Pantothenic acid biosensor as well as preparation method and application thereof

Abstract

The invention relates to the field of biosensors, in particular to a pantothenic acid biosensor and a preparation method and application thereof. The invention provides application of a transcription regulatory factor HLT of bacillus in preparation of a detection product responding to pantothenic acid. A nucleotide sequence of the transcription regulatory factor HLT of bacillus is selected from any one of the following nucleotide sequences (1)-(2): (1) a nucleotide sequence as shown in SEQ ID NO.1; (2) a nucleotide sequence which has the identity of 90% or above with the nucleotide sequence as shown in SEQ ID NO.1 in (1); the invention innovatively discovers that when pantothenic acid exists, the transcriptional regulation factor HLT is induced by the pantothenic acid, the structure of the transcriptional regulation factor HLT is changed, and the transcriptional regulation factor HLT can be combined with a promoter to start the expression of a target gene, so that the transcriptional regulation factor HLT of the bacillus can be used for preparing a detection product responding to the pantothenic acid.

Description

Technical Field

[0001] The present invention relates to the field of biosensors, and in particular to a pantothenic acid biosensor, a preparation method and applications thereof. Background Art

[0002] A biosensor is a device that combines a biological recognition element with a signal output element. It can detect changes in the concentration of specific metabolites in cells and convert them into recognizable signal outputs. Currently, biosensors based on transcriptional regulators have been widely used in pathway optimization and high-throughput screening of various amino acid high-producing strains. However, existing biosensors lack sufficient response strength to certain small molecule metabolites and are limited in the types of metabolites they can respond to, making them difficult to meet the needs of high-throughput screening.

[0003] Pantothenic acid, a water-soluble amino acid, is widely used in feed, nutritional supplements, food fortifiers, and pharmaceuticals. Currently, pantothenic acid production methods are primarily divided into chemical synthesis and biofermentation. Chemical synthesis typically uses isobutyraldehyde and formaldehyde as raw materials. Through a series of chemical reactions, including condensation, cyanation, hydrolysis, and dehydrolactonization, DL-γ-butyrolactone is synthesized. γ-Butyrolactone then reacts with calcium β-alanine in a formaldehyde solution to produce calcium DL-pantothenate. This is then resolved to produce calcium D-pantothenate. While this method is mature, it is complex. In contrast, biofermentation is an emerging green production technology that utilizes microorganisms to directly synthesize calcium D-pantothenate, eliminating the chemical resolution step and resulting in a shorter, more environmentally friendly process. However, the efficiency of currently reported microbial fermentation methods for producing calcium pantothenate is relatively low, and microorganisms that can produce high levels of pantothenate are urgently needed.

[0004] Biosensors have the ability to sense small molecules and can achieve high-efficiency screening of strains. For example, an amino acid biosensor disclosed in patent document CN116731131A selects the F104 position of the NCgl0581 transcriptional regulatory factor from Corynebacterium glutamicum 13032 that can respond to serine and homoserine and undergoes saturation mutation to construct an efficient amino acid biosensor. This mutant not only has improved induction response activity to serine and homoserine, but also has induction response activity to proline and threonine. However, the above-mentioned amino acid biosensor is only applicable to proline and threonine and cannot respond to pantothenic acid, which cannot meet the high-throughput screening requirements of pantothenic acid high-yield strains. Therefore, it is particularly critical to develop a biosensor that can specifically respond to pantothenic acid to achieve accurate detection of pantothenic acid concentration and efficient screening of high-yield strains. It not only has important theoretical significance, but also has significant application value. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a pantothenic acid biosensor, a preparation method and its use. The biosensor can effectively respond to pantothenic acid, realize the qualitative and quantitative detection of pantothenic acid and the efficient screening of high-yield strains, and has broad application prospects.

[0006] To this end, the present invention provides the following technical solutions:

[0007] The present invention discloses the use of a transcriptional regulatory factor HLT of Bacillus in preparing a detection product responsive to pantothenic acid, wherein the nucleotide sequence of the transcriptional regulatory factor HLT of Bacillus is selected from any one of the following nucleotide sequences (1)-(2):

[0008] (1) having the nucleotide sequence shown in SEQ ID NO.1;

[0009] (2) A nucleotide sequence having an identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% or more to the nucleotide sequence represented by SEQ ID NO. 1 described in (1).

[0010] As used herein, "identity" with respect to nucleic acid sequences refers to the degree to which the two nucleic acid sequences have the same nucleotide residue at the same position when the two nucleic acid sequences are aligned for maximum identity, expressed as a percentage. The "identity" between the sequences of two nucleic acid molecules can be determined using a known computer algorithm, such as at least one of the GCG program packages, BLASTN, or FASTA. Other commercially or publicly available programs may include, for example, the DNAStar "MegAlign" program.

[0011] The embodiments of the present invention disclose the use of the Bacillus transcriptional regulatory factor HLT in any of the following:

[0012] (1) Use in detecting pantothenic acid or preparing products for detecting pantothenic acid;

[0013] (2) Use in screening pantothenic acid-producing strains or preparing products for screening pantothenic acid-producing strains;

[0014] (3) Use in screening proteins or protein-encoding genes related to pantothenic acid synthesis or in preparing products for screening proteins or protein-encoding genes related to pantothenic acid synthesis;

[0015] (4) Use in regulating the transcription level of a target gene, or in preparing a product for regulating the transcription level of a target gene.

[0016] In some embodiments, the products include probes, biosensors, pantothenic acid high-producing strains, and strains that utilize pantothenic acid biosensors to produce substances. Preferably, the strains that utilize pantothenic acid biosensors to produce substances can be strains that utilize pantothenic acid biosensors to high-produce pantothenic acid.

[0017] The embodiment of the present invention provides a pantothenic acid biosensor fusion gene, wherein the fusion gene includes the Bacillus transcriptional regulatory factor HLT, an inducible promoter, a constitutive promoter, and a reporter protein encoding gene;

[0018] Wherein, the constitutive promoter is operably connected to the Bacillus transcriptional regulatory factor HLT, and the inducible promoter is operably connected to the reporter protein coding gene.

[0019] In some embodiments, the inducible promoter has a nucleotide sequence as shown in SEQ ID NO. 2, or a nucleotide sequence having an identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% or more to the nucleotide sequence as shown in SEQ ID NO. 2;

[0020] In some embodiments, the constitutive promoter is selected from any one of a trc promoter, a T7 promoter, and a tac promoter; preferably, the trc promoter has a nucleotide sequence as shown in SEQ ID NO.3, or a nucleotide sequence having an identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% or more to the nucleotide sequence as shown in SEQ ID NO.3;

[0021] In some embodiments, the reporter protein encoding gene includes a fluorescent protein encoding gene; preferably, the fluorescent protein encoding gene includes at least one of a green fluorescent protein encoding gene, a red fluorescent protein encoding gene, an orange fluorescent protein encoding gene, a yellow fluorescent protein encoding gene, a green fluorescent protein encoding gene, and a blue fluorescent protein encoding gene.

[0022] In some embodiments, the pantothenate biosensor fusion gene further includes a 5'UTR sequence and a terminator sequence, which can be conventional sequences. Furthermore, the 5'UTR sequence is 718-818 bp in SEQ ID NO. 4, or 1046-1204 bp in SEQ ID NO. 4. The terminator sequence is 964-1015 bp in SEQ ID NO. 4.

[0023] And / or, the pantothenic acid biosensor fusion gene has the nucleotide sequence shown in SEQ ID NO.4, or a nucleotide sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% or more identical to the nucleotide sequence shown in SEQ ID NO.4.

[0024] An embodiment of the present invention discloses a biomaterial, comprising any one of the following:

[0025] 1) A nucleic acid molecule encoding the pantothenic acid biosensor fusion gene; optionally, the nucleic acid molecule is DNA or RNA;

[0026] 2) an expression cassette, recombinant vector, recombinant microorganism, or transgenic cell line expressing the pantothenate biosensor fusion gene;

[0027] 3) an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the nucleic acid molecule described in 1);

[0028] 4) a recombinant vector, recombinant microorganism or transgenic cell line containing the expression cassette described in 2) or 3);

[0029] 5) A host cell containing the recombinant vector described in 2) or 3) or 4).

[0030] In some embodiments, the initial plasmid vector of the recombinant vector is selected from any one of pRSFDuet-1 vector, pETDuet-1 vector, pACYCDuet-1 vector, pTrc99a vector or pET28a vector;

[0031] And / or, the host cell is selected from Escherichia, Corynebacterium glutamicum, Saccharomyces cerevisiae or Yarrowia lipolytica.

[0032] The embodiment of the present invention discloses a pantothenic acid biosensor, comprising the gene encoding the transcriptional regulatory factor HLT, the pantothenic acid biosensor fusion gene, and the biomaterial.

[0033] The embodiments of the present invention disclose the use of the pantothenic acid biosensor fusion gene, the biomaterial, and the pantothenic acid biosensor in any of the following:

[0034] (1) Use in detecting pantothenic acid or preparing products for detecting pantothenic acid;

[0035] (2) Use in screening pantothenic acid-producing strains or preparing products for screening pantothenic acid-producing strains;

[0036] (3) Use in screening proteins or protein-encoding genes related to pantothenic acid synthesis or in preparing products for screening proteins or protein-encoding genes related to pantothenic acid synthesis;

[0037] (4) Use in regulating the transcription level of a target gene, or in preparing a product for regulating the transcription level of a target gene.

[0038] In some embodiments, the product comprises a reagent or a kit.

[0039] The embodiment of the present invention discloses a method for detecting pantothenic acid, comprising using the Bacillus transcriptional regulatory factor HLT encoding gene, the pantothenic acid biosensor fusion gene, the biological material, and the pantothenic acid biosensor for qualitative or quantitative detection;

[0040] Optionally, in the qualitative detection, the biosensor is mixed with a sample to be tested and incubated to generate a signal in response to pantothenic acid, and the sample to be tested contains pantothenic acid;

[0041] Optionally, in the quantitative detection, the biosensor is mixed and incubated with a pantothenic acid standard of known gradient concentrations to generate a signal responding to pantothenic acid. A standard curve is drawn based on the gradient concentrations and the signal responding to pantothenic acid. The signal responding to pantothenic acid of the sample to be tested is substituted into the standard curve to calculate the pantothenic acid concentration.

[0042] The embodiment of the present invention discloses a method for screening a high-producing pantothenic acid strain, comprising using the Bacillus transcriptional regulatory factor HLT encoding gene, the pantothenic acid biosensor fusion gene, the biomaterial, and the pantothenic acid biosensor for screening, including the following steps:

[0043] S1. Cultivate the strain to be evolved;

[0044] S2. inoculating the strain cultured in step S1 into a medium containing pantothenic acid for cultivation;

[0045] S3, inoculating the strain cultured in step S2 into a medium containing pantothenic acid for cultivation;

[0046] S4. Repeat steps S2-S3 until the OD of the strain reaches nm Reach 5-6;

[0047] S5, making the cells of step S4 competent for electroporation, transferring the pantothenate biosensor to the competent state, and performing plate culture;

[0048] S6. Pick the colonies cultured in step S5 and culture them in a multi-well plate, detect the signal produced by each bacterial strain in response to pantothenic acid, and screen the pantothenic acid high-producing strain based on the signal intensity.

[0049] In some embodiments, in step S2 or S3, the concentration of pantothenic acid in the culture medium containing pantothenic acid ranges from 0.1 to 150 g / L.

[0050] And / or, in step S2 or S3, the incubation time is 4-24 hours;

[0051] And / or, in step S6, the culture time is 3-36 hours.

[0052] The technical solution of the present invention has the following advantages:

[0053] 1. The present invention provides a use of the transcriptional regulatory factor HLT of Bacillus in preparing a detection product responsive to pantothenic acid, wherein the nucleotide sequence of the transcriptional regulatory factor HLT of Bacillus is selected from any one of the following nucleotide sequences (1)-(2): (1) having a nucleotide sequence as shown in SEQ ID NO.1; (2) having a nucleotide sequence with an identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% or more with the nucleotide sequence as shown in SEQ ID NO.1 in (1); the present invention innovatively discovers that when pantothenic acid is present, the transcriptional regulatory factor HLT is induced by pantothenic acid to change its structure, thereby being able to bind to a promoter and initiate the expression of a target gene. Therefore, the transcriptional regulatory factor HLT of Bacillus can be used to prepare a detection product responsive to pantothenic acid.

[0054] 2. The pantothenic acid biosensor provided by the present invention comprises a recombinant expression vector encoding a gene encoding the transcriptional regulatory factor HLT of Bacillus, an inducible promoter, a constitutive promoter, and a fluorescent protein encoding gene. It has good responsiveness and high specificity to pantothenic acid, can sensitively detect changes in pantothenic acid concentration, and improves detection accuracy and stability.

[0055] Furthermore, the pantothenic acid biosensor can be used for detection based on an enzyme reader, which is easy to operate and has high throughput. It can quickly screen for high-producing strains of pantothenic acid, meeting the needs of high-throughput screening.

[0056] Furthermore, the pantothenic acid biosensor has a high response intensity and a wide response range, and can detect pantothenic acid, thus expanding the application range of the biosensor.

[0057] Furthermore, the pantothenic acid biosensor has high industrial application value and can be used for the detection of environmental pollutants and high-throughput screening of pantothenic acid-high-producing strains, providing a powerful tool for research and application in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0059] Figure 1 The recombinant plasmid pACYC-HLT prepared in Example 1 of the present invention;

[0060] Figure 2 This is the standard curve drawn in Example 2 of the present invention. DETAILED DESCRIPTION

[0061] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0062] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0063] The culture medium involved in the following examples is:

[0064] LB solid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, agar powder 15 g / L, pH 7.0. Sterilize at 121°C for 20 min.

[0065] LB medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, pH 7.0. Sterilize at 121°C for 20 min.

[0066] Trace elements: FeCl₃·6H₂O 2.4 g / L, CoCl₂·6H₂O 0.3 g / L, CuCl₂·2H₂O 0.15 g / L, ZnCl₂ 0.3 g / L, Na₂MO₄·2H₂O 0.3 g / L, H₃BO₃ 0.075 g / L, MnCl₂·4H₂O 0.495 g / L. Sterilize at 121°C for 30 min.

[0067] M9 medium: glucose 40 g / L (separated), Na2HPO4·12H2O 15.11 g / L, KH2PO4 3 g / L, NH4Cl 1 g / L, NaCl 0.5 g / L, sterilized at 121°C for 20 min, 1 / 1000 volume of MgSO4 (1 mol / L) and 1 / 1000 volume of trace elements (FeCl3·6H2O 2.4 g / L, CoCl2·6H2O 0.3 g / L, CuCl2·2H2O 0.15 g / L, ZnCl2 0.3 g / L, Na2MO4·2H2O 0.3 g / L, H3BO3 0.075 g / L and MnCl2·4H2O 0.495 g / L) were added before fermentation began.

[0068] The genes involved in the following examples were synthesized by GeneWeiZhi.

[0069] Example 1

[0070] This embodiment provides a method for constructing a pantothenic acid biosensor, comprising the following steps:

[0071] (1) Synthesis of fusion genes

[0072] The transcriptional regulatory factor HLT encoding gene (as shown in SEQ ID NO.1), the inducible promoter (as shown in SEQ ID NO.2), the constitutive promoter (as shown in SEQ ID NO.3), and the green fluorescent protein encoding gene GFP from Bacillus were all fully synthesized. The specific promoter used was the trc promoter, and the synthesized fusion sequence was SEQ ID NO.4 (in the sequence, the 1st bp-717bp are the green fluorescent protein encoding gene GFP, the 718-818bp are the 5'UTR sequence, the 819bp-963bp are the inducible promoter, the 964-1015bp are the terminator, the 1016bp-1045bp are the trc promoter, the 1046-1204bp are the 5'UTR sequence, and the 1205bp-1504bp are the transcriptional regulatory factor HLT encoding gene).

[0073] (2) Construction of recombinant vectors and recombinant bacteria

[0074] The DNA fragment shown in SEQ ID NO. 4 was used to replace the sequence between the NcoI and EcoRI restriction sites of pACYCDuet-1, while the remaining sequence remained unchanged, to obtain the recombinant plasmid pACYC-HLT. Specifically, the extracted pACYCDuet-1 plasmid was double-digested with NcoI and EcoRI to obtain a linearized plasmid. The enzyme digestion system consisted of 1 μg of plasmid, 2 μL of buffer, 1 μL each of NcoI and EcoRI, and then added sterile water to 20 μL. After mixing, the system was incubated at 37°C for 1 hour and then at 65°C for 15 minutes. The system was then run on a gel and the target band was recovered. After adding NcoI and EcoRI restriction sites to both ends of the DNA fragment shown in SEQ ID NO. 4, the system was also run on a gel and the target band was recovered.

[0075] Ligation system: Take 100 ng of linearized vector, add the DNA fragment shown in SEQ ID NO.4 in a molar ratio of 4:1 to the linearized fragment, add 2 μL of buffer, 0.5 μL of T4 DNA ligase (commercially available), and use water to make up to 20 μL. The ligation reaction conditions are 22°C for 1 hour and 70°C for 5 minutes. Then use the conventional electroporation method to transform into electrocompetent cells. Subsequently, recovery, plating, and picking are performed to obtain the recombinant plasmid pACYC-HLT. The clones were picked and sent for sequencing, and the results were consistent with expectations. The obtained recombinant plasmid pACYC-HLT has a map as shown below. Figure 1 As shown, the gene sequence is shown as SEQ ID NO.8.

[0076] The obtained recombinant plasmid pACYC-HLT was transformed into electrocompetent Escherichia coli W3110 (commercially available) using a conventional electroporation method to obtain the recombinant bacteria pACYC-HLT containing pACYC-HLT.

[0077] The pantothenic acid biosensor recombinant plasmid pACYC-HLT or the recombinant bacteria pACYC-HLT containing pACYC-HLT was obtained above.

[0078] Example 2

[0079] This example provides a method for detecting pantothenic acid response using the pantothenic acid biosensor obtained in Example 1, comprising the following steps:

[0080] (1) Activation of strains: The recombinant strain pACYC-HLT constructed in Example 1 and stored at -80°C was streaked onto LB solid medium and then placed in an incubator at 37°C for activation culture for 12 h. A single colony was then picked and inoculated into the seed culture medium.

[0081] (2) Seed liquid culture: The seed culture medium is LB medium. The seed culture medium after inoculation is cultured in a shaker at 220 r / min and 37°C for 10 h to make the OD of the seed culture medium 600nm Reach 2 or above.

[0082] (3) The culture medium (35 mL) in step (2) was added to 5 shake flasks containing fresh M9 medium. The volume ratio of the culture medium to the M9 medium was 1:10. Pantothenic acid was added to the 5 shake flasks at final concentrations of 0, 4, 8, 10, and 15 g / L, respectively. The shake flasks were then incubated in a shaker at 220 r / min and 37°C for 1 h.

[0083] (4) Take the culture solution in step (3) and put it into the enzyme plate, and use the enzyme plate reader to detect the fluorescence intensity. The detection conditions are excitation wavelength 484nm and emission wavelength 507nm. At the same time, detect the OD 600nm The fluorescence value detected by the enzyme-labeled instrument is compared with the corresponding OD 600nm The standard curve was drawn with the ratio of the values ​​as the ordinate and the final concentration of pantothenic acid added as the abscissa.

[0084] After testing, the results are as follows Figure 2 As shown in Figure 2, the addition of pantothenic acid can significantly induce the expression of green fluorescent protein, and the fluorescence value per unit cell (RFU / OD 600 ) and pantothenic acid concentration range has a good correlation, and the standard curve equation is y = 7.2805x + 116.52, R 2 =0.9293, demonstrating that the pantothenic acid biosensor of the present invention detects pantothenic acid response with high accuracy and reliability. The R² value of the standard curve equation is close to 1, indicating a good linear relationship between fluorescence intensity and pantothenic acid concentration, further verifying the feasibility of this biosensor in practical applications. This standard curve enables precise quantification of pantothenic acid production, providing strong technical support for subsequent fermentation optimization and industrial production.

[0085] Example 3 Screening of Pantothenic Acid High-Producing Strains

[0086] The initial pantothenic acid strain used in this example was obtained by integrating panC, panB, and panE into the genome of Escherichia coli W3110 using the trc promoter, and the initial pantothenic acid production was 0.3 g / L.

[0087] The strain obtained by integrating panC, panB, and panE into the Escherichia coli W3110 genome using the trc promoter was prepared as follows:

[0088] Knock-in of panC (SEQ ID NO. 5), panB (SEQ ID NO. 6), and panE (SEQ ID NO. 7) into the Escherichia coli W3110 genome was completed using the cripsr / cas9 method, and the plasmids used were pEccas (addgene: 73227) and pEcgRNA (addgene: 166581).

[0089] pEccas was transformed into Escherichia coli W3110 using electroporation to obtain strain wpa1.

[0090] The insertion site of the panC, panB, and panE genes is the ldha gene, and the gRNA used is: 5'-AAACGATGACGGCAGCCGCC-3' (SEQ ID NO.9). For screening, the gRNA needs to be constructed into the pEcgRNA and constructed using the homologous recombination kit (Novozymes, Cat. No.: C117-01). The primers used in the construction process are primer 1-primer 4 (see Table 1 below). After construction is completed, the plasmid pEcgRNA-ldha can be obtained.

[0091] The knocked-in panC, panB, and panE genes are all derived from endogenous genes of Escherichia coli W3110, with nucleic acid sequences of SEQ ID NO.5-7, respectively. They are constructed into pEcgRNA using homologous recombination. The primers used are primer 5-primer 22 (see Table 1 below). After the plasmid construction is completed, pECgRNA-donor is obtained, and then primer 1 and primer 2 are used to obtain the fragment for knock-in.

[0092] Take the electroporation competent state of strain wpa1, add 200 ng of plasmid pEcgRNA-ldha and 100 ng of knock-in fragment, and after electroporation, recovery screening and elimination of the plasmid, a strain with knock-in panC, panB, and panE can be obtained. The strain name is wpa2.

[0093] Table 1. Primers

[0094]

[0095]

[0096]

[0097] This example provides a method for screening pantothenic acid high-producing strains using the pantothenic acid biosensor obtained in Example 1, comprising the following steps:

[0098] (1) Strain wpa2 was cultured in M9 medium at 220 rpm and 37°C in a shaker for 12 h until the stationary phase;

[0099] (2) The strain pre-cultured in step (1) was inoculated into M9 medium containing 150 g / L pantothenic acid at a volume ratio of 1:10. The pH value of the medium was adjusted to 7 with ammonia water. The OD value was measured after culturing at 37°C for 12 h. 600nm value;

[0100] (3) The culture solution of step (2) was inoculated into M9 medium containing 150 g / L pantothenic acid at a volume ratio of 1:10. The pH value of the medium was adjusted to 7 with ammonia water. The OD value was measured after culturing at 37 °C for 12 h. 600nm value;

[0101] (4) Repeat steps (2)-(3) until the OD of the strain is 600nm Reach around 5-6;

[0102] (5) The cells from step (4) were made into electrocompetent cells, the pantothenic acid biosensor plasmid (pACYC-HLT) was electrotransferred into the competent cells, and plated (the plate culture medium was LB solid culture medium containing chloramphenicol resistance);

[0103] (6) Pick the colonies from the plate culture dish and transfer them to a 96-well deep-well plate (using M9 medium) and culture at 37°C for 12 h;

[0104] (7) The liquid in the deep-well plate was aspirated into the ELISA plate, and the fluorescence intensity of each bacterial strain was tested using an ELISA instrument. The detection conditions were an excitation wavelength of 484 nm and an emission wavelength of 507 nm. At the same time, the OD 600nm .

[0105] (8) Compare the fluorescence intensity with the corresponding OD 600nm The ratio of the values ​​is substituted into the standard curve in Example 2 to obtain the yield of pantothenic acid.

[0106] After screening 1,000 strains of bacteria, a strain was obtained that had a three-fold increase in pantothenic acid production, reaching 0.9g / L, while the original strain's production was only 0.3g / L.

[0107] In summary, the pantothenic acid biosensor designed in the present invention has good responsiveness to pantothenic acid and can sensitively detect the concentration of pantothenic acid. In addition, due to the use of an enzyme marker, high-producing strains of pantothenic acid can be quickly screened, which has high industrial application value.

[0108] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of the transcriptional regulatory factor HLT of Bacillus in preparing a detection product responsive to pantothenic acid, characterized in that: The nucleotide sequence of the Bacillus transcriptional regulatory factor HLT is selected from any one of the following nucleotide sequences (1)-(2): (1) having the nucleotide sequence shown in SEQ ID NO.1; (2) A nucleotide sequence having an identity of 90% or more to the nucleotide sequence shown in SEQ ID NO.1 described in (1); preferably, a nucleotide sequence having an identity of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more to the nucleotide sequence shown in SEQ ID NO.1 described in (1).

2. The use according to claim 1, characterized in that The uses include any of the following: (1) Use in detecting pantothenic acid or preparing products for detecting pantothenic acid; (2) Use in screening pantothenic acid-producing strains or preparing products for screening pantothenic acid-producing strains; (3) Use in screening proteins or protein-encoding genes related to pantothenic acid synthesis or in preparing products for screening proteins or protein-encoding genes related to pantothenic acid synthesis; (4) Use in regulating the transcription level of a target gene, or in preparing a product for regulating the transcription level of a target gene; And / or, the products include probes, biosensors, pantothenic acid high-producing strains, and strains that utilize pantothenic acid biosensors to produce substances.

3. A pantothenic acid biosensor fusion gene, characterized in that The fusion gene comprises the Bacillus transcriptional regulatory factor HLT, an inducible promoter, a constitutive promoter and a reporter protein encoding gene as described in claim 1 or 2; Wherein, the constitutive promoter is operably connected to the Bacillus transcriptional regulatory factor HLT, and the inducible promoter is operably connected to the reporter protein coding gene.

4. The pantothenic acid biosensor fusion gene according to claim 3, characterized in that The inducible promoter has a nucleotide sequence as shown in SEQ ID NO.2, or a nucleotide sequence having an identity of 90% or more to the nucleotide sequence as shown in SEQ ID NO.2; preferably, a nucleotide sequence having an identity of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% or more to the nucleotide sequence as shown in SEQ ID NO.1 in (1); And / or, the constitutive promoter is selected from any one of a trc promoter, a T7 promoter, and a tac promoter; preferably, the trc promoter has a nucleotide sequence as shown in SEQ ID NO.3, or a nucleotide sequence having an identity of more than 90% with the nucleotide sequence as shown in SEQ ID NO.3; And / or, the reporter protein encoding gene includes a fluorescent protein encoding gene; preferably, the fluorescent protein encoding gene includes at least one of a green fluorescent protein encoding gene, a red fluorescent protein encoding gene, an orange fluorescent protein encoding gene, a yellow fluorescent protein encoding gene, a green fluorescent protein encoding gene, and a blue fluorescent protein encoding gene; And / or, the pantothenic acid biosensor fusion gene has the nucleotide sequence shown in SEQ ID NO. 4, or a nucleotide sequence having an identity of more than 90% with the nucleotide sequence shown in SEQ ID NO. 4; preferably, a nucleotide sequence having an identity of more than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% with the nucleotide sequence shown in SEQ ID NO. 4; And / or, the pantothenic acid biosensor fusion gene further includes an untranslated region (UTR) and a ribosome binding site sequence.

5. A biomaterial, characterized in that Includes any of the following: 1) A nucleic acid molecule encoding the pantothenic acid biosensor fusion gene according to any one of claims 3 to 4; optionally, the nucleic acid molecule is DNA or RNA; 2) an expression cassette, recombinant vector, recombinant microorganism, or transgenic cell line expressing the pantothenate biosensor fusion gene according to any one of claims 3 to 4; 3) an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the nucleic acid molecule described in 1); 4) a recombinant vector, recombinant microorganism or transgenic cell line containing the expression cassette described in 2) or 3); 5) A host cell containing the recombinant vector described in 2) or 3) or 4); Optionally, the initial plasmid vector of the recombinant vector is selected from any one of pRSFDuet-1 vector, pETDuet-1 vector, pACYCDuet-1 vector, pTrc99a vector or pET28a vector; And / or, the host cell is selected from Escherichia, Corynebacterium glutamicum, Saccharomyces cerevisiae or Yarrowia lipolytica.

6. A pantothenic acid biosensor, characterized in that The invention comprises the transcriptional regulatory factor HLT encoding gene according to claim 1 or 2, the pantothenic acid biosensor fusion gene according to any one of claims 3 to 4, and the biomaterial according to claim 5.

7. Use of the pantothenic acid biosensor fusion gene according to any one of claims 3 to 4, the biomaterial according to claim 5, or the pantothenic acid biosensor according to claim 6 in any one of the following: (1) Use in detecting pantothenic acid or preparing products for detecting pantothenic acid; (2) Use in screening pantothenic acid-producing strains or preparing products for screening pantothenic acid-producing strains; (3) Use in screening proteins or protein-encoding genes related to pantothenic acid synthesis or in preparing products for screening proteins or protein-encoding genes related to pantothenic acid synthesis; (4) Use in regulating the transcription level of a target gene, or in preparing a product for regulating the transcription level of a target gene; Optionally, the product comprises a reagent or a kit.

8. A method for detecting pantothenic acid, characterized in that: The method comprises performing qualitative or quantitative detection using the Bacillus transcriptional regulatory factor HLT encoding gene according to claim 1 or 2, the pantothenic acid biosensor fusion gene according to any one of claims 3 to 4, the biomaterial according to claim 5, and the pantothenic acid biosensor according to claim 6; Optionally, in the qualitative detection, the biosensor is mixed with a sample to be tested and incubated to generate a signal in response to pantothenic acid, and the sample to be tested contains pantothenic acid; Optionally, in the quantitative detection, the biosensor is mixed and incubated with a pantothenic acid standard of known gradient concentrations to generate a signal responding to pantothenic acid. A standard curve is drawn based on the gradient concentrations and the signal responding to pantothenic acid. The signal responding to pantothenic acid of the sample to be tested is substituted into the standard curve to calculate the pantothenic acid concentration.

9. A method for screening a strain with high pantothenic acid production, characterized in that: The method comprises screening using the transcriptional regulatory factor HLT encoding gene of Bacillus according to claim 1 or 2, the pantothenic acid biosensor fusion gene according to any one of claims 3-4, the biomaterial according to claim 5, and the pantothenic acid biosensor according to claim 6, comprising the following steps: S1. Cultivate the strain to be evolved; S2. inoculating the strain cultured in step S1 into a medium containing pantothenic acid for cultivation; S3, inoculating the strain cultured in step S2 into a medium containing pantothenic acid for cultivation; S4. Repeat steps S2-S3 until the OD of the strain reaches 600nm Reach 5-6; S5, making the cells of step S4 competent for electroporation, transferring the pantothenate biosensor to the competent state, and performing plate culture; S6. Pick the colonies cultured in step S5 and culture them in a multi-well plate, detect the signal produced by each bacterial strain in response to pantothenic acid, and screen the pantothenic acid high-producing strain based on the signal intensity.

10. The method for screening a high-producing strain of pantothenic acid according to claim 9, characterized in that: In step S2 or S3, the concentration of pantothenic acid in the pantothenic acid-containing culture medium is in the range of 0.1-150 g / L; And / or, in step S2 or S3, the incubation time is 4-24 hours; And / or, in step S6, the culture time is 3-36 hours.

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