A freeze-drying preservation method for whole-cell biosensors

Abstract

The application discloses a freeze-drying preservation method of a whole-cell biosensor and belongs to the technical field of whole-cell biosensor construction and preservation. The nitrate response type histidine kinase NarX mutant and the whole-cell biosensor are constructed first, and the freeze-drying method of the whole-cell biosensor is provided on the basis. The freeze-dried bacteria powder prepared by the method can be stably preserved under normal temperature conditions, and still can maintain good sensing performance after rehydration, and has stable response capacity to target analytes within a certain concentration range. The method is simple in operation, low in cost, and is beneficial to long-term preservation and rapid detection application of the biosensor.

Description

Technical Field

[0001] This invention relates to a freeze-drying preservation method for whole-cell biosensors, belonging to the field of whole-cell biosensor construction and preservation technology. Background Technology

[0002] Biosensors, as detection tools built upon biorecognition elements and signal transduction mechanisms, have broad application prospects in fields such as environmental monitoring, food safety, and clinical analysis. Among them, biosensors based on whole-cell microorganisms are gradually becoming important detection methods due to their advantages such as simple construction, high response sensitivity, and low cost. Taking nitrate detection as an example, quantitative analysis of the target analyte can be achieved by coupling the sensing element with a reporter gene.

[0003] However, existing whole-cell sensors typically rely on live bacterial strains, which have significant limitations in terms of preservation and use. On the one hand, sensor strains generally need to be preserved under low-temperature conditions, and their activity is prone to decline or even inactivation during long-term storage. On the other hand, in practical detection applications, it is often necessary to reculture single colonies to a certain growth stage before they can be used, resulting in prolonged detection cycles, complex operations, and hindering rapid detection and field applications.

[0004] To improve the stability of microbial preparations, freeze-drying is a common method for preserving microorganisms. Adding preservatives, such as sugars or polyols, during the freeze-drying process can improve cell survival to some extent. Studies have shown that preservatives such as trehalose are effective in maintaining the activity of some engineered bacteria. However, these methods primarily focus on cell survival or general metabolic activity. For whole-cell biosensors with specific functional outputs, the core challenge lies in maintaining induced response capabilities and signal output stability.

[0005] Current technologies lack research on freeze-drying protection methods for whole-cell biosensors, particularly in ensuring that freeze-dried cells maintain good response sensitivity, dynamic range, and linearity after rehydration. Furthermore, the mechanisms by which different protective agents and their concentrations affect sensor performance are complex, and the lack of systematic optimization leads to unstable sensing performance after freeze-drying, making it difficult to meet practical detection needs.

[0006] Therefore, developing a freeze-drying protection method for whole-cell biosensors that can be preserved for a long time at room temperature, and can be rapidly restored and maintain good sensing performance after rehydration is of great significance for promoting the portability and field application of biosensors. Summary of the Invention To address the aforementioned issues, this invention first constructs a nitrate-responsive histidine kinase NarX mutant and a whole-cell biosensor, and then provides a freeze-drying method for the whole-cell biosensor.

[0007] The first objective of this invention is to provide a nitrate-responsive histidine kinase NarX mutant, wherein the nitrate-responsive histidine kinase NarX mutant has one or more mutations at positions 562, 490 or 418, based on the amino acid sequence of the nitrate-responsive histidine kinase NarX shown in SEQ ID NO.1.

[0008] In one embodiment, the nitrate-responsive histidine kinase NarX mutant, based on the amino acid sequence shown in SEQ ID NO.1, has the following modifications: serine at position 562 is mutated to arginine, leucine at position 490 is mutated to methionine, and methionine at position 418 is mutated to isoleucine, i.e., NarX. S562R / L490M / M418I .

[0009] This invention also provides another nitrate-responsive histidine kinase NarX mutant, wherein the nitrate-responsive histidine kinase NarX mutant, based on the amino acid sequence as shown in SEQ ID NO.1, has leucine at position 507 mutated to serine, arginine at position 446 mutated to histidine, and asparagine at position 439 mutated to lysine, i.e., NarX L507F / R446H / N439K .

[0010] This invention also provides another nitrate-responsive histidine kinase NarX mutant, wherein the nitrate-responsive histidine kinase NarX mutant has the following amino acid sequence as shown in SEQ ID NO.1: leucine at position 507 is mutated to phenylalanine and serine at position 562 is mutated to arginine, i.e., NarX. L507F / S562R .

[0011] This invention also provides another nitrate-responsive histidine kinase NarX mutant, wherein the nitrate-responsive histidine kinase NarX mutant, based on the amino acid sequence as shown in SEQ ID NO.1, has the following mutations: leucine at position 507 is mutated to phenylalanine; serine at position 562 is mutated to arginine; proline at position 492 is mutated to alanine; valine at position 541 is mutated to alanine; leucine at position 490 is mutated to methionine; methionine at position 418 is mutated to isoleucine; arginine at position 446 is mutated to histidine; and asparagine at position 439 is mutated to lysine, i.e., NarX. L507F / S562R / P492A / V541A / L490M / M418I / R446H / N439K .

[0012] A second objective of this invention is to provide a polynucleotide encoding the aforementioned nitrate-responsive histidine kinase NarX mutant.

[0013] A third objective of this invention is to provide cells expressing any of the above-mentioned nitrate-responsive histidine kinase NarX mutants, characterized in that the cells are Escherichia coli, Bacillus subtilis, or Corynebacterium glutamicum.

[0014] A fourth objective of this invention is to provide a genetically engineered Escherichia coli strain that expresses any of the above-mentioned nitrate-responsive histidine kinase NarX mutants.

[0015] In one embodiment, the genetically engineered Escherichia coli also expresses the response regulatory protein NarL and a fluorescent protein; The amino acid sequence of the response regulatory protein NarL is shown in SEQ ID NO.2, and the amino acid sequence of the fluorescent protein is shown in SEQ ID NO.3.

[0016] In one implementation, promoter P is used. narG365 Fluorescent proteins were expressed using promoter J23109 to express the NarX mutant and promoter J23101 to express NarL. Among them, promoter P narG365 The nucleotide sequence of promoter J23109 is shown in SEQ ID NO.4, the nucleotide sequence of promoter J23101 is shown in SEQ ID NO.5, and the nucleotide sequence of promoter J23101 is shown in SEQ ID NO.6.

[0017] In one implementation, promoter P narG365 In the promoter P narG515 It is obtained by truncating 150 bp from (SEQ ID NO.7).

[0018] In one embodiment, the genome of the genetically engineered E. coli was also knocked out. narX (Amino acid sequence as shown in SEQ ID NO.1) narL (Amino acid sequence as shown in SEQ ID NO.2) narU (Amino acid sequence as shown in SEQ ID NO.8) narK (Amino acid sequence as shown in SEQ ID NO.9) napA (Amino acid sequence as shown in SEQ ID NO.10) napB One or more of the amino acid sequences shown in SEQ ID NO.11 are used to enhance the detection response for low concentrations of nitrate.

[0019] In one embodiment, NarX and NarL are knocked out of the genome of the genetically engineered Escherichia coli, and one or both of NarU and NarK are further knocked out.

[0020] The fifth objective of this invention is to provide a method for freeze-drying a whole-cell biosensor, wherein the whole-cell biosensor is pretreated by mixing it with a freeze-drying protectant and then freeze-dried to obtain a freeze-dried bacterial powder of the whole-cell biosensor. Among them, the whole-cell biosensor is any of the above-mentioned genetically engineered Escherichia coli strains; The freeze-drying protectant is one or more of sucrose, trehalose, lactose, glycerin, and skim milk powder; The pretreatment is carried out at -80℃ to -20℃ for 4 to 24 hours.

[0021] In one embodiment, the concentration of the freeze-drying protectant is 50-400 g / L, preferably 150-300 g / L.

[0022] The sixth object of the present invention is to provide a freeze-dried bacterial powder for a whole-cell biosensor prepared by the method described above.

[0023] In one embodiment, the freeze-dried bacterial powder of the whole-cell biosensor is activated in water for 0.5 to 6 hours, so that the whole-cell biosensor recovers its detection capability after rehydration.

[0024] In one embodiment, the freeze-dried bacterial powder of the whole-cell biosensor can be stably stored at room temperature for several days to tens of days, and still maintains good response performance after rehydration. It can be directly used for the detection of target analytes, and its output signal includes, but is not limited to, fluorescence, luminescence, colorimetry, electrochemistry or other detectable signals.

[0025] A seventh object of the present invention is to provide the application of any of the above-mentioned NarX mutants, polynucleotides encoding the above-mentioned NarX mutants, cells expressing the above-mentioned NarX mutants, any of the above-mentioned genetically engineered Escherichia coli bacteria, and the above-mentioned whole-cell biosensor lyophilized bacterial powder in the detection of nitrates.

[0026] Beneficial effects of the present invention This invention first constructs a NarX mutant and a whole-cell biosensor, and then provides a freeze-drying method for the whole-cell biosensor.

[0027] This invention optimizes the promoter; when using promoter P... narG365 When expressing fluorescent proteins, with promoter J23109 expressing the NarX mutant and promoter J23101 expressing NarL, the biosensor has a linear range of 1–10 mM and a detection limit of 0.9 mM.

[0028] Furthermore, mutations were performed on the histidine kinase NarX. The resulting NarX mutant improved the response performance of the NarX-NarL two-component biosensor. Specifically, NarX expression... S562R / L490M / M418I (Linear range 0.5~4 mM, detection limit 0.5 mM), NarX L507S / R446H / N439K (Linear range 0.5~4 mM, detection limit 0.5 mM), NarX L507F / S562R (Linear range 1.6~8 mM, detection limit 1.6 mM), NarX L507F / S562R / P492A / V541A / L490M / M418I / R446H / N439K The performance of the biosensor (linear range 0.4~8 mM, detection limit 0.4 mM) has been significantly improved.

[0029] Furthermore, by optimizing the type and concentration of the freeze-drying protectant, the response performance of the biosensor is effectively maintained while ensuring cell survival. The prepared freeze-dried bacterial powder can be stored for a long time at room temperature, significantly reducing storage and transportation costs. When using it, there is no need to reculture the strain; only rehydration and short-term activation are required for detection, significantly shortening the detection cycle and improving the ease of application. At the same time, the protectant used in this invention is widely available, low in cost, and highly safe, and has good prospects for application and promotion. Attached Figure Description

[0030] Figure 1 Optimization of the sensor output promoter; where A: Schematic diagram of the gene circuit of the natural NarX-NarL TCSB biosensor; B: NarX-NarL TCSB at different concentrations of NO3 - induced sfGFP expression level change over time curve; C:P narG515 The output and dynamic range of the promoter-truncated NarX-NarL TCSB, where the dynamic range is the ratio of the maximum output to the minimum output, is indicated above each bar chart.

[0031] Figure 2 Fluorescence output of NarX-NarL TCSB constructed for 36 promoter combinations; where A: 6 promoter sequences replace the promoters in NarX-NarL TCSB respectively; B, C: 36 NarX-NarL TCSB in NO3 - The maximum and minimum outputs are at 0 mM and 20 mM.

[0032] Figure 3The effects of NarX and NarL expression levels on sensor performance are shown in section A: the effect of relative NarX and NarL mRNA expression levels on dynamic range, where Pn and Pm represent the promoters controlling NarX and NarL, respectively; and section B: analysis of the detection limit and detection range of 5-1XL. The red dashed line indicates the detection limit is three times the minimum output of NO3 corresponding to the TCSB signal. - concentration.

[0033] Figure 4 For NarX S562R / L490M / M418I NarX L507S / R446H / N439K NarX P492A / V541A NarX L507F / S562R Maximum output, minimum output, and dynamic range.

[0034] Figure 5 Analysis of the response characteristics of mutants; where A; NarX S562R / L490M / M418I - Analysis of the detection limit and linear detection range of NarL TCSB; B: NarX L507S / R446H / N439K -Analysis of the detection limit and linear detection range of NarL TCSB; C: NarX P492A / V541A Analysis of the detection limit and linear detection range of NarL TCSB; D: NarX L507F / S562R -Analysis of the detection limit and linear detection range of the NarL TCSB. The black curve represents the location of the detection limit, defined as the NO3 required for the sensor output to reach three times the minimum output. - concentration.

[0035] Figure 6 Characterization of the biosensor performance of NarX combined mutants; A: Spatial distribution of the 9 amino acid mutation sites on the NarX kinase domain, the NarX-ATP complex structure was predicted by AlphaFold 3; B: Box plot of the dynamic range of libraries with different numbers of mutation sites; C: Analysis of significant differences in the dynamic range of the combined mutants, significant differences were assessed using a two-tailed, unpaired t-test; D: Schematic diagram of the 2-30XL plasmid; E: Detection characteristic analysis of 2-30XL, the black curve represents the detection limit, which is defined as the NO3 required for the sensor output to reach 3 times the minimum output. - concentration.

[0036] Figure 7 This is a schematic diagram comparing the response curves of a biosensor to nitrate after lyophilization under different protective agent conditions.

[0037] Figure 8 The effect of different concentrations of lyophilization protectant on the response performance of biosensors.

[0038] Figure 9 This is a schematic diagram comparing the response of a biosensor to nitrates obtained by rehydration and activation of a lyophilized bacterial powder prepared by the method of this invention after storage at different temperature conditions for a certain period of time, with that of a freshly cultured bacterial strain.

[0039] Figure 10 This is a flowchart illustrating the freeze-drying preservation method for whole-cell biosensors, showing the steps of cell culture, collection and washing, addition of freeze-drying protectant, pre-freezing treatment, freeze-drying, and rehydration and activation.

[0040] Figure 11 For 2-30XL sensors, NO3 knockout - NO3 in the chassis of the transport and reduction pathway - The response characteristics; where A; dose-response curves for N1~N7. The control refers to the sensor chassis cells as... ΔnarX / ΔnarL A green background indicates 0–1 mM NO3. - The low concentration range; B: N1~N7 in 1 mM NO3 - Comparison of dynamic range under the given conditions, with a dynamic range of 1 mM NO3 - The sensor output under induced conditions is related to 0 mM NO3. - The ratio of sensor outputs under induced conditions. Detailed Implementation

[0041] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0042] In this invention, the dose-response curve is a characteristic curve for evaluating the performance of TCSB. Taking an activation-type two-component sensor as an example, it reflects the correspondence between inducer concentration (input signal) and reporter gene expression level (output signal). This curve can typically be described by the following quantitative parameters: (1) Baseline output (also known as minimum output) refers to the baseline signal output of TCSB in the uninduced state; (2) Dynamic range, which is the ratio of maximum output to minimum output, is often used as a measure of signal-to-noise ratio; (3) Detection threshold (EC50) refers to the concentration of inducer required for the sensor output to reach half of its maximum value; (4) Limit of detection (LOD) indicates the lowest concentration at which the target signal can be reliably monitored. These parameters are key indicators for TCSB performance adjustment.

[0043] In this invention, whole-cell biosensors containing target response elements are inoculated into liquid culture medium and cultured at suitable temperature conditions until they reach the logarithmic growth phase, stationary growth phase, or stable expression phase. The bacterial cells are then collected by centrifugation and washed with buffer to remove residual components of the culture medium. The washed bacterial cells are then resuspended in buffer to form a homogeneous bacterial suspension.

[0044] A lyophilization protectant is added to the bacterial suspension and mixed thoroughly. The lyophilization protectant is selected from one or more of the following: saccharide protectants, polyol protectants, protein protectants, or dairy protectants, such as sucrose, trehalose, lactose, glycerol, or skim milk powder. The mass-volume concentration of the lyophilization protectant can be selected in the range of 50–400 g / L, preferably 150–300 g / L, and more preferably about 240 g / L. By adjusting the type and concentration of the lyophilization protectant, the structural stability and functional retention of cells during the freeze-drying process can be improved.

[0045] The mixed bacterial suspension was pre-frozen at low temperature until the system was completely frozen. Then, it was transferred to a freeze-drying device for sublimation drying under low temperature and vacuum conditions to obtain freeze-dried bacterial powder.

[0046] In use, the lyophilized bacterial powder is added to a liquid culture medium for rehydration and activated for a short time under suitable conditions to restore the cells' metabolic activity and sensing function. The activated sensor cells can be used to detect target analytes and output corresponding signals.

[0047] The freeze-dried bacterial powder prepared by the above method can maintain good sensing response performance after being kept at room temperature for several to several tens of days and then rehydrated, making it suitable for rapid detection applications.

[0048] Raw materials used in the examples: pJKR-H-CdaR and pACYCDuet-1 are both commercially available. The primers used in the examples are shown in Table 1.

[0049] Table 1 Primers

[0050] Test method: Nitrate-induced fluorescence detection method Escherichia coli MG1655 and its derived knockout strains ΔnarX / narL , ΔnarU , ΔnarK , ΔnarU / narK , ΔnapA , ΔnapB , ΔnapA / napB and ΔnarU / narK / napA / napB As a chassis cell, it was used for performance characterization of a nitrate two-component sensor.

[0051] The biosensor plasmid was transformed into competent cells of these strains and plated on LB agar plates containing chloramphenicol resistance. The plates were incubated overnight at 37°C. Single colonies were then picked and inoculated into LB liquid medium and cultured at 37°C with shaking at 250 rpm for 10–12 h. The seed culture was then transferred at 5% to 96-well plates containing fresh LB liquid medium and cultured at 250 rpm for 2–3 h to allow the strains to reach the logarithmic growth phase. A gradient concentration of sodium nitrate solution (final concentration 0–20 mM) was then added, and the plates were covered with an airtight sealing film. After incubation at 37°C for 3 h, the cells were collected by centrifugation at 3900 rpm, washed three times with pre-cooled sterile PBS, and resuspended in PBS. The OD600 was appropriately diluted to approximately 0.5. 200 μL of the bacterial culture was placed in a 96-well plate, and fluorescence intensity was detected using a Biotec HT microplate reader at excitation wavelength 485 nm and emission wavelength 528 nm. Simultaneously, fluorescence intensity was measured at 600 nm. The absorbance OD600 of the bacterial culture was detected at nm. The normalized fluorescence intensity was expressed as the ratio of the detected fluorescence intensity to OD600, in au. The normalized fluorescence intensity can be used to evaluate the output of TCSB (natural two-component system-based biosensor).

[0052] Example 1: Construction of biosensor plasmids 1. Gene knockout To remove interference from NarX and NarL expression in the *E. coli* genome, CRISPR-Cas9 technology was used to target the expression of NarX and NarL in *E. coli* MG1655. narX / narL Operator knockout, construct MG1655 ( ΔnarX / narL ) strain.

[0053] In MG1655 ( ΔnarX / narL Based on this, individual and combined NO3 knockout - Transporter protein-related genes narU / narK NO3 - reductase encoding gene napA / napB strain MG1655 was obtained. ΔnarU ), MG1655 ΔnarK ), MG1655 ΔnapA ), MG1655 ΔnapB ), MG1655 ΔnarU / narK ), MG1655 ΔnapA / napB ) and MG1655 ( ΔnarU / narK / napA / napB ).

[0054] 2. Constructing plasmids Use narX-F / narX-R, narL-F / narL-R, P narG515 Three primer pairs were used to amplify genes encoding two-component proteins from Escherichia coli MG1655. narX , narL and the controlled promoter P of NarL narG515 ; Use primer pairs sfgfp -F / sfgfp -R was amplified from the pJKR-H-CdaR plasmid containing a fluorescent reporter gene. sfgfp Gene fragments including terminators; Simultaneously, using pACYCDuet-1 as a template, PCR amplification was performed using primer pairs VXF / VXR, VLF / VLR, and VGF / VGR to obtain vector fragments linked to different genes; The purified gene fragment and vector fragment were mixed sequentially at a molar ratio of 3:1, and Gibson assembly was performed with the addition of homologous recombinase. Ligation was carried out at 50°C for 1 h. The ligation product was transformed into E. coli JM109 competent cells, and after overnight culture, positive transformants were screened by colony PCR and Sanger sequencing to construct the plasmid pACYC-P. narG515 - sfgfp and pACYC- narX - narL - sfgfp Sensor plasmid.

[0055] Example 2: Truncation of non-coding regions In plasmid pACYC-P narG515 - sfgfp Based on this, the promoter P narG515 The segments are truncated at 50 bp intervals and named sequentially according to their length, P. narG465 P narG415 P narG365 and P narG315 .

[0056] pACYC-P narG515 Using -sfgfp as a template, the gene fragments were amplified using primer pairs Tru465-F / Tru465-R, Tru415-F / Tru415-R, Tru365-F / Tru365-R, and Tru315-F / Tru315-R, respectively. The amplified gene fragments were then assembled and cloned into pACYC-P using Gibson assembly. narG515 - sfgfp In the carrier sfgfp Upstream replacement P narG515 The non-coding region truncated plasmid pACYC-P was obtained. narG465 - sfgfp, pACYC-P narG415 - sfgfp, pACYC-P narG365 - sfgfp, pACYC-P narG315 - sfgfp .

[0057] The plasmid with the non-coding region truncated was transferred into E. coli MG1655. ΔnarX / narL The sensor, which detects plasmids carrying different truncated segments, operates in the range of 0–20 mM NO3. - The output and dynamic range after 10 hours of conditioned induction are shown in the following figures. Figure 1 As shown.

[0058] The results show that after deleting 151 b upstream of the promoter, P narG365 Its dynamic range is the highest, which is P of the original length. narG515 The dynamic range was increased from 13 times to 21 times; while based on P narG365 Further removal of adjacent RstA binding sites significantly reduced the dynamic range to 6-fold. Therefore, subsequent selection of P... narG365 It serves as the output promoter of the sensor.

[0059] Example 3: Different promoter combinations To adjust the expression levels of NarX and NarL, six promoters were selected: BBa_J23109 (J23109), BBa_J23114 (J23114), BBa_J23105 (J23105), BBa_J23107 (J23107), BBa_J23108 (J23108), and BBa_J23101 (J23101), obtained from the Anderson promoter library (http: / / parts.igem.org / Promoters / Catalog / Anderson). Primers were designed based on promoter sequences, using plasmid pACYC- narX - narL - sfgfp Using this as a template, whole-plasmid PCR was performed. After purifying the PCR product with a purification kit, it was digested with Dpn I enzyme at 37°C for 15 min, and then transformed into E. coli JM109 competent cells. Positive clones were screened by Sanger sequencing, and 36 NarX-NarL expression intensities (6 NarX and 6 NarL expression intensities) were successfully constructed.narG365 TCSB plasmid expressing the fluorescent protein sfgfp.

[0060] 36 plasmids were transformed into E. coli MG1655. ΔnarX / narL The chassis contains 0~20 mM NO3 - The sensor performance was tested under certain conditions, and the results are as follows: Figure 2 As shown.

[0061] The results show that when NarX is expressed under the control of J23114 or J23107, simultaneously increasing NarL expression significantly amplifies the sensor output, with the maximum output increasing from 252 au to 24776 au and the minimum output increasing from 306 au to 1847 a.u. However, the maximum output of other promoter combinations did not show an increase with increasing NarL expression. This indicates that NarL expression is not "the stronger the better," and a suitable expression ratio between NarX and NarL is required to induce a high signal output from the TCSB.

[0062] The dynamic range of the sensor was further calculated based on the maximum and minimum output of these 36 promoter combinations, and the dynamic range of the sensor was measured respectively. narX and narL The transcription level was used to calculate its relative transcription ratio, and the results are as follows: Figure 3 As shown.

[0063] The results showed that when the relative transcriptional levels of NarX and NarL were below 0.5, the dynamic range generally exceeded 4-fold; however, higher expression ratios often led to a decrease in the sensor's dynamic range, and even reduced its sensitivity to NO3. - There was no response. This phenomenon may be due to NarX overexpression, which leads to a large amount of NarX being in phosphatase activity, thereby inhibiting NarL phosphorylation and its activation of downstream genes.

[0064] Among all combinations, the combination with J23109 controlling NarX and J23101 controlling NarL had the highest dynamic range (14-fold) and the lowest relative expression ratio of NarX to NarL (approximately 0.003). The combination plasmid was named 5-1XL (i.e., plasmid pACYC-P). J23109 - narX -P J23101 - narL - sfgfp -P narG365 The dose-response curve results showed that the expression plasmid 5-1XL in Escherichia coli MG1655 ( ΔnarX / narL Biosensors for NO3 - The linear detection range is 1~10 mM, and the detection limit is 0.9 mM.

[0065] Example 4: Construction of NarX mutants In plasmid pACYC-P J23109 - narX- P J23101 - narL - sfgfp -P narG365 Based on this, NarX was subjected to multiple point mutations to obtain the mutant NarX. S562R / L490M / M418I NarX L507S / R446H / N439K NarX P492A / V541A NarX L507F / S562R NarX mutant plasmids were constructed separately.

[0066] The NarX mutant plasmid was transformed into E. coli MG1655. ΔnarX / narL A biosensor strain was constructed; TCSB was detected at 0 and 20 mM NO3. - The maximum output, minimum output, and dynamic range under the given conditions are as follows: Figure 4 As shown.

[0067] The results show that NarX S562R / L490M / M418I NarX L507S / R446H / N439K NarX L507F / S562R The dynamic ranges of NarX reached 17, 16, and 19 times, respectively, representing improvements of 21%, 14%, and 36% compared to the control; while NarX... P492A / V541A While the maximum output increased by 135%, the minimum output increased by 200% simultaneously, resulting in a decrease in dynamic range of approximately 30%.

[0068] The results of dose response curve analysis are as follows: Figure 5 As shown, NarX S562R / L490M / M418I With NarX P492A / V541A The detection limit was reduced by approximately 80% (0.5 mM), while NarX... P492A / V541A With NarX L507F / S562R The detection limits were increased to 16 mM and 1.6 mM, respectively.

[0069] Based on the nine amino acid mutation sites involved in the above combined mutations, nine single-point mutants were constructed and further combined with mutations to obtain the mutant NarX. L507F / S562R / P492A / V541A / L490M / M418I / R446H / N439K pACYC-P containing this mutant J23109 - narX -P J23101 - narL - sfgfp -P narG365 The plasmid was named 2-30XL, and the results are as follows: Figure 6 As shown.

[0070] The results showed that none of the single-point mutations significantly improved the dynamic range, meaning that individual changes were insufficient to improve sensor performance, and there may be synergistic effects between different sites; while the expression of 2-30XL in E. coli MG1655 ( ΔnarX / narL The biosensor's detection limit is reduced to 0.4 mM, and it has a linear detection range of 0.4 to 8 mM.

[0071] Example 5: Fabrication of a biosensor Plasmids 2-30XL were transformed into E. coli MG1655, respectively. ΔnarU Biosensors N1 and MG1655 were constructed and obtained. ΔnarK Biosensors N2 and MG1655 were constructed and obtained. ΔnarU / narK Biosensors N3 and MG1655 were constructed and obtained. ΔnapA Biosensors N4 and MG1655 were constructed and obtained. ΔnapB Biosensors N6 and MG1655 were constructed and obtained. Δ napA / napB Biosensors N6 and MG1655 were constructed and obtained. ΔnarU / narK / napA / napB The biosensor N7 was constructed, and different biosensors were used to detect NO3. - Response characteristics, results as follows Figure 11 As shown.

[0072] The results showed that knocking out the transporter protein encoding gene narU or narK ,as well as narU / narK Both knockouts resulted in the sensor output being at a low concentration of NO3. - The response range becomes steeper and saturates earlier, leading to an increase in minimum output. The dynamic range of N1, N2, and N3 decreases from 21x to 10x, 5x, and 8x, respectively. However, N3 exhibits a higher dynamic range in 1 mM NO3. - The output signal under induction changed by 7 times, which was significantly improved by 4 times compared with the control, and its detection limit was reduced to 0.1 mM (linear range 0.1~1 mM).

[0073] In contrast, knocking out the periplasmic reductase gene napA / napB (N4~N6) failed to improve the sensor's response to low concentrations. Further simultaneous blocking of transport and reduction (N7) revealed that the sensor's response to NO3... - There was no obvious response, indicating a constitutive activated state. These results suggest that simultaneous knockout... narU and narK It can significantly reduce the detection limit. The mechanism may originate from perimass NO3. - The increase in net accumulation boosted NarX and NO3.- The binding rate drives NarX conformational changes and autophosphorylation, thereby enhancing sensing and amplifying downstream output signals.

[0074] Subsequent performance tests all used the N3 biosensor.

[0075] Example 5: Preparation of freeze-dried biosensors N3 was cultured at 37°C for approximately 12 hours to obtain an overnight culture. The bacterial culture was centrifuged at 4000 rpm for 5 minutes at room temperature to collect the cells, and the supernatant was discarded. The cells were washed twice with phosphate-buffered saline (PBS) and then resuspended in PBS.

[0076] Different types of freeze-drying protectants were added to the resuspended bacterial solutions to achieve a final concentration of 12% (w / v). These protectants included lactose, sucrose, skim milk powder, glycerol, and trehalose. After mixing, the resulting mixtures were pre-frozen at -80°C for approximately 14 hours.

[0077] After pre-freezing, the sample was placed in a freeze dryer and freeze-dried at -50°C and under vacuum for about 12 hours to obtain freeze-dried bacterial powder.

[0078] The lyophilized bacterial powder was rehydrated in LB liquid medium and activated by shaking at 37°C and 250 rpm for 2 hours. Subsequently, different concentrations of sodium nitrate were added to the culture system, and fluorescence signals were measured after further incubation for a certain period.

[0079] The results are as follows Figure 7 As shown, different protective agents have a significant impact on the performance of the freeze-dried sensor. When sucrose is used as the protective agent, the signal output of the rehydrated strain is closest to that of the freshly cultured strain, exhibiting superior sensing performance.

[0080] Example 6: Optimization of Lyophilization Protectant Concentration Based on Example 5, sucrose was selected as the freeze-drying protectant, and different concentration gradients were set at 120, 180, 240, and 300 g / L. The bacterial cell treatment, pre-freezing, and freeze-drying operations were performed according to the same method as in Example 5 to obtain freeze-dried bacterial powders under different sucrose concentrations. After rehydration and activation, different concentrations of sodium nitrate were added for induction culture, and their fluorescence response was measured.

[0081] The results are as follows Figure 8 As shown, within the tested range, sucrose concentration has a significant impact on the sensing performance after freeze-drying. Specifically, when the sucrose concentration is 240 g / L, the response curve of the rehydrated freeze-dried bacterial powder is closest to that of the freshly cultured strain, maintaining a good linear relationship and a high signal output level within the detection range.

[0082] Example 7: Storage stability of freeze-dried bacterial powder The freeze-dried bacterial powder prepared using 240 g / L sucrose as a preservative in Example 6 was selected and stored at 4°C, -20°C, -80°C, and room temperature, respectively. After a certain storage period, samples from each group were taken for rehydration and activation treatment (the process is as follows). Figure 10 As shown in the figure, a sodium nitrate induction experiment was performed under the same conditions to measure the fluorescence output signal.

[0083] The results are as follows Figure 9 As shown, the freeze-dried bacterial powder maintained its sensing function for a certain period of time under different storage temperature conditions. Among them, after being stored at room temperature for at least 28 days, the rehydrated strain still maintained relatively stable response performance, and its signal output did not decrease significantly compared with the freshly cultured strain.

[0084] Sequences used in the examples Nitrate-responsive histidine kinase NarX starting amino acid sequence SEQ ID NO.1: * Response regulator protein NarL amino acid SEQ ID NO. 2: MSNQEPATILLIDDHPMLRTGVKQLISMAPDITVVGEASNGEQGIELAESLDPDLILLDLNMPGMNGLETLDKLREKSLSGRIVVFSVSNHEEDVVTALKRGADGYLLKDMEPEDLLKALHQAAAGEMVLSEALTPVLAASLRANRATTERDVNQLTPRERDILKLIAQGLPNKMIARRLDITESTVKVHVKHMLKKMKLKSRVEAAVWVHQERIF* The amino acid sequence of fluorescent protein is set forth in SEQ ID NO. 3: MRIGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKDACQF* Promoter P nar G365 Nucleotide SEQ ID NO. 4: CGGTTTTCTCCTGTGGGAGCCTGTCGGCCCTCTGATAAATCGACATTGCTAATGTTTAAGGGCAAGGTAACGCTCTGAAACAGCGTGGGAATTGATAACGATCAAGAGTGATGGGGATGAAAAATAAAGTAAATTCCTAAGATACCCCTTTAGGATAATTAACAGCCACCACATAACAGACTGTAAATTAAGACGTTTTATACCTGTAGTTATTTAAACTATTAAGATTTTTATGCAGATGGGTATTAAGGAGTATTCCCCATGGGTAACATATTAATGGCTCTTAATGAAATCCGGCGAAATAAACCATATAACATTAGTATGTTGTAACTAAATGACGCATAAAAAAAGCGCGACATCATGCC Promoter J23109 nucleotide SEQ ID NO. 5: TTTACAGCTAGCTCAGTCCTAGGGACTGTGCTAGC Promoter J23101 nucleotide SEQ ID NO. 6: TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGC Promoter P nar G515 Nucleotide SEQ ID NO. 7: ACCGTTACTCGTCATACTTCGGGTTACATGTGCTGCGGCTGCGTTCATTCACCCCAGTCACTTACTTTAGTAAGCTCCTGGGATTCATTCACTTGCCGCCTTCCTGTAAACCGAATTATATAGAGTAAAATATTTGATTATCCTTTGCGCGGCATGATGTCGCGCTTTTTTTATGCGTCATTTAGTTACAACATACTAATGTTATATGGTTTATTTCGCCGGATTTCATTAAGAGCCATTAATATGTTACCCATGGGGAATACTCCTTAATACCCATCTGCATAAAAATCTTAATAGTTTAAATAACTACAGGTATAAAACGTCTTAATTTACAGTCTGTTATGTGGTGGCTGTTAATTATCCTAAAGGGGTATCTTAGGAATTTACTTTATTTTTCATCCCCATCACTCTTGATCGTTATCAATTCCCACGCTGTTTCAGAGCGTTACCTTGCCCTTAAACATTAGCAATGTCGATTTATCAGAGGGCCGACAGGCTCCCACAGGAGAAAACCG The amino acid sequence of NarU is shown in SEQ ID NO. 8 MALQNEKNSRYLLRDWKPENPAFWENKGKHIARRNLWISVSCLLLAFCVWMLFSAVTVNLNKIGFNFTTDQLFLLTALPSVSGALLRVPYSFMVPIFGGRRWTVFSTAILIIPCVWLGIAVQNPNTPFGIFIVIALLCGFAGANFASSMGNISFFFPKAKQGSALGINGGLGNLGVSVMQLVAPLVIFVPVFAFLGVNGVPQADGSVMSLANAAWIWVPLLAIATIAAWSGMNDIASSRASIADQLPVLQRLHLWLLSLLYLATFGSFIGFSAGFAMLAKTQFPDVNILRLAFFGPFIGAIARSVGGAISDKFGGVRVTLINFIFMAIFSALLFLTLPGTGSGNFIAFYAVFMGLFLTAGLGSGSTFQMIAVIFRQITIYRVKMKGGSDEQAHKEAVTETAAALGFISAIGAVGGFFIPQAFGMSLNMTGSPVGAMKVFLIFYIVCVLLTWLVYGRRKFSQK* The amino acid sequence of NarK is shown in SEQ ID NO. 9 MSHSSAPERATGAVITDWRPEDPAFWQQRGQRIASRNLWISVPCLLLAFCVWMLFSAVAVNLPKVGFNFTTDQLFMLTALPSVSGALLRVPYSFMVPIFGGRRWTAFSTGILIIPCVWLGFAVQDTSTPYSVFIIISLLCGFAGANFASSMANISFFFPKQKQGGALGLNGGLGNMGVSVMQLVAPLVVSLSIFAVFGSQGVKQPDGTELYLANASWIWVPFLAIFTIAAWFGMNDLATSKASIKEQLPVLKRGHLWIMSLLYLATFGSFIGFSAGFAMLSKTQFPDVQILQYAFFGPFIGALARSAGGALSDRLGGTRVTLVNFILMAIFSGLLFLTLPTDGQGGSFMAFFAVFLALFLTAGLGSGSTFQMISVIFRKLTMDRVKAEGGSDERAMREAATDTAAALGFISAIGAIGGFFIPKAFGSSLALTGSPVGAMKVFLIFYIACVVITWAVYGRHSKK* The amino acid sequence of NapA is set forth in SEQ ID NO. 10 MKLSRRSFMKANAVAAAAAAAGLSVPGVARAVVGQQEAIKWDKAPCRFCGTGCGVLVGTQQGRVVACQGDPDAPVNRGLNCIKGYFLPKIMYGKDRLTQPLLRMKNGKYDKEGEFTPITWDQAFDVMEEKFKTALKEKGPESIGMFGSGQWTIWEGYAASKLFKAGFRSNNIDPNARHCMASAVVGFMRTFGMDEPMGCYDDIEQADAFVLWGANMAEMHPILWSRITNRRLSNQNVTVAVLSTYQHRSFELADNGIIFTPQSDLVILNYIANYIIQNNAINQDFFSKHVNLRKGATDIGYGLRPTHPLEKAAKNPGSDASEPMSFEDYKAFVAEYTLEKTAEMTGVPKDQLEQLAQLYADPNKKVISYWTMGFNQHTRGVWANNLVYNLHLLTGKISQPGCGPFSLTGQPSACGTAREVGTFAHRLPADMVVTNEKHRDICEKKWNIPSGTIPAKIGLHAVAQDRALKDGKLNVYWTMCTNNMQAGPNINEERMPGWRDPRNFIIVSDPYPTVSALAADLILPTAMWVEKEGAYGNAERRTQFWRQQVQAPGEAKSDLWQLVQFSRRFKTEEVWPEDLLAKKPELRGKTLYEVLYATPEVSKFPVSELAEDQLNDESRELGFYLQKGLFEEYAWFGRGHGHDLAPFDDYHKARGLRWPVVNGKETQWRYSEGNDPYVKAGEGYKFYGKPDGKAVIFALPFEPAAEAPDEEYDLWLSTGRVLEHWHTGSMTRRVPELHRAFPEAVLFIHPLDAKARDLRRGDKVKVVSRRGEVISIVETRGRNRPPQGLVYMPFFDAAQLVNKLTLDATDPLSKETDFKKCAVKLEKV* The amino acid sequence of NapB is set forth in SEQ ID NO. 11 MKSHDLKKALCQWTAMLALVVSGAVWAANGVDFSQSPEVSGTQEGAIRMPKEQDRMPLNYVNQPPMIPHSVEGYQVTTNTNRCLQCHGVESYRTTGAPRISPTHFMDSDGKVGAEVAPRRYFCLQCHVPQADTAPIVGNTFTPSKGYGK* 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 nitrate-responsive histidine kinase NarX mutant, characterized in that, The nitrate-responsive histidine kinase NarX mutant has one or more mutations at positions 562, 490, or 418, based on the amino acid sequence as shown in SEQ ID NO.

1.

2. The nitrate-responsive histidine kinase NarX mutant according to claim 1, characterized in that, The NarX mutant has the following mutations: serine at position 562 is mutated to arginine, leucine at position 490 is mutated to methionine, and methionine at position 418 is mutated to isoleucine, based on the amino acid sequence as shown in SEQ ID NO.

1.

3. A polynucleotide encoding the nitrate-responsive histidine kinase NarX mutant of claim 1, or a cell expressing the nitrate-responsive histidine kinase NarX mutant of claim 1, characterized in that, The cells include Escherichia coli, Bacillus subtilis, and Corynebacterium glutamicum.

4. A genetically engineered Escherichia coli bacterium, characterized in that, Express the nitrate-responsive histidine kinase NarX mutant as described in any one of claims 1 to 2.

5. The genetically engineered Escherichia coli according to claim 4, characterized in that, It also expresses the response regulatory protein NarL and the fluorescent protein sfGFP; The amino acid sequence of the response regulatory protein NarL is shown in SEQ ID NO.2, and the amino acid sequence of the fluorescent protein sfGFP is shown in SEQ ID NO.

3.

6. The Enterobacter genetically engineered bacterium according to claim 5, characterized in that, Using promoter P narG365 Expressing fluorescent protein, promoter J23109 expressing nitrate-responsive histidine kinase NarX mutant, promoter J23101 expressing response regulator NarL; Among them, promoter P narG365 The nucleotide sequence of promoter J23109 is shown in SEQ ID NO.4, the nucleotide sequence of promoter J23101 is shown in SEQ ID NO.5, and the nucleotide sequence of promoter J23101 is shown in SEQ ID NO.

6.

7. The genetically engineered Escherichia coli according to claim 4, characterized in that, The genome of the genetically engineered E. coli was knocked out narX and narL and further knocked out narU and narK One or two of these methods can be used to enhance the detection response for low concentrations of nitrate.

8. A method for freeze-drying whole-cell biosensors, characterized in that, The whole-cell biosensor was pretreated by mixing it with a freeze-drying protectant and then freeze-dried to obtain freeze-dried bacterial powder of the whole-cell biosensor. The whole-cell biosensor is the Escherichia coli genetically engineered bacterium as described in any one of claims 4 to 7; The freeze-drying protectant is one or more of sucrose, trehalose, lactose, glycerin, and skim milk powder; The pretreatment is carried out at -80℃ to -20℃ for 8 to 16 hours.

9. The whole-cell biosensor freeze-dried bacterial powder prepared by the method of claim 8.

10. The application of the nitrate-responsive histidine kinase NarX mutant of any one of claims 1-2, or the polynucleotide or cell of claim 3, or the Escherichia coli genetically engineered bacteria of any one of claims 5-7, or the lyophilized bacterial powder of the whole-cell biosensor of claim 9 in the detection of nitrate.

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