WO2019076262A1 - Biosensor for detecting target molecular compound, and system comprising biosensor - Google Patents

Abstract

A biosensor for detecting a target molecular compound, and a system comprising the biosensor. The biosensor is a fusion protein, and comprises a target molecule synthase, a DNA-binding domain and a transcriptional activation domain respectively ligated to both ends of the target molecule synthase, and a degradation determinant. When there is no precursor substance of the target molecule, the degradation determinant degrades the biosensor; when there is a precursor substance of the target molecule, the target molecule synthase recognizes and binds the precursor substance to stabilize the biosensor, so that the degradation determinant fails and is automatically detached, the DNA-binding domain binds to a promoter region of a reporter gene, and the transcriptional activation domain activates reporter gene expression. The present invention can implement accurate and high-throughput screening of various molecular metabolites, greatly reduce complicated manual operations, and lower the costs of detection using high-precision instruments, and can be widely applied.

Description

Biosensor for detecting a target molecular compound and a system including the same Technical field

The present invention relates to the field of compound detection technology, and in particular to a biosensor for detecting a target molecular compound and a system comprising the biosensor.

Background technique

Natural products are components of organisms or their metabolites. They usually have special biological activities and functions. They are the main pathway for the discovery of new drugs and are also a hot spot in current metabolic research. Since the natural product usually has a complicated structure due to containing more active groups, the chemical synthesis step is cumbersome and the yield is extremely low, the main acquisition route is extraction from plants, etc. However, the plant has a long growth cycle and a large number of extraction processes. Loss, as well as the effects of the process, make the acquisition of active natural products far from satisfactory. Therefore, the biosynthesis of natural products has become the main way to achieve industrial production.

The most successful example of biosynthesis of natural products is the artemisinin, an anti-malarial drug with important pharmaceutical value. Keasling et al. chose Saccharomyces cerevisiae as a host to accumulate farnesyl pyrophosphate and introduce corresponding exogenous synthetase, so that Saccharomyces cerevisiae can directly synthesize artemisinic acid. In 2013, Paddon et al. transformed the three-step reaction from Amorphadiene to artemisinic acid catalyzed by a single cytochrome oxidase CYP71AV1 in artemisinic acid biosynthesis into multiple enzymes (CYP71AV1). The three-step reaction catalyzed by CPR1, CYB5, ADH1, ALDH1) reconstituted the biosynthetic pathway of artemisinic acid in yeast, which greatly increased the yield of artemisinic acid. Stephanopoulos et al. introduced the synthetic pathway of taxadiene into E. coli and optimized the whole metabolic pathway to obtain high-yield taxadiene-producing Escherichia coli cells, which is a biosynthesis study of the new anticancer drug paclitaxel. Significant progress. In 2013, Zhang Xueli and others cooperated with Huang Qiqi and others to jointly carry out research on the synthesis of medicinal terpenoids by artificial cells, which significantly improved the ability of artificial yeast cells to synthesize diterpenoids and triterpenoids, and synthesized tanshinone IIA into prodrugs. The yield of miltiradiene increased to 488 mg/L, the yield of squalene was increased to 852 mg/L, and the yield of lycopene was increased to 21.17 mg/L. In recent years, Liu Tianqi and others have used synthetic biology methods to construct efficient metabolic pathways in model organisms, effectively producing rare-source drugs and prodrugs, including various high-value-added natural substances such as lycopene, astaxanthin, and paclitaxel. Compound.

As people continue to explore the process of microbial synthesis of natural products, biosensors of small molecule compounds have been widely used in the study of metabolic pathways. As early as 2000, Gil et al. designed biosensors to monitor environmental toxins; In 2012, Paige et al. designed a biosensor capable of detecting the solubility of the corresponding metabolite. In recent years, many researchers have made relevant research on biosensors of various metabolites and made some progress, but the current method does not propose a simple strategy that can be designed to be very effective and broad. Applied biosensors.

At present, the design process of the LBD (ligand-binding domain) module required for the identification of small molecule compounds is very cumbersome. In 2013, scientists used steroid-binding proteins as an example to locate the amino acid side chains around the optimal orientation ligand by computer calculations, and to map possible binding sites on the scaffold protein structure, according to the nature and structure of these side chains. To optimize the interaction between some additional proteins and ligands. This design is based on these typical natural complexes or protein structures with low Boltzmann-weighted values in the free state and side chain interactions already present. Based on the high complementarity of these methods and phenotypes, 17 proteins with potential binding capabilities were designed for experimental screening. In the course of the experiment, in order to screen for LBD with high binding ability, yeast surface display and flow cytometry were used for detection. At the same time, Isothermal titration calorimetry is used to confirm the interaction relationship (Boltzmann-weighted value), and the final optimization of LBD also requires site-saturation mutagenesis, while using yeast surface display technology. And sorting by fluorescence activated cell sorting (FACS). Only the LBD mining process has undergone a very complicated preliminary theory design and a cumbersome experimental screening optimization process.

Recently, Jester et al. optimized the screening through a series of computer model design, protease engineering, and molecular biotechnology to obtain LBD that can highly identify target small molecules. Taking Digoxin as an example, the whole process of designing a biosensor is described. Firstly, a plurality of LBDs capable of effectively combining digoxin are obtained by means of computer simulation design and introduction of mutations. On the basis of this, a DBD (DNA-binding domain) is connected to the N-terminus of the LBD, and a TAD is connected to the C-terminus (C-terminal). Transcriptional activation domain), and a degenerate determinant (degron) is ligated at one end of the DBD to form a fusion protein. The three fusion proteins have been shown to significantly increase the activity of the target molecule digoxin (10 times, DBD mutation). The body is 60 times). The fusion protein is used as a biosensor, and finally an LBD capable of highly recognizing the target molecule digoxin is obtained. However, the design of the biosensor still needs to rely on the high specificity of the computer designed, constructed and optimized to combine the LBD of the target small molecule, and the whole excavation process is very cumbersome. Moreover, as the target small molecule changes, it is necessary to perform complicated calculations such as computer calculation, construction, and screening. Therefore, current biosensor design strategies are designed and constructed only for a certain metabolite, and do not achieve a very effective and broadly applicable biosensor.

Summary of the invention

The invention provides a biosensor for detecting a target molecular compound and a system comprising the biosensor, which can conveniently construct a biosensor of various molecular compounds with high specificity and effective recognition capability, so that various molecular metabolites Achieving accurate, high-throughput screening greatly reduces cumbersome manual operations and reduces the cost of using high-precision instrumentation for a wide range of applications.

According to a first aspect, in one embodiment, a biosensor for detecting a target molecular compound is provided, the biosensor being a fusion protein, the biosensor comprising a target molecule synthetase, and two synthase enzymes respectively linked to the target molecule a DNA binding domain, a transcriptional activation domain, and a degradation determinant; when the precursor of the target molecule is absent, the degradation determinant degrades the biosensor; when the precursor of the target molecule is present, the target The molecular synthetase recognizes and binds to the precursor substance to stabilize the biosensor, such that the degradation determinant fails to automatically detach, and the DNA binding domain binds to the promoter region of the reporter gene, and the transcription activation domain activates the reporter gene. expression.

Further, the biosensor described above sequentially connects a degradation determinant, a DNA binding domain, a target molecule synthetase, and a transcriptional activation domain from a nitrogen terminal to a carbon terminal.

Further, the above target molecule compound is selected from a natural product, and accordingly, the target molecule synthetase is an enzyme that synthesizes the natural product. It should be noted that the term "natural product" is a chemical substance which is usually produced by living organisms in nature and which is usually pharmacologically or biologically active, and mainly includes proteins, polypeptides, amino acids, nucleic acids, various enzymes, monosaccharides, Oligosaccharides, polysaccharides, glycoproteins, resins, colloids, lignin, vitamins, fats, oils, waxes, alkaloids, volatile oils, flavonoids, glycosides, terpenoids, phenylpropanoids, organic acids, phenols, anthraquinones Naturally occurring chemical constituents such as lactones, steroids, tannins, and antibiotics are commonly used in pharmaceutical drug discovery and drug design.

Further, the above-mentioned target molecule synthetase is a wild type enzyme which synthesizes the above natural product.

Further, the above-mentioned target molecular compound is selected from a drug and/or a prodrug compound, and accordingly, the above-mentioned target molecule synthetase is an enzyme which synthesizes the above-mentioned drug and/or prodrug compound.

Further, the target molecule is lycopene, and the target molecule synthetase is lycopene synthase; preferably, the lycopene synthase is encoded by a CrtI gene; preferably, the precursor of the target molecule is octahydrogen Lycopene.

Further, the target molecule is resveratrol, and the target molecule synthetase is a p-diphenylene synthase; preferably, the p-diphenylene synthase is encoded by an STS gene; preferably, the precursor substance of the target molecule It is p-coumaroyl-CoA.

Further, the above degradation determinant is the degradation determinant MATα2 in yeast.

Further, the above reporter gene is a resistance screening gene and/or a fluorescent marker gene. Preferably, the above resistance screening gene is selected from the group consisting of ampicillin, kanamycin, spectinomycin, and/or tetracycline; or the fluorescent marker gene is selected from the group consisting of a green fluorescent protein GFP gene, a red fluorescent protein RFP gene, and/or yellow Fluorescent protein YFP gene.

According to a second aspect, an embodiment provides a method of constructing a biosensor of the first aspect, comprising: first, a nucleic acid fragment encoding a target molecule synthetase, and a DNA respectively ligated to both ends of the target molecule synthetase The nucleic acid fragment of the binding domain, the nucleic acid fragment of the transcriptional activation domain, and the nucleic acid fragment of the degradation determinant are assembled; and the assembled nucleic acid fragment is then ligated into the plasmid vector.

Further, the above method further comprises: constructing an expression plasmid of the reporter gene, wherein the DNA binding domain binds to a promoter region of the reporter gene, and the transcription activation domain activates the expression of the reporter gene.

Further, assembly of the above nucleic acid fragments is achieved by PCA assembly and/or Gibson assembly.

According to a third aspect, an embodiment provides a nucleotide sequence encoding the biosensor of the first aspect.

Further, the above nucleotide sequence includes the sequence shown as SEQ ID NO: 134.

Further, the above nucleotide sequence includes the sequence shown as SEQ ID NO: 171.

Further, the above nucleotide sequence further includes a first vector sequence.

Further, the first vector sequence described above is the sequence of the vector backbone pRS415.

Further, an expression sequence encoding the above reporter gene is also included.

Further, the above reporter gene expression sequence comprises the sequence shown as SEQ ID NO:133. Further, the above reporter gene expression sequence further comprises a second vector sequence.

Further, the second vector sequence described above is the sequence of the vector backbone pRS413.

According to a fourth aspect, an embodiment provides a method for detecting a target molecule compound, comprising: introducing the biosensor of the first aspect or the nucleotide sequence of the third aspect into a host cell for expression together with a reporter gene; Qualitative and/or quantitative analysis of the target molecule compound by detecting the expression of the above reporter gene.

According to a fifth aspect, an embodiment provides a biosensing system for detecting a target molecule compound, comprising a nucleotide sequence encoding the biosensor of the first aspect, a reporter gene expression sequence, and optionally, further comprising The nucleotide sequence of the biosensor and the host cell in which the reporter gene expression sequence is expressed.

Further, the nucleotide sequence of the above biosensor comprises the sequence shown as SEQ ID NO: 134, and the above reporter gene expression sequence comprises the sequence shown as SEQ ID NO: 133.

Further, the nucleotide sequence of the above biosensor comprises the sequence shown as SEQ ID NO: 171, and the reporter gene expression sequence comprises the sequence shown as SEQ ID NO: 133.

Further, the nucleotide sequence of the above biosensor further includes a first vector sequence, and the reporter gene expression sequence further includes a second vector sequence, and the host cell is a yeast cell.

Further, the first vector sequence is the sequence of the vector backbone pRS415, and the second vector sequence is the sequence of the vector backbone pRS413.

According to a sixth aspect, an embodiment provides the use of the biosensor of the first aspect or the nucleotide sequence of the third aspect or the biosensing system of the fifth aspect for detecting a target molecule compound.

Further, the above uses include relatively qualitative, quantitative analysis, and/or screening of host cells producing the above-described target molecular compound.

The design of the biosensor for detecting a target molecular compound of the present invention, if only for the screening of the final product, selecting the reporter gene as a resistance screening gene, will greatly reduce the costly batch screening process, if it is necessary to obtain a strain bank of different yields. The reporter gene can be designed into various fluorescent marker genes, and the metabolic products can be qualitatively and quantitatively analyzed by detecting the intensity of fluorescence.

The biosensor for detecting a target molecular compound of the present invention is designed according to a naturally occurring target metabolic pathway, and various target molecular synthetases are used as recognition groups, and the above-mentioned target molecule synthetase is naturally occurring and is front of the target molecule. The bulk material has a high degree of specificity and effective recognition ability, omitting the cumbersome excavation process for computer design, construction and optimization screening of the target molecule LBD, which significantly reduces the time cost. In addition, the biosensor of the present invention selects a target molecular synthetase as a group for identifying a precursor substance of a target molecule, and the components of the designed fusion biosensor are relatively independent, the single element replacement is relatively simple, and all natural in nature Compound molecules can find groups that can recognize their precursors. Even if the target molecules are replaced, they can quickly assemble their fusion biosensors, and have a relatively wide range of applications.

DRAWINGS

1 is a schematic diagram showing a model structure and a function of a biosensor according to an embodiment of the present invention;

2 is a schematic view showing the synthesis of a lycopene body according to an embodiment of the present invention;

3 is a schematic view showing the synthesis of a resveratrol body according to an embodiment of the present invention;

4 is an integrated plasmid map in an embodiment of the present invention;

5 is an electropherogram showing the construction of an integrated plasmid in an embodiment of the present invention;

6 is a diagram showing fluorescence results observed on a fluorescent display device after transformation of a yeast strain by a lycopene biosensor according to an embodiment of the present invention;

7 is a diagram showing fluorescence results of a lycopene biosensor after detecting a yeast strain on a microplate reader according to an embodiment of the present invention;

8 is a diagram showing fluorescence results observed on a fluorescent display device after a yeast strain is transformed by a resveratrol biosensor according to an embodiment of the present invention;

Figure 9 is a graph showing the fluorescence results of a resveratrol biosensor after detection of a yeast strain on a microplate reader in accordance with an embodiment of the present invention.

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings. In the following embodiments, many of the details are described in order to provide a better understanding of the invention. However, those skilled in the art can easily realize that some of the features may be omitted in different situations, or may be replaced by other components, materials, and methods. In some instances, some of the operations related to the present invention have not been shown or described in the specification in order to avoid that the core portion of the present invention is overwhelmed by excessive description, and those skilled in the art will describe these in detail. Related operations are not necessary, they can fully understand the relevant operations according to the description in the manual and the general technical knowledge in the field.

The whole process of identifying the target molecular group in the current biosensor is mainly the technology of protein engineering, and the binding relationship between the protein and the ligand molecule is calculated by a computer operation model to predict the binding ability between the two. It is necessary to construct a universal and simple biosensor, and the purpose is to eliminate the cumbersome work of computer calculation, construction, screening, etc., along with the change of the target molecule. The interaction of many enzymes is required in the metabolic pathway to achieve the expression of the target molecule. The formation of the target molecule is the result of the action of the enzyme responsible for the step of identifying the substrate of the previous step. Then, the synthetase of this step should have a high recognition ability or binding ability to the precursor substance.

1 shows a model structure and function of a biosensor of a molecular compound in the embodiment of the present invention, the biosensor is a fusion protein including a target molecule synthetase (as a "recognition group"), and respectively A DNA binding domain (DBD), a transcriptional activation domain (TAD), and a degradation determinant (eg, ligated upstream of the DBD) attached to both ends of the target molecule synthetase.

The reporter gene can select a resistance screening marker or a fluorescent display marker, and finally introduce the constructed fusion protein (ie, a biosensor) into the cell. When the precursor substance of the target molecule is not present, the recognition group cannot bind to the premise substance of the target molecule, thereby causing the biosensor to be unstable, and the degradation determinant degrades the unstable biosensor; when the precursor substance of the target molecule When generated in the body, the target molecule synthetase highly recognizes the precursor substance and binds it effectively. The biosensor becomes more stable due to the effective binding of the substrate, and the degradation determinant is thus automatically detached. The DBD will be able to effectively combine the downstream report. In the promoter region of the gene, TAD will effectively activate the reporter gene for expression. Therefore, the host cell producing the target molecule can be finally screened by the resistance screening marker, or the target molecule can be subjected to relative qualitative/quantitative analysis by detecting the expression level of the reporter gene.

The experimental procedures described in the following examples are merely illustrative of the feasibility of the present invention, and the application of the present invention is not limited thereto. The experimental procedures mentioned in the examples are routine experimental methods unless otherwise specified; the reagent consumables mentioned are conventional reagent consumables unless otherwise specified.

Example 1 Biosensor for detecting lycopene

As shown in Fig. 2, taking the construction of lycopene yeast engineering bacteria as an example, lycopene is formed by synthesizing phytoene into tomato red under the action of lycopene synthase CrtI. Prime. Therefore, CrtI can be used as a recognition group for a biosensor. According to the literature, the optimal mutant of DBD is Gal4, and the two mutants L77F and R60S obtained in the Gal4 dimer can improve the recognition ability of the recognition group. Therefore, G _L77F or G _R60S can be used as a mutant of DBD to construct a biosensor of lycopene, and in this embodiment, G _L77F is selected. According to the literature, the optimal mutant of TAD is VP16 or VP64, both of which can drive the expression of yEGFP by controlling the downstream GAL1 promoter. Therefore, VP16 or VP64 can be used as a mutant of TAD to construct a biosensor of lycopene, and VP16 is selected in this embodiment. The N-terminal of DBD is linked to the degradation determinant MATα2 commonly found in the yeast system, and the downstream reporter gene selects the fluorescent protein yEGFP in yeast, which can quantitatively quantify the target metabolite by detecting the expression level of the fluorescent protein. The sequence fragments of the fusion fragments were synthesized by gene synthesis, and then the fusion fragments were inserted into the plasmid by Gibson assembly, and the plasmids having the fusion protein were assembled into a yeast strain capable of metabolizing lycopene, and the expression of the fluorescent protein was observed and detected.

The experimental process was mainly divided into two major modules: reporter plasmid construction (pRS413+GYC) and GIV (Gal4-crtI-VP16) plasmid construction (pRS415+MGIV). The reporter plasmid contains yEGFP and selects the GAL1 promoter as the promoter, the CYC1 terminator as the terminator, and the vector backbone pRS413; the GIV (Gal4-crtI-VP16) plasmid contains the degradation determinant MATα2, the DBD optimal mutant G _L77F , tomato The erythrin synthase CrtI and the TAD-optimal mutant VP16, as well as the vector backbone pRS415, are shown in Figure 4.

The 5'-GAL1 promoter + yEGFP + CYC1-3' terminator fragment was synthesized by DNA column (1467 bp, cut into 3 segments: GYC-F1 581 bp; GYC-F2 491 bp; GYC-F3 565 bp) and MATα2-G _L77F- IV Fragment (2610 bp, split into 5 segments: MGIV-F1 567 bp; MGIV-F2 583 bp; MGIV-F3 558 bp; MGIV-F4 577 bp; MGIV-F5 580 bp), oligonucleotides resolved by each fragment (see Table 1 below) The PCA was assembled separately to obtain sub-fragments, for example, GYC-F1-1 to GYC-F1-16 were assembled by PCA to obtain sub-fragment GYC-F1 (SEQ ID NO: 125), and the others were deduced to obtain sub-fragments GYC-F2. (SEQ ID NO: 126), GYC-F3 (SEQ ID NO: 127), MGIV-F1 (SEQ ID NO: 128), MGIV-F2 (SEQ ID NO: 129), MGIV-F3 (SEQ ID NO: 130) ), MGIV-F4 (SEQ ID NO: 131), MGIV-F5 (SEQ ID NO: 132); further TA cloning, PCR screening, sequencing identification; sequencing the correct sub-fragments to continue Gibson one-step assembly into the final large Fragment, 5'-GAL1 promoter + yEGFP + CYC1-3' terminator fragment (SEQ ID NO: 133), MATα2-G _{L77F -} IV fragment (SEQ ID NO: 134), and ligated into the vector backbone; Good plasmids were digested and sequenced, and sequencing was selected. Two plasmids were constructed engineering yeast strain transformed lycopene, the observed results. Lycopene yeast engineering bacteria: from the National Gene Bank of Shenzhen Huada Gene Life Science Research Institute, which is based on the Saccharomyces cerevisiae strain SynYII (Accession No. CTCCC NO: M 2014434) as the chassis cell, the lycopene biosynthesis pathway The three foreign genes (CrtE, CrtB, CrtI) were spliced to form a recombinant plasmid, which was introduced into the chassis cells for metabolite detection. The strain capable of expressing lycopene was lycopene yeast engineering bacteria.

Table 1 Sequences required to construct a lycopene biosensor

The specific experimental process is as follows:

1. PCA two-step assembly to obtain sub-fragments:

Shenzhen Huada Gene Technology Service Co., Ltd. was commissioned to synthesize all the oligonucleotides in Table 1, and the synthesized oligonucleotides were diluted to 10 μM, respectively, and 2.5 μL of each was taken out, and 2 μL was taken as a template for the first assembly of SOE PCA. 0.2 μL of Q5 DNA polymerase, 4 μL of 5× buffer, 1.6 μL of 2.5 mM dNTPs, and 20 μL of water were added. The reaction procedure was 98 ° C for 30 sec, 98 ° C for 10 sec, 55 ° C for 1 sec, 60 ° C for 30 sec (-0.5 ° C / cycle), 72 ° C for 30 sec, 20 cycles, 72 ° C for 2 min, and 12 ° C. Step 2: Configure the following reaction system, Ex Taq DNA polymerase (TAKARA) 0.5 μL, 10× buffer 5 μL, 2.5 mM dNTPs 4 μL, first step PCR product 10 μL, first and last oligos of each subfragment Nucleotides were used as primers, each taking 2 μL, and ddH ₂ O was supplemented with 50 μL. The reaction procedure was 94 ° C for 5 min, 94 ° C for 30 sec, 55 ° C for 30 sec, 72 ° C for 30 sec, 29 cycles, 72 ° C for 2 min, and 12 ° C.

Electrophoretic detection: Prepare 1% agarose gel, take 5μL PCR product for electrophoresis detection, 3μL DL 2000 DNA ladder, 180V voltage for 30min, electropherogram shown in Figure 5(a), and obtain the final spliced sub-fragment of about 500bp. .

2.TA clone

The PCR product in step 1 was purified by gel purification using a gel purification kit, and the product was purified by PCR using a TA cloning kit (TAKARA).

3. Bacterial fluid PCR

The monoclonal clones were selected from the TA cloning plate in step 2. After the overnight culture, 2 μL of the bacterial liquid was taken as a template for PCR, 0.5 μL of M13-F and M13-R, 5 μL of 2× PCR Mix, and 10 μL of ddH ₂ O. The reaction procedure was 94 ° C for 5 min, 94 ° C for 30 sec, 55 ° C for 30 sec, 72 ° C for 30 sec, 29 cycles, 72 ° C for 2 min, and 12 ° C. 5 μL of the digested product was subjected to electrophoresis, and electrophoresis was carried out using a 1% agarose gel at 180 V for 30 min. The electrophoresis results are shown in Figure 5(b), and the correct strip size is indicated by the arrow.

4. Sequencing analysis

The correct bacterial extract plasmid of the bacterial solution was selected for sanger sequencing, and the plasmid with the correct assembly sequence was analyzed.

5. Gibson assembles in one step to obtain the final integration plasmid

The correct subfragment cloning plasmid obtained in step 4 and the vector backbone were assembled in one step. The 20 μL reaction system was as follows: 4×buffer 5 μL, the subfragment cloning plasmid and the vector backbone were added in a molar ratio of 5:1, and the endonuclease was 1 μL. DDH ₂ O was supplemented with 20 μL and warmed for 1 h. The endonuclease of the reporter construct was BswiI, and the reaction temperature was 55 ° C. The endonuclease of the GIV (Gal4-crtI-VP16) plasmid construct was TspMI, and the reaction temperature was 75. °C, 10 μL was taken after the warm bath for clonal transformation.

6. Enzyme digestion identification

The monoclonal clone was picked from the TA cloning plate in the step 5, and after the overnight culture, the plasmid was extracted using a kit, and the restriction enzyme digestion was used to identify the plasmid. The enzyme digestion system: 0.2 μL of XbaI (NEB), 0.2 μL of XhoI/PstI (NEB), 1 μL of buffer, 3 μL of plasmid DNA, and 10 μL of ddH ₂ O. The enzyme was digested at 37 ° C for 1 h. 5 μL of the digested product was subjected to electrophoresis, and electrophoresis was carried out using a 1% agarose gel at 180 V for 30 min. The electrophoresis results are shown in Figure 5(c) with a schematic diagram of the simulated digestion.

7. Sequencing analysis

The correct plasmid was selected for sequencing, and the plasmid with the correct assembly sequence was analyzed.

8. Microplate reader detects fluorescence value

The two integrated plasmids with the correct sequencing were mixed and mixed with 1:1, and the concentration was measured. 200 ng of the transformed lycopene yeast engineering bacteria (as a positive yeast strain) was taken out, and the corresponding auxotrophic medium was coated and picked. The colonies were cultured overnight in a liquid medium, and the lycopene yeast engineering strain (as a negative yeast strain) which was not transformed with any plasmid was cultured overnight, as shown in Fig. 6, a tomato containing a biosensor was found on a fluorescent display device. The erythromycin yeast strain (A) has a more pronounced green fluorescence display than the lycopene yeast strain (B) without a biosensor.

In order to obtain more accurate data to illustrate the detection function of the lycopene biosensor, the above-mentioned strains as positive and negative were cultured, the OD value was detected in a time period, and the fluorescence value was read by a microplate reader (the excitation wavelength of yEGFP) It is 488 nm, the emission wavelength is 575 nm), and the data is plotted as the abscissa and the fluorescence value as the ordinate, as shown in Fig. 7. It can be seen from the figure that the positive yeast strain has a significantly higher fluorescence value than the negative yeast strain, which proves that the lycopene biosensor is successfully constructed in this embodiment, and the biosensor can be used to detect whether the strain produces lycopene. .

Example 2 Biosensor for detecting resveratrol

Resveratrol is formed by the synthesis of p-coumaroyl-CoA by resveratrol under the action of p-diphenylene synthase (STS) (Fig. 3). Therefore, the STS can be used as a recognition group of the biosensor, and the oligonucleotide sequence used for construction is as shown in Table 2. The other construction elements are the same as those in Embodiment 1, and the experimental construction method is the same as that in Embodiment 1. For specific information, see Example 1. The final assembled large fragment was the 5'-GAL1 promoter + yEGFP + CYC1-3' terminator fragment (SEQ ID NO: 133) and the Mata2-GL77F-SV fragment (SEQ ID NO: 171), which were ligated into the vector. Skeleton (same as in Example 1).

Finally, the assembled plasmid was subjected to restriction enzyme digestion and sequencing, and the two constructed plasmids with the correct sequencing were co-transformed into the engineering bacteria of resveratrol yeast, and the experimental results were observed.

Resveratrol yeast engineering bacteria: from the National Gene Bank of Shenzhen Huada Gene Life Science Research Institute, which is based on the Saccharomyces cerevisiae strain SynYII (Accession No. CTCCC NO: M 2014434) as the chassis cells, synthesizing resveratrol The foreign genes (Z26250, CYP81B1C, TAL, 4CL3, STS and PAL) required for the pathway are spliced to form a recombinant plasmid, which is introduced into the chassis cells for metabolite detection, and the strain capable of expressing resveratrol is a resveratrol yeast engineering strain. The recombinant strain of resveratrol transformed with two integrated plasmids was used as a positive strain, and the recombinant strain of resveratrol yeast which had not transformed any plasmid was used as a negative strain, and the monoclonal medium of each of the two strains was cultured overnight. As shown in Fig. 8, it can be found on the fluorescent display that the resveratrol yeast strain (C) containing the biosensor has more obvious green fluorescence than the resveratrol yeast strain (D) without the biosensor. display.

Moreover, the OD value of the positive and negative strains was detected by time period, and the fluorescence value was read by a microplate reader (the excitation wavelength of yEGFP was 488 nm, the emission wavelength was 575 nm), and the OD value of the bacterial solution concentration was taken as the abscissa and fluorescence value. As the ordinate draws the data into a graph, as shown in Fig. 9, it can be seen from the figure that the positive yeast strain has a significantly higher fluorescence value than the negative yeast strain, which proves that the resveratrol biosensor is successfully constructed in this embodiment. And the biosensor can be used to detect whether the strain produces resveratrol.

Table 2 Sequences required to construct a resveratrol biosensor

The invention has been described above with reference to specific examples, which are merely intended to aid the understanding of the invention and are not intended to limit the invention. For the person skilled in the art to which the invention pertains, several simple derivations, variations or substitutions can be made in accordance with the inventive concept.

Claims (36)

1. A biosensor for detecting a target molecule compound, wherein the biosensor is a fusion protein, the biosensor comprising a target molecule synthetase, and a DNA binding structure respectively linked to the target molecule synthetase a domain, a transcriptional activation domain, and a degradation determinant; the degradation determinant degrades the biosensor when a precursor material of the target molecule is absent; the target molecule is present when a precursor substance of the target molecule is present A synthetase recognizes and binds to the precursor substance to stabilize the biosensor such that the degradation determinant fails to automatically detach, the DNA binding domain binding to a promoter region of a reporter gene, the transcriptional activation domain The reporter gene expression is activated.
2. The biosensor according to claim 1, wherein the biosensor sequentially connects a degradation determinant, a DNA binding domain, a target molecule synthetase, and a transcriptional activation domain from a nitrogen terminal to a carbon terminal.
3. The biosensor according to claim 1 or 2, wherein the target molecule compound is selected from a natural product, and accordingly, the target molecule synthetase is an enzyme that synthesizes the natural product.
4. The biosensor according to claim 3, wherein the target molecular synthetase is a wild type enzyme that synthesizes the natural product.
5. The biosensor according to claim 1 or 2, wherein the target molecular compound is selected from the group consisting of a drug and/or a prodrug compound, and accordingly, the target molecule synthetase is a synthetic drug and/or An enzyme of a prodrug compound.
6. The biosensor according to claim 1 or 2, wherein the target molecule is lycopene and the target molecule synthetase is lycopene synthase.
7. The biosensor according to claim 6, wherein the lycopene synthase is encoded by a CrtI gene.
8. The biosensor according to claim 6, wherein the precursor substance of the target molecule is phytoene.
9. The biosensor according to claim 1 or 2, wherein the target molecule is resveratrol and the target molecule synthetase is p-diphenylene synthase.
10. The biosensor according to claim 9, wherein the p-diphenylene synthase is encoded by an STS gene.
11. The biosensor according to claim 9, wherein the precursor substance of the target molecule is p-coumaroyl-CoA.
12. The biosensor according to claim 1 or 2, wherein the degradation determinant is a degradation determinant MATα2 in yeast.
13. The biosensor according to claim 1 or 2, wherein the reporter gene is a resistance screening gene and/or a fluorescent marker gene.
14. The biosensor according to claim 13, wherein the resistance screening gene is selected from the group consisting of ampicillin, kanamycin, spectinomycin, and/or tetracycline.
15. The biosensor according to claim 13, wherein the fluorescent marker gene is selected from the group consisting of a green fluorescent protein GFP gene, a red fluorescent protein RFP gene, and/or a yellow fluorescent protein YFP gene.
16. A method of constructing a biosensor according to any one of claims 1 to 15, characterized in that the method comprises first first ligating a nucleic acid fragment encoding a target molecule synthetase, and respectively connecting to the target molecule synthetase The nucleic acid fragment of the DNA binding domain at both ends, the nucleic acid fragment of the transcriptional activation domain, and the nucleic acid fragment of the degradation determinant are assembled; and the assembled nucleic acid fragment is then ligated into the plasmid vector.
17. The method according to claim 16, further comprising: constructing an expression plasmid of a reporter gene, said DNA binding domain binding to a promoter region of a reporter gene, said transcriptional activation domain activating said Report gene expression.
18. The method of claim 16 wherein the assembly of the nucleic acid fragments is accomplished by PCA assembly and/or Gibson assembly.
19. A nucleotide sequence encoding the biosensor of any of claims 1-15.
20. The nucleotide sequence according to claim 19, wherein the nucleotide sequence comprises the sequence set forth in SEQ ID NO:134.
21. The nucleotide sequence according to claim 19, wherein the nucleotide sequence comprises the sequence set forth in SEQ ID NO: 171.
22. The nucleotide sequence according to any one of claims 19 to 21, wherein the nucleotide sequence further comprises a first vector sequence.
23. The nucleotide sequence according to claim 22, wherein the first vector sequence is the sequence of the vector backbone pRS415.
24. The nucleotide sequence according to claim 19, further comprising the expression sequence of the reporter gene according to any one of claims 1, 13-15.
25. The nucleotide sequence according to claim 24, wherein the reporter gene expression sequence comprises the sequence set forth in SEQ ID NO:133.
26. The nucleotide sequence according to claim 24 or 25, wherein the reporter gene expression sequence further comprises a second vector sequence.
27. The nucleotide sequence according to claim 26, wherein the second vector sequence is the sequence of the vector backbone pRS413.
28. A method for detecting a compound of a target molecule, the method comprising: the biosensor according to any one of claims 1 to 15 or the nucleotide sequence of any one of claims 19 to 23, and a report The gene is co-introduced into the host cell for expression; the target molecule compound is qualitatively and/or quantitatively analyzed by detecting the expression of the reporter gene.
29. A biosensing system for detecting a target molecule compound, characterized in that the biosensor system comprises a nucleotide sequence encoding the biosensor according to any one of claims 1 to 15, and a reporter gene expression sequence.
30. The biosensing system according to claim 29, further comprising a host cell in which the nucleotide sequence of the biosensor and the reporter gene expression sequence are expressed.
31. The biosensing system according to claim 29 or 30, wherein the nucleotide sequence of the biosensor comprises a sequence as shown in SEQ ID NO: 134, and the reporter gene expression sequence comprises SEQ ID NO :133 shows the sequence.
32. The biosensing system according to claim 29 or 30, wherein the nucleotide sequence of the biosensor comprises a sequence as shown in SEQ ID NO: 171, and the reporter gene expression sequence comprises SEQ ID NO :133 shows the sequence.
33. The biosensing system according to any one of claims 29 to 32, wherein the nucleotide sequence of the biosensor further comprises a first vector sequence, and the reporter gene expression sequence further comprises a second vector sequence, The host cell is a yeast cell.
34. The biosensor system according to claim 33, wherein the first vector sequence is a sequence of a vector backbone pRS415 and the second vector sequence is a sequence of a vector backbone pRS413.
35. The biosensor according to any one of claims 1 to 15 or the nucleotide sequence according to any one of claims 19 to 27 or the biosensing system according to any one of claims 29 to 34 for detecting a target molecular compound Use in.
36. The use according to claim 35, wherein the use comprises performing a relative qualitative analysis, quantitative analysis, and/or screening of a host cell producing the target molecule compound.

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