Bacterial strains
Lactococcus lactis NZ900 was used as an intermediate host for plasmid construction. The mutant strain (hyaluronidase-free and haemolytic negative) S. equi subsp. zooepidemicus BA06 strain was obtained as previously described (Schiraldi et al. 2010). Plasmid pNZ8148 was obtained from Nizo (Netherlands).
Materials
Genomic DNA and plasmid DNA were isolated using Qiagen DNeasy kit, Qiagen miniprep kit, (Qiagen, Valencia, CA) respectively according to the manufacturer’s instructions. Restriction endonuclease digestions, DNA ligations, agarose gel electrophoresis were performed using standard techniques (Sambrook and Russel 2001).
Construction of the kfoA and kfoC overexpressing strains
The kfoA and kfoC genes were amplified from E. coli K4 genomic DNA. kfoA was amplified with primers kfoA_Up: 5′- GGACGGTGCCATGGATGAATATATTAGTTACAGGTGGAG-3′containing the NcoI recognition site, and KfoA_Dw 5′-TCTCTAGACGCGGATCCTTAAATATAACCATTTGGGTTTTTCA-3′ containing the BamHI and XbaI restriction sites. kfoC was amplified with primers kfoC_ Up 5′-CGGGATCCAGTGAGGAGTTACTGATGAGTATTCTTAATCAAG-3′ and kfoC_Dw 5′-GAGCTCTTATAAATCATTCTCTATTTTTTCC-3′, containing the BamHI and SacI restriction sites respectively. Polymerase chain reaction (PCR) was carried out with Expand High fidelity PCR System (Roche, Monza. Italy) according to the manufacturer’s protocol. DNA fragments were recovered from agarose gels using the Qiaquick gel extraction kit (Qiagen, Valencia, CA). The kfoA and kfoC genes were sequentially cloned into plasmid pNZ8148 using L. lactis as intermediate host to improve transformation efficiency (Additional file 1).
Efficiency of sub-cloning was verified by performing double digestions with BamHI and SacI on the recombinant plasmid extracted from colonies of L. lactis growing on selective medium. Restriction endonucleases were purchased from New England Biolabs and ligases were purchased from Invitrogen (Carlsband, CA). Nucleotide sequencing of all PCR fragments cloned was carried out at BMR Genomics (Padova, Italy) to check whether any mutation was introduced.
pNZ8148kfoAkfoC was isolated from L. lactis-pNZ8148kfoAkfoC and electroporated into S. equi subsp. zooepidemicus BA06 slightly changing the protocol described by Marcellin et al. (2010). Briefly, cells were grown on THY supplemented with 2 M glycine. About 250 mL of fresh medium were inoculated with an o/n pre-culture and grown for 3 h at 37 °C until OD530 reached a value of about 0.6. Prior to harvesting, 0.2 mg/mL of hyaluronidase (H3506, Sigma-Aldrich) were added to the broth and incubated for 30 min at the same temperature. After centrifugation the pellet was resuspended in 0.5 M sucrose and washed twice; finally it was resuspended in about 600 μL of the same solution. The recombinant plasmid pNZ8148kfoAkfoC was added to the ice cold cell aliquot and electroporation was carried out on a Biorad Bio-Rad Gene Pulser (2 mm cuvettes, 2.0 kV, 200 Ω, 25 μF) according to the protocol suggested by the manufacturer. Selection was performed on THY supplemented with 5 μg/mL of chloramphenicol. Plates were incubated o/n at 37 °C.
Shake flask experiments
All cultivations were conducted in 3 L flasks filled with 0.6 L of medium in order to keep a 5:1 air–liquid ratio. Growth temperature was set at 37 °C and the culture was agitated at 140 rpm, in a rotary shaker incubator (model Minitron, Infors, Bottmingen, Switzerland). Experiments were performed on the medium containing per L: sucrose 17 ± 1 g, yeast extract 10 g, KH2PO4 2 g, K2HPO4 9.7 g/L, MnSO4·4H2O 0.1 g, and 1 mL of microelement solution (CaCl2 2 g/L, MnSO4·4H2O 0.05 g/L, CuSO4·5H2O 0.019 g/L, ZnCl2 0.046 g/L). pH was adjusted to 7.2 before strain inoculation. The medium also contained 5 μg/mL of chloramphenicol to avoid plasmid loss. Twenty ng/mL of nisin were added about 2–3 h after inoculating the culture to induce expression of recombinant proteins.
Experiments with the addition of 0.5 mM GalNAc, or 2 mM phosphatidylcholine after induction were also performed.
Determination of sucrose and acids produced during growth was performed by HPLC (UHPLC Dionex Ultimate 3000; Thermofisher) on a Alltech IOA-2000 column (250 mm × 6.5 mm ID). Three mL of broth collected at T0 and at the end of the process were centrifuged and supernatants were ultrafiltered on 3 kDa centricon devices (Millipore, Bedford, MA, USA) at 5000×g and the flow through was used for analyses. Runs were performed at 40 °C with 0.1% v/v sulphuric acid in water as mobile phase at a flow rate of 0.6 mL/min. Detection was performed via UV absorbance at 200 nm and refraction index (Shodex RI-101 detector, Max auto step 5.1 s, temperature 32 °C, rise time 1 s, polarity plus, record range 512 µRIU, integrator range 500 µRIU/UV).
Extraction of intracellular polysaccharides
In order to extract intracellular chondroitin and HA, about 10 g of wet cells were resuspended in 20 mL of distilled water and autoclaved at 120 °C for 15 min. After centrifugation the supernatant was recovered and precipitated on ice with 4 volumes of cold ethanol 96% v/v, and stored at 4 °C o/n. The pellet, recovered after centrifugation was treated with 1 mg/mL DNase I (Applichem) in a buffer containing 100 mM Tris pH 7.5, 50 mM MgCl2 and 10 mM CaCl2 for 1 h at 37 °C and successively digested with 2.5 mg/mL of Protease K from Aspergillus (Sigma-Aldrich) for 2 h at 56 °C. A second precipitation was repeated on the sample o/n at 4 °C and the resulting pellet was dried and used for: (a) hydrodynamic characterization (b) quantification of uronic acids through carbazole assay (Bitter and Muir 1962) (c) determination of relative ratios of HA and chondroitin through high performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) monosaccharide determinations after hydrolysis.
The powder obtained from the control sample was further purified to verify the production of chondroitin in S. equi subsp. zooepidemicus-pNZ8148kfoAkfoC, through structure determination by NMR analysis.
Purification of intracellular chondroitin from S. equi subsp. zooepidemicus-pNZ8148kfoAkfoC by fast protein liquid chromatography (FPLC)
About 100 mg of powder were dissolved in 5 mL of buffer A (20 mM sodium acetate, 0.5 M sodium chloride, pH 7.4) and loaded on an anion exchange column (HiPrep Q Sepharose 16/10 HP, 1.6 × 10.0 cm, GE Healthcare, Milan, Italy), previously equilibrated with the same buffer, using an ÄKTA Explorer 100 purifier system (GE Healthcare, Milan, Italy), connected to the software UNICORN (GE Healthcare, Milan, Italy). The samples were eluted by applying a three steps gradient at 5, 10.5 and 100% of buffer B (20 mM sodium acetate, 3 M sodium chloride, pH 7.4) in 3 column volumes, at a flow rate of 3 mL/min and collected in 2 mL fractions. The chromatographic profiles were registered detecting the signal at 215 nm. The fractions containing a single peak were pooled together and each pool was loaded on a desalting column (HiPrep 26/10 Desalting, 2.6 × 10.0 cm, GE Healthcare, Milan, Italy) previously equilibrated with pure water. Elution was performed by using the same ÄKTA purifier system, at flow rate of 1 mL/min, in 1.5 column volumes; the chromatographic profiles were detected at 215 nm, the eluted peaks were collected and then freeze-dried (Christ Epsilon 2-6D, Martin Christ, Germany). After lyophilisation the peak fractions were analyzed by Size exclusion chromatography-triple detector array (SEC-TDA), NMR and hydrolysed for HPAE-PAD sugar nucleotide determinations.
Precipitation of extracellular HA and chondroitin
After growth in shakeflask the broth was centrifuged at 6000×g, 4 °C for 30 min. The supernatant was precipitated with 1.8 volumes of cold ethanol (99.9% v/v) o/n at 4 °C to remove HA. The precipitate was recovered by centrifugation and vacuum dried at 40 °C for 18 h. The supernatant underwent a second precipitation, to recover chondroitin, with ethanol up to 4 volumes at 4 °C o/n and the recovered precipitate was dried at 40 °C under vacuum.
Fermentation experiments
Fermentation experiments were carried out on a Biostat C plus reactor (Sartorius Stedim; Melsungen, Germany), with 1.6 L working volume. Agitation was provided by 1 rushton impeller and 2 paddle impellers, suitable for viscous liquid mixing. Fermentation medium contained per L: sucrose 70 g, MgSO4·7H2O 2 g, yeast extract 20 g, Na2HPO4·12H2O 2.23 g, K2SO4 1.3 g, arginin 0.05 g and 2.5 mL of microelement solution. The preculture was performed in 1 L shakeflasks filled with 100 mL of medium, reported in the previous section, and grown on a rotary shaker at 200 rpm and 37 °C for 12–14 h. When pH dropped to 5 the flask culture was aseptically used to inoculate the fermenter: inoculum size was 5% of the volume of fermentation medium. A constant pH was maintained at 7.2 by automated addition of 6 M NaOH and 30% v/v sulfuric acid. Stirring and airflow rate were set at 200 rpm and 1.2/1.4 vvm for the duration of the experiment. The medium also contained 5 μg/mL of chloramphenicol and addition of 20 ng/mL of nisin was performed about 3 h after fermentation start. A pulse of sucrose re-establishing a concentration of 20 g/L in the broth was performed after 10 h of growth. At least four samples were withdrawn during the initial batch phase and at the end of the experiments to measure substrate consumption and production of lactic acid by HPLC as described in “Shakeflask experiments” section. The overall process lasted 18 h.
Fermentation broth downstream processing to obtain heteropolysaccharides (HA and chondroitin)
The recovered fermentation broth was added with 5% v/v of trichloroacetic acid (from a 50% w/v solution) before centrifugation at 6000×g for 60 min. The pH of the clarified supernatant was adjusted to 6.5 ± 0.2 with 6 M NaOH. A first ultrafiltration (UF) step was performed on a tangential flow filtration system (Sartoflow Alpha crossflow system, Sartorius) by using a polyethersulfone membrane (0.1 m2) with a cut-off of 100 kDa. The supernatant was concentrated about eightfold, diafiltered with 2 volumes of bidistilled water and the retentate was collected. The cassette was washed with 100 mL of water and the recovered solution was added to the concentrated supernatant and precipitated with 1.8 volumes of cold ethanol (4 °C) o/n. The precipitate was vacuum dried at 40 °C for 18 h.
The 100 kDa permeate was further concentrated on UF polyethersulfone membrane (0.1 m2) with a cut-off of 10 kDa, concentrated tenfold and diafiltered with 300 mL of bidistilled water. The cassette was washed with 100 mL of purified water. Conductivity of the 10 kDa retentate and wash fraction was adjusted to about 15 mS/cm with NaCl before precipitation with 4 volumes of cold ethanol (4 °C) o/n. The recovered precipitate was vacuum dried at 40 °C for 18 h. The dried powder obtained from the 100 and 10 kDa retentate samples were analysed by SEC-TDA, HPAE-PAD and carbazole assay.
Intracellular polysaccharides were extracted as described in the previous paragraph (“Extraction of intracellular polysaccharides” section), and quantified as described above.
Molecular weight determination
The molecular mass determinations of HA and chondroitin were carried out using the SEC-TDA 305 equipment by Viscotek (Malvern Instruments, Italy). Dried samples obtained after fermentation downstream treatments were dissolved in water and analysed whereas samples obtained from shakeflask experiments were dissolved in water and diafiltered with 2 volumes of bidistilled water on 3 kDa centrifugal filter devices (YM-10 Centricon, Millipore, Bedford, MA, USA) at 5000×g to remove salts and low molecular weight contaminants. Analyses were performed at concentrations ranging from 0.1 to 0.4 g/L for HA, and from 0.5 to 4 g/L for chondroitin, to have a column load for each sample (injection volume × sample concentration × intrinsic viscosity) of approximately 0.2 dL, and runs were performed at 40 °C with a running time of 50 min. The fragment molecular weight distribution, molecular size distribution, polydispersity, hydrodynamic radius, and intrinsic viscosity were determined as described by La Gatta et al. (2010) and Restaino et al. (2017).
HPAE-PAD
The aminosugars were identified in extracellular and intracellular polysaccharide samples by using the high-performance chromatographic system equipped with a pulsed amperometric detector (PAD, Thermo Fisher Scientific, Italy) on an anion exchange column (Carbopac PA1, Thermo Fisher Scientific, Italy) as previously demonstrated by Marcellin et al. (2009). About 7 mg of powder (obtained after lyophilisation) were hydrolyzed in 1 M HCl for 18 h at 100 °C and analysed as described by Restaino et al. (2017).
NMR analysis
NMR spectra were recorded with a Bruker DRX-600 (1H: 600 MHz, 13C: 150 MHz) instrument equipped with a cryo probe, in D2O (acetone as internal standard, 1H: (CH3)2CO at δ = 2.22 ppm; 13C: (CH3)2CO at δ = 31.5 ppm). The distorsionless enhancement by polarization transfer-heteronuclear single-quantum correlation (DEPT-HSQC) experiments were measured in the 1H-detected mode by single quantum coherence with proton decoupling in the 13C domain, by using data sets of 2048 × 256 points and typically 32 increments.