A method for designing a fluorescent sensor protein, its product and detection method

Abstract

The present invention relates to a fluorescent sensor protein design method, its product and detection method, which belong to the field of biotechnology. The fluorescent sensor protein design method is characterized in that the distance between the amino acids at both ends of the allosteric domain of the protein is less than 10Å, and the distance change is more than 2Å. Based on the design method, the present invention also provides a fluorescent sensor protein and its gene, recombinant expression vector, transformant and fluorescent sensor, and a method for high-throughput real-time detection of intracellular metabolites. The fluorescent sensor protein includes a sterol O-acyltransferase after allosteric transformation and a cyclic fluorescent protein cpEGFP; the sterol O-acyltransferase after allosteric transformation has an amino acid sequence as shown in SEQ ID NO. 1; the cyclic fluorescent protein cpEGFP has an amino acid sequence as shown in SEQ ID NO. 2. The fluorescent sensor protein is used to detect the concentration of intracellular sterol substances, and the detection is more real-time, providing a more accurate tool for the analysis of related metabolic flows.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a fluorescent sensor protein design method, a product thereof, and a detection method. Background Art

[0002] Sterols are a class of lipid compounds derived from a cyclopentahydrophenanthrene nucleus. By introducing different functional groups at different positions within the nucleus, compounds with varying functional activities can be obtained. For example, cholesterol and 7-dehydrocholesterol in the human body not only possess diverse physiological activities and anti-cancer properties, but are also important precursors for several important drugs, such as adrenocortical hormones and sex hormones.

[0003] With the development of synthetic biology, the de novo synthesis of sterols and some of their derivatives can be achieved by constructing complete sterol metabolic pathways in microbial cells, such as Saccharomyces cerevisiae. Furthermore, exploring and analyzing metabolic pathways and optimizing key genes within these pathways can effectively increase the production of metabolites.

[0004] However, when constructing metabolic pathways and optimizing key enzymes, the detection of sterol compounds is often crucial for efficient construction. Currently, commonly used detection methods include liquid-gas chromatography-mass spectrometry, electrochemical detection, colorimetry, and molecular luminescence. However, these methods are in vitro assays that require complex sample preparation and are unable to perform high-throughput, real-time detection of intracellular metabolites. Summary of the Invention

[0005] The present invention provides a fluorescent sensor protein design method, its product and detection method, aiming to solve the above-mentioned problem in the prior art that it is impossible to perform high-throughput real-time detection of intracellular metabolites.

[0006] The present invention adopts the following technical solutions:

[0007] A method for designing a fluorescent sensor protein, wherein the distance between the amino acids at both ends of the allosteric domain of the protein is less than 10 Å, and the distance variation is more than 2 Å.

[0008] The protein is a sterol protein;

[0009] Preferably, the sterol protein is selected from the group consisting of: sterol O-acyltransferase, cholesterol oxidase ChoR 1, cholesterol 7α-hydroxylase CYP7A, 7-dehydrocholesterol reductase DHCR7, lecithin cholesterol acyltransferase LCAT, intestinal cholesterol acyltransferase SOAT2.

[0010] The allosteric domain of the sterol O-acyltransferase is selected from the group consisting of: L132-E134, T411-W420, V135-I138, L437-K445, T419-V424;

[0011] Preferably, the sterol O-acyltransferase has the amino acid sequence shown in SEQ ID NO.1.

[0012] A fluorescent sensor protein comprising a sterol O-acyltransferase after conformational change using the fluorescent sensor protein design method and a cyclic fluorescent protein cpEGFP; the sterol O-acyltransferase after conformational change has the amino acid sequence shown in SEQ ID NO. 1; the cyclic fluorescent protein cpEGFP has the amino acid sequence shown in SEQ ID NO. 2.

[0013] Preferably, the fluorescent sensor protein is obtained by replacing the amino acid sequence of the cyclic fluorescent protein cpEGFP with that of the threonine at position 419 to the valine at position 424 of the allosteric domain of the sterol O-acyltransferase after the allosteric change;

[0014] In a preferred embodiment, linker amino acids are added to both ends of the amino acid sequence of the circulating fluorescent protein cpEGFP;

[0015] In a more preferred embodiment, the linker amino acid at the N-terminus is GG, and the linker amino acid at the C-terminus is GGAAA;

[0016] In a more specific embodiment, the fluorescent sensor protein has an amino acid sequence as shown in SEQ ID NO.3.

[0017] A gene for a fluorescent sensor protein having a nucleotide sequence as shown in SEQ ID NO. 4.

[0018] A recombinant expression vector is connected to the gene sequence of the fluorescent sensor protein.

[0019] A transformant is transformed with the recombinant expression vector.

[0020] A fluorescent sensor, characterized by being selected from: the fluorescent sensor protein, and / or the gene of the fluorescent sensor protein, and / or the recombinant expression vector, and / or the transformant.

[0021] A method for high-throughput real-time detection of intracellular metabolites, which uses the fluorescent sensor to ferment a substrate.

[0022] Fermentation products were collected on different fermentation days for fluorescence detection;

[0023] Preferably, the fluorescence detection includes: performing GC-MS detection using a gas chromatography-mass spectrometer and performing fluorescence intensity detection using an enzyme marker.

[0024] The amino acid sequence of the sterol O-acyltransferase involved in the fluorescent sensor protein of the present invention is shown in (SEQ ID NO. 1).

[0025] Under the stimulation of 7-dehydrocholesterol, its allosteric domains are L132-E134 (12-16); T411-W420 (291-302);

[0026] More preferred allosteric domains are V135-I138 (15-20); L437-K445 (317-329);

[0027] The more preferred allosteric domain is T419-V424 (299-306);

[0028] The present invention provides a design standard for the change of the distance between the amino acids at both ends of the allosteric domain.

[0029] Considering the intensity of fluorescence induced by allosteric changes, the distance between the amino acids at both ends of the allosteric domain should be designed to be less than 10 Å, and the distance change should be more than 2 Å.

[0030] The present invention provides a method for constructing a fluorescent sensor protein. The sensor can perform fluorescence characterization on the concentration of 7-dehydrocholesterol in cells.

[0031] The invention provides a recombinant Pichia pastoris, comprising a constructed detection system based on sterol O-acyltransferase, and characterizing its detection performance in vitro.

[0032] The present invention provides a recombinant Saccharomyces cerevisiae, comprising a synthetic pathway for 7-dehydrocholesterol, a constructed detection system based on sterol O-acyltransferase, and in vivo verification of the detection performance.

[0033] The beneficial effects of the present invention are as follows: Compared with existing technologies, this invention provides, for the first time, a fluorescent sensing system and design method capable of real-time detection of steroid metabolites in cells. This design method and the resulting fluorescent sensor protein used to construct the detection system are applicable to the in vivo reconstitution and synthesis of steroid products, making detection more convenient and providing a more efficient tool for studying key enzymes and rate-limiting steps. Furthermore, this fluorescent sensor protein is applicable to the detection of intracellular steroid concentrations, providing more real-time detection and a more precise tool for analyzing related metabolic fluxes. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the change in the distance between the amino acids at both ends of the allosteric domain and the fluorescence response to 7-dehydrocholesterol in Experimental Example 1 of the present invention.

[0035] Figure 2Schematic diagram of the construction of the fluorescent sensor protein of Experimental Example 2 of the present invention.

[0036] Figure 3 This is a gel image of nucleic acid gel detection of the 299-306 fluorescent sensor protein in Experimental Example 3 of the present invention.

[0037] Figure 4 This is the in vitro detection curve of the 299-306 fluorescent sensor protein in Experimental Example 4 of the present invention.

[0038] Figure 5 This is a graph showing the specific detection results of the 299-306 fluorescent sensor protein in Experimental Example 4 of the present invention.

[0039] Figure 6 Schematic diagram of the fluorescence response of the fluorescent sensor protein of Experimental Example 6 of the present invention to intracellular 7-dehydrocholesterol.

[0040] Figure 7 Schematic diagram of the fluorescence response of the fluorescent sensor protein of Experimental Example 6 of the present invention to different intracellular concentrations of 7-dehydrocholesterol. DETAILED DESCRIPTION

[0041] The present invention will be further described in detail below in conjunction with specific embodiments and experimental examples so that those skilled in the art can more clearly understand the present invention. The following experimental examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following experimental examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following experimental examples are commercially available products well known to those skilled in the art unless otherwise specified.

[0042] The first group of embodiments, the fluorescent sensor protein design method of the present invention

[0043] This group of embodiments provides a method for designing fluorescent sensor proteins. All embodiments in this group share the following common features: the distance between the amino acids at both ends of the allosteric domain of the protein is less than 10 Å, and the distance variation is greater than 2 Å.

[0044] Under the induction of 7-dehydrocholesterol molecules, the distance between the amino acids at both ends of the allosteric domain of the protein changes. This change is the key to the generation of fluorescence. The structural diagram of the allosteric domain of the protein is shown in FIG. Figure 2 By molecular dynamics simulation, the present invention calculates the distance change between the amino acids at both ends of the allosteric domain of the protein before and after the induction of 7-dehydrocholesterol molecules to obtain the distance change value (such as Figure 1 As shown), and further by constructing a sensor, the intensity of fluorescence was verified experimentally ( Figure 4-Figure 7), and then the distance change value with the best fluorescence intensity, which can realize high-throughput real-time detection of intracellular metabolites, is above 2Å.

[0045] In a preferred embodiment, the protein is a sterol protein;

[0046] In a specific embodiment, the sterol proteins herein refer to the sum of a class of proteins that use sterol compounds (such as cholesterol, 7-dehydrocholesterol, plant sterols and other lipid compounds with a cyclopentane polyhydrophenanthrene nucleus) as response molecules.

[0047] Preferably, the sterol protein is selected from the group consisting of: sterol O-acyltransferase, cholesterol oxidase ChoR 1, cholesterol 7α-hydroxylase CYP7A, 7-dehydrocholesterol reductase DHCR7, lecithin cholesterol acyltransferase LCAT, intestinal cholesterol acyltransferase SOAT2.

[0048] In a specific embodiment, the allosteric domain of the sterol O-acyltransferase is selected from the group consisting of: L132-E134, T411-W420, V135-I138, L437-K445, T419-V424;

[0049] In a specific embodiment, L132-E134 represents the region of sterol O-acyltransferase that can undergo significant structural changes under the induction of 7-dehydrocholesterol, namely, leucine at position 132 to glutamate at position 134, threonine at position 411 to tryptophan at position 420, valine at position 135 to isoleucine at position 138, leucine at position 437 to lysine at position 445, and threonine at position 419 to valine at position 424.

[0050] Preferably, the sterol O-acyltransferase has the amino acid sequence shown in SEQ ID NO.1.

[0051] The second group of examples, the fluorescent sensor protein of the present invention

[0052] This group of embodiments provides a fluorescent sensor protein. All embodiments in this group share the following common features: the fluorescent sensor protein comprises a modified sterol O-acyltransferase and a circulating fluorescent protein cpEGFP; the modified sterol O-acyltransferase has the amino acid sequence set forth in SEQ ID NO. 1; and the circulating fluorescent protein cpEGFP has the amino acid sequence set forth in SEQ ID NO. 2.

[0053] Any act of cloning, amplifying, enriching, expressing, connecting, transforming, synthesizing, culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, transforming, selling, or offering for sale the amino acid sequence shown in SEQ ID NO.1 after conformational change, and / or combining the amino acid sequence shown in SEQ ID NO.1 after conformational change with a fluorescent protein (including but not limited to cyclic fluorescent proteins cpEGFP and cpERFP) to construct a fluorescent sensor or fluorescent sensor protein or a bioluminescent sensor or a bioluminescent sensor protein, and / or using the amino acid sequence shown in SEQ ID NO.1 after conformational change to sense and monitor intracellular metabolites including but not limited to 7-dehydrocholesterol, falls within the scope of protection of the present invention.

[0054] The other fluorescent sensors or fluorescent sensor proteins or bioluminescent sensors or bioluminescent sensor proteins include but are not limited to: fluorescent sensor proteins described in CN102952181A and fluorescent sensors described in CN118104133A.

[0055] Those skilled in the art can, based on actual production needs, combine conventional technical means or basic common sense of production processes in the field of molecular biology or genetic engineering (for example, "Practical Molecular Biology Operation Guide", "Molecular Biology Experimental Techniques Experimental Operation Guide", "Molecular Cloning Experimental Guide", "Concise Molecular Biology Experimental Guide", etc.) to reverse compile the amino acid sequence of the fluorescent sensor protein to obtain its gene sequence, design specific amplification primers to obtain its gene sequence, and ligate the gene sequence with an expression vector to obtain a recombinant expression vector that can express a fluorescent sensor protein, or further transform the recombinant expression vector into competent cells to obtain transformants that can express a fluorescent sensor protein, and then propagate and culture the transformants under conditions suitable for their growth to efficiently produce the fluorescent sensor protein. This is possible and easy for those skilled in the art to do without any technical obstacles.

[0056] In a specific embodiment, the allosteric domain of the allosteric sterol O-acyltransferase is replaced by the amino acid sequence of the circulating fluorescent protein cpEGFP to obtain Figure 2 The cyclic fluorescent protein cpEGFP of the structure shown is inserted into the fluorescent sensor protein inside the sterol O-acyltransferase after the conformational change;

[0057] More specifically, the amino acid sequence of the cyclic fluorescent protein cpEGFP is replaced by the threonine at position 419 to the valine at position 424 of the allosteric domain of the sterol O-acyltransferase to obtain the 299-306 fluorescent sensor protein, whose amino acid sequence is shown in SEQ ID NO.3 and the corresponding gene sequence is shown in SEQ ID NO.4.

[0058] In some embodiments, the amino acid sequence of the cyclic fluorescent protein cpEGFP is substituted from leucine at position 132 to glutamate at position 134 of the allosteric domain of the allosteric sterol O-acyltransferase to obtain a 12-16 fluorescent sensor protein, the corresponding gene sequence of which is shown in SEQ ID NO.5.

[0059] In other embodiments, the amino acid sequence of the cyclic fluorescent protein cpEGFP is substituted from threonine at position 411 to tryptophan at position 420 in the allosteric domain of the allosteric sterol O-acyltransferase to obtain the 291-302 fluorescent sensor protein, the corresponding gene sequence of which is shown in SEQ ID NO.6.

[0060] In some embodiments, the amino acid sequence of the cyclic fluorescent protein cpEGFP is substituted from valine 135 to isoleucine 138 in the allosteric domain of the allosteric sterol O-acyltransferase to obtain a 15-20 fluorescent sensor protein, the corresponding gene sequence of which is shown in SEQ ID NO.7.

[0061] In other embodiments, the amino acid sequence of the cyclic fluorescent protein cpEGFP is substituted from leucine 437 to lysine 445 in the allosteric domain of the allosteric sterol O-acyltransferase to obtain a 317-329 fluorescent sensor protein, the corresponding gene sequence of which is shown in SEQ ID NO.8.

[0062] In a preferred embodiment, linker amino acids are added to both ends of the amino acid sequence of the circulating fluorescent protein cpEGFP;

[0063] In a more preferred embodiment, the linker amino acid at the N-terminus is GG, and the linker amino acid at the C-terminus is GGAAA;

[0064] In a more specific embodiment, the fluorescent sensor protein has an amino acid sequence as shown in SEQ ID NO.3, or its gene has a nucleotide sequence as shown in SEQ ID NO.4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 or SEQ ID NO. 8.

[0065] The third group of examples, the gene of the fluorescent sensor protein of the present invention

[0066] This group of embodiments provides a gene for a fluorescent sensor protein. All embodiments in this group share the following common feature: the gene for the fluorescent sensor protein is selected from the nucleotide sequence shown in SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, or SEQ ID NO. 8.

[0067] Any act of cloning, amplifying, enriching, expressing, connecting, transforming, synthesizing, culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, transforming, selling, or offering for sale a gene sequence as shown in SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, or SEQ ID NO. 8, and / or combining an amino acid sequence obtained by transcribing and translating a gene sequence as shown in SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, or SEQ ID NO. 8 with other fluorescent sensors or fluorescent sensor proteins or bioluminescent sensors or bioluminescent sensor proteins, and / or using an amino acid sequence obtained by transcribing and translating a gene sequence as shown in SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, or SEQ ID NO. 8 to sense and monitor intracellular metabolites including but not limited to 7-dehydrocholesterol falls within the scope of protection of the present invention. In addition to 7-dehydrocholesterol, those skilled in the art can also develop and design corresponding fluorescent sensors or fluorescent sensor proteins or bioluminescent sensors or bioluminescent sensor proteins based on the teachings of the present invention for intracellular metabolites.

[0068] The other fluorescent sensors or fluorescent sensor proteins or bioluminescent sensors or bioluminescent sensor proteins include but are not limited to: fluorescent sensor proteins described in CN102952181A and fluorescent sensors described in CN118104133A.

[0069] Those skilled in the art can, based on actual production needs, combine conventional technical means or basic common sense of production processes in the field of molecular biology or genetic engineering (for example, "Practical Molecular Biology Operation Guide", "Molecular Biology Experimental Techniques Experimental Operation Guide", "Molecular Cloning Experimental Guide", "Concise Molecular Biology Experimental Guide", etc.) to reverse compile the amino acid sequence of the fluorescent sensor protein to obtain its gene sequence, design specific amplification primers to obtain its gene sequence, and ligate the gene sequence with an expression vector to obtain a recombinant expression vector that can express a fluorescent sensor protein, or further transform the recombinant expression vector into competent cells to obtain transformants that can express a fluorescent sensor protein, and then propagate and culture the transformants under conditions suitable for their growth to efficiently produce the fluorescent sensor protein. This is possible and easy for those skilled in the art to do without any technical obstacles.

[0070] The fourth group of embodiments, the recombinant expression vector of the present invention

[0071] This group of embodiments provides a recombinant expression vector. All embodiments in this group have the following common feature: the recombinant expression vector is connected to the gene sequence of a fluorescent sensor protein described in any one of the third group of embodiments.

[0072] In some embodiments, the recombinant expression vector is an expression vector connected to the gene sequence of a fluorescent sensor protein described in any one of the third group of embodiments;

[0073] In a specific embodiment, the expression vector is selected from: pGAPZαC vector, pESC vector.

[0074] Any act of cloning, amplifying, enriching, expressing, connecting, transforming, synthesizing, culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, transforming, selling, or offering for sale a recombinant expression vector, and / or combining a fluorescent sensor protein expressed by the recombinant expression vector with other fluorescent sensors or fluorescent sensor proteins or bioluminescent sensors or bioluminescent sensor proteins, and / or using a fluorescent sensor protein expressed by the recombinant expression vector to sense and monitor intracellular metabolites including but not limited to 7-dehydrocholesterol, falls within the scope of protection of the present invention.

[0075] The other fluorescent sensors or fluorescent sensor proteins or bioluminescent sensors or bioluminescent sensor proteins include but are not limited to: fluorescent sensor proteins described in CN102952181A and fluorescent sensors described in CN118104133A.

[0076] Those skilled in the art can, based on actual production needs, combine conventional technical means or basic common sense of production processes in the field of molecular biology or genetic engineering (for example, "Practical Molecular Biology Operation Guide", "Molecular Biology Experimental Techniques Experimental Operation Guide", "Molecular Cloning Experiment Guide", "Concise Molecular Biology Experiment Guide", etc.), use the recombinant expression vector to express a recombinant expression vector of a fluorescent sensor protein, or transform the recombinant expression vector into competent cells to obtain transformants that can express a fluorescent sensor protein, and then expand and culture the transformants under conditions suitable for their growth to enable them to efficiently produce the fluorescent sensor protein. This does not pose any technical obstacles to those skilled in the art and is both possible and easy to do.

[0077] The fifth group of examples, transformants of the present invention

[0078] This group of embodiments provides a transformant. All embodiments in this group have the following common feature: the transformant is transformed with a recombinant expression vector as described in any one of the fourth group of embodiments.

[0079] In some embodiments, the transformant is a host cell transformed with a recombinant expression vector according to any one of Group 4 embodiments;

[0080] In a specific embodiment, the host cell is selected from: Escherichia coli TOP10 competent cells, Pichia pastoris cells, and Saccharomyces cerevisiae cells.

[0081] Any act of cloning, amplifying, enriching, expressing, connecting, transforming, synthesizing, culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, transforming, selling, or offering for sale the transformant, and / or the act of combining a fluorescent sensor protein expressed by the transformant with other fluorescent sensors or fluorescent sensor proteins or bioluminescent sensors or bioluminescent sensor proteins, and / or the act of using a fluorescent sensor protein expressed by the transformant to monitor intracellular metabolites including but not limited to 7-dehydrocholesterol, falls within the scope of protection of the present invention.

[0082] Those skilled in the art can, based on actual production needs, combine conventional technical means or basic common sense of production processes in the field of molecular biology or genetic engineering (for example, "Practical Molecular Biology Operation Guide", "Molecular Biology Experimental Technology Experimental Operation Guide", "Molecular Cloning Experiment Guide", "Concise Molecular Biology Experiment Guide", etc.), cultivate, expand, ferment, enrich, produce, prepare, use, and inoculate the transformant to express, secrete, produce, and obtain a fluorescent sensor protein. This does not pose any technical obstacles to those skilled in the art and is possible and easy to do.

[0083] Sixth embodiment, fluorescence sensor of the present invention

[0084] This group of embodiments provides a fluorescence sensor. All embodiments in this group share the following common features: the fluorescence sensor is selected from: a fluorescence sensor protein described in any one of the second group of embodiments, and / or a gene for a fluorescence sensor protein described in any one of the third group of embodiments, and / or a recombinant expression vector described in any one of the fourth group of embodiments, and / or a transformant described in any one of the fifth group of embodiments.

[0085] Group 7 Example, detection method of the present invention

[0086] This group of embodiments provides a method for high-throughput real-time detection of intracellular metabolites. All embodiments in this group have the following common feature: a substrate is fermented using a fluorescent sensor as described in any one of the sixth group of embodiments.

[0087] In a specific embodiment, the fermentation products are collected on different fermentation days for fluorescence detection;

[0088] Preferably, the fluorescence detection includes: performing GC-MS detection using a gas chromatography-mass spectrometer and performing fluorescence intensity detection using an enzyme marker.

[0089] Experimental Example 1: Molecular dynamics simulation of the allosteric domain of human sterol O-acyltransferase

[0090] In this experimental example, human sterol O-acyltransferase 1 (SOAT1) was selected, and its amino acid sequence is shown in SEQ ID NO. 1.

[0091] Calculate the distance change between target amino acid residue pairs before and after allosteric activation of SOAT1 by 7-dehydrocholesterol. Screen for domains with allosteric properties: select amino acid residue pairs with a distance less than 10 Å and a distance change of at least 2 Å.

[0092] The distance changes between the amino acids at both ends of the allosteric domain were detected before and after the reaction. The distance changes of the five allosteric domains are as follows:

[0093] a.291-302: 18.25Å-13.5Å;

[0094] b.12-16: 10.5Å-8.5Å;

[0095] c.15-20: 7.75Å-5.25Å;

[0096] d.317-329: 8.25Å-5.25Å;

[0097] e. 299-306: 10Å-5Å;

[0098] The changes in the distance between the amino acids at both ends of the above five allosteric domains and their fluorescence responses to 7-dehydrocholesterol are shown in Figure 2. Figure 1 a, b, c, d, and e are shown in the figure; the fluorescence intensity comparison of the five allosteric domains is shown in the figure. Figure 1 As shown in f.

[0099] Experimental Example 2: Construction of Fluorescent Sensor Protein

[0100] This experimental example provides a method for constructing a fluorescent sensor protein capable of detecting steroid products, comprising the following steps:

[0101] The SOAT1 gene was synthesized by the company after optimization of the codon preference of Saccharomyces cerevisiae. After correct sequencing, the target gene was double-digested with restriction endonucleases. At the same time, the pGAPZαC plasmid was double-digested with restriction endonucleases. The reaction system and conditions are shown in Table 1:

[0102] Table 1. Double enzyme digestion system

[0103]

[0104] The reaction was continued at 37°C for 30 min, and the linearized vector and fragments were recovered by agarose gel electrophoresis.

[0105] The purified PCR fragment was ligated with the plasmid. The composition and reaction conditions of the 10 μL reaction system are shown in Table 2:

[0106] Table 2. Enzyme-linked systems

[0107]

[0108] React at 20°C for 2 hours. The resulting product can be used to transform competent E. coli TOP10 cells. Mix the DNA to be transformed with competent cells (DNA: competent cells ratio <1 / 10) and place on ice for 30 minutes. Heat shock at 42°C for 90 seconds. Quickly transfer the tube to an ice bath and allow the cells to cool for 3-5 minutes. Add 500µL of LB medium and pre-incubate at 37°C for 45-60 minutes. Spread an appropriate volume (200µL) evenly on a solid plate containing bleomycin-containing LB. Invert the plate and incubate at 37°C for 12-16 hours until colonies appear.

[0109] A single colony on the culture plate was inoculated into bleomycin-resistant LB medium and cultured overnight. The plasmid pGAPZαC-SOAT1 was extracted and sent to a sequencing company for sequencing.

[0110] Based on the gene sequence of the SOAT1 allosteric domain 299-306, the upstream primer 299-306-HF and the downstream primer 299-306-HR of loop P were designed at both ends (primers are shown in Table 7). Then, a PCR system was configured to amplify the gene fragment without the allosteric domain 299-306 using a PCR instrument. The composition of the 50µL PCR system is shown in Table 3:

[0111] Table 3. PCR amplification system

[0112]

[0113] PCR reaction program: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 1 min, 30 amplification cycles, final extension at 72°C for 10 min, and storage at 16°C.

[0114] After the PCR reaction is completed, the reaction product is taken and subjected to 1% agarose gel electrophoresis to detect whether the target band size is correct. If the detection is correct, agarose gel electrophoresis is performed to recover the PCR product and purify it to obtain the vector fragment.

[0115] The laboratory previously stored a cyclic fluorescent protein (cpEGFP) fragment, whose amino acid sequence is as SEQ ID NO.2.

[0116] Using this gene fragment as a template and Phanta®Max DNA polymerase, we designed upstream primers SO-FP-F and downstream primers SO-FP-R (see Table 7 for primers). These primers introduced GG at the N-terminus and GGAAA at the C-terminus, respectively, to connect the linkers at both ends of SOAT1. A PCR system was then configured to amplify the linker-containing cyclic fluorescent protein gene fragment using a PCR instrument. The composition of the 50 µL PCR system is shown in Table 3.

[0117] PCR reaction program: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 1 min, 30 amplification cycles, final extension at 72°C for 10 min, and storage at 16°C.

[0118] After the PCR reaction is completed, the reaction product is taken and subjected to 1% agarose gel electrophoresis to detect whether the target band size is correct. If the detection is correct, agarose gel electrophoresis is performed to recover the PCR product and purify the target DNA fragment.

[0119] The purified vector fragment and target DNA fragment were assembled by Gibson. The 10µL Gibson assembly system is shown in Table 4:

[0120] Table 4. Gibson assembly system

[0121]

[0122] Assemble at 50°C for 1 hour, and the resulting product can be used to transform competent E. coli TOP10 cells. Mix the DNA to be transformed with competent cells (DNA: competent cells ratio <1 / 10), incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, quickly transfer the tube to an ice bath, and allow the cells to cool for 3-5 minutes. Add 500µL of LB medium and pre-incubate at 37°C for 45-60 minutes. Spread an appropriate volume (200µL) evenly on a solid plate containing ampicillin-resistant LB. Invert the plate and incubate at 37°C for 12-16 hours until colonies appear.

[0123] A single colony from the culture plate was inoculated into bleomycin-resistant LB medium and cultured overnight. The plasmid was then extracted and sent to a sequencing company for sequencing. This yielded the plasmid vector pGAPZαC-SOAT1-299-306 for the 299-306 fluorescence detection system. The schematic diagram of the structural construction of the 299-306 fluorescence detection system (fluorescent sensor protein) provided in this experimental example is shown below. Figure 2 shown.

[0124] Experimental Example 3: Construction of Pichia pastoris expression vector

[0125] This experimental example provides a method for constructing a Pichia pastoris expression vector, comprising the following steps:

[0126] Linearized expression plasmid was transformed into Pichia pastoris cells: The expression plasmid pGAPZαC-SOAT1 was linearized. The reaction system composition and reaction conditions are shown in Table 5:

[0127] Table 5. Linearization system

[0128]

[0129] After incubation at 37°C for 1 h, the DNA was purified using a DNA recovery kit.

[0130] Linearized expression plasmid was transformed into Pichia pastoris cells:

[0131] 1. Preparation of Pichia pastoris competent cells: Pick from the plate A single colony was inoculated into a test tube containing 5 mL of YPD medium and cultured overnight at 30°C and 200 rpm; 1 mL of the bacterial solution was transferred to a test tube containing 20 mL of YPD medium and cultured at 30°C and 200 rpm for 4-6 hours until the OD 600The bacterial suspension was transferred to a 50 mL centrifuge tube, centrifuged at 5000 rpm and 4°C for 5 min, and the supernatant was discarded. The bacterial suspension was resuspended in 25 mL of pre-cooled sterile water, centrifuged at 5000 rpm and 4°C for 5 min, and the supernatant was discarded. The previous step was repeated once. The bacterial suspension was resuspended in 25 mL of pre-cooled 1 M sorbitol, centrifuged at 5000 rpm and 4°C for 5 min, and the supernatant was discarded. Finally, the bacterial suspension was resuspended in about 200-400 μl of 1 M pre-cooled sorbitol and aliquoted into 90 μl tubes for use.

[0132] 2. Pichia pastoris electroporation: Add 10 μl of the linearized plasmid fragment to 90 μl of Pichia competent cells, mix thoroughly, and transfer the culture to a pre-chilled electroporation cuvette. Incubate on ice for 5 min. Place the cuvette in an electroporation instrument and perform electroporation using the following parameters: voltage 1.8 kV, capacitance 25 μF, and resistance 200 Ω. Immediately after electroporation, add 1 mL of pre-chilled YPD solution and transfer the culture to a 10 mL centrifuge tube. Incubate at 30°C on a shaker for 1-2 h. Centrifuge at 5000 rpm for 3 min, discard the supernatant, resuspend the culture in 200 μL of 1 M sorbitol, and plate onto a YPD plate containing ≥100 μg / mL bleomycin. Incubate at 30°C for 3 days and observe colony growth.

[0133] 3. Screening of Positive Clones: Randomly pick eight single colonies from the screening plate and streak them onto new bleomycin-containing YPD plates. Lyse the yeast cells with yeast lysis buffer. Using the lysed yeast cells as a template, perform PCR with primers pGAP-F and 3'AOX1 to verify whether the target gene SOAT1 is inserted into the Pichia pastoris genome. The 15 μL PCR system is as follows:

[0134] Table 6. Verification PCR system

[0135]

[0136] The PCR conditions were as follows: 94°C pre-denaturation for 10 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 30 amplification cycles, 72°C final extension for 10 min, and finally storage at 16°C. The amplification results were detected by 1.0% agarose gel electrophoresis. The verification results were as follows: Figure 3 As shown, the theoretical band size is 2642 bp.

[0137] Experimental Example 4: Extracellular Detection of SOAT1-299-306 Sensor

[0138] Preparation of crude SOAT1-299-306 enzyme solution: Take 30 ml of fermentation broth, centrifuge at 5000 rpm for 30 min, remove the supernatant, resuspend with 10 mL of pre-cooled deionized water, take 4 mL into a 5 mL crushing tube, add 2 5 mm crushing beads, 2 3 mm crushing beads and two spoons of a mixture of glass crushing beads and quartz sand, and crush at low temperature in a cryo-grinder.

[0139] Reaction system of SOAT1-299-306 and 7-dehydrocholesterol: SOAT1-299-306 crude enzyme solution was mixed evenly with 7-dehydrocholesterol of different concentrations in a 1:1 ratio, placed in a 96-well microplate and used for fluorescence detection using a microplate reader.

[0140] The selected concentrations of 7-dehydrocholesterol were: 25 mg / L, 50 mg / L, 62.5 mg / L, 125 mg / L, 250 mg / L, and 500 mg / L.

[0141] The measured specific fluorescence intensity showed a linear relationship with the concentration of 7-dehydrocholesterol. The in vitro detection curve of the sensor was obtained by repeated measurements. Figure 4 shown.

[0142] Specificity detection of SOAT1-299-306: SOAT1-299-306 crude enzyme solution was mixed evenly with 1 g / L of interference at a ratio of 1:1, placed in a 96-well microplate, and fluorescence detection was performed using a microplate reader.

[0143] Interfering substances include: glycyrrhetinic acid, glycyrrhizic acid, lanosterol, ergosterol, cholesterol, and water.

[0144] The results of specific tests are as follows Figure 5 shown.

[0145] Experimental Example 5: Construction of SOAT1-299-306 sensor in 7-dehydrocholesterol-producing yeast strain

[0146] Using a yeast strain capable of producing 7-dehydrocholesterol constructed earlier in the laboratory as the base strain, the plasmid vector pGAPZαC-SOAT1-299-306 constructed in Experimental Example 2 as the template, the upstream primer PESC-URA-SOAT1-F and the downstream primer PESC-URA-SOAT1-R were designed (primers are shown in Table 7). Phanta®Max DNA polymerase was used to configure the PCR system, and finally the target gene SOAT1-299-306 was amplified using a PCR instrument. The composition of the 50 µL PCR system is shown in Table 3:

[0147] PCR reaction program: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 3 min, amplification cycles for 30 times, final extension at 72°C for 10 min, and storage at 16°C.

[0148] After the PCR reaction is completed, the reaction product is taken and subjected to 1% agarose gel electrophoresis to detect whether the target band size is correct. If the detection is correct, agarose gel electrophoresis is performed to recover the PCR product and purify it to obtain the DNA fragment SOAT1-299-306.

[0149] Using the PESC plasmid containing the URA selection marker as a template, the homologous recombination plasmid PESC-URA-SOAT1-299-306 was constructed. The construction method was similar to that in Experimental Example 3, and the specific primers are shown in Table 7. Using this recombinant plasmid as a template, the upstream primer URA-SOAT1-F and the downstream primer URA-SOAT1-R were designed. PCR amplification and purification were performed to obtain the URA-tagged target gene expression cassette URA-SOAT1-299-306.

[0150] Using the Saccharomyces cerevisiae genome as a template, primer pairs LPP1up-F / R for the upper homology arm LPP1 gene and LPP1down-F / R for the lower homology arm LPP1 gene were designed (primers are shown in Table 7). Phanta®Max DNA polymerase was used to configure a PCR system, and finally, the upper and lower homology arms LPP1up and LPP1down were obtained by PCR amplification and purification.

[0151] The specific operations for strain construction are as follows:

[0152] (1) Strain activation: Streak the bottom plate strain stored at -80℃ on YPD medium, culture it in a 30℃ constant temperature incubator, pick a single colony, and culture it in a 50 ml centrifuge tube containing 5 ml liquid YPD medium at 30℃ and 200 rpm until the OD reaches 0.8-1.2. Use this as the seed liquid.

[0153] (2) Transformation of Saccharomyces cerevisiae: 200 μL of the activated yeast solution was inoculated into a 100 ml Erlenmeyer flask containing 20 ml of YPD medium, and cultured at 200 rpm in a 30 °C shaker for 5-6 h until the OD600 nm value reached 0.8-1.2; the above bacterial solution was transferred to a 2 ml EP tube, centrifuged at 5000 rpm for 2 min, and the step was repeated to collect 3-5 ml of bacterial solution and discard the supernatant; 1 mL of pre-cooled sterile water was added to resuspend the cells, and the supernatant was discarded after centrifugation at 5000 rpm for 2 min (this step is to wash the residual culture medium in the bacteria and can be repeated 2-3 times); 1 mL of 1 M pre-cooled sorbitol was added to resuspend the cells, and the supernatant was discarded after centrifugation at 5000 rpm for 2 min; 90 μL of 1 M Resuspend the cells again with sorbitol, add the fragment expression cassette URA-SOAT1-299-306 to be transformed, homology arms LPP1 upper and LPP1 lower to the resuspended bacterial solution, and transfer it to a pre-cooled electroporation cup for electroporation; add 1 ml of fresh YPD culture medium to the electroporated bacterial solution; incubate at 30°C for 1-2 hours, then centrifuge at 5000 rpm for 3 minutes, discard the supernatant, resuspend with 200 μL 1 M sorbitol, spread on the defective plate, and culture at 30°C for 3 days to observe the growth of the colonies.

[0154] (4) Positive detection: Pick a single clone and lyse the yeast cells with yeast lysis buffer. Use the lysed yeast cells as a template and perform PCR detection with the primers LPP1-YZ-F and LPP1-YZ-R listed in the table.

[0155] Table 7. Primers used for integration

[0156]

[0157] Table 8. Construction of strains

[0158]

[0159] Experimental Example 6: Intracellular Detection of SOAT1-299-306 Sensor

[0160] (1) Strain fermentation: The target strain constructed in the above experimental example was streaked on the corresponding screening marker plate and cultured in a 30℃ constant temperature incubator for 2 to 3 days. A single colony was picked and cultured in a 50ml test tube containing 5ml of defective culture medium. The culture was placed at 30℃ and 200 rpm until the OD reached 0.8 to 1.2. The seed liquid in the tube was inoculated into a 100ml triangular flask containing 20ml of culture medium (YPD medium: 20g / L peptone, 10g / L yeast powder, 20g / L glucose) with an inoculation volume of 10%. Each bacterium was cultured in triplicate at 30℃ and 220 rpm for 4 days.

[0161] (2) Sample processing and detection: Take 1 ml of fermentation broth, centrifuge at 5000 rpm for 2 min, remove the supernatant, and resuspend in deionized water. Repeat three times to obtain the test sample. Observe the fluorescence state under a 40× microscope. Measure the specific fluorescence intensity using a microplate reader in a 96-well plate.

[0162] (3) Sample processing and detection of 7-dehydrocholesterol:

[0163] Place 1 mL of the fermented recombinant Saccharomyces cerevisiae strain in a 2 mL centrifuge tube and centrifuge at 12,000 rpm for 2 minutes. Discard the supernatant and collect the cells. Add 1 mL of ethyl acetate, two 5 mm crushing beads, two 3 mm crushing beads, and a mixture of glass crushing beads and quartz sand to the centrifuge tube and place in a cryo-grinder for cryo-crushing. Centrifuge the crushed mixture at 12,000 rpm for 10 minutes and collect the supernatant. Filter an appropriate amount of the supernatant (ethyl acetate containing the target compound) through a 0.22 mm organic filter membrane. Transfer 100 μL of the supernatant to a light-proof liquid chromatography vial and evaporate the ethyl acetate solvent in a fume hood. Add 200 μL of the alkylation mixture to the dried product, tighten the cap, and alkylate in an 80°C water bath for 30 minutes. Remove the vial and transfer the alkylation mixture to a liquid chromatography vial sleeve. Tighten the cap for GC-MS analysis.

[0164] The instrument used in this experiment was a Shimadzu GCMS-QP2010 Ultra gas chromatography-mass spectrometer, and the chromatographic column was Ris-5MS (30.0 m × 0.25 mm × 0.25 μm).

[0165] Methods: Column oven temperature was 80°C, inlet temperature was 300°C, carrier gas was 99.9% helium, gas flow rate was 1.0 mL / min, column flow rate was 1.20 mL / min, linear velocity was 40.4 cm / sec, purge flow was 6.0 mL / min, and split ratio was 40:1. The GC program is shown in the table below. MS scan time was 15 to 21 minutes, and the scan range was 50–700 m / z.

[0166] Table 9. GC program settings

[0167]

[0168] From the microscopic examination results, it can be seen that the SOAT1-299-306 sensor can produce a specific fluorescence response to 7-dehydrocholesterol in the Saccharomyces cerevisiae strain, e.g. Figure 6 As shown;

[0169] From the GC-MS test results and the fluorescence test results of the microplate reader, it can be seen that the fluorescence intensity is correlated with the concentration change of 7-dehydrocholesterol produced by the chassis strain, such as Figure 7 shown.

[0170] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A fluorescent sensor protein, characterized in that The amino acid sequence of the fluorescent sensor protein is shown in SEQ ID NO.

3.

2. A gene encoding a fluorescent sensor protein, characterized in that: It encodes the fluorescent sensor protein according to claim 1.

3. A recombinant expression vector, characterized in that: Connected to the coding gene sequence of the fluorescent sensor protein according to claim 2.

4. A transformant, characterized in that A host cell transformed with the recombinant expression vector according to claim 3.

5. A method for real-time detection of intracellular metabolites, characterized in that: The transformant according to claim 4 is used to detect the intracellular metabolite 7-dehydrocholesterol of the host cell, wherein the host cell is selected from Pichia pastoris cells or Saccharomyces cerevisiae cells.

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