH 6.0. Trace element solution (PTM1) for the bioreactor cultivation contained: 6 g L−1 CuSO4·5H2O, 0.08 g L−1 NaCI, 3 g L−1 MnSO4·H2O, 0.2 g L−1 Na2MoO4·2H2O, 0.02 g L−1 H3BO3, 0.5 g L−1 CoCl2, 20 g L−1 ZnCl2, 65 g L−1 FeSO4·7H2O, 0.2 g L−1 d(+)-biotin, 5 mL L−1 95–98% H2SO4. Basal salt medium (BSM) for the bioreactor cultivations contained: 60 g L−1 glycerol, 1.17 g L−1 CaSO4·2H2O, 18.2 g L−1 K2SO4, 14.9 g L−1 MgSO4·7H2O, 4.13 g L−1 KOH, 26.7 mL L−1 85% (v/v) o-phosphoric acid, 0.2 mL L−1 Antifoam Struktol J650, 4.35 mL L−1 PTM1, NH4OH as N-source. pH was maintained by using 12.5% NH3,aq. Throughout all shake-flask cultivations, Zeocin™ was used in a concentration of 50 µg mL−1.
Strain selection
After transformation, 10 Zeo resistant clones were picked and grown overnight in 10 mL BMGY-Zeo medium in 100 mL baffled shake-flasks at 230 rpm and 30 °C. Then, cells were harvested by centrifugation (1800×g, 4 °C, 10 min) and re-suspended in BMMY-Zeo for adaptation of the cells to MeOH. Again, cells were grown at 230 rpm and 30 °C. Recombinant protein production was induced by adding 1.5% (v/v) pulses of pure MeOH supplemented with 12 mL PTM1/L MeOH to the culture each day, for 5 days. Each day, a sample was taken and analyzed for OD600, total protein content in the cell-free cultivation broth (Bradford assay) as well as the presence of recombinant HRP C1A by SDS-PAGE. A recombinant Pichia pastoris strain carrying the empty pPICZαC vector was included as negative control in all experiments.
Analysis of strain morphology and glycosylation
To understand a possible impact of the genotype and phenotype on overall strain physiology and productivity, an initial shake-flask screening, including morphological analysis was performed. The strain morphology was analyzed under inducing conditions for the wt, ∆OCH1 and SuperMan5 strain. In parallel, growth and product formation were monitored to assure product presence for later glycosylation pattern analysis.
Shake-flask screening
A fresh cryo tube (− 80 °C) was thawed for each HRP C1A containing strain, added to 200 mL BMGY-Zeo medium in a 1000 mL shake-flask and incubated at 28 °C and 230 rpm overnight. The next day, 50 mL of each culture were transferred to 450 mL BMMY-Zeo, including also 10 µM Hemin (Heme) to ease HRP C1A induction [41]. Induced cultures were grown in 2.5 L baffled flasks and a working volume of 500 mL. For comparability, HRP C1A induction was performed at 28 °C for all 3 strains. To assure complete depletion of the initial C-source (glycerol) and accurate adaptation to the inducing C-source in the shake-flasks (MeOH), cells were grown for 23 h in BMMY-Zeo-Heme before the first MeOH pulse was given. MeOH pulses were given each day as 1% (v/v) with PTM1 (12 mL L−1 MeOH). Sampling of the cultures was done approximately every 12 h. After 47 h of induction, 100 mL of each culture were harvested, centrifuged (4000×g, 10 min, 4 °C), the cell free supernatant was concentrated 20× with a 10 kDa centrifugal filter membrane (Amicon®Ultra—15) tube (Merck Millipore Ltd., Carrigtwohill, IRL) and stored at − 20 °C for further analysis. Enzyme activity and total protein content of the concentrates were measured and aliquots of the concentrates were used for identification of the respective HRP C1A glycosylation pattern of each strain. However, the total induction time of the shake-flask cultures was 71 h to further monitor growth and the morphological behavior of the different strains.
Microscopy
Twenty microlitres of the cultivation broth were pipetted onto a standard glass slide (25 × 75 mm) and then covered with an extra-large cover slide (24 × 60 mm). Images were recorded at a 40× magnification with a five megapixel microscopy CCD colour camera (Olympus, Austria). These images were used as a rough estimation of cell agglomerate formation and agglomerate diameter.
Flow cytometry
Samples of the shake-flask screening were diluted in phosphate buffered saline (PBS) (2.65 g L−1 CaCl2 solution, 0.2 g L−1 KCl, 0.2 g L−1 KH2PO4, 0.1 g L−1 MgCl·6 H2O, 8 g L−1 NaCl and 0.764 g L−1 Na2HPO4·2H2O at pH 6.5) to an OD600 of 1. Then, 0.5 µL of 20 mM propidium iodide stock in dimethyl sulfoxide (both from Sigma Aldrich, St. Louis, United States) and 5 µL of 12 mM fluorescein diacetate (Sigma Aldrich, St. Louis, United States) stock in acetone were added to 0.5 mL of the cell suspension. After 10 min incubation in the dark at room temperature, the sample was further diluted (1:10 in PBS) for flow cytometric analysis.
A CytoSense flow cytometer (CytoBuoy, Woerden, Netherlands) with two forward scatter (FSC), one sideward scatter (SSC) and two fluorescence channels (green, red) was used for single cell analysis. The implemented laser had a wavelength of 488 nm. The configuration of the emission wavelength filter set was 515–562 ± 5 nm for the green fluorescence channel (used for fluorescein diacetate) and 605–720 ± 5 nm for the red fluorescence channel (used for propidium iodide). The flow cytometer was equipped with a PixeLINK PL-B741 1.3 MP monochrome camera for image-in-flow acquisition, which made real-time imaging of cell agglomerates possible. For data evaluation, the software CytoClus3 (CytoBuoy, Woerden, Netherlands) was used.
The CytoSense flow cytometer provides multiple spatially resolved data points per channel per particle. This signal is achieved for both scatter channels as well as for green and red fluorescence channels [29], which is the basis for multiple curve parameters. Except for length parameters in µm, all parameters are in arbitrary units, as the user can set the sensitivity of the detector. The following parameters were used for the distinction of morphological classes: maximum (maximum of signal curve), total (area under curve), length (length of the signal) and sample length (length of signal above trigger level). Furthermore, image-in-flow feature enabled visual identification of yeast agglomerates, termed clusters. It should be noted that while FSC signals are closely linked to particle size (sample length), FSC length signals do not always correspond entirely to sample length due to overlays of other signals, which was seen during calibration with defined beads.
Glycosylation analysis
A glycopeptide analysis using an LC–ESI–MS system was performed, as we have reported before [12, 42]. The concentrated samples of the shake-flask screening were digested in solution. The proteins were S-alkylated with iodoacetamide and digested with trypsin (Promega, Madison, United States). The peptide mixtures were loaded on a BioBasic C18 column (BioBasic-18, 150 × 0.32 mm, 5 µm; ThermoFisher Scientific, Vienna, Austria) using 80 mM ammonium formiate buffer as the aqueous solvent. A gradient from 5% B (B: 80% acetonitrile) to 40% B in 45 min was applied, followed by a 15 min gradient from 40% B to 90% B that facilitates elution of large peptides, at a flow rate of 6 µL min−1. Detection was performed with a QTOF MS (Bruker maXis 4G) equipped with the standard ESI source in positive ion, DDA mode (= switching to MSMS mode for eluting peaks). MS-scans were recorded (range 150–2200 Da) and the 3 highest peaks were selected for fragmentation. Instrument calibration was performed using an ESI calibration mixture (Agilent, Santa Clara, United States). The nine possible glycopeptides were identified as sets of peaks consisting of the peptide moiety and the attached N-glycan varying in the number of HexNAc, hexose and phosphate residues. The theoretical masses of these glycopeptides were determined with a spreadsheet using the monoisotopic masses for amino acids and monosaccharides. Manual glycopeptide searches were made using DataAnalysis 4.0 (Bruker, Billerica, United States).
Bioreactor cultivations
After conducting the shake-flask screening, we characterized the recombinant SuperMan5 strain in terms of physiology, biomass growth and productivity using a dynamic strategy of conducting MeOH pulses during batch cultivations in the controlled environment of a bioreactor, which we have described several times before [12, 33,34,35,36]. This cultivation was used for subsequent purification to perform product kinetics and thermal stability analysis.
Preculture
Frozen stocks (− 80 °C) from working cell banks were incubated in 100 mL of YNBM-Zeo in 1 L shake-flasks at 30 °C and 230 rpm for 24 h. The preculture was transferred aseptically to the respective culture vessel. The inoculation volume was 10% of the final starting volume.
Cultivation
Batch cultivation was carried out in a 5 L working volume Labfors glass bioreactor (Infors, Bottmingen, Switzerland). BSM was sterilized in the bioreactor and pH was adjusted to pH 5.0 by using 12.5% NH3,aq after autoclavation. Sterile filtered PTM1 was transferred to the reactor aseptically. pH and dissolved oxygen probes were calibrated prior to cultivation start. Dissolved oxygen (dO2) was measured with a sterilizable polarographic dissolved oxygen electrode (Mettler Toledo, Vienna, Austria) and maintained above 20% throughout the cultivation. The pH was measured with a sterilizable electrode (Mettler Toledo, Vienna, Austria) and maintained constant at pH 5.0 with a step controller using 12.5% NH3,aq. Base consumption was determined gravimetrically. Agitation was fixed to 1495 rpm. The culture was aerated with 2.0 vvm dried air and offgas of the culture was measured by using an infrared cell for CO2 and a paramagnetic cell for O2 concentration (Servomax, Egg, Switzerland). Temperature, pH, dO2, agitation as well as CO2 and O2 in the offgas were measured on-line and logged in a process information management system (PIMS Lucullus; Applikon Biotechnology, Delft, Netherlands).
The end of the initial batch phase at 30 °C and therefore complete glycerol consumption was indicated by an increase in dO2, a drop in offgas CO2 and an increase in offgas O2. The first MeOH pulse (adaptation pulse) of a final concentration of 0.5% (v/v) was conducted with MeOH supplemented with 12 mL PTM1 per 1 L of added MeOH (MeOH/PTM1 pulse). Subsequently, at least two MeOH/PTM1 pulses were given to 1% (v/v) at 30 °C, then 25 °C, 20 °C and finally 15 °C. For each pulse, at least two samples were taken to determine the concentrations of MeOH and product as well as dry cell weight (DCW) and OD600 to calculate the strain specific physiological parameters. Induction was carried out in the presence of 1 mM hemin, which was added prior to the adaptation pulse [43].
Sample analysis
Analysis of growth- and expression-parameters
Dry cell weight (DCW) was determined by centrifugation of 5 mL culture broth (4000×g, 4 °C, 10 min), washing the pellet twice with 5 mL water and subsequent drying for 72 h at 105 °C. Determination was performed in triplicates. OD600 of the culture broth was measured in duplicates using a spectrophotometer (Genesys 20; ThermoFisher Scientific, Vienna, Austria). The activity of HRP C1A in the cell free supernatant was determined with a CuBiAn XC enzymatic robot (Optocell, Bielefeld, Germany) in duplicates. Cell free samples (60 µL) were added to 840 µL of 1 mM ABTS in 50 mM potassium phosphate buffer, pH 6.5. The reaction mixture was incubated for 5 min at 37 °C and was started by the addition of 100 µL of 0.078% H2O2. Changes of absorbance at 420 nm were measured for 180 s and rates were calculated. Calibration was done by using commercially available horseradish peroxidase (Type VI-A, P6782; Sigma-Aldrich, Vienna, Austria,) as standard at six different concentrations (0.02; 0.05; 0.1; 0.25; 0.5 and 1.0 U mL−1). Protein concentration of cell free supernatant was determined at 595 nm using the Bradford Protein Assay Kit (Bio-Rad Laboratories GmbH, Vienna, Austria) with bovine serum albumin (protein standard; micro standard, liquid; P0914; Sigma Aldrich, Vienna, Austria) as standard.
Substrate concentrations
Concentration of glycerol and MeOH was determined in cell free samples of the bioreactor cultivation by HPLC (Agilent Technologies, Santa Clara, United States) equipped with a Supelco guard column, a Supelco gel C-610H ion-exchange column (Sigma-Aldrich, Vienna, Austria) and a refractive index detector (Agilent Technologies, Santa Clara, United States). The mobile phase was 0.1% H3PO4 with a constant flow rate of 0.5 mL min−1 and the system was run isocratically. Calibration was done by measuring standard points in the range of 0.1–10 g L−1 glycerol and MeOH.
Data analysis
Strain characteristic parameters of the bioreactor cultivation were determined at a carbon dioxide evolution rate (CER) above 2.5 mmol g−1 h−1 during each MeOH pulse. Along the observed standard deviation for the single measurement, the error was propagated to the specific rates (qs and qp) as well as to the yield coefficients. The error of determination of the specific rates and the yields was therefore set to 10% and 5%, respectively for single measurement-derived values like seen in the batch phase [34]. For the pulse experiments, the average value and the standard deviation were used as two pulses were given for each temperature.
Enzyme characterization
Kinetic constants
A cell free bioreactor supernatant with HRP C1A from the SuperMan5 strain was twofold concentrated and diafiltrated with buffer (500 mM NaCl, 20 mM NaOAc, pH 6.0) [44, 45]. Protein concentration of the HRP C1A preparation was determined at 595 nm using the Bradford Protein Assay Kit (Bio-Rad Laboratories GmbH, Austria) with bovine serum albumin as standard. The kinetic constants for ABTS and H2O2 were determined. The reaction was started by adding 10 μL enzyme solution (1.0 mg mL−1) to 990 μL reaction buffer containing either ABTS in varying concentrations (0.01–5 mM) and 1 mM H2O2 or H2O2 in varying concentrations (0.001–0.5 mM) and 5 mM ABTS in 50 mM potassium phosphate buffer at pH 6.5. The change in absorbance at 420 nm was recorded in a spectrophotometer UV-1601 (Shimadzu, Japan) at 30 °C. Absorption curves were recorded with a software program (UVPC Optional Kinetics; Shimadzu, Japan). Measurements were performed in triplicates.
Thermal stability
The purified enzyme solution was incubated at 60 °C. At different time points, aliquots were withdrawn, the solutions were immediately cooled and centrifuged (20,000×g, 15 min) to pellet precipitated proteins and the remaining catalytic activity in the supernatants was measured [46].