Pyridoxal phosphate-responsive biosensor and application thereof

Abstract

This invention relates to a pyridoxal phosphate-responsive biosensor and its application, belonging to the field of synthetic biology biosensor technology. This invention is the first to discover and identify the relationship between pyridoxal phosphate and Cgl1185-P. cgl2838 The transcriptional regulation and response relationship between these factors was investigated, and this relationship was used as the basis for constructing a biosensor responsive to pyridoxal. The transcription factor regulatory system Cgl1185-P derived from Corynebacterium glutamicum was used. cgl2838 A biosensor for the specific detection of pyridoxal phosphate was constructed by assembling a reporter gene as the main component onto a plasmid, enabling effective detection of pyridoxal phosphate concentrations in intracellular or culture environments. This biosensor possesses advantages such as clear structure, high sensitivity, and good host adaptability, and can be used for fermentation process monitoring, cofactor metabolism research, and screening of relevant strains, showing promising application prospects in the fields of cofactor concentration monitoring and metabolic regulation.

Description

Technical Field

[0001] This invention relates to a pyridoxal phosphate-responsive biosensor and its application, belonging to the field of synthetic biology biosensor technology. Background Technology

[0002] Biosensors are a class of technologies that utilize biomolecules to specifically recognize target substances and convert the recognition process into detectable signals. They have significant application value in metabolic engineering, synthetic biology, and biomanufacturing. Compared with traditional physicochemical detection methods, biosensors offer advantages such as ease of operation, real-time detection capabilities, and applicability to in vivo cell monitoring, making them suitable for fermentation process monitoring and metabolic regulation research.

[0003] Pyridoxal phosphate (PLP) is the main active form of vitamin B6 and an essential cofactor for many metabolic enzyme reactions, widely involved in amino acid and related metabolic processes. Intracellular PLP levels directly affect cell growth and metabolic efficiency; both insufficient and excessive PLP levels can adversely affect microbial metabolism. Therefore, effective monitoring of PLP concentration is of great significance.

[0004] Currently, the detection of pyridoxal phosphate mainly relies on high-performance liquid chromatography (HPLC). Although this method is highly accurate, it is usually complex to operate, has high detection costs, and is difficult to monitor the concentration of PLP in living cells in real time, which limits its application in online monitoring and dynamic control of the fermentation process. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the first objective of this invention is to provide a pyridoxal phosphate biosensor with high responsiveness. The second objective is to provide an application for the aforementioned pyridoxal phosphate biosensor in detecting pyridoxal phosphate content in the fermentation environment. This solves the problems of a lack of pyridoxal phosphate biosensors and a lack of real-time monitoring in existing technologies.

[0006] To achieve the aforementioned objectives, the present invention employs the following technical solution:

[0007] One of the technical solutions provided by this invention is a biosensor responsive to pyridoxal phosphate, the biosensor comprising: a transcription factor Cgl1185 expression cassette and a promoter P cgl2838 and reporter genes and other elements; the promoter P cgl2838 The reporter gene is regulated by the transcription factor Cgl1185 and is regulated by the promoter P. cgl2838 Regulation; Furthermore, the biosensor also includes a gene encoding the repressor protein LacI and a promoter P. trc The gene encoding the repressor protein LacI is located in promoter P. cgl2838 Downstream and affected by Pcgl2838 Regulation of expression, promoter P trc It is located downstream of the gene encoding the repressor protein LacI and is used to regulate the expression of its downstream reporter genes; Furthermore, the transcription factor Cgl1185 expression cassette contains the gene encoding the transcription factor Cgl1185 and the promoter P. cgl1185 and terminator; Furthermore, the biosensor also includes a replicon gene for controlling the initiation of replication of the entire biosensor; Furthermore, the biosensor also includes an resistance gene for screening transformants containing the biosensor; Furthermore, the replicon includes, but is not limited to, […]. pBL1, ori, ColE1 wait; Furthermore, the resistance gene includes, but is not limited to, [other than] ... Cm, Amp, Kan wait; Furthermore, P cgl2838 The promoter ribosome binding site RBS is replaced with R36, R48, R33, R13, or R18, etc.; where: The nucleotide sequence of R13 is: AGCATGAAAGGGAAAGGACCCC; The nucleotide sequence of R18 is: AGCATGGAAGGAGAGAAGCCCC; The nucleotide sequence of R33 is: AGCATGGAAGGGGGGGGACCCC; The nucleotide sequence of R36 is: AGCATGAAAAGGAAAGGACCCC; The nucleotide sequence of R48 is: AGCATGGGGGAAAAAGAGCCCC; Furthermore, the reporter gene includes, but is not limited to, […]. gfp , rfp , CFP , sfgfp , egfp , yfp , ecfp Isogenes; Furthermore, the transcription factor Cgl1185 has the nucleotide sequence shown in SEQ ID NO.5; Furthermore, the promoter P cgl1185 The nucleotide sequence is shown in SEQ ID NO.6; Furthermore, the promoter P cgl2838 The nucleotide sequence is shown in SEQ ID NO.7; Furthermore, the reporter gene gfpThe nucleotide sequence is shown in SEQ ID NO.1; Furthermore, the repressor protein LacI encodes a gene with the nucleotide sequence shown in SEQ ID NO.8; Furthermore, the promoter P trc The nucleotide sequence is shown in SEQ ID NO.9; Preferably, the biosensor contains a promoter P. cgl1185 Transcription factor Cgl1185 encoding gene, terminator T1, promoter P cgl2838 And recombinant plasmids containing elements such as reporter genes; more preferably, the expression vectors that can be used for the recombinant plasmids include, but are not limited to, expression vectors commonly used in the art, such as pEC-XK99E, pXMJ19, pEP2, etc. More preferably, using pEC-XK99E plasmid as an expression vector, a structure containing promoter P is constructed. cgl1185 Transcription factor Cgl1185 encoding gene, terminator T1, promoter P cgl2838 and reporter genes gfp biosensor pECXK99E-Cgl1185-P cgl2838 -GFP, the nucleotide sequence of the biosensor is shown in SEQ ID NO.2; More preferably, using pEC-XK99E plasmid as an expression vector, a structure containing promoter P is constructed. cgl1185 Transcription factor Cgl1185 encoding gene, terminator T1, promoter P cgl2838 Ribosome binding site R36, gene encoding repressor protein LacI, promoter P trc and reporter genes gfp biosensor pECXK99E-Cgl1185-P cgl2838 - lacI -P trc -GFP, the nucleotide sequence of the biosensor is shown in SEQ ID NO.3.

[0008] The second technical solution provided by the present invention is the application of the biosensor described in the first technical solution, particularly its application in detecting pyridoxal phosphate or products containing pyridoxal phosphate. Furthermore, the biosensor is introduced into pyridoxal phosphate producing strains, such as Corynebacterium glutamicum, to detect the production and concentration changes of pyridoxal phosphate in a culture environment containing the producing strain. Furthermore, the biosensor is introduced into a separate host (such as Escherichia coli MG1655, Escherichia coli BL21, Bacillus subtilis 168, etc.) and then added to the detection system to detect pyridoxal phosphate in the system.

[0009] The third technical solution provided by this invention is a biosensor for regulating pyridoxal phosphate concentration, wherein the biosensor sequentially comprises: a transcription factor Cgl1185 expression cassette, a promoter P cgl2838 The gene encoding the repressor protein LacI and the promoter P trc And elements such as the pyridoxal phosphate synthase gene; Furthermore, the pyridoxal phosphate synthase is a pyridoxal phosphate synthase complex composed of the PdxS gene and the PdxT gene; PdxS gene, Gene ID: 939988, PdxT gene, Gene ID: 939971; Preferably, the sensor is plasmid pECXK99E-Cgl1185-P Cgl2838 -lacI-P trc -BspdxST, the nucleotide sequence of the biosensor is shown in SEQ ID NO.10.

[0010] The fourth technical solution provided by this invention is a recombinant microorganism comprising the biosensor described in one or three technical solutions, wherein the host cell used by the recombinant microorganism includes, but is not limited to, Corynebacterium glutamicum, with the preferred Corynebacterium glutamicum being (…). Corynebacterium glutamicum ATCC 13032.

[0011] The fifth technical solution provided by the present invention is the application of the recombinant microorganisms described in the fourth technical solution, particularly in the production of pyridoxal phosphate or 5-aminolevulinic acid (5-ALA).

[0012] Beneficial effects: This invention is the first to discover and identify pyridoxal phosphate and Cgl1185-P cgl2838 The transcriptional regulation and response relationship between these factors was investigated, and this relationship was used as the basis for constructing a biosensor responsive to pyridoxal. The transcription factor regulatory system Cgl1185-P derived from Corynebacterium glutamicum was used. cgl2838 A biosensor for the specific detection of pyridoxal phosphate was constructed by assembling a reporter gene as the main component onto a plasmid, enabling effective detection of pyridoxal phosphate concentrations in intracellular or culture environments. This biosensor possesses advantages such as clear structure, high sensitivity, and good host adaptability, and can be used for fermentation process monitoring, cofactor metabolism research, and screening of relevant strains, showing promising application prospects in the fields of cofactor concentration monitoring and metabolic regulation. Attached Figure Description

[0013] Figure 1 pECXK99E-Cgl1185-P cgl2838 -GFP plasmid map.

[0014] Figure 2 For Cgl1185-P cgl2838 The response to pyridoxal phosphate.

[0015] Figure 3 Schematic diagram for RBS optimization of response element.

[0016] Figure 4 The result diagram shows the optimization of the RBS response element.

[0017] Figure 5 This is a schematic diagram illustrating the principle of pyridoxal phosphate concentration in a negative feedback biosensor response gradient.

[0018] Figure 6 The fluorescence density represents the response gradient of the negative feedback biosensor based on the concentration of pyridoxal phosphate.

[0019] Figure 7 This is a graph showing the production of 5-ALA. Detailed Implementation

[0020] The embodiments described below are exemplary descriptions of key experimental evidence and are not intended to limit the core content and application scope of this invention due to the amount of evidence. It should be noted that all accompanying drawings and corresponding descriptions only exemplarily illustrate the concept, principles, and representative experimental evidence of the disclosed embodiments of this invention. Where the chain of evidence is complete, it is unnecessary to show all the specific details and extended details of the various embodiments listed in this invention.

[0021] The biosensor provided by this invention is based on pyridoxal phosphate and Cgl1185-P cgl2838 A biosensor relating transcriptional regulation and response, the sensor comprising a transcription factor Cgl1185 expression cassette, a Cgl1185-regulated promoter P cgl2838 and by promoter P cgl2838 Driven reporter genes. Under low PLP conditions, transcription factor cgl1185 can bind to P. cgl2838 The promoter region is inhibited and its transcriptional activity is suppressed, thereby maintaining reporter gene expression at a low level. When PLP concentration increases, the interaction between PLP and cgl1185 weakens its effect on P... cgl2838 The promoter binding ability is enhanced, thereby relieving transcriptional repression and promoting reporter gene expression, ultimately manifested as an increase in fluorescence signal with increasing PLP concentration. Those skilled in the art can select a reporter gene from existing technologies based on practical considerations. Multiple reporter genes can be chosen; commonly used protein molecules capable of generating visual detection signals or detectable small molecules, such as fluorescent proteins and color proteins, can all achieve the response of the biosensor described in this invention. Preferably, such as… gfp , rfp , CFP , sfgfp , egfp , yfp , ecfp The aforementioned sensors also include necessary elements for expression, such as replication origin sites, preferably, such as... colE1 Replication origin sites, etc. The aforementioned sensors may also contain markers such as resistance genes, for example... kan r This facilitates screening. Those skilled in the art can also add other components to the above-mentioned sensors according to actual needs, such as constructing the above-mentioned components onto expression vectors in the prior art, such as pEC-XK99E, pXMJ19, pEP2, etc., to obtain recombinant plasmids that can be used as sensors.

[0022] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0023] 1. The source and acquisition method of some of the biological materials involved in this invention The original strain Corynebacterium glutamicum used in this invention ( Corynebacterium glutamicum ATCC13032 is sourced from ATCC (The Global Bioresource Center) and was purchased in October 2012.

[0024] The Corynebacterium glutamicum A40 strain is prior art, and this strain has been disclosed in Chinese invention patent application CN118652920 A.

[0025] The plasmid pEC-XK99E was purchased from Fenghui Biotechnology Co., Ltd.

[0026] The primers used were synthesized by Genewiz.

[0027] The Gibson assembly reagent used was purchased from Ibotech.

[0028] Other biochemical reagents used were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0029] 2. The composition and preparation of some of the culture media involved in this invention BHI liquid medium: 37 g / L brain heart broth powder; BHI solid medium with 2% agar powder added.

[0030] CGIII medium: tryptone 10 g / L, yeast extract 10 g / L, NaCl 2.5 g / L, pH adjusted to 7.0.

[0031] CGXII medium: ammonium sulfate 20 g / L, urea 5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate trihydrate 1.3 g / L, 3-(N-morpholino)propanesulfonic acid (MOPS) 42 g / L, calcium chloride 0.01 g / L, magnesium sulfate heptahydrate 0.25 g / L, ferrous sulfate heptahydrate 0.002 g / L, manganese sulfate monohydrate 0.002 g / L, zinc sulfate heptahydrate 0.0002 g / L, copper sulfate 0.0004 g / L, nickel chloride hexahydrate 0.00004 g / L, protocatechuic acid 0.006 g / L, biotin 0.0004 g / L, vitamin B1 0.00002 g / L, adjusted to pH 7 with 5 mol / L NaOH aqueous solution.

[0032] The antibiotic concentration in the culture medium for the strain containing plasmid pEC-XK99E and its extended plasmid was 50 μg / mL kanamycin.

[0033] The present invention will be further explained and illustrated below through specific embodiments.

[0034] Example 1: Construction of Biosensors Using the pEC-XK99E plasmid as a template, the pEC-XK99E fragment with homologous arms was amplified using primers pEC-F and pEC-R (primers are shown in Table 1). Using the GFP-encoding gene as a template (SEQ ID NO.1), the GFP-encoding gene fragment with homologous arms for Gibson assembly was amplified using primers GFP-F and GFP-R. The above fragment was then subjected to fusion PCR and seamless cloning to obtain the plasmid pECXK99E-GFP.

[0035] Table 1 Primers used to construct the screening backbone plasmid

[0036] Example 2: Screening of transcription factor regulatory combinations responding to pyridoxal phosphate concentrations based on transcriptome data. 1. The strain Corynebacterium glutamicum ATCC13032 was cultured in an environment containing different concentration gradients of pyridoxal phosphate. Specifically, after the strain was activated, it was inoculated at a 1% inoculum into 50 mL of CGXII medium containing pyridoxal phosphate at final concentrations of 0 mmol / L and 8 mmol / L, respectively, and cultured at 30℃ and 220 rpm.

[0037] Cell samples were collected during the logarithmic growth phase for transcriptome analysis. Transcriptome analysis showed that, compared to the culture environment without pyridoxal phosphate, the expression levels of 1000 genes changed under pyridoxal phosphate conditions (8 mmol / L), with 571 genes upregulated and 429 genes downregulated, among which the cgl2838 gene was significantly upregulated. Using the CoryneRegNet7 database, the expression of P... cgl2838 The promoter is regulated by the transcription factor Cgl1185. Therefore, it is speculated that P cgl2838 The response effect of PLP comes from the control of Cgl1185.

[0038] 2. To further verify the above hypothesis, Cgl1185-P cgl2838 The transcriptional regulatory relationship serves as the basis for subsequent sensor construction, thereby testing its response to PLP.

[0039] (1) Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, and Cgl1185-F and Cgl1185-R as primers (primers are shown in Table 2), the genome containing P was amplified. cgl1185 The transcription factor cgl1185 gene and fragments of the terminator.

[0040] (2) Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, P Cgl2838 -F、P Cgl2838 -R is a primer, amplifying P Cgl2838 Excerpt.

[0041] (3) Using the basic plasmid pECXK99E-GFP as a template, and GFP-F / C-pEC-VR primers, a linear plasmid with homologous regions was amplified.

[0042] (4) The fragments and linear plasmids obtained in steps (1), (2) and (3) above are connected by Gibson assembly to obtain plasmid pECXK99E-Cgl1185-P Cgl2838 -GFP. A schematic diagram of the constructed plasmid is shown below. Figure 1 This plasmid also carries P cgl1185 transcription factor cgl1185 and P Cgl2838 Plasmid pECXK99E-Cgl1185-P Cgl2838 The full-length nucleotide sequence of the -GFP plasmid is shown in SEQ ID NO.2.

[0043] 3. To verify P cgl1185 and P cgl2838 To investigate the promoter's response to changes in PLP concentration, two control plasmids were constructed.

[0044] (1) Construction of plasmid pEC-P cgl1185 -GFP pECXK99E-Cgl1185-P cgl2838 -GFP is used as a template, and primer P is used. cgl1185 -F and P cgl1185 -R amplification yielded a linear fragment, which was then ligated via Gibson assembly to obtain the plasmid pEC-P. cgl1185 -GFP. Plasmid pEC-P cgl1185 -GFP is in pECXK99E-Cgl1185-P cgl2838 -Based on the GFP plasmid, it mainly retains P cgl1185 The promoter, the GFP portion of the green fluorescent protein, and the plasmid backbone.

[0045] (2) Construction of plasmid pEC-P cgl2838 -GFP pECXK99E-Cgl1185-P cgl2838 -GFP was used as a template, and primers Huan-P were used. Cgl2838 -F and Huan-P Cgl2838 -R amplification yields a linear fragment, which is then ligated via Gibson assembly to obtain the plasmid pEC-P. cgl2838 -GFP. Plasmid pEC-P cgl2838 -GFP is in pECXK99E-Cgl1185-P cgl2838 -Based on the GFP plasmid, it mainly retains P cgl2838 The promoter, the GFP portion of the green fluorescent protein, and the plasmid backbone.

[0046] The target plasmid was further obtained through chemical transformation with E. coli.

[0047] Table 2 Primers used to construct the screening test plasmid

[0048] Example 3: Construction and testing of a combined sensor for transcription factor regulation in response to pyridoxal phosphate concentration. The pECXK99E-Cgl1185-P successfully constructed in Example 2 Cgl2838 -GFP, pEC-P cgl1185 -GFP, pEC-P cgl2838 -GFP plasmids were electroporated into Corynebacterium glutamicum ATCC 13032 to obtain strain Cg-pECXK99E-Cgl1185-P Cgl2838 -GFP, Cg-pEC-P cgl1185 -GFP, Cg-pEC-P cgl2838 -GFP.

[0049] These strains were subjected to high-throughput screening in environments containing pyridoxal phosphate at concentrations of 0 mmol / L, 4 mmol / L, and 8 mmol / L, respectively. Details are as follows: Single colonies were picked and cultured overnight at 30°C and 220 rpm in 5 mL of BHI medium containing kanamycin antibiotic to obtain seed culture. 100 μL of the seed culture was then transferred to (1) 5 mL of CGXII medium containing kanamycin antibiotic; (2) 5 mL of CGXII medium containing kanamycin antibiotic with a final concentration of 4 mmol / L pyridoxal phosphate; and (3) 5 mL of CGXII medium containing kanamycin antibiotic with a final concentration of 8 mmol / L pyridoxal phosphate. After culturing at 30°C and 500 rpm for 24 h, 200 μL of the culture was diluted 20 times to determine the fluorescence intensity (GFP) and absorbance at 600 nm (OD). 600 ), to obtain a uniform relative fluorescence intensity (GFP / OD) 600 The excitation wavelength is 488 nm and the absorption wavelength is 520 nm.

[0050] The results are as follows Figure 2 As shown in the figure. Cgl1185-P cgl2838 P cgl1185 P cgl2838 These represent strains Cg-pECXK99E-Cgl1185-P. Cgl2838 -GFP, Cg-pEC-P cgl1185 -GFP, Cg-pEC-P cgl2838 -GFP.

[0051] Depend on Figure 2 It can be seen that Cgl1185-P cgl2838 The transcriptional regulation combination exhibited a stepwise response at different PLP concentrations. With increasing PLP concentration (0 mM to 8 mM), the fluorescence intensity significantly increased, indicating that the combination effectively responded to changes in PLP concentration. In contrast, using P alone... cgl1185 or P cgl2838 The promoter response to changes in PLP and its concentration was not significant.

[0052] The above results further prove that Cgl1185 and P cgl2838 The combination showed a significant response to PLP, meaning that under low PLP conditions, the transcription factor cgl1185 could bind to P. cgl2838 The PLP region inhibits its transcriptional activity, thereby maintaining GFP expression at a low level. When PLP concentration increases, the interaction between PLP and cgl1185 weakens its effect on GFP expression. cgl2838The promoter binding ability is enhanced, thereby relieving transcriptional repression and promoting GFP expression, ultimately manifested as an increase in fluorescence signal with increasing PLP concentration. Therefore, the plasmid pECXK99E-Cgl1185-P constructed in this invention... Cgl2838 -GFP can be used as a biosensor to detect PLP and reflect its relative concentration.

[0053] Example 4: Optimization of the sensitivity of the transcription factor biosensor pECXK99E-Cgl1185-P Cgl2838 Using GFP as a template, amplification was performed using primers R36-F, R48-F, R33-F, R13-F, R18-F, and RBS-R (primers are shown in Table 4), yielding pECXK99E-Cgl1185-P. Cgl2838 The -Rxx-GFP fragment (with the corresponding RBS sequence inserted upstream of the GFP gene, where Rxx represents R36, R48, R33, R13, and R18 respectively) was then ligated using Gibson assembly to obtain the corresponding plasmid pECXK99E-Cgl1185-P. Cgl2838 -R13-GFP, pECXK99E-Cgl1185-P Cgl2838 -R18-GFP, pECXK99E-Cgl1185-P Cgl2838 -R33-GFP, pECXK99E-Cgl1185-P Cgl2838 -R36-GFP, pECXK99E-Cgl1185-P Cgl2838 -R48-GFP. The above plasmid will be pECXK99E-Cgl1185-P Cgl2838 -GFP in P Cgl2838 The original RBS in the promoter is replaced with R36, R48, R33, R13, or R18, respectively, as shown in the structure. Figure 3 As shown.

[0054] The above plasmids were electroporated into Corynebacterium glutamicum ATCC 13032 to obtain the following recombinant bacteria: Cg-pECXK99E-Cgl1185-P Cgl2838 -R13-GFP, Cg-pECXK99E-Cgl1185-P Cgl2838 -R18-GFP, Cg-pECXK99E-Cgl1185-P Cgl2838 -R33-GFP, Cg-pECXK99E-Cgl1185-P Cgl2838 -R36-GFP, Cg-pECXK99E-Cgl1185-P Cgl2838-R48-GFP. Simultaneously, Cg-pECXK99E-Cgl1185-P Cgl2838 -GFP was used as a control.

[0055] The above-mentioned strains were subjected to high-throughput screening in environments containing pyridoxal phosphate at concentrations of 0 mmol / L, 4 mmol / L, and 8 mmol / L, respectively. Details are as follows: Single colonies were picked and cultured overnight at 30°C and 220 rpm in 5 mL of BHI medium containing kanamycin antibiotic to obtain seed culture. 100 μL of the seed culture was then transferred to (1) 5 mL of CGXII medium containing kanamycin antibiotic; (2) 5 mL of CGXII medium containing kanamycin antibiotic with a final concentration of 4 mmol / L pyridoxal phosphate; and (3) 5 mL of CGXII medium containing kanamycin antibiotic with a final concentration of 8 mmol / L pyridoxal phosphate. After culturing at 30°C and 500 rpm for 24 h, 200 μL of the culture was diluted 20 times to determine the fluorescence intensity (GFP) and absorbance at 600 nm (OD). 600 ), to obtain a uniform relative fluorescence intensity (GFP / OD) 600 The excitation wavelength is 488 nm, and the absorption wavelength is 520 nm. GFP / OD 600 The results are as follows Figure 4 As shown.

[0056] The response sensitivity to PLP is defined as: GFP / OD at 4mM PLP. 600 GFP / OD at 0mM 600 The ratio of the values ​​is shown in Table 3.

[0057] It can be seen that the sensor sensitivity of wild-type RBS is 2.07; the transcription factor biosensor with RBS R36 has the highest sensitivity, reaching 15.16, representing a 7.32-fold increase in sensitivity and exhibiting a good trend. Therefore, RBS R36 was selected as the P transcription factor biosensor. Cgl2838 RBS.

[0058] That is, select pECXK99E-Cgl1185-P Cgl2838 Further research will be conducted on R36-GFP as a PLP-responsive biosensor.

[0059] Table 3. Candidate RBS sequences and the sensitivity of sensors containing these RBS sequences.

[0060] Table 4 Primers used to construct the RBS optimized sensor

[0061] Example 5: Performance evaluation of the pyridoxal phosphate biosensor before and after optimization To further evaluate the impact of RBS optimization on sensor performance, the original pECXK99E-Cgl1185-P was subjected to further testing. cgl2838 -GFP sensor and R36 replacement for RBS optimized sensor pECXK99E-Cgl1185-P cgl2838 -R36-GFP was analyzed using dose-response curves.

[0062] strain Cg-pECXK99E-Cgl1185-P Cgl2838 -GFP and Cg-pECXK99E-Cgl1185-P Cgl2838 -R36-GFP was inoculated into CGXII medium containing mM PLP concentration gradients of 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, and 10 mM and cultured for 24 h. OD was then measured. 600 And fluorescence intensity, and calculate relative fluorescence intensity (GFP / OD) 600 Key performance parameters were obtained by nonlinear fitting of the experimental data using the Hill equation (Table 5).

[0063] The results showed that by optimizing the RBS intensity, the maximum fluorescence intensity of the sensor was significantly improved, and its fluorescence dynamic range increased from 13.25 times to 24 times (fluorescence dynamic range is the multiple by which the fluorescence intensity of the sensor changes between the lowest and highest signal values, used to represent the range of the sensor's response from no signal to the maximum signal. A larger range indicates that the sensor can resolve more significant signal changes and has higher detection sensitivity). Meanwhile, the EC of the PLP... 50 The value decreased from 4.30 mM to 3.09 mM (EC). 50 The Hill coefficient represents the PLP concentration at which the sensor's fluorescence signal reaches half of its maximum signal. A lower value indicates a more sensitive sensor to PLP (meaning a significant signal response can be generated even at low concentrations). This suggests that the optimized sensor can produce a significant response even under low PLP concentrations, thus improving detection sensitivity. Furthermore, a higher Hill coefficient indicates stronger synergy and a steeper response curve, which is beneficial for improving signal resolution.

[0064] Table 5 Key performance parameters for dose-response curve fitting

[0065] Example 6: Construction of a negative feedback-regulated pyridoxal phosphate biosensor The PLP-responsive biosensors constructed in this invention are all positive feedback sensors, meaning that the higher the PLP concentration in the system, the better the response of Cgl1185-P.Cgl2838 The stronger the response, the higher the intensity of GFP expression will be initiated.

[0066] The repressor protein LacI and the promoter P with a LacI binding site trc There is a negative regulatory relationship between them. The repressor protein LacI prevents RNA polymerase from binding to P through steric hindrance. trc The binding of the promoter region, or the inhibition of transcriptional elongation of the bound RNA polymerase, effectively blocks P. trc Transcription of downstream target genes shuts down the expression system. Furthermore, the aforementioned negative feedback regulatory mechanism exhibits a significant concentration dependence.

[0067] Therefore, LacI and P trc Introducing this negative regulatory relationship into the aforementioned constructed PLP-responsive biosensor yields a negative feedback regulatory biosensor, where LacI expression is controlled by P... Cgl2838 Controlled, the expression of GFP in the sensor is affected by P trc Therefore, GFP expression is negatively correlated with the PLP concentration in the system. That is, when PLP is lacking in the system, Cgl1185-P... Cgl2838 The system cannot generate a response, therefore it cannot initiate the expression of downstream LacI proteins; when LacI is deficient, P... trc Initiating transcription of the downstream GFP-encoding gene, resulting in high GFP expression; when there is sufficient PLP in the system, Cgl1185-P Cgl2838 This will generate a response and initiate the expression of the downstream LacI protein, while the presence of LacI protein inhibits P. trc This initiates transcription of the downstream GFP-encoding gene, resulting in low GFP expression. The principle is as follows: Figure 5 As shown.

[0068] The construction process of the negative feedback-regulated biosensor is as follows: (1) pECXK99E-Cgl1185-P Cgl2838 -R36-GFP was used as a template, with primers Cgl1185-P Cgl2838 -R36-F、Cgl1185-P Cgl2838 -R36-R (primers are shown in Table 6), amplified pECXK99E-Cgl1185-P with homologous arms. Cgl2838 -GFP linear vector fragment.

[0069] (2) Using pEC-XK99E as a template, primer lacI-P trc -F、lacI-P trc -R amplification yielded lacI-P containing a homologous arm. trc Excerpt.

[0070] (3) The two fragments were ligated using Gibson assembly to obtain the biosensor plasmid pECXK99E-Cgl1185-P that responds to PLP. Cgl2838 -lacI-P trc -GFP, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0071] The plasmid pECXK99E-Cgl1185-P Cgl2838 -lacI-P trc -GFP was electroporated into Corynebacterium glutamicum ATCC13032 to obtain strain Cg-Cgl1185-P Cgl2838 -lacI-P trc -GFP.

[0072] The strains were subjected to a gradient concentration response test of pyridoxal phosphate, with seven gradient concentrations of pyridoxal phosphate: 0 mmol / L, 0.5 mmol / L, 1 mmol / L, 1.5 mmol / L, 2.0 mmol / L, 2.5 mmol / L, and 3 mmol / L.

[0073] Single colonies were picked and cultured overnight at 30°C and 220 rpm in 5 mL of BHI medium containing kanamycin antibiotic to create a seed culture. 100 μL of this seed culture was then transferred to 4.9 mL of CGXII medium (containing kanamycin antibiotic) with pyridoxal phosphate concentrations of 0 mmol / L, 0.5 mmol / L, 1 mmol / L, 1.5 mmol / L, 2.0 mmol / L, 2.5 mmol / L, and 3 mmol / L, respectively. After 18 h of incubation at 30°C and 500 rpm, 200 μL of each culture, diluted 20-fold, was used to measure the fluorescence intensity (GFP) and absorbance at 600 nm (OD). 600 (The excitation wavelength is 488nm and the absorption wavelength is 520nm).

[0074] The results are as follows Figure 6 As shown in the figure (pEC-Cgl1185-PCgl2838-lacI-Ptrc-GFP represents strain Cg-Cgl1185-P),... Cgl2838 -lacI-P trc -GFP). It can be seen that the fluorescence intensity of this biosensor has a negative linear relationship with the concentration of pyridoxal phosphate in the culture environment, and can respond as low as 0.5 mM PLP, with a low response limit, making it suitable for intracellular regulation.

[0075] Table 6 Primers required for sensor construction

[0076] Example 7: Application of a negative feedback-regulated pyridoxal phosphate biosensor 5-Aminolevulinic acid (5-ALA) is a precursor to important compounds such as porphyrin, chlorophyll, and heme, and is widely used in medicine, agriculture, and animal husbandry.

[0077] In the biosynthesis of 5-aminolevulinic acid (5-ALA), PLP acts as an important cofactor, and its synthesis and regulation play a crucial role in the production performance of the strain. Both excessively low and excessively high PLP concentrations negatively impact the production process. Although the PLP-responsive biosensor constructed in the foregoing embodiments of this invention can visualize and reflect PLP concentration, this static sensing method is limited to real-time monitoring of PLP levels and cannot regulate PLP concentration.

[0078] Based on the currently constructed PLP-responsive biosensor, automatic regulation of PLP concentration can be achieved by introducing a negative feedback control circuit. The negative feedback mechanism enables the system to inhibit further synthesis of PLP when the concentration is too high, preventing excessive PLP from causing cytotoxicity, while activating the synthesis mechanism when the PLP concentration is insufficient, to ensure a stable supply of intracellular PLP.

[0079] The negative feedback-regulated pyridoxal phosphate biosensor pECXK99E-Cgl1185-P constructed in Example 6 was used. Cgl2838 -lacI-P trc The fluorescent protein GFP in -GFP is replaced with the PLP synthase gene (specifically, the PLP synthase complex BspdxST, composed of the PdxS and PdxT genes; PdxS gene, Gene ID: 939988, PdxT gene, Gene ID: 939971). The PLP synthase complex catalyzes the synthesis of pyridoxal phosphate (PLP) from ribose-5-phosphate (R5P), glyceraldehyde-3-phosphate (G3P), and glutamine. PdxT provides the ammonia source, and PdxS completes the PLP synthesis. This combines PLP synthesis in the 5-ALA producing strain with the strain's PLP requirement. That is, when there is sufficient PLP in the system, Cgl1185-P Cgl2838 This will generate a response and initiate the expression of the downstream LacI protein, while the presence of LacI protein inhibits P. trc It initiates transcription of the downstream PLP synthase gene, thus inhibiting further PLP synthesis by PLP synthase; when PLP is lacking in the system, Cgl1185-P Cgl2838 The system cannot generate a response, therefore it cannot initiate the expression of downstream LacI proteins; when LacI is deficient, P... trcIt initiates the transcription of downstream PLP synthase genes, thereby prompting PLP synthase to further synthesize PLP, thus ensuring a stable supply of PLP in the cell.

[0080] Specific applications are as follows: (1) Using pEC-XK99E as a template and pEC-ZF / pEC-ZR as primers (primers are shown in Table 7), the linear vector fragment was amplified; The BspdxST gene was synthesized according to the sequence SEQ ID NO.4, and the BspdxST gene fragment was amplified using BspdxST-F / BspdxST-P primers.

[0081] The two fragments were ligated using Gibson assembly to obtain the corresponding plasmid pEC-lacI P. trc -BspdxST, the plasmid was electroporated into the A40 chassis strain as a control strain G1 for static expression of PLP synthase.

[0082] (2) pECXK99E-Cgl1185-P Cgl2838 -lacI-P trc -GFP was used as a template, ZF / ZR was used as primers, and the negative feedback regulatory circuit vector fragment was amplified by PCR. The BspdxST fragment and the vector fragment were ligated using Gibson assembly to obtain the corresponding plasmid pECXK99E-Cgl1185-P. Cgl2838 -lacI-P trc -BspdxST (the nucleotide sequence of the plasmid is shown in SEQ ID NO.10), the plasmid was electroporated into the A40 chassis strain to obtain the dynamically expressing PLP synthase strain G2.

[0083] Single colonies of both control strain G1 and experimental strain G2 were picked and cultured in 5 mL of BHI medium containing kanamycin for 12 hours at 30°C and 220 rpm. Then, 1 mL of the seed culture was transferred to 50 mL of CGIII medium and cultured for another 12 hours at 30°C and 220 rpm. Finally, the secondary seed culture was inoculated into a 500 mL Erlenmeyer flask containing 50 mL of CGXII medium. Initial OD 600 =0.5, initial glucose concentration was 30 g / L, glycine concentration was 13.5 g / L, and G1 strain required a final concentration of 1 mM IPTG. The mixture was cultured at 30℃ and 220 rpm for 72 h, and 5-ALA yield was measured.

[0084] The results are as follows Figure 7As shown, the yield of strain G1 was 6.49 ± 0.33 g / L, and that of strain G2 was 7.92 ± 0.30 g / L. Dynamic regulation of PLP supply significantly enhanced 5-ALA synthesis. Dynamic regulation of BspdxST expression could adjust PLP supply according to cellular metabolic status. Maintaining lower expression during cell growth reduced metabolic burden; while increasing expression during product synthesis enhanced PLP supply and increased ALAS activity, thereby promoting 5-ALA synthesis. Therefore, compared to static expression with sustained high expression, dynamic expression is more beneficial for increasing 5-ALA yield.

[0085] Table 7

[0086] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these modifications and combinations all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent should be determined by the claims.

Claims

1. A biosensor responsive to pyridoxal phosphate, characterized in that, The biosensor includes: a transcription factor Cgl1185 expression cassette and a promoter P. cgl2838 and reporter genes, the promoter P cgl2838 The reporter gene is regulated by transcription factor Cgl1185 and is regulated by promoter P. cgl2838 Regulation; The transcription factor Cgl1185 expression cassette contains the gene encoding transcription factor Cgl1185 and promoter P. cgl1185 and terminator; The transcription factor Cgl1185 has the nucleotide sequence shown in SEQ ID NO.5; The promoter P cgl1185 The nucleotide sequence is shown in SEQ ID NO.6; The promoter P cgl2838 The nucleotide sequence is shown in SEQ ID NO.

7.

2. A biosensor responsive to pyridoxal phosphate as described in claim 1, characterized in that, The biosensor also contains a gene encoding the repressor protein LacI and a promoter P. trc The gene encoding the repressor protein LacI is located in promoter P. cgl2838 Downstream of the promoter P trc It is located downstream of the gene encoding the repressor protein LacI.

3. A biosensor responsive to pyridoxal phosphate as described in claim 1, characterized in that, P cgl2838 The promoter ribosome binding site RBS is replaced with R36, R48, R33, R13, or R18; where: The nucleotide sequence of R13 is: AGCATGAAAGGGAAAGGACCCC; The nucleotide sequence of R18 is: AGCATGGAAGGAGAGAAGCCCC; The nucleotide sequence of R33 is: AGCATGGAAGGGGGGGGACCCC; The nucleotide sequence of R36 is: AGCATGAAAAGGAAAGGACCCC; The nucleotide sequence of R48 is: AGCATGGGGGAAAAAGAGCCCC; The reporter genes include, but are not limited to, those mentioned above. gfp , rfp , CFP , sfgfp , egfp , yfp , ecfp Gene.

4. A biosensor responsive to pyridoxal phosphate as described in claim 1, characterized in that, The biosensor further includes a replicon gene for controlling the initiation of replication of the entire biosensor; and / or the biosensor further includes an resistance gene for screening transformants containing the biosensor.

5. A biosensor responsive to pyridoxal phosphate as described in claim 1, characterized in that, The biosensor is pECXK99E-Cgl1185-P cgl2838 -GFP plasmid, pECXK99E-Cgl1185-P cgl2838 The nucleotide sequence of the GFP plasmid is shown in SEQ ID NO.2; or, the biosensor is pECXK99E-Cgl1185-P cgl2838 - lacI -P trc -GFP plasmid, pECXK99E-Cgl1185-P cgl2838 - lacI -P trc The nucleotide sequence of the -GFP plasmid is shown in SEQ ID NO.

3.

6. The use of the biosensor according to any one of claims 1-5 in detecting pyridoxal phosphate or products containing pyridoxal phosphate.

7. A biosensor for regulating pyridoxal phosphate concentration, characterized in that, The biosensor sequentially comprises: a transcription factor Cgl1185 expression cassette, a promoter P, and other components. cgl2838 The gene encoding the repressor protein LacI and the promoter P trc And the pyridoxal phosphate synthase gene.

8. A biosensor for regulating pyridoxal phosphate concentration as described in claim 7, characterized in that, The biosensor is pECXK99E-Cgl1185-P Cgl2838 -lacI-P trc -BspdxST plasmid, pECXK99E-Cgl1185-P Cgl2838 -lacI-P trc The nucleotide sequence of the -BspdxST plasmid is shown in SEQ ID NO.

10.

9. A recombinant microorganism, characterized in that, It includes the biosensor described in any one of claims 1-5 or 7-8.

10. The use of the recombinant microorganism of claim 9 in the production of pyridoxal phosphate or 5-aminolevulinic acid.

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