Biosensor responding to beta-alanine and application

Abstract

The invention discloses a biosensor responding to beta-alanine and application, and belongs to the technical field of bioengineering. A ligand binding pocket residue of BsGabR is subjected to mutation modification on the basis of a GABA biosensor, a BsGabR mutant capable of specifically recognizing and responding to beta-alanine and activating reporter gene expression is obtained through screening and iterative combinatorial mutation, the mutant is used for constructing a new biosensor, pregabalin can be efficiently and specifically responded, and the biosensor has a good application prospect. 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 synthetase or strains, and has a relatively great application prospect.

Description

Technical Field

[0001] This invention relates to a biosensor that responds to β-alanine and its application, 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, 5-aminovaleric acid, pregabalin and so on. 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 seriously 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 applied 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 detect changes in enzyme activity by detecting catalytic products, enabling rapid screening of highly active mutants. Therefore, biosensors can be developed into high-throughput screening platforms for modified enzymes or strains, thereby significantly 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 one amino acid, GABA, limiting its application scenarios.

[0005] β-Alanine is an important non-protein amino acid, not only used directly in energy supplements but also as a crucial intermediate in the synthesis of vitamin B5, carnosine, and their derivatives. Currently, its biosynthesis mainly follows two pathways: one utilizes decarboxylases to generate β-alanine from L-aspartic acid via α-decarboxylation; the other uses acrylic acid as a substrate and undergoes amino addition via amino-addition via amino-lyases. However, a high-throughput detection method for β-alanine is currently lacking, hindering efficient screening of β-alanine-producing biomaterials. Therefore, developing high-performance biosensors for rapid β-alanine detection is crucial for screening related enzymes and for the metabolic regulation and modification of bacterial strains. Summary of the Invention

[0006] This invention provides a BsGabR mutant, which, based on the parent shown in SEQ ID NO.5, has one or more mutations among M208V, F250T, F431Y, and F431W.

[0007] In one embodiment, the BsGabR mutant is based on the amino acid sequence shown in SEQ ID NO.5, with methionine at position 208 mutated to threonine, phenylalanine at position 250 mutated to threonine, and methionine at position 431 mutated to tryptophan.

[0008] In one embodiment, the BsGabR mutant is based on the amino acid sequence shown in SEQ ID NO.5, with methionine at position 208 mutated to valine, phenylalanine at position 250 mutated to threonine, and methionine at position 431 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 M208V / F250T / F431W is shown in SEQ ID NO.6.

[0011] This invention also provides a biosensor (named SenBA) responsive to β-alanine (β-Ala, BA), containing the coding gene of the BsGabR mutant and the constitutive promoter P. con promoter P gabT and 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.6.

[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-24a(+) as a plasmid backbone.

[0014] The present invention also provides recombinant microorganisms containing the aforementioned biosensor.

[0015] In one embodiment, the microorganisms include, but are not limited to, Escherichia coli.

[0016] In one embodiment, the Escherichia coli is Escherichia coli JM109.

[0017] The present invention also provides the application of the biosensor in the screening of amino acid decarboxylases, wherein the screening includes, but is not limited to, high-throughput screening.

[0018] The present invention also provides amino acid decarboxylase mutants obtained by screening using the biosensor described above.

[0019] In one embodiment, the amino acid decarboxylase mutant is based on the parental line and has a D59N mutation.

[0020] In one embodiment, the amino acid decarboxylase mutant is based on the parental line and has mutations of D59N / S134G, C38L / D59N / S134G, C38P / D59N / S134G, or M37F / C38S / D59N / S134G.

[0021] In one embodiment, the amino acid sequence of the parent is shown in SEQ ID NO.8.

[0022] Beneficial effects: 1. This invention modifies the ligand binding pocket of BsGabR to screen and obtain a BsGabR mutant that can specifically recognize β-alanine and activate sfGFP expression. This mutant can be used to construct a highly efficient biosensor for detecting the genetic code of β-alanine, with high induction rate and strong specificity.

[0023] 2. The present invention uses the constructed biosensor that responds to β-alanine to screen amino acid decarboxylases, which significantly improves the screening efficiency.

[0024] 3. This biosensor can be used for the dynamic regulation of amino acid metabolic pathways, as well as a high-throughput screening platform for amino acid synthases or strains, and has great application prospects. Attached Figure Description

[0025] Figure 1 ligand binding pocket analysis for BsGabR.

[0026] Figure 2 Screening for BsGabR mutants in response to β-alanine; where a: residue grouping screening; b: iterative mutation screening.

[0027] Figure 3 The performance of the biosensor in response to β-alanine was characterized; where a: determination of the ligand response range of SenBA; b: determination of the ligand specificity of SenBA.

[0028] Figure 4 Construction of the MjMfnA high-throughput screening system; where a: schematic diagram of MjMfnA high-throughput screening; b: determination of MjMfnA high-throughput screening conditions.

[0029] Figure 5 Screening for mutations that enable efficient β-alanine production catalyzed by MjMfnA; wherein, a: MjMfnA catalytic pocket residue analysis; b: residue grouping screening; c: iterative combination mutation screening; d: pure enzyme specific activity determination. Detailed Implementation

[0030] 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.

[0031] 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.

[0032] 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.

[0033] Antibiotic concentrations used during culture: kanamycin (50 μg / mL), chloramphenicol (17 μg / mL).

[0034] 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 Fluorescence. When detecting fluorescence, the excitation wavelength is 485 nm and the emission wavelength is 528 nm.

[0035] 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.

[0036] (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 BA (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 BA, respectively, 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. The fluorescence induction rate was calculated. The fluorescence induction rate refers to the FI / OD ratio of the BA-supplemented culture medium. 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 BA, respectively, 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 and streaked onto LB agar plates containing the corresponding antibiotics. Single colonies were then 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. 600 And 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 concentrations of BA in test tubes and continue incubation at 37°C and 200 rpm for 24 h. Detect the OD of the culture medium. 600 And FI.

[0040] 6. Database creation and filtering for MjMfnA: Library construction and screening of MjMfnA were performed on vectors containing chloramphenicol resistance genes and p15A replication origin sites. The enzyme-substrate complex structure of MjMfnA was constructed via molecular docking to determine the substrate catalytic pocket. Residues within 5 Å of the substrate molecule, excluding the active site, were selected as key pocket residues. One to two residues, along with those with similar sequences, were grouped together, and random mutations were introduced via NNK design on primers and whole-plasmid PCR amplification to construct combined saturated mutant libraries for each group of residues. SenBA was used for high-throughput screening of the mutant library; the screening process for highly active MjMfnA 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 SenBA and evenly spread on LB agar plates containing kanamycin, chloramphenicol, 20 mM L-aspartic acid, and 5 mM arabinose. The plates were incubated at 37°C for 12 h. To ensure effective coverage of the theoretical mutation, the total number of transformants on the plates should exceed 4000. The transformant plates were observed under a blue light spectrometer, and transformants with strong fluorescence were selected and transferred to 96-well plates containing 800 μL of LB medium (kanamycin and chloramphenicol). The plates were 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, 20 mM L-aspartic acid, and 5 mM arabinose). The plates were 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 value was calculated using wild-type MjMfnA as a control. Transformants with higher fluorescence values ​​were selected for further validation. During residue grouping screening, 5 mM arabinose was used to induce enzyme expression. In iterative combination screening, the concentration of arabinose used decreased accordingly as the activity of MjMfnA increased.

[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 seed culture was then transferred to test tubes containing 5 mL of LB medium (containing kanamycin, chloramphenicol, 20 mM substrate, and 5 mM or lower concentration of arabinose) and cultured at 37°C and 200 rpm for 24 h. 200 μL of 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 wild-type MjMfnA as a control. During residue grouping screening, 5 mM arabinose was used to induce enzyme expression. In iterative combination screening, the concentration of arabinose used decreased accordingly as the activity of MjMfnA increased.

[0043] 7. Overexpression and purification of MjMfnA and its mutant enzymes: Recombinant bacteria expressing MjMfnA and its mutant enzyme were inoculated into test tubes containing 5 mL of LB medium and cultured at 37 °C 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 °C 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.

[0044] 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 C-terminally fused MjMfnA target protein with a His×6 tag was purified using a Ni-NTA-Sefinose pre-packed gravity column (Shanghai Sangon Biotech Co., Ltd.).

[0045] 8. 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.

[0046] (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.

[0047] (5) The purity of the enzyme was determined by SDS-PAGE and the concentration was determined by the Bradford method.

[0048] 9. HPLC determination of the activity of MjMfnA: All reaction systems for determining MjMfnA activity were 0.5 mL, containing 0.05 mg / mL protein, 100 mM sodium aspartate, and 0.5 mM PLP. The reaction was incubated at 70°C for 3 minutes, and the reaction was terminated by adding 0.5 mL of 1 M sodium hydroxide solution.

[0049] HPLC was used to quantitatively detect the product BA. 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. After derivatization in the dark for 40 min, 750 μL of n-hexane was added 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.

[0050] Enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of product per minute.

[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 subtilisThe 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 is cloned into the 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 1 The 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 Residue grouping of GabR ligand binding pockets

[0055] Example 3: Screening for BA-responsive residue grouping mutations Using plasmid pEV-sfGFP-gabR (published in 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.) 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 incubated on LB plates containing 50 mM BA at 37 °C for 12 h.

[0056] The plates were observed under a blue light spectrometer. Colonies exhibiting significant green fluorescence were picked and transferred to 96-well deep-well plates, where they were cultured in LB medium containing 10 mM BA for initial screening. Transformants showing significant BA induction were then transferred to test tubes containing 10 mM BA for secondary screening and verification, ultimately yielding mutants with high induction rates. Fluorescence results from test tube screening after 12 hours of culture are shown below. Figure 2 a and Table 3.

[0057] Table 2. BsGabR ligand binding pocket residues grouping, mutation, and library construction primers

[0058] Table 3. Residue grouping fluorescence screening (10 mM BA)

[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 in M208, F250, and F431 residues could induce BsGabR to respond to BA, with the mutations in F250 and F431 showing the best effect, followed by M208. Therefore, a random saturation mutant library was constructed from residues in the CAST10 and CAST8 groups, using primers PgabR-CAST10-1 / PgabR-CAST10-2 and PgabR-CAST8-1 / PgabR-CAST8-2 (primer sequences are shown in Table 2). Following the method in Example 3, the mixture was cultured and fluorescence intensity was detected. After screening, four combined mutants, F250V / F431W, F250A / F431G, F250M / F431G, and F250T / F431W, were found to respond to BA, with F250T / F431W showing the best effect, achieving an induction rate of 3.1%. Figure 2 (b, Table 4). Further, using F250T / F431W as a template, a saturated mutant library of M208 residues was constructed. Finally, four three-point combination mutations, M208I / F250T / F431W, M208T / F250T / F431W, and M208V / F250T / F431W, were screened and found to respond to BA. Among them, M208V / F250T / F431W showed the best effect, with an induction rate of 4.1 (b). Figure 2 b, Table 4). The sensor pEV-sfGFP-gabR corresponding to this mutant. M208V / F250T / F431W It was named SenBA.

[0060] Table 4 Iterative evolutionary screening of BsGabR ligand binding pocket residues (10 mM BA)

[0061] Example 5: SenBA response range and ligand specificity detection The recombinant bacteria containing SenBA constructed in Example 4 were cultured statically at 37°C for 12 h on LB plates containing different concentrations of BA. BA concentrations of 0, 1, 2, 5, 10, 20, and 50 mM were added to the culture medium, and the fluorescence intensity of the sensor in response to different concentrations of BA was measured. The results are as follows: Figure 3 As shown in a and Table 5, the fluorescence signal of the sensor increases with increasing BA concentration. The relationship between BA concentration and fluorescence intensity can be expressed by the function y = -375.64x. 2 +35155x+116027 describes, and R 2 It reached 0.994.

[0062] Table 5. Response effects of recombinant bacteria containing sensor SenBA to different concentrations of BA.

[0063] Recombinant bacteria containing SenBA were cultured using the aforementioned method, and glycine (Gly), BA, or γ-aminobutyric acid (GABA) at concentrations of 10 mM were added to the culture medium. The fluorescence intensity of the culture medium with different added amino acids and the fluorescence intensity of the blank control were measured. The specificity of SenBA 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. The mutant SenBA only responds to BA and not to other amino acids.

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

[0065] Example 6: Construction of a high-throughput screening system for amino acid decarboxylase MjMfnA Source Methanocaldococcus jannaschii The amino acid decarboxylase of ATCC 43067 exhibits excellent thermostability and can catalyze the decarboxylation of various amino acids, including L-aspartic acid to BA; however, its activity is very low. A high-throughput screening system for MjMfnA was developed based on SenBA, and MjMfnA underwent directed evolution to verify the effectiveness of SenBA in high-throughput enzyme screening systems.

[0066] The gene of MjMfnA (nucleotide sequence shown in SEQ ID NO.7) was cloned into the p15A vector (disclosed in "Construction and Application of a High-Throughput In Vivo Screening Platform for the Evolution of Nitrile Metabolism-Related Enzymes Based on a Desensitized Repressive Biosensor"). BAD Downstream of the promoter, expression was induced by arabinose to obtain plasmid p15A-MjMfnA. Primers used are shown in Table 7. The constructed recombinant plasmid p15A-MjMfnA was transformed into the SenBA-containing recombinant bacteria constructed in Example 4 to construct a high-throughput screening system for MjMfnA, such as... Figure 4As shown in Figure a. The working principle of this system is as follows: by adding arabinose to the culture medium to induce the expression of MjMfnA, MjMfnA can catalyze the added L-aspartic acid to BA; the generated BA can be responded to by SenBA, thereby outputting a fluorescence signal. Based on this, the fluorescence signal can be correlated with the activity of MjMfnA, that is, the higher the activity of MjMfnA, the stronger the output fluorescence signal, thereby achieving the purpose of rapidly screening mutants with high MjMfnA activity by detecting the fluorescence signal intensity.

[0067] Since the concentration of arabinose affects the expression level of MjMfnA, suitable screening conditions were first determined. Recombinant bacteria containing p15A-MjMfnA and SenBA were inoculated into test tubes containing 3 mL of LB medium (containing kanamycin and chloramphenicol) and cultured at 37℃ and 200 rpm for 12 h. 50 μL of seed culture was then transferred to test tubes containing 5 mL of LB medium (containing kanamycin, chloramphenicol, 20 mM L-aspartic acid, and different concentrations of arabinose), with final arabinose concentrations of 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 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 at a arabinose concentration of 5 mM; therefore, a 5 mM arabinose concentration was used as the first-round screening condition.

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

[0069] Table 8. Determination of MjMfnA screening conditions based on SenBA

[0070] Example 7: Screening for highly active mutants of amino acid decarboxylase MjMfnA The structures of MjMfnA (shown in SEQ ID NO. 8) and PLP-Asp were analyzed, with amino acid residues within 5 Å of PLP-Asp serving as the catalytic pocket. Figure 5a) The catalytic pocket of MjMfnA includes: G35, S36, M37, C38, N56, G58, D59, G93, G94, T95, E96, N98, A131, H132, F133, S134, F135, K137, I177, G179, T180, T181, D206, A208, F209, T240, D242, H244, G254, Y273, L274, Q279, G284, T285, R371, and V373, totaling 36 amino acid residues. Considering the sequence distances of the residues in the catalytic pocket and their interactions with PLP-Asp, the 36 residues were grouped, with each group containing 1-2 residue sites, resulting in 21 groups. The grouping is shown in Table 9.

[0071] Using plasmid p15A-MjMfnA 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, and transformed into the *E. coli* strain JM109 containing SenBA constructed in Example 4. Transformants were cultured on LB agar plates containing 20 mM L-Asp and 5 mM arabinose at 37°C for 12 h. The plates were observed under a blue light microscope, and colonies with obvious green fluorescence were picked and cultured in 96-well deep-well plates in LB medium containing 20 mM L-Asp and 5 mM arabinose for initial screening. Transformants with high sfGFP expression levels were then transferred to test tubes containing LB medium containing 20 mM L-Asp and 5 mM arabinose for secondary screening and verification. The fluorescence results of the test tube screening are shown in [Table 10]. Figure 5 b and Table 10.

[0072] The results showed that, in the catalytic pocket residue grouping screening, five mutations with significantly increased fluorescence intensity were obtained: T240G, C38L, S134C, S134G, and D59N. Figure 5(b and Table 11). Among them, the C4 group residue mutation showed the most significant improvement (D59N), followed by the C9 group residue mutation (S134C, S134G), and then the C2 group mutation (C38L). Therefore, using the optimal C4 group mutation D59N as a template, random saturation mutations of C9 group residues were introduced (using primers PMjMfnA-C9-1 and PMjMfnA-C8 / 9-2, see Table 10) to construct a mutant library, which was then transformed into E. coli strain JM109 containing SenBA. The transformants were incubated statically at 37°C for 12 h on LB agar plates containing 20 mM L-Asp and 5 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 20 mM L-Asp and 0.5 mM arabinose. Fluorescence intensity was detected for initial screening. Transformants with high sfGFP expression levels were then transferred to LB medium containing 20 mM L-Asp and 0.5 mM arabinose for further screening and verification. The results showed that the double-point mutant D59N / S134G (SfGFP expression level) exhibited significantly higher fluorescence intensity compared to D59N. Figure 5 (c and Table 11). Continuing with D59N / S134G as a template, random saturation mutations of C2 group residues were introduced (using primers PMjMfnA-C2-1 and PMjMfnA-C1 / 2-2, see Table 10) to construct a mutant library, which was then transformed into *E. coli* strain JM109 containing SenBA. Transformants were incubated statically at 37°C for 12 h on LB agar plates containing 20 mM L-Asp and 5 mM arabinose. The plates were observed under a blue light microscope, and colonies with obvious green fluorescence were picked and transferred to 96-well deep-well plates for initial screening in LB medium containing 20 mM L-Asp and 0.5 mM arabinose. Transformants with high sfGFP expression levels were then transferred to test tubes containing LB medium containing 20 mM L-Asp and 0.5 mM arabinose for secondary screening and verification. Figure 5 As shown in c and Table 11, combined mutations that further enhanced fluorescence intensity were obtained: C38L / D59N / S134G, C38P / D59N / S134G, and M37F / C38S / D59N / S134G. Among them, M37F / C38S / D59N / S134G had the highest fluorescence intensity and was the optimal mutation.

[0073] Wild-type MjMfnA and mutant M37F / C38S / D59N / S134G were purified separately, and their specific activities were measured at 70℃. The results are as follows: Figure 5As shown in Figure d, the specific activity of the wild-type enzyme was 0.11 ± 0.02 U / mg, while that of the M37F / C38S / D59N / S134G enzyme was 2.57 ± 0.05 U / mg, showing a significant increase. The specific activity of the M37F / C38S / D59N / S134G enzyme was 24.3 times that of the wild-type enzyme. This result demonstrates the promising application of SenBA in screening enzymes that efficiently catalyze the conversion of L-Asp to BA.

[0074] Table 9 Residue grouping of the MjMfnA catalytic pocket

[0075] Table 10 GabR ligand-binding pocket residue grouping and mutation primers

[0076] Table 11 Screening of residue combinations for MjMfnA

[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 amino acid sequence shown in SEQ ID NO.5, it has one or more mutations among M208V, F250T, F431Y, and F431W.

2. The BsGabR mutant according to claim 1, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.5, methionine at position 208 was mutated to valine, phenylalanine at position 250 was mutated to threonine, and methionine at position 431 was mutated to tryptophan.

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

4. A biosensor responsive to β-alanine, 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 encoding gene of the BsGabR mutant is shown in SEQ ID NO. 6; 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 plasmids 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 biosensor according to any one of claims 4 to 6 in the screening of amino acid decarboxylases.

10. Amino acid decarboxylase mutants obtained by screening using any one of the biosensors described in claims 4 to 6.

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