Biosensing tool responding to 5-aminovaleric acid and application

Abstract

The invention discloses a biological sensing tool responding to 5-aminovaleric acid and application, and belongs to the technical field of biological engineering. A ligand binding pocket residue of BsGabR is subjected to mutation modification on the basis of a GABA biosensor, a BsGabR mutant capable of recognizing a drug 5-aminovaleric acid and activating expression of a reporter gene is obtained through screening and iterative combination mutation, the mutant is used for constructing a new biosensor, the new biosensor can efficiently and specifically respond to 5-aminovaleric acid, and the biosensor has the advantages of high sensitivity, high sensitivity, high sensitivity and the like. The method can be used for dynamic regulation and control of an amino acid metabolic pathway and a high-throughput screening platform of amino acid oxidase or strains, and has a relatively great application prospect.

Description

Technical Field

[0001] This invention relates to a biosensing tool and its application in response to 5-aminovaleric acid, belonging to the field of bioengineering technology. Background Technology

[0002] Amino acids are important bioactive substances and are widely used in food, feed and pharmaceutical synthesis. In addition to the natural amino acids that make up proteins, there are many kinds of non-natural amino acids, many of which are high-value drugs, pharmaceutical intermediates, and precursors for biomaterial synthesis, such as β-alanine, γ-aminobutyric acid, and 5-aminovaleric acid. Amino acids can be prepared by bio-fermentation, enzyme catalysis, chemical synthesis and other methods. With the rapid development of biotechnology, the bio-preparation of amino acids is rapidly replacing chemical synthesis. Highly active amino acid synthases and high-yield amino acid strains are necessary conditions for the efficient preparation of amino acids. However, traditional amino acid detection methods are inefficient and severely limit the screening and modification of related enzymes and strains. Genetically encoded biosensors can convert the concentration signals of specific metabolites in cells into gene expression level signals and dynamically regulate gene expression. They are mainly used in the following two aspects: (1) Using biosensors that respond to intermediate metabolites to realize the dynamic regulation of pathway genes and maximize the efficiency of cell factories. (2) Constructing a high-throughput screening platform for enzymes to help the high-activity evolution of enzymes. Biosensors reflect changes in enzyme activity by detecting catalytic products, thereby rapidly screening highly active mutants. Therefore, biosensors can be developed into high-throughput screening platforms for modified enzymes or strains, thereby greatly accelerating the development of amino acid biosynthesis.

[0003] Bacillus subtilis Bacillus subtilis In this process, the transcription factor BsGabR can specifically bind to GABA and activate the target promoter P. gabT The transcription of GABA transaminase gene, thereby regulating its transcription. gabT BsGabR is a homodimeric protein composed of two subunits linked in a centrosymmetric manner. The N-terminus is a DNA-binding domain (DBD), and the C-terminus is an effector-binding domain (EBD). BsGabR uses PLP as a cofactor for ligand binding. In the absence of GABA, PLP covalently binds to Lys312 of BsGabR in the form of an inner aldolimine. When GABA enters the ligand-binding pocket, the covalent bond between PLP and Lys312 breaks, and PLP forms an outer aldolimine with GABA, thereby triggering an allosteric effect in BsGabR and activating transcription.

[0004] The inventors previously identified the gene encoding BsGabR and its target promoter P. gabTHeterogeneously constructed into *E. coli*, a GABA biosensor system was built using green fluorescent protein (sfGFP) as a reporter gene and applied for high-throughput screening of glutamate α-decarboxylase (Song C, Luo J, Qiao J, Liu Z, Cheng Z, Zhou Z, Han L. pH-adaptive evolution of glutamate decarboxylase enables gamma-aminobutyric acid biosynthesis without pH control. Bioresour Technol. 2025 Oct 1;440:133432.). However, wild-type BsGabR only responds to the amino acid GABA, limiting its application scenarios.

[0005] 5-Aminopentanoic acid (5PA) is an important platform chemical, a precursor for the synthesis of novel engineering plastics such as nylon 5 and nylon 5,6, and can also be used as an additive in perovskite solar cells and a raw material for the synthesis of artificial antigens. The main limiting factor in the biocatalytic synthesis of 5PA is the low activity of the enzymes involved in the synthetic pathway. However, the lack of efficient detection methods for 5PA restricts the modification of enzymes that catalyze its synthesis to achieve high activity. Summary of the Invention

[0006] This invention provides a BsGabR mutant, which, based on the parent shown in SEQ ID NO.5, has mutations of H114G and M115W.

[0007] In one embodiment, the mutant contains mutations in H114G, M115W, and F431Y.

[0008] In one embodiment, the BsGabR mutant is based on the amino acid sequence shown in SEQ ID NO.5, with histidine at position 114 mutated to glycine and methionine at position 115 mutated to tryptophan.

[0009] The present invention also provides a gene encoding the BsGabR mutant.

[0010] In one embodiment, the nucleotide sequence of the gene encoding the BsGabR mutant H114G / M115W is shown in SEQ ID NO. 6.

[0011] This invention also provides a biosensor responsive to 5-aminovaleric acid (5PA), containing the coding gene of the BsGabR mutant and the constitutive promoter P. con promoter P gabTand green fluorescent protein sfGFP; the promoter P gabT Regulates the expression of green fluorescent protein sfGFP; promoter P gabT and promoter P con The transcription direction is reversed; the nucleotide sequence of the BsGabR mutant is shown in SEQ ID NO.7.

[0012] In one implementation, the promoter P gabT The nucleotide sequence is shown in SEQ ID NO.2; the promoter P con The nucleotide sequence is shown in SEQ ID NO.1.

[0013] In one embodiment, the biosensor uses pET series vectors as a plasmid backbone.

[0014] In one embodiment, the biosensor uses pET24a(+) as a plasmid backbone.

[0015] The present invention also provides recombinant microorganisms containing the aforementioned biosensor.

[0016] In one embodiment, the microorganisms include, but are not limited to, Escherichia coli.

[0017] In one embodiment, the Escherichia coli is Escherichia coli JM109.

[0018] The present invention also provides the application of the biosensor that responds to 5-aminovaleric acid in the biological field.

[0019] In one embodiment, the application includes, but is not limited to, screening of amino acid oxidase DavB or dynamic regulation of the 5PA anabolic pathway.

[0020] In one embodiment, the application includes, but is not limited to, high-throughput screening of the amino acid oxidase DavB.

[0021] The present invention also provides amino acid oxidase mutants obtained by screening using the biosensor described above.

[0022] Beneficial effects: 1. This invention modifies the ligand binding pocket of BsGabR to screen and obtain a BsGabR mutant that can recognize 5-aminovaleric acid (5PA) and activate sfGFP expression. This mutant can be used to construct a highly efficient genetically encoded biosensor for detecting 5PA (Sen5PA), which has a high induction rate and strong specificity for 5PA.

[0023] 2. The 5-aminovaleric acid-responsive sensor Sen5PA constructed in this invention can be used for dynamic regulation of the 5PA biosynthetic pathway, as well as a high-throughput screening platform for related synthetic enzymes or strains, and has great application prospects. Attached Figure Description

[0024] Figure 1 ligand binding pocket analysis for BsGabR.

[0025] Figure 2 In response to 5PA, BsGabR mutant screening was performed; where a: residue grouping screening; b: iterative mutation screening.

[0026] Figure 3 The performance of the biosensor in response to 5PA was characterized; where a: determination of the ligand response range of Sen5PA; b: determination of the ligand specificity of Sen5PA.

[0027] Figure 4 The construction of the DavB high-throughput screening system is shown in Figure 1; where a: schematic diagram of DavB high-throughput screening; b: determination of DavB high-throughput screening conditions.

[0028] Figure 5 Screening for highly active DavB mutations; where a: DavB catalytic pocket residue analysis; b: screening for DavB mutants. Detailed Implementation

[0029] 1. Gene cloning and plasmid construction: (1) Preparation of PCR reaction system (50μL): 25μL PrimeSTAR Max Premix (2×) (Takara), 2μL each of upstream and downstream primers (10 μM), 0.5μL DNA template, 20.5μL ddH2O. (2) PCR reaction program: 98℃ for 1min pre-denaturation; 98℃ for 30s, 55℃ for 15s, 72℃ for 1min, 30 cycles; 72℃ for 5min extension. (3) DpnI digestion to remove template DNA: Add 1μL restriction endonuclease DpnI (Takara) and 5μl 10×Buffer to the PCR reaction product, and react in a 37℃ metal bath for 1h. Purify the digested reaction solution using the Gel Extraction Kit (Kangwei Century) to obtain high-purity PCR amplification products. (4) Seamless cloning. Prepare a DNA seamless ligation reaction system (10 μL): 5 μL 2X MultiFSeamless Assembly Mix (ABclonal), 2.5 μL each of DNA gene fragment and vector fragment. React in a 55℃ metal bath for 30 min to complete fragment assembly. (5) Transformation. Transform the seamless cloning reaction solution into Escherichia coli JM109 competent cells and plate them onto selection agar plates containing the corresponding antibiotics. Incubate overnight at 37℃ to obtain transformants. Pick single colonies for further verification.

[0030] 2. Culture medium: LB medium (L -1 ): 10 g tryptone, 10 g NaCl, 5 g yeast extract, pH 7.0, add 20 g agar powder when preparing solid culture medium.

[0031] ZYM5052 culture medium (L) -1 ): Tryptone 10 g, yeast extract 5 g, Na2HPO4 3.55 g, KH2PO4 3.4 g, NH4Cl 2.66 g, Na2SO4 0.71 g, MgSO4 0.24 g, glycerol 5 g, glucose 0.5 g, lactose 2 g.

[0032] Antibiotic concentrations used during culture: kanamycin (50 μg / mL), chloramphenicol (17 μg / mL).

[0033] 3. Detection method for sfGFP fluorescence intensity: Transfer 200 μL of bacterial culture medium to a 96-well black-walled transparent-bottom ELISA plate and detect the OD using a Synergy™ H4 ELISA reader. 600 And fluorescence. When detecting fluorescence, the excitation wavelength is 485 nm and the absorption wavelength is 528 nm.

[0034] 4. BsGabR database construction and filtering: BsGabR library construction and screening were performed on E. coli vectors containing kanamycin resistance and the ori replication origin site. Based on the three-dimensional structure of BsGabR, the ligand-binding pockets for PLP and amino acids were determined, and residues within 5 Å of the ligand were selected as ligand-binding pocket residues. Residues with 1-2 residues or similar sequences were grouped together, and random mutations were introduced at the target residue positions via whole-plasmid PCR amplification using degenerate codons NNK designed on primers, thereby constructing combined saturated mutant libraries for each group of residues. The screening process for BsGabR mutants included primary screening in 96-well plates and secondary screening in test tubes.

[0035] (1) Initial screening in 96-well plates. Plasmids containing the combined saturated mutant library were transformed into JM109 competent cells and plated onto LB plates supplemented with 50 mM 5PA (kanamycin resistant), and cultured overnight at 37°C. Transformants were observed under blue light, and fluorescent transformants were selected and transferred to 96-well deep-well plates containing 800 μL of LB medium (kanamycin resistant), and cultured at 37°C and 300 rpm for 12 h. 10 μL of seed culture was transferred to 1 mL of LB medium and 1 mL of LB medium supplemented with 10 mM target amino acids, and cultured at 37°C and 300 rpm for 24 h. 200 μL of culture medium was transferred to an ELISA plate, and the fluorescence intensity was detected using an ELISA reader to calculate the induction rate.

[0036] Induction rate refers to the FI / OD ratio of culture medium supplemented with amino acids. 600 FI / OD of culture medium without added amino acids 600 The ratio.

[0037] (2) Test tube rescreening. The positive mutants that passed the initial screening in 96-well plates were inoculated into test tubes containing 3 mL of LB medium and cultured at 37°C and 200 rpm for 12 h. 50 μL of seed culture was then transferred to test tubes containing 5 mL of LB medium and 5 mL of LB medium supplemented with 10 mM 5 PA, and cultured at 37°C and 200 rpm for 24 h. 200 μL of culture medium was transferred to an ELISA plate, and the fluorescence intensity was detected using an ELISA reader to calculate the fluorescence induction rate.

[0038] 5. Detection of biosensor ligand specificity: Recombinant bacterial strains containing different amino acid biosensors were taken from a -80℃ freezer, streaked on LB agar plates containing the corresponding antibiotics, and single colonies were picked and cultured in test tubes containing 5 mL of LB medium at 200 rpm. -1 Incubate overnight at 37°C. Transfer 2% inoculum to 5 mL LB medium containing 10 mM of different amino acids in test tubes and incubate for another 24 h at 37°C and 200 rpm. Detect the OD of the culture medium. 600And fluorescence intensity (FI).

[0039] Characterization of ligand concentration-dependent fluorescence signals of biosensors. Recombinant bacterial strains containing different amino acid biosensors were taken from a -80°C freezer and streaked onto LB agar plates containing the corresponding antibiotics. Single colonies were picked and cultured in test tubes containing 5 mL of LB medium at 200 rpm. -1 Incubate overnight at 37°C. Transfer 2% inoculum to 5 mL LB medium containing different amino acid concentrations in test tubes and incubate for another 24 h at 37°C and 200 rpm. Detect the OD of the culture medium. 600 And FI.

[0040] 6. DavB database creation and filtering: Library construction and screening of DavB were performed on vectors containing chloramphenicol resistance genes and the p15A replication origin site. The enzyme-substrate complex structure of DavB was constructed via molecular docking, and the key loop structure involved in substrate binding was identified and focused on for modification. Adjacent residues on the loop structure were grouped into sets, and random mutations were introduced via NNK design on primers and whole-plasmid PCR amplification to construct a combined saturated mutant library of each residue set. Sen5PA was used for high-throughput screening of the mutant library; the screening process for highly active DavB mutants included fluorescence-based primary and secondary screening.

[0041] (1) Initial screening in 96-well plates. Plasmids containing the mutant library were transformed into competent cells containing Sen5PA and evenly spread on LB agar plates containing kanamycin, chloramphenicol, 20 mM substrate L-Lys, and 1 mM arabinose. The plates were incubated overnight at 37°C and 300 rpm. To ensure effective coverage of the theoretical mutation, the total number of transformants on the plate should exceed 4000. The transformant plates were observed under a blue light spectrometer. Transformants with strong fluorescence were selected and transferred to 96-well plates containing 800 μL of LB medium (kanamycin and chloramphenicol) and incubated at 37°C and 300 rpm for 12 h. Then, 10 μL of the seed culture was transferred to another 96-well plate containing 1 mL of LB medium (containing kanamycin, chloramphenicol, 10 mM L-Lys, and 0.5 mM arabinose) and incubated at 37°C and 300 rpm for 24 h. Transfer 200 μL of culture medium to a black-walled, transparent-bottomed microplate, and use a microplate reader to detect the FI / OD ratio. 600 The relative fluorescence values ​​were calculated using the wild type as a control. Transformants with higher fluorescence values ​​were selected for further validation.

[0042] (2) Test tube re-screening. The positive transformants verified by the initial screening in 96-well plates were inoculated into test tubes containing 3 mL of LB medium (kanamycin and chloramphenicol) and cultured at 37°C and 200 rpm for 12 h. 50 μL of the seed culture was then transferred to test tubes containing 5 mL of LB medium (containing kanamycin, chloramphenicol, 10 mM L-Lys, and 0.5 mM arabinose) and cultured at 37°C and 200 rpm for 24 h. 200 μL of the culture was transferred to a black-walled, transparent-bottomed microplate and analyzed using a microplate reader. The FI / OD ratio was calculated. 600 The relative fluorescence values ​​were calculated using the wild type as a control.

[0043] (3) Determination of pure enzyme cascade reaction. The selected DavB mutant was cloned into the pET-24a(+) vector, and the recombinant plasmid was transformed into the expression host BL21(DE3). DavB was induced to express using self-inducing medium ZYM5052. After purification by metal affinity chromatography and dialysis to remove salts, the specific activity of the pure enzyme was measured. Mutations with increased specific activity were identified as positive mutations.

[0044] 7. Enzyme expression and purification: Wild-type DavB and the mutant DavA were cloned into the pET-24a(+) vector, and the recombinant plasmid was transformed into the expression host BL21(DE3). The recombinant bacteria were inoculated into test tubes containing 5 mL of LB medium and cultured at 37 ℃ and 200 rpm for 12 h. 1 mL of seed culture was then transferred to an Erlenmeyer flask containing 50 mL of ZYM5052 medium and cultured at 37 ℃ and 200 rpm. When OD... 600 When the coefficient of performance is 0.8, the temperature is lowered to 30 °C for 12 h to induce induction.

[0045] After culturing, the bacterial culture was centrifuged at 8000 rpm for 10 min, and the cells were collected and resuspended in 10 mL Ni-Native-0 buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0). The cell resuspended cells were sonicated and the target protein with a C-terminal His×6 tag was purified using a Ni-NTA-Sefinose pre-packed gravity column (Shanghai Sangon Biotech Co., Ltd.).

[0046] 8. Specific steps for protein purification: (1) Pre-equilibrate the gravity column using 5-10 column volumes of Ni-Native-0 buffer; Add crude enzyme supernatant, and the target enzyme binds to the resin carrier; (2) Use 5-10 column volumes of Ni-Native-20 buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) to wash away non-specifically adsorbed proteins; (3) Use 5 column volumes of Ni-Native-500 buffer (50 mM NaH2PO4, 300 mM NaCl, 500 mM imidazole, pH 8.0) to elute the target protein.

[0047] (4) The elution buffer was dialyzed in PBS (8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4·12H2O, 0.24 g / L KH2PO4, pH 7.4) buffer for 8 h to remove high concentrations of salt.

[0048] (5) The purity of the enzyme was determined by SDS-PAGE and the concentration was determined by the Bradford method.

[0049] 9. Assay of the activity of pure enzyme in vitro cascade catalysis of L-Lys to 5PA: An in vitro reaction system for catalyzing the conversion of L-Lys to 5PA was constructed using purified DavB and DavA enzymes. The catalytic system had a volume of 0.5 mL and contained 0.2 mg / mL DavB, 0.2 mg / mL DavA, and 100 mM L-Lys. The reaction was incubated at 37 °C for 20 min, and the reaction was terminated by adding 0.05 mL of 1 M sodium hydroxide solution.

[0050] 5PA was quantitatively detected by HPLC. The sample underwent pre-column derivatization with phenyl isothiocyanate (PITC): the reaction solution was diluted 10-fold with sterile water, and 250 μL of triethylamine-acetonitrile (14:86) and 250 μL of PITC-acetonitrile (1:84) were added sequentially after each 500 μL dilution. Derivatization was performed in the dark for 40 min, followed by the addition of 750 μL of n-hexane to terminate the derivatization. The lower layer obtained from the extraction was filtered through a 0.22 μm organic filter membrane for subsequent HPLC analysis. HPLC analysis was performed using a Diamonsil 5 μm C18 column (250*4.6 mm) from Dima Technologies, with a detection wavelength of 254 nm. Mobile phase A: 80% acetonitrile aqueous solution. Mobile phase B: 3% acetonitrile-0.1M sodium acetate aqueous solution. The flow rate of the mobile phase was 0.6 mL / min. Gradient elution was used: 0-5 min, 5% mobile phase A, 95% mobile phase B; 5-30 min, 30% mobile phase A, 70% mobile phase B; 30-35 min, 5% mobile phase A, 95% mobile phase B.

[0051] Example 1: Construction of Biosensors The construction method of plasmid pEV-sfGFP-gabR refers to the paper "pH-adaptive evolution of glutamate decarboxylase enables gamma-aminobutyric acid biosynthesis without pH control". The plasmid pEV-sfGFP-gabR includes components derived from... Bacillus subtilis The transcription factor gabR (nucleotide sequence shown in SEQ ID NO.4), denoted as 168, is composed of the constitutive promoter P. con (nucleotide sequence as shown in SEQ ID NO.1) Expression; promoter P gabT (nucleotide sequence as shown in SEQ ID NO.2) and promoter P con The transcription direction is reversed; the reporter gene sfGFP (nucleotide sequence shown in SEQ ID NO.3) is cloned into promoter P. gabT Downstream; promoter P gabT It can be activated by BsGabR.

[0052] The biosensor works by activating the promoter P in the absence of GABA. gabT BsGabR exhibits low activity, with extremely low sfGFP expression levels. Upon the addition of GABA, BsGabR binds to GABA and activates the promoter P. gabT The expression level of sfGFP was increased, thus establishing a coupling relationship between the expression level of sfGFP and the concentration of GABA.

[0053] Example 2: GabR structural analysis and determination of ligand-binding pocket residues The crystal structure of Bacillus subtilis GabR (NCBI Accession: CAB12197.1) has been resolved. It is a homodimer containing two domains: an N-terminal DNA-binding domain (DBD) and a C-terminal ligand-binding domain (EBD). The crystal structure of the GabR EBD-PLP-GABA complex is given by PDB accession number 5T4J. Based on the crystal structure of 5T4J, the catalytic pocket of GabR was analyzed, with amino acid residues within 5 Å of the PLP-GABA ligand defined as the ligand-binding pocket. Figure 1The ligand-binding pocket of GabR includes: H114, M115, M145, G180, T181, Q182, Y205, R207, M208, T245, F250, D279, Y281, L340, R430, and F431, totaling 16 amino acid residues. Considering the sequence distances of the residues in the binding pocket and their interactions with the ligands, the 16 residues were grouped into 10 groups, each containing 1-2 residue sites. The grouping is shown in Table 1.

[0054] Table 1. CAST grouping of GabR ligand binding pockets

[0055] Example 3: Mutation screening of ligand-binding pocket residues in response to different amino acids Using plasmid pEV-sfGFP-gabR (published in "pH-adaptive evolution of glutamated ecarboxylase enables gamma-aminobutyric acid biosynthesis without pH control. Bioresour Technol") as a template, PCR amplification was performed using primers containing the codons NNK (N = A / C / G / T, K = G / T, specific primers are shown in Table 2) to construct combinatorial mutant libraries with different groups. After digestion and purification, the PCR products were seamlessly cloned and assembled, and transformed into Escherichia coli strain JM109. The transformants were then incubated on LB plates containing 50 mM 5PA at 37°C for 12 h.

[0056] The plates were observed under a blue light spectrometer. Colonies exhibiting obvious green fluorescence were picked and transferred to 96-well deep-well plates. These plates were then incubated in LB medium containing 10 mM 5 PA at 37 ℃ and 200 rpm for initial screening. Transformants showing significant sfGFP expression induced by the added specific amino acids were then transferred to test tubes containing 10 mM 5 PA for secondary screening and verification, ultimately yielding mutants with high induction rates. The fluorescence results of the test tube screening after 12 h of incubation are shown in the figure. Figure 2 a and Table 3.

[0057] Table 2 GabR ligand binding pocket residue grouping and mutation primers

[0058] Table 3. CAST grouping fluorescence screening (10 mM 5 PA)

[0059] Example 4: Iterative evolution of key residues in the GabR ligand binding pocket The residue grouping and screening results in Example 3 showed that mutations responsive to 5PA were obtained from the CAST1, CAST3, CAST4, and CAST10 groups. Among them, the H114G / M115W mutation in the CAST1 group had the highest induction rate; the F431Y mutation in the CAST10 group was second, with an induction rate of 2.7. To further improve the sensor's response to 5PA, a saturated mutant library for the CAST10 group was constructed using H114G / M115W as a template, using primers PgabR-CAST10-1 / PgabR-CAST10-2 (primer sequences are shown in Table 3). Following the method in Example 3, the mutants were cultured and fluorescence intensity was detected. After screening, no mutants with a further increased induction rate compared to H114G / M115W were obtained; the highest induction rate, H114G / M115W / F431Y, was only 7.0 (…). Figure 2 (b, Table 4). Therefore, H114G / M115W was selected as the optimal mutation for responding to 5PA, and the sensor pEV-sfGFP-gabR containing this mutant was used. H114G / M115W It was named Sen5PA.

[0060] Table 4. Iterative evolutionary screening of residues in ligand binding pockets (10 mM 5PA)

[0061] Example 5: Sen5PA response range and ligand specificity detection The recombinant bacteria containing Sen5PA constructed in Example 4 were cultured, and 0, 1, 2, 5, 10, 20, and 50 mM concentrations of 5PA were added to the culture medium, respectively. The fluorescence intensity was measured for each concentration of 5PA added. The results are as follows: Figure 3 As shown in a and Table 5, the fluorescence signal of the sensor increases with increasing 5PA concentration. Within the 0-50 mM 5PA concentration range, the relationship between 5PA concentration and fluorescence intensity can be expressed by the function y = -253.95x. 2 +32677x+30564 describes, and R 2 It reached 0.9999.

[0062] Table 5. Response effects of recombinant bacteria containing the sensor Sen5PA to different concentrations of 5PA.

[0063] Recombinant bacteria containing Sen5PA were cultured using the aforementioned method, and 10 mM concentrations of glycine (Gly), β-alanine (BA), γ-aminobutyric acid (GABA), or 5PA were added to the culture medium, respectively. The fluorescence intensity of each amino acid added and the fluorescence intensity of the blank control were measured. The specificity of Sen5PA for different amino acid ligands was determined by calculating the induction rate. The results are as follows: Figure 3 As shown in b and Table 6, Sen5PA shows almost no response to Gly and GABA, a weak response to BA, and a highly sensitive response to 5PA.

[0064] Table 6. Validation of Sen5PA's ligand recognition specificity

[0065] Example 6: Construction of a high-throughput screening system for amino acid oxidase DavB Source Pseudomonas putida DavB (ATCC 47054) is an amino acid oxidase that catalyzes the conversion of L-Lys to 5-aminopentanamide; DavA is an amide hydrolase that catalyzes the conversion of 5-aminopentanamide to 5PA. DavB has the lowest activity and is the rate-limiting enzyme in the conversion of L-Lys to 5PA. A high-throughput screening system for DavB was developed based on Sen5PA, and directed evolution of DavB was performed to verify the effectiveness of Sen5PA in the high-throughput screening of 5PA synthases.

[0066] The genes of DavB (nucleotide sequence as shown in SEQ ID NO.7) and DavA (nucleotide sequence as shown in SEQ ID NO.8) were cloned into the p15A vector (published in the paper "Construction and Application of a High-ThroughputIn Vivo Screening Platform for the Evolution of Nitrile Metabolism-RelatedEnzymes Based on a Desensitized Repressive Biosensor"). BAD Downstream of the promoter, their expression was induced by arabinose to obtain plasmid p15A-DavB-DavA. The primers used are shown in Table 7. Plasmid p15A-DavB-DavA was transformed into the recombinant bacteria containing the sensor Sen5PA constructed in Example 4 to construct a high-throughput screening system for DavB (e.g., ...). Figure 4(As shown in a). The working principle of this system is as follows: by adding arabinose to the culture medium to induce the expression of DavB and DavA, DavB can catalyze the added L-Lys to 5-aminopentanamide; subsequently, it is further catalyzed by DavA to 5PA. The generated 5PA can be responded to by Sen5PA, thereby outputting a fluorescence signal. Based on this, the fluorescence signal can be correlated with the activity of DavB, that is, the higher the activity of DavB, the stronger the output fluorescence signal, thereby achieving the purpose of rapidly screening for highly active DavB mutants by detecting the intensity of the fluorescence signal.

[0067] Since the concentration of arabinose affects the expression levels of DavB and DavA, suitable screening conditions were first determined. Recombinant bacteria containing p15A-DavB-DavA and Sen5PA were inoculated into test tubes containing 3 mL of LB medium (containing kanamycin and chloramphenicol) and cultured at 37℃ and 220 rpm for 12 h. Then, 50 μL of seed culture was transferred to test tubes containing 5 mL of LB medium (containing kanamycin, chloramphenicol, 10 mM L-lysine, and different concentrations of arabinose), with final arabinose concentrations of 0, 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.5, or 2 mM, and cultured at 37℃ and 200 rpm for 24 h. The fluorescence intensity was then measured. Results are as follows: Figure 4 As shown in Tables b and 8, the fluorescence intensity gradually increases with increasing arabinose concentration, demonstrating the effectiveness of the screening system. The fluorescence intensity is moderate when the arabinose concentration is 0.4–0.6 mM; therefore, a 0.5 mM arabinose concentration was used as the screening condition.

[0068] Table 7 Primers used to construct plasmid p15A-DavB-DavA

[0069] Table 8. Determination of DavB screening conditions for the amino acid biosensor Sen5PA.

[0070] Example 7: Screening for highly active mutants of DavB Analysis of the structure of the DavB and L-Lys complex revealed that the D238 and E245 residues of DavB form hydrogen bonds and salt bridges with the ε-amino group of the substrate L-Lys; W514 and the α-amino group form π-π interactions ( Figure 5a) These three residues are located on two loop structures, namely Q227-L244 and F504-V517. Therefore, it is speculated that these two loop structures may be very important for enzyme-substrate binding. These two loops contain a total of 32 residues, which were grouped into 16 groups, each containing two adjacent residue sites. The grouping is shown in Table 9.

[0071] Using plasmid p15A-DavB-DavA as a template, PCR amplification was performed using primers containing the codon NNK (N = A / C / G / T, K = G / T, specific primers are shown in Table 10) to construct a random saturation mutant library with different groupings of residues. After digestion and purification, the PCR products were seamlessly cloned and assembled, then transformed into *E. coli* strain JM109 containing Sen5PA. Transformants were cultured statically at 37°C on LB agar plates containing 20 mM L-Lys and 1 mM arabinose. The plates were observed under a blue light spectrometer, and colonies with obvious green fluorescence were picked and cultured in 96-well deep-well plates in LB medium containing 10 mM L-Lys and 0.5 mM arabinose for initial screening. Transformants with high sfGFP expression levels were then transferred to test tubes containing LB medium containing 10 mM L-Lys and 0.5 mM arabinose for secondary screening and verification. The fluorescence results of the tube screening are shown in [Table 10]. Figure 5 b and Table 11.

[0072] The results showed that in the catalytic pocket residue grouping screening, mutations with enhanced fluorescence intensity were obtained in the residues of groups L14 and L15, namely W512F / T513M and W512F / T513C in group L14, and P514G / A515G and A515G in group L15. Figure 5 (b and Table 11). Among them, A515G showed the most significant enhancement. Therefore, using A515G as a template, a saturated mutant library of L14 grouped residues was constructed and transformed into the *E. coli* strain JM109 containing Sen5PA constructed in Example 4. The transformants were incubated statically at 37°C for 12 h on LB plates containing 20 mM L-Lys and 1 mM arabinose. The plates were observed under a blue light spectrometer, and colonies with obvious green fluorescence were picked and cultured in 96-well deep-well plates in LB medium containing 10 mM L-Lys and 0.5 mM arabinose for initial screening. Transformants with high sfGFP expression levels were then transferred to test tubes containing LB medium containing 10 mM L-Lys and 0.5 mM arabinose for secondary screening and verification. The fluorescence results of the test tube screening are shown in [Figure 11]. Figure 5 b and Table 11. Among the obtained iterative combination mutations, W512F / T513G / A515G showed the most significant increase in fluorescence intensity and was the optimal mutation.

[0073] Wild-type DavB and its mutants W512F / T513G / A515G, as well as DavA, were purified separately. An in vitro dual-enzyme catalytic system for the production of 5PA from L-Lys was set up, containing 0.2 mg / mL DavB, 0.2 mg / mL DavA, and 100 mM L-Lys. The reaction was terminated after 20 min at 37 °C, and the amount of 5PA produced was measured. The results showed that wild-type DavB and DavA catalyzed the production of 4.6 ± 0.4 mM of 5PA; the mutants W512F / T513G / A515G of DavB and DavA catalyzed the production of 18.7 ± 1.3 mM of 5PA. The product concentration using the DavB mutant was 4.1 times that using the wild-type, indicating a significant increase in the activity of the DavB mutant compared to the wild-type. This result demonstrates the promising application of Sen5PA in screening for highly efficient enzymes catalyzing 5PA.

[0074] Table 9. Residue grouping of DavB substrate binding loops

[0075] Table 10 DavB substrate binding Loop residue grouping and mutation primers

[0076] Table 11 Screening of DavB mutants

[0077] 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. The BsGabR mutant, characterized by, Based on the parent shown in SEQ ID NO.5, it has mutations of H114G and / or M115W.

2. The BsGabR mutant according to claim 1, characterized in that, The BsGabR mutant is based on the amino acid sequence shown in SEQ ID NO. 5, with histidine at position 114 mutated to glycine and methionine at position 115 mutated to tryptophan.

3. The gene encoding the BsGabR mutant as described in claim 1 or 2.

4. A biosensor responsive to 5-aminovaleric acid, characterized in that, The coding gene containing the BsGabR mutant, constitutive promoter P con promoter P gabT and fluorescent proteins; the promoter P gabT Regulation of fluorescent protein expression; promoter P gabT and promoter P con The transcription direction is reversed; the nucleotide sequence of the BsGabR mutant is shown in SEQ ID NO.7; the promoter P gabT The nucleotide sequence is shown in SEQ ID NO.2; the promoter P con The nucleotide sequence is shown in SEQ ID NO.

1.

5. The biosensor according to claim 4, characterized in that, The biosensor uses pET series vectors as its plasmid backbone.

6. The biosensor according to claim 4 or 5, characterized in that, The fluorescent proteins include, but are not limited to, green fluorescent protein.

7. A recombinant microorganism containing any one of the biosensors described in claims 4 to 6.

8. The recombinant microorganism according to claim 7, characterized in that, The microorganisms include, but are not limited to, Escherichia coli.

9. The application of the 5-aminovaleric acid-responsive biosensor according to any one of claims 4 to 6 in the screening of the amino acid oxidase DavB.

10. The application of the biosensor according to any one of claims 4 to 6 in the dynamic regulation of 5-aminovaleric acid synthesis.

Images