Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti
© The Author(s). 2018
Received: 21 June 2017
Accepted: 30 April 2018
Published: 11 May 2018
As a very important coenzyme in the cell metabolism, Vitamin B12 (cobalamin, VB12) has been widely used in food and medicine fields. The complete biosynthesis of VB12 requires approximately 30 genes, but overexpression of these genes did not result in expected increase of VB12 production. High-yield VB12-producing strains are usually obtained by mutagenesis treatments, thus developing an efficient screening approach is urgently needed.
By the help of engineered strains with varied capacities of VB12 production, a riboswitch library was constructed and screened, and the btuB element from Salmonella typhimurium was identified as the best regulatory device. A flow cytometry high-throughput screening system was developed based on the btuB riboswitch with high efficiency to identify positive mutants. Mutation of Sinorhizobium meliloti (S. meliloti) was optimized using the novel mutation technique of atmospheric and room temperature plasma (ARTP). Finally, the mutant S. meliloti MC5–2 was obtained and considered as a candidate for industrial applications. After 7 d’s cultivation on a rotary shaker at 30 °C, the VB12 titer of S. meliloti MC5–2 reached 156 ± 4.2 mg/L, which was 21.9% higher than that of the wild type strain S. meliloti 320 (128 ± 3.2 mg/L). The genome of S. meliloti MC5–2 was sequenced, and gene mutations were identified and analyzed.
To our knowledge, it is the first time that a riboswitch element was used in S. meliloti. The flow cytometry high-throughput screening system was successfully developed and a high-yield VB12 producing strain was obtained. The identified and analyzed gene mutations gave useful information for developing high-yield strains by metabolic engineering. Overall, this work provides a useful high-throughput screening method for developing high VB12-yield strains.
Vitamin B12 (cobalamin, VB12) is one of the most structurally complex natural Vitamins. VB12 is only produced by some bacteria and archaea and it requires more than 30 enzymes for the de novo synthesis of VB12 [1, 2]. VB12 plays an important role as an essential co-factor in various biochemical processes such as DNA synthesis, fatty acid synthesis, amino acid metabolism and energy production [3, 4]. VB12 has been widely used in food and medicine fields , and the global demand for VB12 in these applications has been growing rapidly [6, 7]. A considerable number of enhancement and development of natural products mainly rely on mutagenesis and screening strategies , while targeted engineering strategies by gene overexpression or deletion do not always generate desired results due to the complexity of highly regulated metabolic networks.
In order to screen libraries with high genetic diversity, a key problem is developing smart screening methods. Traditional methods suffer from low dynamic range, low throughput and unwanted sensitivity to variable experimental parameters . Fluorescence-activated cell sorting (FACS) allows ultrahigh throughput in cell sorting and requires a sortable signal output by biosensors in individual cells that respond to various intracellular concentrations of the target molecule with a correlation . Developing a biosensor sensitive to VB12 concentration is thus very important.
Riboswitches are small structured mRNA elements that regulate gene expression and translation in response to specific small molecule ligands . They act as relatively simple and direct metabolite sensors that are capable of both molecular recognition and transducing this information by altering protein expression [12–14]. Natural riboswitches are found with the highest frequency in the 5’-UTR of bacterial mRNAs, where they regulate the expression of downstream genes through structural changes undergone in response to the binding of a specific target molecule.
VB12 riboswitch has been found to widely regulate VB12 biosynthesis in bacteria . Expression of cob operon in cobalamin biosynthetic pathway and transporter encoding gene btuB are repressed by the presence of vitamin B12, in particular. The VB12 riboswitch consists of a binding region (aptamer domain) and an expression platform that modulates gene expression [16, 17]. Upon ligand binding, the aptamer region undergoes a conformational change that is interpreted by the expression platform (Fig. 1), resulting in repressed gene expression [18, 19]. Comparative analysis of the genes and regulatory regions from different bacterial genomes is a powerful approach allowing the identification of regulatory patterns . Using this approach, Vitreschak et al. have summarized approximately 200 VB12-elements from 67 bacterial genomes , providing candidates for biosens or development.
Strains and culture conditions
The initial strain, S. meliloti 320, was stored in our laboratory . S. meliloti 320 was grown in LB medium supplemented with MgCl2 (2.5 mM) and CaCl2 (2.5 mM) (LB/MC medium), or minimal M9 medium supplemented with 1 μg/mL biotin and 10 ng/mL CoCl2, as well as 0.1% sucrose for liquid medium or 0.2% sucrose for solid medium (unless indicated otherwise), respectively, (named M9/sucrose) . And the strain was grown at 30 °C with shaking at 200 rpm. Antibiotics were used as follows: spectinomycin (Sm; 200 μg/mL for E. coli and 600 μg/mL for S. meliloti), gentamicin (Gm; 10 μg/mL for E. coli, 20 μg/mL for S. meliloti), and kanamycin (Km; 50 μg/mL for E. coli and 100 μg/mL for S. meliloti).
For construction of Sm320-N8 strain, genetic deletions in S. meliloti 320 genome were generated by homologous recombination and allelic replacement. Two fragments of cobI-left and -right flanking DNA sequences (Additional file 1: Table S1) were ligated by overlap extension polymerase chain reaction (PCR), and the PCR product was cloned into a suicide vector pJQ200SK carrying sacB and gentamicin resistance gene. The construct was introduced into S. melilothi by triparental mating with help of the mobilizing strain MT616  carrying pRK600, and the culture was plated onto LB/MC-agar containing gentamicin. A second reciprocal recombination event was selected on LB/MC-agar containing 5% sucrose, and then isolates with the desired deletions were subsequently screened by PCR verification in which primers hybridizing outside the cloned flanking sequences were used to amplify the deletion region.
Riboswitch sensor construction and validation
To construct riboswitch sensor plasmids, the promoter PmelA was amplified from pSymB megaplasmid, the approximately 200 bp VB12 riboswitch sequence containing a riboswitch and RBS from different strains were obtained by artificial synthesis (riboswitch sequences were provided in Additional file 1: Table S2), and the genes were amplified by PCR. Fragments containing PmelA promoter, VB12 riboswitch sequence and GFP reporter gene were fused by PCR. Next, the pBHR1 plasmid and the fused PCR fragments were ligated by Gibson assembly at 50 °C for 1 h, and then cloned in E. coli strain DH5α. Detailed constructions of the riboswitch-containing plasmids are shown in Additional file 1: Figure S2 and Additional file 1: Table S2. The plasmids were then separately transferred into Sm320, Sm320-N8 and Sm320-L6 (engineered mutants with different production capacities) by triparental mating. S. meliloti containing PmelA-riboswitch-gfp plasmid was grown at 30 °C for 12 h, and then transferred into shake flask fermentation with 10% (v/v) of inoculation for 3 days. The samples were harvested with centrifuging at 4000 g for 8 min, followed by washing in PBS for three times. The cells were collected to measure their GFP fluorescence by flow cytometry .
Strains and plasmids used and constructed in this study
Strain or plasmid
Sources of reference
Sinorhizobium meliloti 320, VB12 producing strain.
Sm320 derivative, carrying plasmid pBR-OhemE to reduce production capacity
Sm320 derivative, knocking out cobI gene
Sm320 derivative, carrying plasmid pBR- SYB-lacI and plasmid pMB-gfp was used in the ARTP treat
MT607/pRK600; Escherichia coli helper strain for mobilizing RK2/RP4-derived plasmids
Vector for construction of deletions, sacB,GmR
Broad-host-range vector, PtauA promoter,AmpR
Broad-host-range vector, Derivative of PBBR122,KmR
pBHR1 plasmid carrying EC-butB riboswitch were cloned in front of a gfp reporter gene driven by the constitutive promoter PmelA
pBHR1 plasmid carrying PF-cbiB riboswitch were cloned in front of a gfp reporter gene driven by the constitutive promoter PmelA
pBHR1 plasmid carrying SM-bluB riboswitch were cloned in front of a gfp reporter gene driven by the constitutive promoter PmelA
pBHR1 plasmid carrying SM-cobU riboswitch were cloned in front of a gfp reporter gene driven by the constitutive promoter PmelA
pBHR1 plasmid carrying MLO-bluB riboswitch were cloned in front of a gfp reporter gene driven by the constitutive promoter PmelA
pBHR1 plasmid carrying MLO-metE riboswitch were cloned in front of a gfp reporter gene driven by the constitutive promoter PmelA
pBHR1 plasmid carrying SY-btuB riboswitch were cloned in front of a gfp reporter gene driven by the constitutive promoter PmelA
pBHR1 plasmid carrying BI-cbiW riboswitch were cloned in front of a gfp reporter gene driven by the constitutive promoter PmelA
pBHR1 plasmid carrying SY-btuB riboswitch were cloned in front of a lacI reporter
pMB393 containing lacO and carrying gfp reporter
pBHR1 plasmid carrying hemE gene to reduce production capacity
Mutagenesis by ARTP and determination of optimal treatment time
Pure helium was used as the plasma working gas for ARTP mutation and the operating parameters were as follows: (1) the radio frequency power input was 100 W; (2) the distance between the plasma torch nozzle exit and the sample plate was 4 mm; and (3) the temperature of the plasma jet was below 30 °C . Under these operating conditions, the ARTP mutagenesis dosage was only dependent on the treatment time.
Whole genome sequencing
The genomic DNA of strain S. meliloti 320 was extracted from bacterial cells grown overnight in LB/MC medium at 30 °C and 200 rpm. The DNA was extracted using OMEGA Bacterial DNA kits (Omega bio-tek, Feyou Biotechnology, China) following the manufacturer’s protocol [26, 27]. To verify DNA quality, PCR amplifications were performed in a final volume of 50 μL, containing 10 μM each primer, 1.25 units of Q5 High-Fidelity DNA polymerase (New England Biolabs, USA), dNTP mixture (250 μM each dNTP), and approximately 50 ng of template DNA. The PCR amplification conditions consisted of an initial denaturation for 10 min at 94 °C, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min with a final extension of 10 min at 72 °C. The quality and quantity of DNA were analyzed using 1% agarose gel electrophoresis and spectrophotometric method, respectively. The verified genomic DNA was selected for further analysis.
Biomass was determined with a spectrophotometer at 600 nm. The sample for measuring cell concentrations was obtained as follows: 1 mL of broth was centrifuged at 10,000×g for 10 min, the supernatant was discarded, and the cell pellets were resuspended in 1 mL of 0.85% NaCl buffer. The concentrations of the principal fermentation products were determined by high performance liquid chromatography (HPLC) . The sample (25 mL) was mixed with 2.5 mL of NaNO2 8% (w/v) and 2.5 mL of glacial acetic acid. The mixtures were boiled for 30 min, and then centrifuged. The supernatants were filtrated through a 0.22-μm membrane filter. Then, 20 μl of NaCN 10% (w/v) was added to 1 mL of the aqueous phase. 20 μL of the final sample was injected into an Agilent 1260 HPLC with a 5C18-250A column (Agilent, 4.6 mm id × 250 mm, 5 μm) thermostated at 35 °C. The mobile phase consists of water and acetonitrile (70:30 [v/v]) at 0.8 mL/min . Samples were detected at 361 nm. All values presented in this study were averages of at least three independent trials.
Preparation of cell extracts and detection of the amount of intracellular VB12
Centrifugation was used to sediment 500 μL of cell suspension through 100 μL silicone oil on the bottom of the tube. Cell pellets were then resuspended by 200 μL perchloric acid (20%, v/v) by gentle vortexing after disruption by sonication. After breaking up the cells, extracts were neutralized by addition of 200 μL Na2CO3 and the membrane materials and denatured proteins were removed by centrifugation. Then, 100 μL of NaNO2 8% (w/v) and 100 μL of glacial acetic acid were added to 1 mL of aqueous phase, and the mixture was boiled for 30 min, centrifuged and the supernatant was filtrated through a filter membrane (0.22 μm), and then 2 μl of NaCN 10% (w/v) was added to 100 μl of the aqueous phase. The samples were stored in − 20 °C for VB12 analysis by HPLC. The amount of intracellular VB12 was calculated as the mill grams of intracellular VB12 per liter of culture.
The fermentation medium contained (per liter of deionized water): sucrose 60 g/L; corn steep liquor (CSL) 20 g/L; betaine 6 g/L; ammonium sulfate 2 g/L; potassium dihydrogen phosphate 2.6 g/L; cobalt chloride 0.06 g/L; and 0.1 g/L DMB was added to the medium. The pH before autoclaving was adjusted to 7.0–7.4. The stock sugar solution was autoclaved separately before mixing with the rest of the medium.
For cultivating VB12-producing strains in microtiter plates, colonies were cultivated in 100 μL LB/MC medium overnight using 96-well microtiter plates (Corning Costar 3930, square U-bottom, 350 μL) and then 10% (v/v) inocula was transferred to 2 mL fermentation medium using 24-well deep microtiter plates (Corning Costar 3524, square U-bottom, 5 mL) at 30 °C, 700 rpm, 80% humidity in a Microtron shaker (Infors, China).
For shake flask fermentation, the seed culture was grown at 30 °C for 12 h and was used to provide 10% (v/v) inocula for fermentation. Experiments were conducted in 250 mL Erlenmeyer flasks containing 30 mL of each medium at 30 °C at an agitation rate of 200 rpm in rotary shakers for 7 days. The samples were used to determine cell mass (OD600 nm), residual sugars every 24 h, and product at 7 days.
Engineered mutants with different production capacities of VB12
Construction of riboswitch-based VB12 sensors
Conserved RNA elements upstream of some B12-regulated genes
The start codon
Proximal downstream genes
Coding of enzyme protein
Propionibacterim freudenreichii (PF)
VB12 biosynthesis protein
VB12 biosynthesis protein
Vitamin B12 transport
VB12 transporter BtuB
VB12 transporter BtuB
B12-dependent or alternative metabolic pathways
Examination of the SY-btuB riboswitch sensor
In order to verify the response behavior of the new sensor system to the amount of intracellular VB12 level, GFP fluorescence was observed by fluorescence microscope (DM5000B, Leica, Germany). A very weak fluorescence value was detected for the samples without VB12, and there was a corresponding increase in GFP fluorescence value along with the increase amount of intracellular VB12 (Fig. 5b). This result indicated that this sensor could work in different strains to sense the different levels of the amount of intracellular VB12 by producing corresponding levels of fluorescence intensity. Moreover, a corresponding rise in GFP fluorescence values of Sm320-N8 strains carrying SY-btuB sensor double plasmids could be observed by increasing VB12 concentration in the medium (Additional file 1: Figure S3).
Next, we simulated a screening process to verify the reliability of the sensor-reporter. The three strains with different production capacities were mixed together at equal optical density and the mixture of strains was used for screening with FCAS. Colonies exhibiting the top 1% of fluorescence values were analyzed to evaluate the sorting performance of the SY-btuB sensor double plasmid (Summit 5.2 analysis). As expected, nearly 84% of sorted cells were identified as the high-producing Sm320 strain (Fig. 5c). Overall, Sm320-Mybi represented the best regulatory device, and the strain Sm320-Mybi was used in the subsequent experiments.
High throughput screening of mutants using riboswitch
ARTP-irradiation was used to induce random mutations, and high VB12-producing mutants were selected. Growing cells were treated with helium-based ARTP for 15, 30, 60, 75, 90 and 120 s, respectively. The lethality rate was over 80% when the exposure time exceeded 60 s, and there was no cell survival after 120 s treatment (Fig. S4). Therefore, the optimal exposure time used in subsequent studies was set at 60 s with lethality ranged from 80 to 85%.
We then attempted to screen hyperproducing strains from a library of variant strains using the engineered riboswitch. After treatment of ARTP, selection was carried out using the “dual-repression” SY-btuB riboswitch. Colonies exhibiting the top 0.1% of fluorescence signals were selected from the genetic library. The wild type strain and the mutants were cultured in LB/MC medium in a 96-well microtiter plate at 30 °C for 12 h and then transferred to fresh medium for fermentation in 24-well deep plate at 30 °C with shaking for 7 days.
Whole genome sequencing analyses
To identify the gene mutations contributing to the high VB12 production, we sequenced the genomic DNA of the mutant Sm320MC5–2. The gene mutations were identified from the sequence data. A total of 17 SNP (single nucleotide polymorphism) and 1 Indel (insertion and deletion) were identified in the Sm320MC5–2 genome (Additional file 1: Table S3). Genome-scale gene expression analysis of Sm320 and Sm320MC5–2 revealed that the mutant genes were located in the VB12 metabolic pathway, heme branch pathway, ABC transporters, VB12 precursor synthesis pathway, tricarboxylic acid cycle and other metabolic pathways. These SNPs functioned through altering the transcription level or enzyme activities and may have resulted in enhanced metabolic flux towards production of VB12 or by weakening the competing branches. This study demonstrates a new paradigm of revealing unexpected gene mutations to guide metabolic engineering for increasing VB12 yield.
In the past few years, various mRNA elements containing engineered riboswitches and reporter genes have been described and applied in modulating genetic switches, discovering the genetic pathway and optimizing cellular behaviors. However, none of simple and direct metabolite sensors has been applied in screening to increase the yield and productivity of target molecules. In this study, we reported successful application of a riboswitch sensor as a sensitive and novel detection method for VB12 screening. Moreover, we identified a VB12 riboswitch element that is responsible for expression of the VB12 transporter, btuB, in the genome of Salmonella typhimurium . We validated the applicability of this riboswitch by screening higher VB12 producers from a genetic DNA library.
It is necessary to emphasize that the titer of VB12 was heavily dependent on the culture conditions. In our previous study, we did some research to select the nitrogen source, considering the sediment of CSL may interfere the accuracy of the experiments, we replaced the nitrogen sources “2% CSL” by “2% AYP, 1% peptone” in the fermentation medium when the strains were screened by FACS, and the comparison was shown in Additional file 1: Figure S5. When the strains were cultivated on shake flasks, VB12 was produced at millimolar concentrations. But when the strains were screened by FACS, the amount of intracellular VB12 was only about 1.06 μg/mL (micromolar levels of B12). So, the levels of VB12 produced could be detected by riboswitch normally.
In this study, 156 ± 4.2 mg/L of VB12 was produced by the selected mutant MC5–2. In order to identify gene mutations that contributed to the high production of VB12, we sequenced the genomic DNA of the mutant Sm320MC5–2 Many gene mutations in Sm320MC5–2 genome may play key roles in the VB12 hyperproduction. The SNPs in hemin, hemN, sdhA, betB, ribH, and metH were not reported in S. meliloti. These SNPs may alter transcription levels and enzyme activities to enhance metabolic flux towards producing VB12 or weakening the competing branches to improve productivity.
There were two changes identified in the heme branch pathway, mutation of hemin (c.C1228T: p.H410Y) and hemN (c.C632T: p.T211I). The mutation of hemN is speculated to decrease urinary porphyrins original III metabolic flow to the synthesis of hemoglobin leading to increasing productivity of VB12. The gene hemin encodes an iron complex transport system substrate-binding protein, and its mutation may decrease the active efflux of hemoglobin, reducing the heme anabolic flow to maintain the cell intracellular requirement.
In addition, we speculate that the mutation of sdhA (c.G1846A:p.A616T) in the tricarboxylic acid cycle conversion of succinate as carbon sources is necessary but insufficient for the VB12 metabolic pathways. The mutant of betB (c.T301C:p.W101R) gene may enhance the utilization of betaine to improve VB12 productivity. The ribH (c.T95C:p.L32P) encodes riboflavin kinase in riboflavin metabolism and may play a key role in 5, 6-dimethylbenzimidazo-le (DMB) synthesis, a precursor of VB12 biosynthesis.
Finally, one cobalamin-dependent enzyme in S. meliloti, methionine synthase (MetH c.G733A:p.G245S), catalyzes the final step in methionine biosynthesis and is required for growth . In future work, the contribution of these enzymes to VB12 production needs to be determined, which may help elucidate the underlying mechanism and analyze metabolic circuits for further improvement of VB12 production in S. meliloti.
We developed a Vitamin B12 high-throughput screening system by riboswitch sensor for the selection of VB12hyperproducing strains. The S. meliloti mutant library was generated by ARTP. Numerous mutants were obtained, and Sm320MC5–2 exhibited 21.82% higher VB12 yield than the wild type strain. The genomic DNA of the hyperproducing mutant was subjected to whole genome sequencing, and the potential contributions of the gene mutations were discussed. This study provides a new paradigm in high throughput screening and elucidating the unexpected gene mutations which would be helpful for rewiring metabolic pathway to raise the yield of VB12.
Tianjin Science Fund for Distinguished Young Scholars (17JCJQJC45300), Nature Science Foundation of Tianjin (CN) (16JCYBJC23500, 15JCQNJC09500), Tianjin Science and Technology project (15PTCYSY00020), the Key Projects in the.
Tianjin Science & Technology Pillar Program (14ZCZDSY00058), and the Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences. All the findings mentioned above have no roles in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
YYC and DZ conceived of the manuscript. YYC, MMX, HD and YQ performed the experiment. YYC, MMX, HD, TCZ, BWZ, JCW and DZ wrote the manuscript. All authors have read and approved the final manuscript.
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