Bacterial strains, media, and growth conditions
E. coli Top10 was cultivated in Luria–Bertani (LB) broth or on LB plates with 2 % (w/v) agar at 37 ℃ as a host for transformation. C. ammoniagenes ATCC 6871 grown in NCM medium at 30 ℃ was used as the transformation host for E. coli/Corynebacteria vectors used in this study. After transformation by electroporation (1.8 kV), transformants were plated on BHIS plates with 20 μg/mL chloramphenicol at 30 ℃. Fermentation medium (glucose 100 g/L, peptone 12 g/L, yeast powder 8 g/L, MgSO4·7H2O 10 g/L, KH2PO4 10 g/L, K2HPO4 10 g/L, urea 7 g/L, pH 7.5-7.8) was used to cultivate strains producing CoA. Cultivation for expression analysis was performed in at least biological duplicates.
Recombinant DNA techniques
C. ammoniagenes genomic DNA was isolated using a genomic DNA isolation kit (Tiangen, Beijing, China). Kits for plasmid isolation, extraction of DNA from agarose gels and PCR product purification were also purchased from Tiangen (Beijing, China). I-5 2 × High-Fidelity Master Mix and Trelief™ SoSoo Cloning Kit were purchased from Tsingke (Beijing, China) and used for routine molecular biology applications.
Construction of probe-vector pXMJ190
The shuttle vector pXMJ19 (a kind gift from Professor Dong, Figure 2a) was used as the backbone for vectors constructed in this study. To eliminate interference from the promoter already present in pXMJ19, the tac promoter and lac operator were removed. A gene encoding red fluorescent protein (RFP) was inserted into the MCS to form a probe-vector named pXMJ190 (Figure 2b). Gibson assembly was used to assemble DNA fragments upstream of the reporter gene rfp, resulting in seamless ligation between fragments and the probe-vector [31, 32].
Genetic manipulation of C. ammoniagenes
To increase transformation efficiency, recipients were grown in 100 mL of NCM medium at 30 °C until an OD600 nm of approximately 1.0. Cells were incubated on ice for 20 min and harvested by centrifugation in a polypropylene tube at 4000 rpm for 10 min at 4 °C. After washing twice in cold distilled water and two washes in ice-cold 10% glycerol, cells were resuspended in 1 mL of 10% glycerol. For electroporation, 100 μL of the freshly prepared electro-competent cells were mixed with 3 μL plasmid (50 ng/μL in ddH2O) in a cold sterile electroporation cuvette (1 mm electrode gap) and pulsed immediately with a MicroPulser electroporator (Bio-Rad Laboratories, Inc., Hercules, CA). The electroporator was usually set at a voltage of 1.8 kV. Cells were subsequently resuspended in 0.9 mL of BHIS, heated at 46 °C for 6 minutes and withdrawn immediately for recovery by incubating for 3 h at 30 °C and then plated for selection of transformants.
Construction and analysis of selected promoters
The transcriptional levels of genes can be estimated by Reads Per Kilobase per Million mapped reads (RPKM), so the genes were ranked by RPKM value based on transcriptional data from C. ammoniagenes and the top 20 genes were selected. All of the genes were identified from the genome and their promoters were predicted as below. Six annotated molecular chaperones in the genome were sorted and their promoters were predicted as outlined below. According to previous studies, the promoter-5’-UTR junction influences mRNA and protein levels [23, 33]. Therefore, we integrated the corresponding 5’-UTR into each promoter.
Promoter sequences were predicted using the Neural Network Promoter Prediction online tool (http://www.fruitfly.org/seq_tools/promoter.html) . The promoter prediction score threshold was set to 0.7. Moreover, due to the possibility of tandem promoters, all eligible promoter sequences within 300 bp upstream of the start codon were considered . For correct transcription initiation, the complete “promoter” consisted of 5’-UTR, predicted promoter region and Shine-Dalgarno sequence, which typically correspond to the region 60 bp upstream of a predicted promoter and 1 bp upstream of the start codon. The 60 bp extension upstream of the promoter accounts for potential UP-elements [36, 37].
Promoters were amplified from C. ammoniagenes ATCC 6871 and C. glutamicum ATCC 13032 genomic DNA with the corresponding primers. Oligonucleotide primers used in this work are listed in Table S1. PCR products were ligated into the vector pXMJ190 using Gibson assembly as shown in Figure 2c. All plasmids were constructed in E. coli TOP10 and then transformed into C. ammoniagenes ATCC 6871 for subsequent analysis.
Fluorescence intensity assay
To evaluate RFP expression under the control of the various promoters, C. ammoniagenes strains harboring various vectors were grown overnight on LB plates containing 20 μg/ml chloramphenicol. Fluorescence was observed using a LUYOR-3430 stereo microscope with a fluorescence excitation source (LUYOR, USA) set at 501 nm and matching lenses to detect RFP. Pictures were captured with a camera.
For more accurate comparisons, the fluorescence intensities of bacteria harboring different plasmids were measured using a Synergy H4 microplate reader (BioTek, USA). In order to exclude other interfering factors, harvested cells were washed once with PBS (pH 7.4) and then resuspended in PBS (pH 7.4) at an OD600 nm of approximately 1.0. The excitation wavelength for RFP was set at 554 nm and emission was set at 586 nm. Fluorescence intensities were normalized by OD600 nm and were used to indicate the activities of the promoters. Bacteria harboring pXMJ19-Ptac were induced with 1 mM IPTG.
Construction of the recombinant plasmid pXMJ190-Prpl21-PpcoaA-RFP
To further examine the function of the isolated promoters, a type III pantothenate kinase from P. putida KT2440 (PpcoA) and RFP were co-expressed under the control of the strongest promoter Prpl21. The PpcoaA gene was amplified by PCR from the genomic DNA of P. putida KT 2440, and the Shine-Dalgarno sequence for translation of RFP was calculated using the RBS calculator online tool (https://www.denovodna.com/software/). All primers used in this section are listed in Table S1. The recombinant plasmid pXMJ190-Prpl21-PpcoaA-RFP was constructed with Gibson assembly and positive colonies were confirmed by DNA sequencing (Tsingke, China). Recombinant plasmids were transformed into C. ammoniagenes ATCC 6871 for further experiments
C. ammoniagenes strains were precultured in 10 mL LB medium at 30 °C and shaken at 220 rpm for 24 h. Ten percent of the culture was inoculated in a 250 mL shake flask containing 100 mL fermentation medium. After 24 h cultivation, cell samples were harvested by centrifugation at 12,000 rpm for 10 min. Forty micrograms of cell lysate were loaded per lane. The PpcoaA and RFP expression was analyzed by 15% (v/w) polyacrylamide gel electrophoresis (PAGE) with cell-free extract under denaturing conditions. Mini-Protean III Electrophoresis System (Bio-Rad, USA) was utilized to perform the operation. Coomassie Brilliant Blue R-250 (0.2%, w/v) was utilized to stain protein on the gel.
Analysis of CoA production
Coenzyme A content was determined according to the modified phosphotransacetylase method [38, 39]. All reagents were purchased from National Institutes for Food and Drug Control. Briefly, 3.0 mL of Tris buffer (pH 7.6), 0.1 mL of acetyl phosphate dilithium salt (15.2 g/L) and 0.1 mL of the test solution were added into a 1 cm cuvette and mixed. Absorbance at 233 nm was recorded as E0; and then 0.01 mL of the phosphotransacetylase (30-40 U/mL) solution was added, mixed well and the highest absorbance within 3 to 5 minutes was taken as E1. Finally, another 0.01 mL of phosphotransacetylase solution was added, mixed well and the absorbance was read as E2. The number of CoA units per milliliter was calculated as U= (2E1-E0-E2)×5.55×413.