L-phenylalanine biosensor based on cyclic rearrangement fluorescent protein and application of L-phenylalanine biosensor

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to an L-phenylalanine biosensor based on cyclic rearrangement fluorescent protein and application of the L-phenylalanine biosensor. Specifically, the L-phenylalanine biosensor at least comprises a fusion protein composed of an L-phenylalanine specific signal recognition element and a cyclic rearrangement fluorescent protein. Experiments prove that the biosensor developed by the invention has the advantages of high response amplitude, strong fluorescence brightness, moderate affinity and the like, and can realize qualitative, especially quantitative detection of L-phenylalanine in different biological samples and living bacteria and living mammalian cells in a high-efficiency, high-temporal-spatial-resolution and low-cost manner; therefore, the method has good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology, specifically relating to an L-phenylalanine biosensor based on cyclic rearranged fluorescent proteins and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] L-Phenylalanine is a crucial amino acid essential for normal physiological metabolism in the human body. In the human body, L-phenylalanine is catalyzed by L-phenylalanine hydroxylase to produce L-tyrosine. L-phenylalanine hydroxylase controls more than 75% of the catabolism of L-phenylalanine; abnormal expression of hydroxylase can lead to L-phenylalanine metabolic disorders, resulting in phenylketonuria (PKU). In PKU patients, due to the inhibited hydroxylation of L-phenylalanine, excessive accumulation of L-phenylalanine in the blood occurs. This L-phenylalanine is converted into phenylpyruvic acid, phenylacetic acid, and phenylethylamine glutamine via metabolic bypass and excreted in the urine. Common symptoms include intellectual disability, pale hair and skin, eczema, epilepsy, and extreme hyperactivity. Currently, clinical screening and diagnosis of PKU primarily use blood and urine L-phenylalanine concentrations as key indicators, and efficient detection of these concentrations is of significant clinical importance.

[0004] Conventional methods for L-phenylalanine detection include high-performance liquid chromatography (HPLC), mass spectrometry (MS), and capillary electrophoresis (CEE). However, these methods are typically expensive, time-consuming, and require specialized analytical equipment. Currently, several L-phenylalanine detection methods based on enzyme sensors and electrochemical sensors have been developed. For example, methods using NAD+... + The L-phenylalanine dehydrogenase PADH-mediated L-phenylalanine dehydrogenation reaction combines with the redox fluorescent indicator MPMS. Electrons generated from the PADH-catalyzed L-phenylalanine dehydrogenation are transferred to MPMS, and the resulting reduced form of MPMS exhibits bright fluorescence under UV irradiation. Phenylalanine ammonia-lyase PAL catalyzes the production of trans-cinnamic acid from L-phenylalanine. t -CA) reacts with ammonia, and is produced by oxidation with the addition of potassium permanganate. t -CA can cause changes in the light absorption of the sample solution. While enzyme-linked fluorescence assays are relatively sensitive, they are primarily based on stoichiometric NADH or... tQuantifying L-phenylalanine levels using α-CA is susceptible to interference from NADH or other metabolites that react with potassium permanganate in the sample, and requires cumbersome pretreatment procedures such as protein removal. Furthermore, Kamruzzaman M et al. developed a microfluidic chip-based chemiluminescent detection technique for L-phenylalanine, while ParriLLa M et al. developed a method for detecting L-phenylalanine in biological fluids using a wearable wristband-based electrochemical sensor. However, these microfluidic chip- or electrochemical sensor-based methods are relatively expensive and difficult to widely implement. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, the inventors, through long-term technical and practical exploration, have provided an L-phenylalanine biosensor based on a cyclic rearranged fluorescent protein and its applications. Specifically, the L-phenylalanine biosensor comprises at least a fusion protein consisting of an L-phenylalanine-specific signal recognition element and a cyclic rearranged fluorescent protein. Experiments have demonstrated that the biosensor developed in this invention possesses advantages such as high response amplitude, strong fluorescence brightness, and moderate affinity, enabling efficient, high spatiotemporal resolution, and low-cost qualitative and, especially, quantitative detection of L-phenylalanine in various biological samples, as well as in live bacteria and live mammalian cells. Based on the above research results, this invention has been completed.

[0006] Specifically, to achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a fusion protein comprising an L-phenylalanine-specific signal recognition element and a fluorescent protein; wherein the L-phenylalanine-specific signal recognition element is a prephenylylate dehydratase PheA; and the fluorescent protein may be a cyclic rearranged fluorescent protein.

[0007] In a second aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the aforementioned fusion protein.

[0008] A third aspect of the present invention provides a recombinant expression vector containing the isolated nucleic acid molecules described above.

[0009] In a fourth aspect, the present invention provides a host cell containing the isolated nucleic acid molecule, the recombinant expression vector, or a fusion protein expressed as described above.

[0010] A fifth aspect of the present invention provides a method for preparing the above-described fusion protein, the method comprising: The host cells were cultured, and the fusion protein was isolated and purified to obtain the fusion protein.

[0011] A sixth aspect of the present invention provides the use of the above-described fusion protein, nucleic acid molecule, vector and / or host cell in the preparation of a biosensor for detecting L-phenylalanine.

[0012] A seventh aspect of the present invention provides a biosensor for detecting L-phenylalanine, said biosensor comprising at least the aforementioned fusion protein.

[0013] An eighth aspect of the present invention provides a method for in vitro detection of L-phenylalanine, the method comprising at least: contacting a sample to be tested with the biosensor, and analyzing the concentration or presence of L-phenylalanine in the sample to be tested based on changes in the fluorescence signal of the biosensor.

[0014] A ninth aspect of the present invention provides a method for intracellular detection of L-phenylalanine, the method comprising at least: inducing the expression of the biosensor in the cell, and analyzing the concentration or presence of L-phenylalanine in the cell based on changes in the fluorescence signal of the biosensor.

[0015] A tenth aspect of the present invention provides the application of the above-described fusion protein, biosensor, and / or the above-described detection method in the detection of L-phenylalanine-related diseases and / or the screening of related drugs.

[0016] Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial technical effects: (1) The L-phenylalanine biosensor provided by the above technical solution is to insert a cyclic rearranged superfolded yellow fluorescent protein into the regulatory domain of the L-phenylalanine recognition element PheA (PheA) derived from Escherichia coli. ACT It is formed in the appropriate position within the structure. L-phenylalanine and its regulatory domain PheA ACT The conformational changes induced by the combination lead to conformational changes in the polypeptide backbone and amino acid side chains at the insertion site of the cyclic rearranged hypersheet yellow fluorescent protein, which further significantly alters the fluorescence intensity of the cyclic rearranged hypersheet yellow fluorescent protein. This change in fluorescence intensity can be used as an indicator for detecting L-phenylalanine concentration.

[0017] (2) The L-phenylalanine biosensor Phesor based on cyclic rearranged fluorescent protein provided by the above technical solution has a fluorescence response amplitude of 4028.18 ± 34.31% to L-phenylalanine, and an apparent dissociation constant ( K d The concentration was 1659.85 ± 110.02 μM, which indicates high specificity for L-phenylalanine.

[0018] (3) The L-phenylalanine biosensor Phesor provided by the above technical solution is suitable for quantitative detection of L-phenylalanine in a variety of biological samples, including human serum, urine and dried blood spot samples. It has the advantages of simple preparation, simple composition, low cost, convenient operation and high-throughput detection. It has important clinical application value for the development of diagnosis and treatment technology for L-phenylalanine-related diseases.

[0019] (4) The L-phenylalanine biosensor Phesor provided by the above technical solution is suitable for in vivo detection of L-phenylalanine in live bacteria and mammalian cells. It has the advantages of being non-invasive and having high spatiotemporal resolution. It can realize real-time and in-situ detection of L-phenylalanine in bacterial cells and mammalian cells, which has important scientific value for the development of research on the metabolic mechanism and functional diversity of L-phenylalanine. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 This is a schematic diagram illustrating the structural domain composition and functional analysis of PheA in Embodiment 1 of the present invention.

[0022] Figure 2 The initial sensors PheA-cpSFYFP and PheA in Embodiment 1 of this invention are... ACT Comparison of the response amplitudes of -cpSFYFP to 5 mM branched acids, prephenylacetic acid, phenylpyruvic acid, and L-phenylalanine. Figure A shows the response and specificity analysis of PheA-cpSFYFP; Figure B shows the response amplitude of PheA-cpSFYFP. ACT Response and specificity analysis of -cpSFYFP.

[0023] Figure 3 The circular rearrangement supersheeted yellow fluorescent protein inserted into PheA in Example 1 of this invention ACT Schematic diagrams of L-phenylalanine biosensor insert variants formed at different sites within the domain and comparison of the response amplitude of their crude extracts to L-phenylalanine.

[0024] Figure 4 This is the dose-response curve of Phesor-0.38 to L-phenylalanine in Example 1 of the present invention.

[0025] Figure 5 This study compares the response amplitude of crude extracts of 672 Phesor-0.38 Linker random mutants screened in Example 1 of this invention to L-phenylalanine.

[0026] Figure 6This is the dose-response curve of Phesor to L-phenylalanine in Example 1 of the present invention.

[0027] Figure 7 This is a specificity analysis of Phesor in Example 1 of the present invention.

[0028] Figure 8 This invention illustrates the application of Phesor in the quantitative detection of L-phenylalanine in human serum, urine, and dried blood spot samples in Example 2. Figure A shows a flowchart of the quantitative detection of L-phenylalanine in human body fluid samples based on Phesor; Figure B shows a consistency analysis of the quantitative detection results of L-phenylalanine in human serum samples using Phesor and HPLC; Figure C shows a consistency analysis of the quantitative detection results of L-phenylalanine in human urine samples using Phesor and HPLC; Figure D shows the quantitative detection results of L-phenylalanine in dried blood spot samples from healthy individuals and patients with phenylketonuria based on Phesor.

[0029] Figure 9 This study presents the gradient response and specificity analysis of Phesor expressed in Escherichia coli BL21(DE3) to L-phenylalanine in Example 3 of this invention.

[0030] Figure 10 This invention provides a functional identification of Phesor in Escherichia coli BL21(DE3) endogenous L-phenylalanine detection in Example 3 of the present invention.

[0031] Figure 11 This is an example of the gradient response and specificity analysis of Phesor expressed in human embryonic kidney cells HEK293FT to L-phenylalanine in Example 4 of the present invention. Figure A shows the specificity analysis of Phesor expressed in HEK293FT cells; Figure B shows the gradient response of Phesor expressed in HEK293FT cells to L-phenylalanine.

[0032] Figure 12 This invention provides a functional identification of Phesor used in the detection of endogenous L-phenylalanine in HEK293FT cells in Example 4 of this invention. Detailed Implementation

[0033] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0034] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0035] Terminology Definition In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures used herein, such as those in virology, biochemistry, nucleic acid chemistry, and immunology, are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0036] In this invention, "fusion protein" refers to oligopeptides, peptides, protein sequences or fragments thereof, and specifically to molecules that are actually formed, recombinant, synthetic, or semi-synthetic. It should be noted that the term "fusion protein" and similar terms do not imply that the amino acid sequence is limited to the full-length molecule containing the complete natural amino acid sequence. It should be understood that the various references to "fusion protein" and similar terms in this invention will include full-length sequences as well as any fragments, derivatives, or variants thereof.

[0037] In this invention, a "nucleic acid molecule" or "nucleic acid sequence" is a linear fragment of single- or double-stranded DNA or RNA that can be isolated from any source. In the context of this invention, preferably, a nucleic acid molecule is a DNA fragment. A "nucleic acid molecule" is also called a polynucleotide molecule.

[0038] In this invention, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0039] In this invention, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of compared positions multiplied by 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match).

[0040] In this invention, the twenty common amino acids involved are written in accordance with conventional usage. For example, ImmunoLogy-ASynthesis (2nd Edition, ES GoLub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented as A or ALa.

[0041] As mentioned earlier, there is an urgent need to develop an L-phenylalanine biosensor with high efficiency, high response, high spatiotemporal resolution, and low cost to meet the needs for its quantitative detection in vivo and in vitro.

[0042] In view of this, in a typical embodiment of the present invention, a fusion protein is provided, the fusion protein comprising at least an L-phenylalanine-specific signal recognition element and a fluorescent protein linked to the L-phenylalanine-specific signal recognition element.

[0043] The L-phenylalanine-specific signal recognition element can be derived from Escherichia coli (E. coli). Escherichia coLi The branched acid mutase PheA (NCBI number WP_000200120.1) of MG1655 is regulated by specific allosteric changes of L-phenylalanine. Of course, based on the concept of this invention, other known L-phenylalanine-specific signal recognition elements or PheA homologs are also applicable to the technical solutions of this application and therefore fall within the scope of protection of this invention.

[0044] In this invention, the fluorescent protein is preferably a circular rearranged fluorescent protein, specifically a class of visual reporter gene-encoded proteins, including circular rearranged cyan fluorescent protein (cpTFP), circular rearranged green fluorescent protein (cpGFP), circular rearranged yellow fluorescent protein (cpYFP), and circular rearranged red fluorescent protein (cpRFP). In one specific embodiment of this invention, the circular rearranged fluorescent protein used is a cpYFP variant containing four mutation sites (S30R, Y39N, N105T, Y145F) (circular rearranged hyperfolded yellow fluorescent protein cpSFYFP, which has the characteristic of strong fluorescence intensity). When L-phenylalanine is present, the conformational change induced by the binding of L-phenylalanine to the signal recognition element PheA can lead to a conformational change in cpSFYFP, thereby greatly changing the fluorescence properties of the sensor, thus realizing the detection of L-phenylalanine.

[0045] Furthermore, the fusion protein can be obtained by linking the cyclic rearranged fluorescent protein to the N-terminus or C-terminus of the transcription regulatory factor, or the fusion protein can be obtained by inserting the cyclic rearranged fluorescent protein into the prebenzoic acid dehydratase PheA; specifically, the amino acid sites of the cyclic rearranged fluorescent protein cpSFYFP inserted into the prebenzoic acid dehydratase PheA are as follows: 286K / 287A, 288I / 289N, 289N / 290V, 290V / 291S, 291S / 292D, 292D / 293Q, 293Q / 294V, 294V / 295P, 295P / 296A, 303A / 304T, 3 04T / 305G, 305G / 306Q, 306Q / 307Q, 307Q / 308A, 308A / 309G, 309G / 310A, 320N / 321H, 321H / 322N , 322N / 323L, 323L / 324I, 324I / 325M, 332P / 333I, 333I / 334H, 334H / 335G, 335G / 336N, 336N / 3 37P, 337P / 338W, 338W / 339E, 339E / 340E, 346I / 347Q, 347Q / 348A, 348A / 349N, 349N / 350L, 350L / 351E, 351E / 352S, 352S / 353A, 365I / 366T, 366T / 367R, 367R / 368S, 376P / 377S, 377S / 378E, 378E / 379N, 379N / 380V, 380V / 381V, 381V / 382P, 382P / 383V, 383V / 384D, 384D / 385P, 385P / 386T. It should be noted that cpSFYFP uses linkers with an N-terminus of "serine-alanine-glycine" and a C-terminus of "glycine-glycine-cysteine" to connect with PheA... ACT The upper and lower segments of the domain are connected. In a preferred embodiment of the present invention, the fusion protein is a cyclic rearranged superfolded yellow fluorescent protein cpSFYFP with an N-terminal linker of "alanine-arginine-leucine" and a C-terminal linker of "glutamine-histidine-leucine", inserted between amino acids 286-365 and 366-386 of PheA.

[0046] Specifically, the fusion protein is selected from: (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO.1; (a2) A protein having the same or similar function by substituting, deleting and / or adding one or more amino acid residues of the amino acid sequence shown in (a1). (a3) has an amino acid sequence composition that is 40% or more identical to that shown in (a1) or (a2) and has the same or similar function as the protein shown in (a1) or (a2).

[0047] In (a2), the substitution, deletion and / or addition of one or more amino acid residues generally refers to the substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0048] In one specific embodiment of the invention, the fusion protein further comprises a signal peptide and / or a tag protein. Further, the fusion protein comprises a signal peptide at its N-terminus. In some embodiments, the fusion protein comprises a tag protein, such as a His tag, at its C-terminus. No specific limitations are imposed herein.

[0049] The fusion proteins described in this invention can be prepared using various methods known in the art, such as genetic engineering (recombinant technology) or chemical synthesis (e.g., the Fmoc solid-phase method). The fusion proteins of this invention are not limited by their method of preparation.

[0050] Therefore, in one or more embodiments of the present invention, an isolated nucleic acid molecule is provided, the nucleic acid molecule being capable of encoding the above-mentioned fusion protein.

[0051] Specifically, the nucleic acid molecule has any of the nucleotide sequences described in (b1)–(b4): (b1) The nucleotide sequence as shown in SEQ ID NO.2; (b2) A sequence formed by substitution, deletion and / or addition of one or more nucleotides as shown in (b1); (b3) has 40% or more identity with the nucleotide sequence defined in (b1) or (b2) and is a nucleic acid molecule encoding the fusion protein; (b4) A nucleotide sequence that, under stringent conditions, can hybridize with any of the nucleotide sequences described in (b1)–(b3) and encode a fusion protein with the same function.

[0052] In the above nucleic acid molecules, the stringent conditions can be as follows: hybridization at 50 °C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5 M Na3PO4, and 1 mM EDTA, followed by rinsing at 50 °C in 2×SSC and 0.1% SDS; or hybridization at 50 °C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, followed by rinsing at 50 °C in 1×SSC and 0.1% SDS; or hybridization at 50 °C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, followed by rinsing at 50 °C in 0.5×SSC and 0.1% SDS; or hybridization at 50 °C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, followed by rinsing at 50 °C in a mixed solution of 0.5 M Na3PO4 and 1 mM EDTA. Alternatively, hybridization can be performed at 50 °C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, followed by rinsing at 65 °C in 0.1×SSC and 0.1% SDS; or hybridization can be performed at 65 °C in a solution of 6×SSC and 0.5% SDS, followed by washing once each with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

[0053] The aforementioned 40% or more of identity can be 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% or more of identity.

[0054] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA, etc., without specific limitations.

[0055] In one or more specific embodiments of the present invention, a recombinant expression vector is provided, wherein the recombinant expression vector comprises at least the above-mentioned nucleic acid molecules.

[0056] The recombinant expression vector can be any one or more of a viral vector, plasmid, bacteriophage, phage particle, granule, or artificial chromosome; the viral vector may include adenovirus vector, retrovirus vector, or adeno-associated virus vector; the artificial chromosome includes bacterial artificial chromosome, phage P1-derived vector, yeast artificial chromosome, or mammalian artificial chromosome; more preferably, it is a plasmid; the plasmid includes, but is not limited to, pETDuet-1 and pcDNA3.1. (+) .

[0057] In one or more specific embodiments of the present invention, a host cell is provided, wherein the host cell contains the above-mentioned nucleic acid molecule, the above-mentioned recombinant expression vector, or is capable of expressing the above-mentioned fusion protein.

[0058] The host cells include bacterial cells, fungal cells, and animal cells; The bacteria can be any one or more of Escherichia coli, Agrobacterium, Bacillus, Streptomyces, Pseudomonas, or Staphylococcus.

[0059] In one or more specific embodiments of the present invention, the bacteria include, but are not limited to, Escherichia coli (e.g., BL21(DE3)), Agrobacterium tumefaciens (e.g., GV3101), Agrobacterium rhizogenes, Lactococcus lactis, Bacillus subtilis, Bacillus cereus, or Pseudomonas fluorescens.

[0060] The fungal cells include yeast.

[0061] The animal cells may be mammalian cells, and more specifically, the mammalian cells include, but are not limited to, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, and HEK293FT cells.

[0062] In one or more specific embodiments of the present invention, a method for preparing the above-mentioned fusion protein is provided, the method comprising: culturing the above-mentioned host cells, and isolating and purifying the fusion protein.

[0063] In one or more specific embodiments of the present invention, the application of the above-mentioned fusion protein, nucleic acid molecule, recombinant expression vector and / or host cell in the preparation of a biosensor for detecting L-phenylalanine is provided.

[0064] In one or more specific embodiments of the present invention, a biosensor for detecting L-phenylalanine is provided, wherein the biosensor comprises at least the aforementioned fusion protein.

[0065] That is, the L-phenylalanine biosensor is a biosensor Phesor constructed by inserting cpSFYFP with the N-terminal linker "alanine-arginine-leucine" and the C-terminal linker "glutamine-histidine-leucine" between amino acids 286-365 and 366-386 of PheA (its amino acid sequence is shown in SEQ ID NO.1 and its nucleotide sequence is shown in SEQ ID NO.2).

[0066] Furthermore, the biosensor may also include other reagents, devices, and / or equipment for the detection of L-phenylalanine.

[0067] For example, in this invention, the reagent may contain a detection buffer (such as a fluorescence assay buffer: 50 mM Tris-HCl, pH 7.4).

[0068] In practical applications, the aforementioned biosensors can exist in the form of detection kits, particularly for the detection of L-phenylalanine in the (in vitro) environment.

[0069] When biosensors are used to detect L-phenylalanine in the body (such as within cells), they can be directly induced to express in cells, thereby detecting the presence or concentration changes of intracellular L-phenylalanine.

[0070] Therefore, in one or more specific embodiments of the present invention, a method for in vitro detection of L-phenylalanine is provided, the method comprising at least: contacting the sample to be tested with the biosensor, and analyzing the concentration or presence of L-phenylalanine in the sample to be tested based on the change in fluorescence signal of the L-phenylalanine biosensor.

[0071] The sample to be tested is a sample containing or suspected of containing L-phenylalanine. The sample can be a biological sample or an environmental sample. The biological sample includes, but is not limited to, bacterial culture medium, bacterial lysate, cell culture medium, cell lysate, animal blood (such as whole blood, serum, dried blood spot samples, etc.), animal urine, and animal tissue fluid. The animal may be a mammal, with humans being preferred.

[0072] The environmental samples include, but are not limited to, water bodies (such as surface water and wastewater), soil leachate, food processing liquids, and fermentation liquids. Optionally, the samples may be pretreated, such as by dilution, filtration, centrifugation to remove impurities, and adjustment of pH or ionic strength, to adapt them to the detection environment of the biosensor.

[0073] At this time, the L-phenylalanine may also include other reagents, devices and / or equipment for the detection of L-phenylalanine; The reagents include detection buffers (such as fluorescence assay buffer: 50 mM Tris-HCl, pH 7.4).

[0074] In one or more specific embodiments of the present invention, a method for intracellular detection of L-phenylalanine is provided, the method comprising at least: inducing the expression of an L-phenylalanine biosensor in cells, and analyzing the concentration or presence of L-phenylalanine in cells based on changes in the fluorescence signal of the L-phenylalanine biosensor.

[0075] At this point, the L-phenylalanine biosensor is the aforementioned fusion protein.

[0076] The cells may be bacterial cells, fungal cells, or animal cells, and are not specifically limited thereto. In specific embodiments of the present invention, the cells include, but are not limited to, Escherichia coli BL21(DE3) cells and human embryonic kidney cells (HEK293FT).

[0077] The above methods can be used to achieve qualitative or quantitative detection of L-phenylalanine in vitro or in vivo.

[0078] In another specific embodiment of the present invention, the application of the above-mentioned fusion protein, biosensor and / or detection method is provided in the detection of L-phenylalanine-related diseases and / or screening of related drugs.

[0079] The L-phenylalanine-related diseases include, but are not limited to, hereditary amino acid metabolism disorders (such as pentylosinic ketoacidosis), diseases related to abnormal function of phenylalanine metabolic enzymes (including hereditary diseases caused by gene mutations in phenylalanine hydroxylase, dihydropteridine reductase, etc.), and phenylalanine metabolic pathway disorders caused by other factors, manifested as accumulation of phenylalanine and abnormal metabolites in the blood and tissues, as well as secondary phenylalanine metabolic disorders (including abnormal phenylalanine levels and related neurological, developmental, and metabolic pathological states that may be caused by liver and kidney dysfunction, other metabolic diseases, nutritional abnormalities, drug or exogenous substance interference, etc.). This information can be used to assist or directly contribute to the (newborn) screening, clinical diagnosis, disease monitoring, treatment adjustment, efficacy evaluation, and prognosis of L-phenylalanine-related diseases.

[0080] The relevant drugs include, but are not limited to, L-phenylalanine metabolism regulators, phenylalanine hydroxylase activators, and L-phenylalanine clearance promoters. Specifically, L-phenylalanine metabolism regulators include compounds or preparations capable of reducing the rate of L-phenylalanine production in vivo, promoting the conversion of L-phenylalanine to tyrosine, or accelerating the clearance of L-phenylalanine and its metabolites. Phenylalanine hydroxylase activators include compounds or preparations capable of enhancing the activity of key enzymes in the phenylalanine metabolic pathway (such as phenylalanine hydroxylase) and improving the supply of cofactors (such as tetrahydrobiopterin) to promote the efficient metabolism of L-phenylalanine. L-phenylalanine clearance promoters include compounds that affect the intestinal absorption, renal excretion, or intracellular transport of L-phenylalanine, thereby reducing the level of L-phenylalanine in the blood and tissues.

[0081] The present invention will be further illustrated below with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. In the following embodiments, the expression vectors pETDuet-1 and pcDNA3.1 are used. (+) All materials and reagents used were purchased from Novagen; unless otherwise specified, all other materials and reagents used were obtained commercially. Unless otherwise specified, all experimental methods used were standard methods.

[0082] Example 1: Construction and optimization of L-phenylalanine biosensor (1) Feasibility and specificity modification of PheA for constructing L-phenylalanine biosensors The ACT domain (Pfam ID: PF01842) is a well-studied regulatory domain that can bind to specific amino acids and regulate enzyme activity. It originates from *Escherichia coli* (…). Escherichia coLi The cladization acid mutase / prephenylic acid dehydratase PheA (NCBI No.: WP_000200120.1) of MG1655 is an allosteric enzyme in the L-phenylalanine synthesis pathway. It consists of a cladization acid mutase domain (CM), a prephenylic acid dehydratase domain (PDT), and an ACT domain. Previous studies have found that L-phenylalanine can bind to the ACT domain of PheA and inhibit cladization acid mutase and prephenylic acid dehydratase activities through an allosteric effect (see appendix). Figure 1 ).

[0083] To clarify the feasibility of using PheA to construct an L-phenylalanine biosensor and to optimize the sensor's specificity by truncating the catalytic domain of PheA, a cyclic rearranged supersheeted yellow fluorescent protein cpSFYFP was linked to full-length PheA. ACT After constructing the domain (i.e., lysine 286 to threonine 386 of PheA), the recombinant plasmids pETDuet-PheA-cpSFYFP and pETDuet-PheA were obtained. ACT -cpSFYFP. In the above-mentioned recombinant plasmid, cpSFYFP is linked to the full-length PheA and PheA lines via an N-terminal "serine-alanine-glycine" linker. ACT The structural domains are connected.

[0084] The recombinant plasmids pETDuet-PheA-cpSFYFP and pETDuet-PheA were used. ACT -cpSFYFP was transformed into the expression strain *Escherichia coli* BL21(DE3), and the successfully constructed strains were screened on LB agar plates containing ampicillin. Single colonies were picked, and the strains were preserved after successful verification by PCR and sequencing.

[0085] The initial sensors PheA-cpSFYFP and PheA were obtained through exogenous expression and purification. ACT -cpSFYFP. The initial sensor was mixed with L-phenylalanine and the compounds involved in the catalytic reaction, namely cladonic acid, prephenylic acid, and phenylpyruvic acid. The change in the fluorescence ratio (F) of the initial sensor before and after the addition was measured using a fluorescence microplate reader. 488 nm / F 405 nm The ratio of fluorescence emission intensity at 528 nm under 488 nm excitation to that under 405 nm excitation was measured to determine the sensor's response to the above compounds. The results are attached. Figure 2As shown, PheA-cpSFYFP responds to four compounds, including L-phenylalanine, while the catalytic domain PheA is truncated. ACT -cpSFYFP can specifically respond to L-phenylalanine, but has no obvious response to branched acid, prephenylalanine, or phenylpyruvic acid.

[0086] pETDuet-PheA-cpSFYFP, pETDuet-PheA ACT The construction method of -cpSFYFP is as follows: using the pETDuet-cpSFYFP constructed in the laboratory as a template, reverse PCR amplification was performed to obtain a linearized plasmid backbone containing cpSFYFP; using the genome of E. coli MG1655 as a template, PCR amplification was performed to obtain full-length PheA and PheA... ACT DNA fragments containing structural domains. A DNA assembly method based on the T5 exonuclease was used to ligate the plasmid backbone and full-length PheA and PheA domains. ACT Domain-specific DNA fragments. Specifically, 5 μL of the recombinant target gene fragment and a linearized plasmid backbone are added to a 15 μL ligation system, with a molar ratio of target gene fragment to linearized plasmid of 4:1. Incubate at 30 ºC for 40 minutes, then cool on ice for 10 minutes to obtain recombinant plasmids pETDuet-PheA-cpSFYFP and pETDuet-PheA. ACT -cpSFYFP. Transform it into the expression strain. E. coLi In BL21(DE3), strains successfully constructed were screened by plating on LB agar plates containing ampicillin. Single colonies were picked, and the strains were preserved after successful verification by bacterial culture PCR and sequencing.

[0087] The pETDuet-cpSFYFP plasmid backbone was amplified by reverse PCR, and the primers were designed as follows: Upstream primer: 5'-CATCGGATCCTGGCTGTGG-3'; Downstream primer: 5'-TCTGCAGGCTTCAACAGCGA-3'.

[0088] Full-length PheA and PheA derived from Escherichia coli MG1655 were amplified by PCR. ACT For the DNA fragment containing the structural domain, the primer design is as follows: PheA upstream primer: 5'-ACAGCCAGGATCCGATGACATCGGAAAACCCGTTACTG-3'; PheA downstream primer: 5'-CTGTTGAAGCCTGCAGAGGTTGGATCAACAGGCACTACG-3'; PheA ACTUpstream primer: 5'-ACAGCCAGGATCCGATGAAAGCCATTAACGTGTCTGATCAG-3'; PheA ACT Downstream primer: 5'-CTGTTGAAGCCTGCAGAGGTTGGATCAACAGGCACTACG-3'.

[0089] The purification method for the above biosensor is as follows: The recombinant plasmids pETDuet-PheA-cpSFYFP and pETDuet-PheA are purified... ACT -cpSFYFP E. coLi BL21(DE3) strain was inoculated at a ratio of 2% into 500 mL of LB medium containing ampicillin and cultured at 37 ºC and 180 rpm until OD. 600 nm Approximately 0.6 mg of the solution was added with 1 mM IPTG, and protein expression was induced overnight at 23 °C and 160 rpm. The bacterial cells were collected by centrifugation, washed twice with binding buffer, and resuspended at OD200. 600 nm The bacterial cells were lysed with 1 mM PMSF and 10% glycerol, and centrifuged at 12,000 rpm and 4 ºC for 50 min to remove cell debris, thus obtaining a crude extract. The crude extract was filtered through a 0.22 μm filter and purified using a 5 mL nickel column. The purified biosensor was obtained by elution with different concentrations of elution buffer.

[0090] The fluorescence detection method for the above biosensor response is as follows: Dilute the purified initial sensor to 4 / 3 μM using detection buffer. Prepare 20 mM concentrations of L-phenylalanine, cladonic acid, prephenylic acid, and phenylpyruvic acid using detection buffer. Under light-protected conditions, mix the purified sensor with the above-mentioned specific concentration compounds at a volume ratio of 3:1. Transfer 100 μL of the mixture to a black flat-bottomed 96-well plate. Detect the fluorescence intensity ratio of the sensor using a PerkinElmer Ensight fluorescence microplate reader. R (F) 488 nm / F 405 nm The fluorescence intensity varies. Instrument parameters were set as follows: excitation wavelengths of 488 nm and 405 nm, and emission wavelength of 528 nm. The ratio of fluorescence intensity measured under 488 nm excitation to that under 405 nm excitation (F...) is... 488 nm / F 405 nm The change in level is used to assess the sensor's response.

[0091] (2) Preliminary construction of L-phenylalanine biosensor PCR amplification was derived from PheA in Escherichia coli MG1655. ACT DNA fragments of structural domains, through BamH I / Hind The III restriction site was cloned into the pETDuet-1 expression vector to obtain PheA. ACT Domain expression plasmid pETDuet-PheA ACT Transform it into the expression strain E. coLi In BL21(DE3), strains that were successfully constructed were screened by plating on LB agar plates containing ampicillin. Single colonies were picked, and the strains were preserved after successful verification by PCR and sequencing.

[0092] pETDuet-PheA ACT Based on this, in PheA ACT The following sites are selected in the middle of the domain for insertion into the cyclic rearrangement of the hyperfolded yellow fluorescent protein cpSFYFP: 286K / 287A, 288I / 289N, 289N / 290V, 290V / 291S, 291S / 292D, 292D / 293Q, 293Q / 294V, 294V / 295P, 295P / 296A, 303A / 304T, 3 04T / 305G, 305G / 306Q, 306Q / 307Q, 307Q / 308A, 308A / 309G, 309G / 310A, 320N / 321H , 321H / 322N, 322N / 323L, 323L / 324I, 324I / 325M, 332P / 333I, 333I / 334H, 334H / 33 5G, 335G / 336N, 336N / 337P, 337P / 338W, 338W / 339E, 339E / 340E, 346I / 347Q, 347Q / 348A, 348A / 349N, 349N / 350L, 350L / 351E, 351E / 352S, 352S / 353A, 365I / 366T, 36 Fifty sensor insertion variant coding plasmids were constructed using the following sequences: 6T / 367R, 367R / 368S, 376P / 377S, 377S / 378E, 378E / 379N, 379N / 380V, 380V / 381V, 381V / 382P, 382P / 383V, 383V / 384D, 384D / 385P, and 385P / 386T. In these recombinant plasmids, cpSFYFP was linked to PheA using N-terminal "serine-alanine-glycine" and C-terminal "glycine-glycine-cysteine" linkers. ACT The upper and lower segments of a structural domain are connected.

[0093] The aforementioned sensor insertion variant encoding plasmid was transformed into the expression strain *Escherichia coli* BL21(DE3), and the resulting strains were screened on LB agar plates containing ampicillin. Single colonies were picked, and after successful verification by PCR and sequencing, the strains were preserved.

[0094] The above 50 sensor insert variants and PheA were obtained through exogenous expression and ultrasonic disruption. ACT The crude extract of -cpSFYFP was used. The crude extract was mixed with 0 mM and 20 mM L-phenylalanine at a volume ratio of 3:1, and the fluorescence ratio (F) was measured using a fluorescence microplate reader. 488 nm / F 405 nm ), calculate the above 50 biosensor insert variants with PheA ACT The response amplitude Δ of the crude extract of -cpSFYFP to 5mM L-phenylalanine R (( R 5 mM - R 0 mM ) / R 0 mM (See attached) Figure 3 As shown, three sensors are inserted into the variant Phesor. 338W / 339E Phesor 365I / 366T Phesor 367R / 368S The crude extract had a higher response amplitude than PheA. ACT -cpSFYFP. Following the purification method for the biosensor described in (1) above, the three sensor insert variants were exogenously expressed and purified. The purified biosensor insert variants were mixed with gradient concentrations of L-phenylalanine, and the fluorescence ratio (F) of the sensor insert variants under gradient concentration L-phenylalanine conditions was detected using a fluorescence microplate reader. 488 nm / F 405 nm Plotting L-phenylalanine concentration on the x-axis and fluorescence ratio on the y-axis, dose-response curves of the sensor insert variant to L-phenylalanine were obtained by fitting the data, and the maximum response amplitude to L-phenylalanine was calculated. R max (i.e., the maximum ratio change). See attached. Figure 4 As shown, insert cpSFYFP into PheA ACT The sensor insert variant (named Phesor-0.38) obtained with amino acid sites between 365I and 366T in the structural domain exhibited the highest maximum response amplitude, at 67.16 ± 2.88%, and the apparent dissociation constant ( K d The value was 444.08 ± 79.38 μM.

[0095] pETDuet-PheA ACTThe construction method is as follows: via BamH I / Hind III. Enzyme digestion of the pETDuet-1 expression vector yielded a linearized plasmid backbone (carrying...) BamH I / Hind III restriction enzyme site); using the E. coli MG1655 genome as a template, PheA was obtained by PCR amplification. ACT DNA fragments containing the structural domain. The plasmid backbone and cpSFYFP fragment were ligated using a T5 exonuclease-based DNA assembly method to obtain the recombinant plasmid pETDuet-PheA. ACT Transform it into the expression strain E. coLi In BL21(DE3), strains that were successfully constructed were screened by plating on LB agar plates containing ampicillin. Single colonies were picked, and the strains were preserved after successful verification by PCR and sequencing.

[0096] PheA derived from Escherichia coli MG1655 was amplified by PCR. ACT For the DNA fragment containing the structural domain, the primer design is as follows: Upstream primer: 5'-CACCACAGCCAGGATCCGATGAAAGCCATTAACGTGTCTGATCAG-3', carrying a BamH I site; Downstream primer: 5'-CATTATGCGGCCGCAAGCTTTCAGGTTGGATCAACAGGCACT-3', carrying a Hind Site III.

[0097] PheA ACT Biosensor variants with different insertion sites within the domain were constructed as follows: using the recombinant plasmid pETDuet-PheA ACT Using pETDuet-cpSFYFP as a template, a linearized plasmid backbone was obtained by reverse PCR amplification at the insertion sites listed above. Using the laboratory-constructed pETDuet-cpSFYFP as a template, the cpSFYFP DNA fragment was obtained by PCR amplification. The plasmid backbone and the cpSFYFP fragment were ligated using a DNA assembly method based on T5 exonuclease to obtain the recombinant plasmid pETDuet-PheA. 286-n -cpSFYFP-PheA (n+1)-386 Transform it into the expression strain E. coLi In BL21(DE3), strains that were successfully constructed were screened by plating on LB agar plates containing ampicillin. Single colonies were picked, and the strains were preserved after successful verification by PCR and sequencing.

[0098] pETDuet-PheA was amplified by reverse PCR. ACTThe plasmid backbone was prepared, and the DNA fragment of cpSFYFP was amplified by PCR. The primers were designed as follows: upstream primer for cpSFYFP: 5'-TCTGCAGGCTTCAACAGCGACAACGTCTATATCAT-3'; Downstream primer for cpSFYFP: 5'-ACAGCCACCGTTGTACTCCAGCTTGTGCCCCAGGATGTTGCCG-3'.

[0099] The fluorescence detection method for the response of the crude extract of the above biosensor is as follows: the expression strain carrying the recombinant plasmid of the above biosensor insert variant is inoculated at a ratio of 1% into 50 mL of LB medium containing ampicillin, and cultured at 37 ºC and 180 rpm until OD. 600 nm Approximately 0.6 mg of the solution was added with 1 mM IPTG, and protein expression was induced overnight at 23 °C and 160 rpm. The bacterial cells were collected by centrifugation and resuspended in assay buffer to OD200. 600 nm Add 1 mM PMSF to the sample, sonicate to disrupt the bacterial cells, and centrifuge 1 mL of the disrupted liquid at 12,000 rpm and 4 ºC for 10 minutes to remove cell debris to obtain a crude extract. Mix the crude extract with 0 mM and 20 mM L-phenylalanine at a volume ratio of 3:1, and transfer 100 μL of the mixture to a black flat-bottomed 96-well plate. Detect the fluorescence ratio using a PerkinElmer Ensight fluorescent microplate reader. R (F) 488 nm / F 405 nm The instrument parameters were set as follows: excitation wavelengths of 488 nm and 405 nm, and emission wavelength of 528 nm. The ratio of the fluorescence intensity measured under 488 nm excitation to that measured under 405 nm excitation was calculated. R (F) 488 nm / F 405 nm ), calculate the response amplitude Δ of the crude extract of the biosensor insert variant to 5 mM L-phenylalanine. R (( R 5 mM - R 0 mM ) / R 0 mM ).

[0100] The dose-response curve detection method for the above biosensor is as follows: The purified biosensor is diluted to 4 / 3 μM using detection buffer. A gradient concentration of L-phenylalanine is prepared using detection buffer. Under light-protected conditions, the biosensor and L-phenylalanine solution are mixed at a volume ratio of 3:1. 100 μL of the mixture is transferred to a black flat-bottomed 96-well plate. The fluorescence ratio of the biosensor under the gradient concentration of L-phenylalanine is detected using a PerkinElmer Ensight fluorescence microplate reader. R (F) 488 nm / F 405 nm The instrument parameters were set as follows: excitation wavelengths of 488 nm and 405 nm, and emission wavelength of 528 nm. A dose-response curve of the biosensor to L-phenylalanine was obtained by fitting the L-phenylalanine concentration to the x-axis and the fluorescence ratio to the y-axis, and the maximum response amplitude of the biosensor to L-phenylalanine was calculated. R max (i.e., the maximum ratio change) and the apparent dissociation constant K d .

[0101] (3) Optimization of L-phenylalanine biosensor based on cyclic rearranged fluorescent protein Based on Phesor-0.38, this study targets cpSFYFP and PheA in Phesor-0.38. ACT Linkers that connect the upper and lower segments are used to construct a library of random mutants, and a high-throughput screening strategy is used to further optimize the sensor's response amplitude.

[0102] Using the recombinant plasmid pETDuet-Phesor-0.38 (i.e. pETDuet-Phesor 365I / 366T Using pETDuet-cpSFYFP as a template, a linearized plasmid backbone with breaks at the 365I and 366T insertion sites was obtained by inverse PCR amplification; using pETDuet-cpSFYFP as a template, the cpSFYFP fragment containing a random linker was amplified by degenerate primers (such as ON-mutation and OC-mutation); and a recombinant plasmid encoding a biosensor variant containing a random linker was obtained by DNA assembly based on T5 exonuclease.

[0103] The above recombinant plasmid was transformed into the expression strain. E. coLiIn BL21(DE3), the samples were plated on LB agar plates containing ampicillin for subsequent high-throughput screening. Single colonies with bright fluorescence were picked from the plates, cultured overnight to induce protein expression, and then sonicated using a Scientz-48TD multichannel sonicator. After centrifugation at 4 °C and 12,000 rpm for 10 minutes, crude extracts were obtained. The response amplitude Δ of the crude extracts of each biosensor random mutant to 5 mM L-phenylalanine was determined according to the fluorescence detection method for crude extract response described in (2) above. R (( R 5 mM - R 0 mM ) / R 0 mM (See attached) Figure 5 As shown, among the 672 randomly selected mutants, 7 Linker random mutants exhibited a response amplitude exceeding 80% to 5 mM L-phenylalanine. Following the biosensor purification method described in (1) above, these 7 Linker random mutants were exogenously expressed and purified. The dose-response curves of these 7 Linker random mutants to L-phenylalanine were obtained according to the biosensor dose-response curve determination method described in (2) above, and the maximum response amplitude to L-phenylalanine was calculated. R max (i.e., the maximum ratio change). See attached. Figure 6 As shown, cpSFYFP inserts into PheA with an N-terminal linker of "alanine-arginine-leucine" and a C-terminal linker of "glutamine-histidine-leucine". ACT The L-phenylalanine biosensor variant obtained at amino acid sites 365I / 366T within the domain exhibits the highest Δ R max The value was 4028.18 ± 34.31%. K d The value was 1659.85 ± 110.02 μM, and it was named Phesor.

[0104] Phesor was used to detect the fluorescence response of the biosensor at 1 mM according to the fluorescence detection method in (1), which included 20 L-amino acids, including L-phenylalanine, as well as structural analogs such as d-phenylalanine, phenylpyruvic acid, and 2-phenylacetamide. N The responses of acetyl-L-phenylalanine and d-phenyllactic acid are shown in the attached figure. Figure 7 As shown, Phesor exhibits high specificity for L-phenylalanine, but shows no significant response to other compounds.

[0105] The method for constructing a Linker random mutant library based on Phesor-0.38 is as follows: using the recombinant plasmid pETDuet-Phesor-0.38 (i.e., pETDuet-Phesor...) 365I / 366T Using pETDuet-cpSFYFP as a template, a linearized plasmid backbone with breaks at the 365I and 366T insertion sites was obtained by reverse PCR amplification. Using pETDuet-cpSFYFP as a template, a cpSFYFP fragment containing a random linker was amplified by degenerate primers (such as ON-mutation and OC-mutation). Recombinant plasmids encoding a biosensor variant containing a random linker were obtained using a T5 exonuclease-based DNA assembly method. This plasmid was then transformed into the expression strain. E. coLi BL21(DE3) was applied to LB agar plates containing ampicillin.

[0106] The pETDuet-Phesor-0.38 plasmid backbone was amplified by reverse PCR. The primers were designed as follows: Phesor-0.38 reverse PCR upstream primer: 5'-GATTTCCCCTAACTCTTTCAATGC-3'; Phesor-0.38 reverse PCR downstream primer: 5'-ACCCGTTCAATGAAGGTATTGGG-3'.

[0107] The cpSFYFP fragment containing a random linker was amplified by PCR. The primers were designed as follows: 3N-mutation primer: 5'-AAGAGTTAGGGGAAATCNNBNNBNNBTTCAACAGCGACAACGTCTATATC-3'; 2N-mutation primer: 5'-AAGAGTTAGGGGAAATCNNBNNBTTCAACAGCGACAACGTCTATATC-3'; 1N-mutation primer: 5'-AAGAGTTAGGGGAAATCNNBTTCAACAGCGACAACGTCTATATC-3'; 0N-mutation primer: 5'-AAGAGTTAGGGGAAATCTTCAACAGCGACAACGTCTATATC-3'; 3C-mutation primer: 5'-CTTCATTGAACGGGTVNNVNNVNNGTTGTACTCCAGCTTGTGCCCCA-3'; 2C-mutation primer: 5'-CTTCATTGAACGGGTVNNVNNGTTGTACTCCAGCTTGTGCCCCA-3'; 1C-mutation primer: 5'-CTTCATTGAACGGGTVNNGTTGTACTCCAGCTTGTGCCCCA-3'; 0C-mutation primers: 5'-CTTCATTGAACGGGTGTGTACTCCAGCTTGTGCCCCA-3'.

[0108] The fluorescence detection method for random mutants combined with high-throughput screening of the above biosensor is as follows: pick a bright fluorescent single colony from the plate and induce protein expression overnight in the presence of 1 mM IPTG; take 15 mL of bacterial culture, centrifuge at 4 °C and 6,000 rpm for 10 minutes, collect the bacterial cells, resuspend them with 2.5 mL of detection buffer and transfer them to a 48-well deep plate, add PMSF to a final concentration of 1 mM; use a Scientz-48TD multichannel ultrasonic disruptor to disrupt the bacterial cells, take 1 mL of the disruption liquid and centrifuge at 4 °C and 12,000 rpm for 10 minutes, collect the supernatant; determine the response amplitude of the crude extract of the random mutant to 5 mM L-phenylalanine according to the fluorescence detection method of crude extract response in (2) above, screen random mutants with high response amplitude, and obtain sequence information by sequencing.

[0109] The LB medium formula mentioned in the above steps is as follows: 10 g / L peptone; 5 g / L yeast extract; 10 g / L NaCl, pH 7.0; sterilized at 121 ºC for 20 minutes.

[0110] The formulation of the 15 μL ligation system in the DNA assembly method based on T5 exonuclease described in the above steps is as follows: 4 μL 5× isothermal reaction buffer (0.5 M Tris-HCl, 0.05 M MgCl2, 0.05 M dithiothreitol), 0.004 μL 10 U / μL T5 exonuclease, and 11 μL ddH2O.

[0111] The binding buffer formulation mentioned in the above steps is: 20 mM Na2HPO4, 20 mM imidazole, 500 mM NaCl, adjusted to pH 7.4; the elution buffer formulation is: 20 mM Na2HPO4, 500 mM imidazole, 500 mM NaCl, adjusted to pH 7.4.

[0112] The detection buffer solution described in the above steps is formulated as follows: 50 mM Tris, with the pH adjusted to 7.4 using HCl.

[0113] Example 2: Application of Phesor in the quantitative detection of L-phenylalanine in human serum and urine samples (1) Preparation of human serum, urine and dried blood spot samples Serum samples were collected from healthy adults. Venous blood was placed in a coagulation tube and allowed to stand at room temperature for 2 h. The blood was then centrifuged at 4 ℃ and 2,000 × g for 30 min. The supernatant was filtered through a 0.22 μm filter membrane and stored at -80 ℃ for later use.

[0114] Urine samples were collected from healthy adults. The urine was collected directly into sterile centrifuge tubes, filtered through a 0.22 μm filter membrane, and stored at -80 ℃ for later use.

[0115] Dried blood spot samples were collected from healthy children and children with phenylketonuria. Approximately 25-75 μL of whole blood was adsorbed onto filter paper cards, air-dried at room temperature, and then sealed for storage. To extract L-phenylalanine from the dried blood spots, the blood spot area was completely cut into small pieces, transferred to 500 μL of detection buffer, sonicated for 20 min, vortexed for 2 min, and stirred overnight. The resulting supernatant was centrifuged at 12,000 × g for 10 min, and the supernatant was thoroughly dried using a vacuum centrifuge at 45 °C. The supernatant was resuspended in an appropriate amount of detection buffer, filtered through a 0.22 μm filter membrane, and stored at -80 °C for later use. (2) Phesor and HPLC were used for the quantitative detection and consistency analysis of L-phenylalanine in human serum, urine and dried blood spot samples. The HPLC-based quantitative detection procedure for L-phenylalanine in human serum, urine, and dried blood spot samples is as follows: Serum samples were mixed with methanol at a 1:1 volume ratio, vortexed for 2 min, centrifuged at 12,000 × g for 15 min, filtered through a 0.22 μm filter, and then analyzed by high-performance liquid chromatography. Urine and treated dried blood spot samples were boiled at 105 °C for 15 min, centrifuged at 12,000 × g for 10 min, filtered through a 0.22 μm filter, and then analyzed by high-performance liquid chromatography.

[0116] The procedure for establishing dose-response curves for Phesor in detecting L-phenylalanine in serum and urine samples is as follows. Gradual concentrations of L-phenylalanine (40, 120, 200, 400, 1200, 2000, 4000, 12000, 20000, 40000, 120000 μM) were added to the serum and urine samples. Following the biosensor dose-response curve determination method in Example 1 (2) above, Phesor was diluted to 2 μM with detection buffer. Under light-protected conditions, the purified Phesor was mixed with the treated serum and urine samples at a 1:1 volume ratio. After incubation at room temperature for 10 min, the mixture was transferred to a black flat-bottomed 384-well plate. The fluorescence ratio (F) of Phesor was read using a PerkinElmer Ensight fluorescence microplate reader. 488 nm / F 405 nm The dose-response curves of Phesor for detecting L-phenylalanine in serum and urine samples were obtained by fitting the data.

[0117] The procedure for quantitative detection of L-phenylalanine in human serum and urine samples based on Phesor is attached. Figure 8 As shown in A. Different concentrations of L-phenylalanine were added to serum and urine samples from healthy individuals to simulate samples from patients with hyperphenylalaninemia or phenylketonuria. Phesor was diluted to 2 μM according to the biosensor response determination method in Example 1 (1). Under light-protected conditions, the purified Phesor was mixed with serum samples (real samples from healthy adults and simulated samples from patients with hyperphenylalaninemia or phenylketonuria) and urine samples (real samples from healthy adults and simulated samples from patients with hyperphenylalaninemia or phenylketonuria) at a volume ratio of 1:1. After incubation at room temperature for 10 min, the mixture was transferred to a black flat-bottomed 384-well plate. The fluorescence ratio (F) of Phesor was read using a PerkinElmer Ensight fluorescence microplate reader. 488 nm / F 405 nm The measured Phesor fluorescence ratio was then substituted into the dose-response curves established above for detecting known L-phenylalanine concentrations in serum and urine samples based on Phesor for quantification. The treated dried blood spot samples were then measured and quantified using the same procedure.

[0118] Further analysis of the consistency between Phesor and HPLC in the quantitative detection of L-phenylalanine in human serum and urine samples is shown in the attached figure. Figure 8 B. Appendix Figure 8 As shown in Figure C, the results of physor and HPLC measurements exhibit good correlation and consistency. (Appendix) Figure 8 D indicates that Phesor can reliably quantify L-phenylalanine in dried blood spot samples, and has the potential for use in neonatal phenylketonuria screening and clinical testing.

[0119] The chromatographic conditions for the quantitative determination of L-phenylalanine by HPLC were as follows: ZORBAX SB-C18 column (5 μm, 4.6 mm × 150 mm), detection wavelength 210 nm, column temperature 30 °C, and flow rate 0.6 mL / min. -1 The injection volume was 10 μL, the detection time was 30 min, and the mobile phase was 10% (v / v) acetonitrile.

[0120] Example 3: Application of Phesor in Real-time Detection of L-Phenylalanine in Live Bacteria (1) Functional identification of Phesor in Escherichia coli BL21(DE3) The construction in Example 1 E. coLi BL21(DE3)-pETDuet-Phesor strain was inoculated at a 1% inoculum in 50 mL of solution containing 100 mg L. -1 Ampicillin was cultured in LB medium at 37 °C and 180 rpm until OD. 600 nm Approximately 0.6 mg / L was added to 1 mM IPTG, and Phesor expression was induced overnight at 23 °C and 160 rpm. An appropriate amount of bacterial culture was collected by centrifugation at 6,000 rpm and 4 °C for 2 minutes. The cells were washed twice with phosphate buffer, and then resuspended in phosphate buffer supplemented with 9.5 mM EDTA, 0.1% toluene, and 1% glycerol to OD200. 600 nm The concentration was 5.0. After vortexing for 2 min, the mixture was transferred to 4 ℃ and allowed to stand for 10 h to induce bacterial permeation and excretion of endogenous L-phenylalanine. The permeated bacterial solution was diluted 10-fold with ddH2O. 99 μL of the bacterial solution was mixed with 1 μL of a gradient concentration of L-phenylalanine, L-tyrosine, and L-tryptophan in a black flat-bottomed 96-well plate, and the fluorescence intensity was continuously read using a PerkinElmer Ensight fluorescence microplate reader. The instrument parameters were set as follows: excitation wavelengths of 405 nm and 488 nm, emission wavelengths of 528 nm, temperature of 30 ℃, rotation speed of 180 rpm, and detection interval of 1 minute. (See attached image) Figure 9 As shown, in E. coLi Phesor expressed in BL21(DE3) can respond in real time to the addition of gradient concentrations of exogenous L-phenylalanine in a dose-dependent manner, and has high specificity for L-phenylalanine, with no significant change in fluorescence ratio for L-tyrosine and L-tryptophan.

[0121] (2) Application of Phesor in the detection of L-phenylalanine in Escherichia coli BL21(DE3) exist E. coLiIn BL21(DE3), the biosynthesis and transport of aromatic amino acids are transcriptionally regulated by the transcription factor TyrR. TyrR can recognize three aromatic amino acids and inhibit the expression of DAHP synthase AroF, thereby reducing the carbon flux of the aromatic amino acid synthesis pathway. This was previously achieved in a chassis strain constructed in the laboratory. E. coLi BL21 (DE3) Δ LacZ Δ frdA Δ LdhA Δ poxB Δ pfLB Δ pta Δ ackA Knock off tyrR Furthermore, the introduction of the A294S mutation into phenylalanine-tRNA synthetase led to its restriction of L-phenylalanine. K m The process was repeated, ultimately resulting in engineered E. coli strains with intracellular L-phenylalanine accumulation. E. coLi BL21 (DE3) Δ LacZ Δ frdA Δ LdhA Δ poxB Δ pfLB Δ pta Δ ackA Δ tyrR Δ pheRS:: pheRS A294S .

[0122] To determine whether Phesor can be used for analysis E. coLi BL21(DE3) showed fluctuations in endogenous L-phenylalanine. Phesor was expressed in the engineered *E. coli* strain with intracellular L-phenylalanine accumulation constructed above, and inoculated at a 1% inoculum size into 50 mL containing 100 mg of L-phenylalanine. -1 Ampicillin was cultured in LB medium at 37 °C and 180 rpm until OD. 600 nm Add approximately 0.6 mg of 1 mM IPTG and incubate overnight at 23 °C and 160 rpm to induce Phesor expression. Collect an appropriate amount of bacterial culture, centrifuge at 6,000 rpm and 4 °C for 2 minutes to collect the cells, wash twice with carbon-free M9 medium, and resuspend to OD200. 600 nm The culture was incubated at 5.0 mg / L under carbon-starved conditions for 8 h, followed by the addition of 20 mM glucose to terminate carbon starvation. The fluorescence ratio (F-value) of the Phesor-expressing *E. coli* strain was measured using a PerkinElmer Ensight fluorescence microplate reader before carbon starvation, 8 h after carbon starvation, and 10 min and 20 min after glucose addition. 488 nm / F 405 nm (See attached) Figure 10As shown, the engineered E. coli strain expressing Phesor accumulates higher concentrations of L-phenylalanine intracellularly and is broken down during carbon starvation, while L-phenylalanine can be reaccumulated intracellularly after glucose supplementation.

[0123] The phosphate buffer (1 L) formulation mentioned in the above steps is as follows: 2.26 g KH2PO4, 4.1 g K2HPO4, 2.24 g NaH2PO4·H2O, 3.34 g Na2HPO4, pH adjusted to 7.0 with NaOH, sterilized at 121 ºC for 20 minutes.

[0124] The carbon-free M9 medium formulation described in the above steps is as follows: 34 mM Na2HPO4, 22 mM KH2PO4, 8.5 mM NaCl, 9.3 mM NH4Cl, 1 mM MgSO4, 300 μM CaCl2, 149 μM Na2EDTA, 6 μM ZnCl2, 600 nM CoCl2, 80 nM MnCl2, 31 μM FeCl3, 760 nM CuCl2, 1.62 μM H3BO3, pH adjusted to 7.4, and sterilized at 121 ºC for 20 minutes.

[0125] Example 4: Application of Phesor in the quantitative detection of L-phenylalanine in mammalian cells (1) Functional identification of phosor in human cell HEK293FT Phesor's nucleic acid sequence underwent mammalian codon optimization and whole-genome synthesis by General Biosystems (Anhui) Co., Ltd. A Kozak sequence (5'-GCCACC-3') was added before the start codon and ligated into pcDNA3.1. (+) plasmids, and stored in E. coLi Top 10 strains. Recombinant plasmid pcDNA3.1-Phesor was extracted from this strain and transfected into HEK293FT cells. 26 h after transfection, cells were washed twice with 1× Hank's balanced salt buffer supplemented with 20 mM HEPES. Cells were then subjected to fluorescence imaging at 30-second intervals under a Zeiss 900 laser confocal microscope. Instrument parameters were set as follows: excitation wavelengths of 405 nm and 488 nm, and emission wavelengths from 497 to 617 nm. Cells were first treated with 80 μM digitalis saponin in the imaging medium to induce cell permeation. After 5 min, gradient concentrations of L-phenylalanine, L-tyrosine, and L-tryptophan were added, and the response of Phesor expressed in HEK293FT cells to L-phenylalanine and its structural analogues L-tyrosine and L-tryptophan was continuously imaged. (See attached image.) Figure 11As shown, Phesor can respond in real time to the addition of exogenous L-phenylalanine in a dose-dependent manner, while showing no significant response to L-tyrosine and L-tryptophan, demonstrating its potential for real-time detection of L-phenylalanine in mammalian cells.

[0126] (2) Application of Phesor in the detection of L-phenylalanine in human cells HEK293FT In HEK293FT cells, the L-amino acid transporter LAT1 plays a role in the uptake of L-phenylalanine by human cells. To determine whether Phesor could be used to analyze endogenous L-phenylalanine fluctuations in HEK293FT cells, the LAT1 expression plasmid pcDNA3.1-LAT1 was obtained as described above. 0, 5, and 10 mM L-phenylalanine were added to the culture medium of HEK293FT cells transfected with Phesor alone and co-transfected with Phesor and LAT1. The fluorescence ratio of Phesor expression in HEK293FT cells was imaged 20 h after the addition of L-phenylalanine. (See attached image.) Figure 12 As shown, the fluorescence ratio of Phesor increased after LAT1 overexpression in HEK293FT cells.

[0127] The amino acid / nucleotide sequences involved in this invention Phesor's amino acid sequence: MKAINVSDQVPAKTTLLMATGQQAGALVEALLVLRNHNLIMTRLESRPIHGNPWEEMFYLDIQANLESAEMQKALKELGEIARLFNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNV DGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGTYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNQHLTRSMKVLGCYPSENVVPVDPT (SEQ ID NO.1) Phesor's nucleotide sequence: The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fusion protein, characterized in that, The fusion protein includes an L-phenylalanine-specific signal recognition element and a fluorescent protein; The L-phenylalanine-specific signal recognition element is prephenylacetic acid dehydratase PheA; The fluorescent protein is specifically a cyclic rearranged fluorescent protein, further including any one or more of cyclic rearranged cyan fluorescent protein, cyclic rearranged green fluorescent protein, cyclic rearranged yellow fluorescent protein, and cyclic rearranged red fluorescent protein; Furthermore, the fusion protein is selected from: (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO.1; (a2) A protein with the same or similar function as the amino acid sequence shown in (a1) by substitution, deletion and / or addition of one or more amino acid residues. (a3) has an amino acid sequence composition that is 40% or more identical to that shown in (a1) or (a2) and has the same or similar function as the protein shown in (a1) or (a2).

2. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein of claim 1; Furthermore, the nucleic acid molecule has any of the nucleotide sequences described in (b1)–(b4): (b1) The nucleotide sequence as shown in SEQ ID NO.2; (b2) A sequence formed by substitution, deletion and / or addition of one or more nucleotides as shown in (b1); (b3) has 40% or more identity with the nucleotide sequence defined in (b1) or (b2) and is a nucleic acid molecule encoding the fusion protein; (b4) A nucleotide sequence that, under stringent conditions, can hybridize with any of the nucleotide sequences described in (b1)–(b3) and encode a fusion protein with the same function.

3. A recombinant expression vector, characterized in that, The recombinant expression comprises at least the nucleic acid molecule as described in claim 2.

4. A host cell, characterized in that, The host cell contains the nucleic acid molecule of claim 2, the recombinant expression vector of claim 3, or is capable of expressing the fusion protein of claim 1.

5. The method for preparing the fusion protein according to claim 1, characterized in that, The preparation method includes: culturing the host cells as described in claim 4, and separating and purifying the fusion protein.

6. The use of the fusion protein of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3, and / or the host cell of claim 4 in the preparation of a biosensor for detecting L-phenylalanine.

7. A biosensor for detecting L-phenylalanine, characterized in that, The biosensor comprises at least the fusion protein as described in claim 1.

8. The biosensor as described in claim 7, characterized in that, The biosensor also includes other reagents, devices, and / or equipment for the detection of L-phenylalanine.

9. A method for in vitro detection of L-phenylalanine, characterized in that, The method includes at least: incubating the sample to be tested with the biosensor described in claim 7 or 8, and analyzing the concentration or presence of L-phenylalanine in the sample to be tested based on the change in fluorescence signal of the biosensor; The sample to be tested is any biological or environmental sample containing or suspected of containing L-phenylalanine.

10. A method for intracellular detection of L-phenylalanine, characterized in that, The method includes at least: inducing the expression of the biosensor of claim 7 in cells, and analyzing the concentration or presence of L-phenylalanine in cells based on changes in the fluorescence signal of the biosensor; Furthermore, the cells are bacterial cells, fungal cells, or animal cells.

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