A vitamin b12-responsive biologic element and uses thereof

Abstract

The application discloses a riboswitch element responding to vitamin B12 and construction and application of a vitamin B12 biosensor based on the riboswitch element, and belongs to the fields of genetic engineering technology, molecular detection and biosensing. The biosensor comprises a riboswitch element responding to vitamin B12 and a selectable marker gene. An effective relationship between vitamin B12 concentration in a bacterium and expression of the marker gene is established by using the sensor. The biosensor can be used for selecting a high-yield vitamin B12 strain in a relatively fast and easy way, and has an important application prospect in detection and screening of vitamin B12-producing microorganisms.

Description

Technical Field

[0001] This invention relates to a bio-element that responds to vitamin B12 (Adenosylcobalamin) and its application, belonging to the fields of genetic engineering, molecular detection and biosensing technology. Background Technology

[0002] Vitamin B12, also known as cobalamin, is a corrin-like compound with wide applications in the pharmaceutical and food industries. However, its large molecular weight and complex chemical structure make its chemical synthesis time-consuming, labor-intensive, costly, and time-consuming. Furthermore, the high skill requirements of operators during synthesis hinder its suitability for industrial production. Some microorganisms in nature can synthesize vitamin B12 to meet their growth needs, acting as natural cellular factories for vitamin B12 synthesis. Currently, microbial fermentation is the most cost-effective method for producing vitamin B12 and is widely used in industrial production. Rhizobium, as a natural vitamin B12-producing strain, shows great potential for industrial production.

[0003] Currently, the most effective strategies for increasing vitamin B12 microbial fermentation yield both domestically and internationally are strain evolution engineering and fermentation process optimization. The key to successful strain evolution engineering is obtaining as many mutant libraries as possible and establishing efficient screening strategies. Currently, techniques such as HPLC and LC-MS are commonly used to detect vitamin B12 yield, but these are relatively cumbersome and time-consuming, significantly limiting the efficiency of strain screening. Therefore, developing a biosensor capable of rapidly detecting intracellular vitamin B12 content and applying it to rapid, high-throughput screening in strain evolution engineering can relatively quickly and easily screen for high-yielding strains, improving breeding efficiency and having significant application value for promoting the growth of the vitamin B12 microbial fermentation industry. Summary of the Invention

[0004] The technical problem to be solved by this invention is to discover vitamin B12 response elements and construct a vitamin B12 biosensor to meet the need for rapid detection of vitamin B12.

[0005] The first objective of this invention is to provide a ribo-switching element that responds to vitamin B12 (Adenosylcobalamin), named RS1888 The vitamin B12 riboswitch RS1888 The *Rhizobium sinense* strain CGMCC NO.9638 (also see CN104342390A), a high-vitamin B12-producing bacterium previously screened in our laboratory, is capable of responding to changes in vitamin B12 concentration; the nucleotide sequence of the vitamin B12 riboswitch is shown in SEQ ID NO. 1. This invention also includes homologous sequences of this sequence in *Rhizobium sinense*, which have the same function.

[0006] A second objective of this invention is to provide a biosensor containing a vitamin B12 riboswitch. RS1888 and genes encoding markers; RS1888 It regulates the expression of genes encoding markers in response to changes in vitamin B12 concentration.

[0007] In one embodiment of the present invention, a vitamin B12 riboswitch RS1888 The gene encoding the marker is located on a plasmid vector, meaning the biosensor is a plasmid.

[0008] In one embodiment of the present invention, the plasmid vector is pME6032.

[0009] In one embodiment of the present invention, the marker is green fluorescent protein (GFP). Specifically, the nucleotide sequence of the gene encoding GFP is shown in SEQ ID NO. 6.

[0010] Furthermore, by introducing transcriptional repressors and their regulated promoters, the response characteristics and sensitivity of the sensor are altered, and the intensity of the response signal is enhanced. lacI and lacO Reduce the background leakage expression of the sensor. The resulting optimized biosensor further contains expression lacI Encoding genes and lacO The nucleotide sequence encoding the gene, and lacO Encoding genes placed PtetR After the promoter, lacI The LacI protein, which encodes the gene, expresses through interaction with... lacO Combination will inhibit PtetR Transcription of the promoter reduces the background leakage expression of the sensor.

[0011] In one embodiment of the present invention, the transcriptional repressor and its regulatory promoter are TetR and PtetR The nucleotide sequence of the gene encoding TetR is shown in SEQ ID NO. 2. PtetR The nucleotide sequence is shown in SEQ ID NO. 4.

[0012] In one embodiment of the present invention, the nucleotide sequence of the gene encoding LacI is shown in SEQ ID NO. 3. lacO The nucleotide sequence of the gene encoding the gene is shown in SEQ ID NO. 5.

[0013] The third objective of this invention is to provide a method for screening high-yielding vitamin B12 strains, by converting the above-mentioned vitamin B12 biosensor into *Rhizobium sinense* of alfalfa, thereby achieving a correlation between vitamin B12 yield and fluorescence intensity, and using fluorescence intensity to screen high-yielding strains.

[0014] This invention also claims protection for the application of the biosensor in strain screening and optimization of enzyme activity and expression levels in the vitamin B12 synthesis pathway.

[0015] The beneficial effects of the present invention are as follows: The vitamin B12 biosensor provided by the present invention can achieve a good relationship between vitamin B12 concentration and fluorescence intensity within a certain range, thereby achieving the purpose of rapid detection of vitamin B12. It can be used for rapid screening of high-yield vitamin B12 strains and has a good application prospect. Attached Figure Description

[0016] The accompanying drawings, which constitute a part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0017] Figure 1 The pVS-RS1888-GFP sensor responds to the addition of extracellular vitamin B12.

[0018] Figure 2 The pVS-RS1888-TetR-PtetR-GFP sensor responds to the addition of extracellular vitamin B12.

[0019] Figure 3 The pVS-LacI-RS1888-TetR-PtetR-lacO-GFP sensor responds to the addition of extracellular vitamin B12.

[0020] Figure 4 Vitamin B12 production of strains with different vitamin B12 production capacities.

[0021] Figure 5 Vitamin B12 production of different recombinant strains with different vitamin B12 production capabilities containing the pVS-LacI-RS1888-TetR-PtetR-lacO-GFP sensor.

[0022] Figure 6 The response of the pVS-LacI-RS1888-TetR-PtetR-lacO-GFP sensor in different vitamin B12-producing strains. Detailed Implementation

[0023] The present invention will now be described in detail with reference to specific accompanying drawings and embodiments. The examples described below are merely preferred embodiments of the present invention. It should be noted that the following description is only for explaining the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention.

[0024] Example 1: Vitamin B12 Riboswab RS1888 Acquisition and identification (1) RS1888 Acquisition This invention performed transcriptome sequencing on *Alfalfa rhizobium sinense* CGMCC NO.9638 with and without exogenous vitamin B12 supplementation. Analysis of the 5' non-coding region of the significantly downregulated gene revealed a sequence that responds to vitamin B12 and significantly downregulates downstream gene transcription, located in the 5' non-coding region of the operon orf1885-1888, named... RS1888 The nucleotide sequence is shown in SEQ ID NO. 1.

[0025] (2) RS1888 Identification The genome of *Agrobacterium sinense* CGMCC NO.9638 was extracted from alfalfa. The 5' untranslated region of the operon *orf1885-1888* containing the adapter was amplified using primers RS1888-F (nucleotide sequence shown in SEQ ID NO. 7) and RS1888-R (nucleotide sequence shown in SEQ ID NO. 8). The 5' untranslated region of the operon containing the adapter was amplified using GFPmut1-F (nucleotide sequence shown in SEQ ID NO. 9) and GFPmut1-R (nucleotide sequence shown in SEQ ID NO. 10). gfpmut1 Using pME6032 plasmid as a template, the plasmid backbone pVS1-Gibson with adapter was amplified using primers pVS1-ver-F (nucleotide sequence shown in SEQ ID NO. 11) and pVS1-ver-R (nucleotide sequence shown in SEQ ID NO. 12). The three fragments were recombined using Gibson assembly to obtain plasmid pVS-RS1888-GFP, which was transformed into E. coli DH5α. Positive clones were selected for PCR and sequencing to verify successful plasmid construction.

[0026] The pVS-RS1888-GFP recombinant plasmid was transformed into *Agrobacterium sinense* CGMCC NO.9638. Single colonies were picked and cultured overnight at 30°C and 200 rpm in 5 mL of TYC medium containing spectinomycin (600 mg / L) to prepare a seed culture. 50 μL of the seed culture was then transferred to 5 mL of fresh TYC medium containing spectinomycin (600 mg / L). Additional vitamin B12 was added to the experimental groups at final concentrations of 10 μM and 50 μM, and the cultures were incubated at 30°C and 200 rpm. The fluorescence intensity and absorbance (OD) at 600 nm were measured. 600nm The results showed that the experimental group's unit OD 600nm The fluorescence intensity of the bacteria was significantly lower than that of the control group, as shown in the results. Figure 1 As shown. TYC medium (g / L): tryptone 5, yeast extract 3, calcium chloride 0.67, with an additional 18 g of agar powder added to the solid medium. LB medium (g / L): sodium chloride 10, tryptone 10, yeast extract 5, with an additional 15 g of agar powder added to the solid medium.

[0027] Example 2: Construction of a Vitamin B12 Biosensor by E. coli Using the K12 genome as a template, the tetR fragment with the adapter was amplified using primers TetR-F (nucleotide sequence shown in SEQ ID NO. 13) and TetR-R (nucleotide sequence shown in SEQ ID NO. 14). Using the pVS1 plasmid as a template, the plasmid backbone pVS1-Gibson-1 with the adapter Gibson assembly was amplified using primers pVS1-ver-F1 (nucleotide sequence shown in SEQ ID NO. 15) and pVS1-ver-R1 (nucleotide sequence shown in SEQ ID NO. 16). Using the pVS-RS1888-GFP recombinant plasmid as a template, the fragment with the adapter Gibson assembly was amplified using primers RS1888-F1 (nucleotide sequence shown in SEQ ID NO. 17) and RS1888-R1 (nucleotide sequence shown in SEQ ID NO. 18). RS1888 Fragment; using fragments containing PtetR Primers GFPmut1-F1 (nucleotide sequence shown in SEQ ID NO.19) and GFPmut1-R1 (nucleotide sequence shown in SEQ ID NO.20) amplified a sequence containing the promoter sequence. PtetR promoter and terminator sequences gfpmut1 Fragments. The fragments obtained from the above amplification are sequentially connected, in the order of those containing... PtetR promoter and terminator sequences gfpmut1 Fragment, in reverse RS1888 Fragments and tetRThe fragment, along with the plasmid backbone pVS1-Gibson-1, yielded the recombinant plasmid pVS-RS1888-TetR-PtetR-GFP. The recombinant plasmid was then transformed into... E. coli Positive clones were selected from DH5α cells for PCR and sequencing to verify successful plasmid construction.

[0028] With lacI Using the plasmid as a template, primers LacI-F (nucleotide sequence shown in SEQ ID NO. 21) and LacI-R (nucleotide sequence shown in SEQ ID NO. 22) were used to amplify the promoter-containing gene with adapter. lacI The fragment; using pVS-RS1888-TetR-PtetR-GFP plasmid as a template, the plasmid backbone pVS1-Gibson-2 with adapter Gibson assembly was amplified using primers pVS1-ver-F2 (nucleotide sequence as shown in SEQ ID NO. 23) and pVS1-ver-R2 (nucleotide sequence as shown in SEQ ID NO. 24); using pVS-RS1888-TetR-PtetR-GFP recombinant plasmid as a template, the fragment with adapter Gibson assembly was amplified using primers RS1888-TetR-F (nucleotide sequence as shown in SEQ ID NO. 25) and RS1888-TetR-R (nucleotide sequence as shown in SEQ ID NO. 26). RS1888 and tetR Sequence fragments; using those containing PtetR promoter and lacO The primers GFPmut1-F2 (nucleotide sequence shown in SEQ ID NO. 27) and GFPmut1-R2 (nucleotide sequence shown in SEQ ID NO. 28) amplified sequences containing... PtetR promoter, lacO Sequence and terminating subsequence gfpmut1 Fragments. The fragments obtained from the above amplification are sequentially connected, in the order of those containing... PtetR promoter, lacO and terminating subsequence gfpmut1 Fragment, in reverse RS1888 Fragments tetR Fragments and lacI The fragment and plasmid backbone pVS1-Gibson-2 were used to obtain the recombinant plasmid pVS-LacI-RS1888-TetR-PtetR-lacO-GFP, which was then transformed into... E. coli Positive clones were selected from DH5α cells for PCR and sequencing to verify successful plasmid construction.

[0029] Example 3: Response of Vitamin B12 Biosensor to Extracellular Vitamin B12 Addition Recombinant plasmids pVS-RS1888-TetR-PtetR-GFP and pVS-LacI-RS1888-TetR-PtetR-lacO-GFP were transformed into competent cells of *Agrobacterium sinense* CGMCC NO.9638. Single colonies were picked and cultured overnight at 30°C and 200 rpm in 5 mL of TYC medium containing spectinomycin (600 mg / L) to prepare seed culture. The seed culture was then transferred at a ratio of 1:100 to 5 mL of fresh TYC medium containing spectinomycin (600 mg / L). Additional vitamin B12 was added to the experimental groups at final concentrations of 10 and 50 μM, and the cultures were incubated at 30°C and 200 rpm. The strains transformed with the pVS-LacI-RS1888-TetR-PtetR-lacO-GFP sensor showed positive OD values ​​in the bacterial cells. 600nm When the concentration reached approximately 0.8, IPTG was added to a final concentration of 1 mM. The fluorescence intensity and absorbance at 600 nm were measured. The results showed that with increasing vitamin B12 addition (0 μM, 10 μM, and 50 μM), the OD of the bacterial cells inoculated with the recombinant plasmid pVS-RS1888-TetR-PtetR-GFP increased. 600nm The fluorescence intensities of the bacteria were 671, 980, and 1158 a.u., respectively, showing an increasing trend (e.g. Figure 2 (As shown). Bacterial cell units OD of the recombinant plasmid pVS-LacI-RS1888-TetR-PtetR-lacO-GFP were introduced. 600nm The fluorescence intensities of the bacteria were 176, 1506, and 2239 a.u., respectively, showing an increasing trend (e.g. Figure 3 (As shown).

[0030] contrast Figure 1 , Figure 2 and Figure 3 The results show that the introduction tetR and PtetR Subsequently, the sensor's response characteristics reversed, changing from suppression to activation, resulting in a change in response sensitivity and an increase in response signal strength. lacI and lacO Subsequently, the sensor's background leakage expression decreased from 671 a.u. to 176 au, indicating a reduction in leakage expression.

[0031] Example 4: Construction of recombinant *Rhizobium sinense* strains from alfalfa with different vitamin B12 yields An essential gene in the vitamin B12 synthesis pathway of wild-type alfalfa rhizobium CGMCC NO.9638 (denoted as WT) was identified. cobO ( btuRKnockout yielded a recombinant strain that does not produce vitamin B12, named W4; one operon in the vitamin B12 synthesis pathway in WT was removed. cobPWNO A promoter sequence was inserted into the 5' uncoding region to obtain a recombinant strain, named W7. Recombinant strains W4 and W7 were fermented with WT. The main experimental procedure was as follows: colonies were picked and placed in 24-well plates containing 2 mL of seed culture medium and cultured at 30℃, 600 rpm, and 80% humidity for 2 days to obtain a seed culture. The seed culture was then transferred to 2 mL of fresh fermentation medium at a 1:5 ratio and fermented in a shaker at 30℃, 600 rpm, and 80% humidity. Samples were taken on the second, third, and seventh days, and the vitamin B12 yield was determined by HPLC. The results are as follows: Figure 4 As shown, WT had the highest yield, followed by recombinant strain W7, while recombinant strain W4 produced almost no yield.

[0032] Seed culture medium (g / L): sucrose 40, corn steep liquor 20, betaine 5, (NH4)2SO4 1, (NH4)2HPO4 2, MnSO4·H2O 0.8, CoCl2·6H2O 0.02, MgO 0.3, DMBI 0.01, ZnSO4·7H2O 0.01, CaCO3 1.5, pH controlled at 7.0–7.4 by NaOH.

[0033] Fermentation medium (g / L): sucrose 80, corn steep liquor 30, betaine 15, (NH4)2SO4 2, MgSO4 1.5, K2HPO4 0.75, CoCl2·6H2O 0.14, DMBI 0.075, ZnSO4·7H2O 0.08, CaCO3 1, pH controlled at 7.0–7.4 by NaOH.

[0034] The method for detecting vitamin B12 is as follows: Take 1 mL of fermentation broth, add 0.1 mL each of 8% sodium nitrite solution and glacial acetic acid, shake well, and boil in a 100℃ water bath for 30-60 min. Cool to room temperature, centrifuge at 13,000 rpm for 1 min, and filter the supernatant through a 0.22 μm filter membrane into a sample vial. Use a C18-250A column (Agilent, 4.6 mm Id 9×250 mm, 5 µm), with a mobile phase of 30% organic phase (methanol) and 70% inorganic phase (water), an absorption wavelength of 361 nm, a column temperature of 35℃, a flow rate of 0.8 mL / min, and an injection volume of 15 µL. After the liquid chromatography is completed, calculate the yield according to the vitamin B12 standard curve.

[0035] Example 5: Construction of a recombinant vitamin B12 biosensor with different vitamin B12 yields from *Alfalfa* rhizobia. The constructed pVS-LacI-RS1888-TetR-PtetR-lacO-GFP plasmid was transformed into wild-type *Agrobacterium sinense* CGMCC NO.9638 (WT) and recombinant strains W4 and W7. Colonies were picked and placed in 24-well plates containing 2 mL of seed culture medium containing spectinomycin (600 mg / L). The plates were incubated at 30 °C, 600 rpm, and 80% humidity for 2 days to obtain a seed culture. The seed culture was then transferred at a 1:5 ratio to 2 mL of fresh fermentation medium containing spectinomycin (600 mg / L) and fermented in a shaker at 30 °C, 600 rpm, and 80% humidity. Samples were taken on the second, third, and seventh days, and vitamin B12 yield was determined by HPLC. The results are as follows: Figure 5 As shown, after being transferred to the sensor, the vitamin B12 production of WT and W7 was lower than that of strains that were not transferred to the sensor, but the trend was the same as that of strains that were not transferred to the sensor: vitamin B12 production was WT>W7>W4.

[0036] Example 6: Application of Vitamin B12 Biosensor in Detecting Intracellular Vitamin B12 Production The constructed pVS-LacI-RS1888-TetR-PtetR-lacO-GFP plasmid was transformed into wild-type *Agrobacterium sinense* CGMCC NO.9638 (WT) and recombinant strains W4 and W7. Colonies were picked and cultured in 5 mL of TYC medium containing spectinomycin (600 mg / L) at 30℃ and 200 rpm for 24 h to obtain seed culture. The seed culture was then transferred to 10 mL of fermentation medium containing spectinomycin (600 mg / L) at a 1:100 inoculum and cultured at 30℃ and 200 rpm for 40 h. IPTG was then added to a final concentration of 1 mM for induction. After fermentation, the cell fluorescence intensity and OD were measured. 600nm The OD units of strains WT, W7, and W4 were determined. 600nm The effective relationship between bacterial cell fluorescence intensity and the vitamin B12 production capacity of the strains is shown in Table 1. The biosensor can effectively screen for *Rhizobium sinense* in alfalfa. The OD values ​​of strains WT, W7, and W4 were [data missing]. 600nm The fluorescence intensity results of the bacterial cells are as follows Figure 6 As shown.

[0037] Table 1. Relationship between vitamin B12 production and fluorescence intensity Vitamin B12 (mg / L) <![CDATA[Fluorescence intensity / OD 600nm > 0 193.2 74.04 492.4 114.82 670.8 Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A response to vitamin B 12 The ribose switching element is characterized by: Able to combine with vitamin B 12 It also downregulates the transcription of downstream genes; the nucleotide sequence of the riboswitch element is shown in SEQ ID NO.

1.

2. A biosensor, characterized in that, Contains vitamin B as described in claim 1 12 Riboswitching elements and marker genes.

3. The biosensor as described in claim 2, characterized in that, It is a plasmid.

4. The biosensor as described in claim 3, characterized in that, The plasmid vector is pME6032.

5. The biosensor according to any one of claims 2 to 4, characterized in that: The marker gene is a fluorescent protein encoding gene.

6. A biosensor as described in any one of claims 2 to 4, characterized in that: It also contains a transcriptional repressor and its regulatory promoter, and the ribo-switching element regulates the expression of the transcriptional repressor, which in turn controls the expression of genes encoding markers.

7. The biosensor according to claim 6, characterized in that: The transcriptional repressor is TetR, and its regulatory promoter is PtetR.

8. The biosensor according to claim 7, characterized in that: The nucleotide sequence of the gene encoding TetR is shown in SEQ ID NO. 2, and the nucleotide sequence of the gene encoding PtetR is shown in SEQ ID NO.

4.

9. The biosensor according to claim 7 or 8, characterized in that: Furthermore, it contains nucleotide sequences that express the lacI and lacO coding genes. The lacO coding gene is placed after the PtetR promoter. The LacI protein expressed by the lacI coding gene inhibits the transcription of the PtetR promoter by binding to lacO, thereby reducing the background leakage expression of the sensor.

10. The biosensor according to claim 9, characterized in that: The nucleotide sequence of the lacI encoding gene is shown in SEQ ID NO. 3, and the nucleotide sequence of the lacO encoding gene is shown in SEQ ID NO.

5.

11. A screening method for vitamin B production 12 The method using strains is characterized by: The biosensor according to any one of claims 2 to 10 is transferred into the target material containing vitamin B. 12 Among strains with production capacity, the expression intensity of marker genes was detected to screen for vitamin B production. 12 strains.

12. The method as described in claim 11, characterized in that, The strain in question is *Alfalfa rhizobium*.

13. Using the biosensor according to any one of claims 2 to 10 to detect vitamin B 12 Optimize the amount of vitamin B synthesized 12 Applications related to the expression or enzyme activity of proteins involved in the synthesis pathway.

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