Strains and Culture Conditions
The bacterial strains and plasmids used in this study were listed in Table S1, except for all the bicistronic expression vectors. C. glutamicum CGMCC1.15647, C. glutamicum ATCC13032, E. coli DH5α were stored in our laboratory. E. coli was grown in LB medium or on LB plates containing 1.5% (wt/vol) agar at 37 °C. Unless otherwise indicated, C. glutamate was cultured in LBB broth (LB + 10 g/L brain heart infusion, pH 7.0) at 30 °C for 24 h. The medium for the transformation of C. glutamate was LBHIS medium (5 g/L tryptone, 2.5 g/L yeast extract, 5 g/L NaCl, 18.5 g/L brain heart infusion and 91 g/L sorbitol, pH 7.0). The final concentration of chloramphenicol was 30 mg/L for E. coli and 20 mg/L for C. glutamicum. Isopropyl β-D-Thiogalactoside (IPTG) with a final concentration of 1 mmol/L was added when the OD600 of the cells reached about 0.5.
DNA manipulation and Plasmid construction
C. glutamicum genomic DNA was isolated using a genomic isolation kit (CWBIO, China). Kits for plasmid isolation, DNA gel extraction, and PCR product purification were purchased from Axygen (China). PCR was carried out using PrimerSTAR (TaKaRa, China). T4 ligase and restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA). All the DNA manipulation procedures, including PCR, restriction enzyme digestion, ligation, and agarose gel electrophoresis were carried out following the standard procedures. After successfully constructed in E. coli DH5α, each plasmid was transformed into C. glutamicum CGMCC1.15647 by electroporation as previously described.
All the primers used in this study were listed in Table S2. To obtain the expression vectors harboring different fore-cistronic sequences, a pioneering bicistronic plasmid pbtac-HP-12 was constructed as follows: first, the 62 bp of the N-terminal coding sequence of the candidate gene, NCgl2826, was amplified from C. glutamicum ATCC13032 genome; Primers conferred the amplicon an XhoI cleavage site and one conserved SD sequence (terms as SD1) at the 5' end, as well as the other second SD sequence (terms as SD2) ended with a translation coupling frame (TAATG) at the 3' end. Then the above fore-cistronic amplicon was inserted to the HindⅢ digested original vector pXMJ19 by the homologous recombination kit (Vazyme, China), just before the MCS region of pXMJ19 (Fig. S1).
The remaining 23 bicistronic expression plasmids were constructed by substituting the fore-cistron region in pbtac-HP-12 with the corresponding 23 fore-cistron fragments amplified from C. glutamicum ATCC13032. Here the previously added cleavage site XhoI at the N-terminal of fore-cistron was paired with HindIII for fragments insertion.
To compare the strength of these enhanced expression vectors, PCR products of EGFP were digested with HindⅢ/EcoRⅠ and then inserted into all the 24 bicistronic expression plasmids. Meanwhile, the other EGFP PCR fragments were digested with Xho1/EcoRⅠ and then ligated into pbtac-HP-12 to obtain a monocistronic control pXMJ19-EGFP. To further test the expression ability of the top three strongest bicistronic vectors, PCR products of ADH (GenBank: AGN20313.1), RamA (GenBank: AGN20080.1), GD (GenBank: MH370856.1), BoIFN-α (GenBank: EU276064.1), PΙNP (GenBank: X00820.1) (flanked with HindⅢ/EcoRⅠ adaptors) and ALDH (GenBank: AGN20304.1) (flanked with HindⅢ/ BamH1 adaptors) were cloning to these three plasmids, respectively. All the six genes were appended with a 6 × histidine tag at the C-terminal during the PCR process. ADH, ALDH and RamA were originated from C. glutamicum CGMCC1.15647, while the codon-optimized GD (GenBank: MN.816264), BoIFN-α (GenBank: MN.816265) and PΙNP (contained a cspB signal peptide) (GenBank: MN.816263) genes were synthesized by the company of Shenggong (Shanghai, China). To obtain the monocistronic control vectors expressing the above proteins, each encoding gene was individually amplified again and ligated to pbtac-HP-12. The cloning site here was Xho1/BamH1 for ALDH and Xho1/EcoRⅠ for the rest.
GFP intensity measurement
Cells harboring EGFP plasmids were diluted to a moderate concentration (OD600 ≈ 0.5) and the total fluorescence intensity was measured by a fluorescence spectrophotometer (the excitation wavelength: 488 nm, the emission wavelength: 507 nm). The unit fluorescence intensity of each sample was calculated through normalizing the total fluorescence intensity with OD600. The EGFP expression level was also analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Real-time quantitative PCR (qPCR)
To analyze the transcriptional level of EGFP, strains were grown in 10 mL LBB medium for 24 h, harvested by centrifugation and washed twice with ice-cooled PBS. Total RNA extraction, reverse transcription, and qPCR were then performed using kits from TaKaRa (Dalian, China) according to the manufacturer's instructions. The PCR condition was: 95 °C for 30 s and 45 cycles at 95 °C for 15 s, 62 °C for 30 s, 72 °C for 20 s. The relative EGFP transcription level was analyzed by the 2− ΔΔCt method and the transcript level of housekeeping gene 16S rRNA was used as the endogenous control. Every sample was measured across at least three biological repeats, which had three duplicated wells each.
Protein preparation and western blotting assay
After 24 h of cultivation, the cells were harvested by centrifugation at 12,000 g for 5 min at 4 °C, washed twice with PBS, and then disrupted by sonication on ice. For soluble expressed proteins EGFP, ADH, ALDH, RamA and gD, the lysates were centrifuged at 12,000 g for 15 min and the supernatants were collected for the subsequent protein analysis. For proteins expressed mainly in the inclusion form (BoIFN-α), the whole lysates were used directly. For protein PΙNP, just take the medium supernatant to further analysis. All the protein samples were analyzed by 12% (w/v) SDS-PAGE.
The proteins on the SDS-PAGE gel were electrophoretically transferred onto a polyvinyl difluoride membrane using a Bio-Rad transblot device (USA). The membrane was incubated within 5% non-fat milk powder for 2 h to block nonspecific binding sites. After incubation, replaced the milk with monoclonal horseradish peroxidase (HRP)-conjugated anti-His6 antibody. Washed the membrane three times with TBST after 1 hour of incubation. Finally, the protein was performed using an ECL kit (Amersham Biosciences, America). The strength of protein bands was quantified using Image J software.
Fed-batch cultivation and purification of PΙNP
To validate large-scale expression of EGFP of the top three strongest vectors and achieve large-scale production of PΙNP. After overnight activation, 200 mL of C. glutamicum seed solutions were all transferred to 1.8 L of LBB medium (30 g/L glucose) in a 5 L fermenter (Applikon EZ-control). Throughout the total 48 h of cultivation, the temperature was maintained at 30 °C. The dissolved oxygen was maintained at 30% (v/v). The speed was set to 400–1000 r/min and the pH of the medium was controlled at 7. To avoid glucose starvation, 50 mL glucose solution (300 g/L) was added every four hours after 12 h of inoculation. Glucose concentrations in the culture medium were monitored by a glucose assay kit (Sigma, St. Louis, Missouri, USA).
The protein purification steps were presented below: the medium supernatant contained PΙNP were collected first, protein purification used an AKTA purifier system (GE, Sweden) and a HisTrap HP affinity column. Protein quantity and purity was determined by SDS-PAGE analysis.