eforRed mutant, its encoding gene and expression vector and its application

Abstract

The present invention belongs to the field of genetic engineering technology, and more particularly to a kind of eforRed mutant, its encoding gene and expression vector and its application.The eforRed mutant amino acid sequence is selected from at least one of SEQ ID NO.3-10.The chromogenic protein eforRed mutant provided by the present invention has good visible light excitation activity, and is stronger in fluorescence intensity in the visible light region than the wild-type eforRed and its K201E mutant, and color development is more sensitive and structural stability is also better.In addition, the eforRed mutant of the present invention has no phototoxicity compared to the luciferase reporter gene, does not kill cells, and does not need to add expensive exogenous substrates to develop color. As a reporter gene, it has broad prospects and application advantages in the fields of visual screening of genetic transformation positive transformants, monitoring gene expression activity, protein localization, live cell fluorescence tracing and biological imaging.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to an eforRed mutant, its encoding gene and expression vector and applications thereof. Background Art

[0002] Reporter genes are an important tool for studying gene expression and activity detection. Their expression products can be used as detection markers to indicate certain changes or processes. In genetic transformation, reporter genes are fused with gene expression regulatory elements to form chimeric genes, or fused with other target genes for expression. By detecting the expression of reporter genes, such as reporter proteins or mRNA, the expression of the target gene in cells can be reflected, thereby visualizing the target gene transformation and expression process. Therefore, reporter genes are often used to determine whether exogenous genes have been successfully introduced into recipient cells, tissues, or organs to efficiently screen positive clones. The activity of the reporter gene-encoded product can also be used to monitor the expression activity of the target gene, such as detecting promoter activity, or detecting the expression location of the target gene in plant cells or tissues, as well as performing live cell tracing or bioimaging.

[0003] Traditional reporter genes, such as the kanamycin resistance gene, hygromycin marker gene, luciferase gene, and β-glucuronidase gene, have been gradually replaced by fluorescent protein reporter genes due to the need for expensive exogenous substrate addition, low reaction sensitivity, and cytotoxicity. Fluorescent proteins (FPs) are a broad class of proteins capable of fluorescing. Different FPs emit different colors of fluorescence under different wavelengths of excitation light. When a target gene carries a fluorescent protein reporter gene, it will be expressed in recipient cells after transduction, emitting a visible fluorescent signal. Fluorescent proteins offer advantages such as sensitive reactions, substrate-free fluorescence excitation, easy detection of light signals, lack of cytotoxicity, and the ability to track in vivo. They have become one of the most widely studied and utilized reporter tools in biochemistry and cell biology. For example, Zymomonas mobilis is a strain that produces products such as lactic acid, 2-3-butanediol, and isobutanol. Studies have shown that expressing fluorescent proteins such as GFP or mCherry in Zymomonas mobilis enables visual monitoring of its fermentation production. By monitoring the color value of the fermentation broth or strain, its growth status can be determined, which is very beneficial for screening dominant strains and controlling the fermentation process. However, even though the application of fluorescent proteins has many advantages, detection still requires instruments such as flow cytometers and fluorescence microplate readers to excite fluorescence and then pass the detection sensor through the instrument data processing, making the inspection process more complicated.

[0004] Chromogenic proteins, as homologs of fluorescent proteins, strongly absorb visible light, resulting in distinct colors visible under natural light. This eliminates the need for complex fluorescence detection procedures and offers potential advantages as reporter genes. Previous studies by our group demonstrated that eforRed (chromo-red fluorescent GFP-like protein, NBCI accession number: ACD13196.1), a chromogenic protein from Echinopora forskaliana, and its K201E mutant exhibit visible light color development. Transfection of eforRed into Zymomonas mobilis creates a visualized cell factory that facilitates the detection of various intracellular and extracellular target product levels and rapid assessment of strain growth compliance. However, key limitations in its application lie in low fluorescence signal strength, weak color under visible light, and insufficient protein stability. Therefore, sequence optimization is crucial to generate chromogenic protein reporter genes with deeper colors and stronger fluorescence. Summary of the Invention

[0005] In response to the problems of low fluorescence intensity and light color under visible light of the chromogenic protein eforRed in the existing technology, the present invention provides an eforRed mutant and a nucleic acid molecule and expression vector containing its encoding gene, and further provides the application of the nucleic acid molecule and expression vector as a reporter gene, especially in constructing a visual cell factory.

[0006] The present invention is specifically implemented through the following technical solutions:

[0007] The first aspect of the present invention provides an eforRed mutant, whose amino acid sequence is selected from at least one of SEQ ID NOs. 3-10.

[0008] Furthermore, the amino acid sequence of the eforRed mutant is selected from at least one of SEQ ID NO.3-6 and SEQ ID NO.8-10; further, the amino acid sequence of the eforRed mutant is selected from at least one of SEQ ID NO.8-10; and still further, the amino acid sequence of the eforRed mutant is selected from at least one of SEQ ID NO.9-10.

[0009] The second aspect of the present invention provides a nucleic acid molecule comprising a gene sequence encoding the eforRed mutant as described above.

[0010] Furthermore, the nucleic acid molecule also includes a gene sequence encoding a promoter, and the eforRed mutant is located downstream of the promoter.

[0011] Furthermore, the promoter is selected from Pgap-6M or Pgap-4S promoter, and the nucleotide sequences of Pgap-6M and Pgap-4S promoters are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively.

[0012] The third aspect of the present invention provides an expression vector comprising the nucleic acid molecule described above.

[0013] Furthermore, the expression vector is a pEZ15A vector.

[0014] The fourth aspect of the present invention provides the use of the nucleic acid molecule or the expression vector as described above as a reporter gene.

[0015] Furthermore, the application is as a reporter gene in constructing a visual cell factory, and the visual cell factory is a transgenic engineered bacterium obtained by transferring the above-mentioned nucleic acid molecule or expression vector into a recipient cell.

[0016] Furthermore, the preparation method of the visualized cell factory includes the following steps: connecting the promoter encoding gene and the eforRed mutant encoding gene to form a fusion gene, inserting the fusion gene into the pEZ15A vector, constructing a recombinant expression vector, and transferring the recombinant expression vector into the recipient cell.

[0017] Furthermore, the recipient cell is selected from Escherichia coli or Zymomonas mobilis. Still further, it is selected from Escherichia coli DH5α or Zymomonas mobilis ZMNP.

[0018] The advantages and positive effects of the present invention are:

[0019] The chromogenic protein eforRed mutant provided by the present invention has good visible light excitation activity, and has stronger fluorescence intensity in the visible light region than the wild-type eforRed and its K201E mutant, more sensitive color development, and better structural stability. It is fused with a constitutive strong promoter and expressed in a prokaryotic expression system. The bacterial cells of the genetically modified engineered bacteria are bright in color and obvious in color development under visible light. An obvious pink change can be observed only by the naked eye, which has the advantage of being more intuitive and convenient to monitor. In addition, the eforRed mutant of the present invention has no phototoxicity compared to the luciferase reporter gene, will not kill cells, and does not require the additional addition of expensive exogenous substrates to develop color. As a reporter gene, it has broad prospects and application advantages in the fields of visual screening of positive transformants for genetic transformation, monitoring gene expression activity, protein localization, live cell fluorescence tracing, and biological imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is a fluorescence intensity graph of Escherichia coli transformed with wild-type eforRed and various K201 saturation mutants according to the present invention;

[0022] Figure 2 This is a fluorescence intensity graph of Escherichia coli cells transformed with wild-type eforRed and the T24V-K201E combined mutant according to an embodiment of the present invention;

[0023] Figure 3 This is a fluorescence intensity graph of Zymomonas mobilis transformed with wild-type eforRed and T24V-K201E combined mutant according to an embodiment of the present invention;

[0024] Figure 4 This is a protein energy diagram of the wild-type eforRed and various eforRed mutants in the examples of the present invention;

[0025] Figure 5 This is a plate culture diagram of Zymomonas mobilis overexpressing wild-type eforRed and T24V-K201H mutant in an embodiment of the present invention. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the examples. Unless otherwise specified, the equipment and reagents used in each example and test example can be obtained from commercial sources. The specific examples described herein are only used to illustrate the present invention and are not intended to limit the present invention.

[0027] Based on the information contained in this application, it will be readily apparent to those skilled in the art that various changes can be made to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are provided merely to illustrate specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields would clearly be able to make to the embodiments of the present invention are encompassed within the scope of the appended claims.

[0028] In order to better understand the present invention rather than to limit the scope of the present invention, all numerals representing dosage, percentage, and other numerical values ​​used in this application should be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods. In addition, the meaning of the terms "comprise", "comprising", "containing", "having" and the like is non-restrictive, and other steps and other ingredients that do not affect the results can be added.

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below.

[0030] The chromogenic protein eforRed (chromo-red fluorescent GFP-like protein, NBCI accession number: ACD13196.1) derived from Echinopora forskaliana exhibits high visible light absorption. Previous studies (see patent "CN116426448A Visualization of Zymomonas mobilis, Construction Method, and Application (Publication Date: July 14, 2023)") using Z. mobilis as a recipient cell for genetic transformation investigated the luminescence properties of this protein and its K201E mutant. The results showed that the recombinant strain exhibited a visible pink color after fermentation, requiring no exogenous substrate for color development and exhibiting no cytotoxicity. This enabled visual monitoring of the fermentation process of Z. mobilis under visible light. Furthermore, under natural light observation, the K201E mutant exhibited a stronger color than wild-type eforRed, consistent with the fluorescence signal detected by flow cytometry. This patent reveals the potential advantages of the chromogenic protein eforRed and its K201E mutant as reporter genes, providing a monitoring basis for their use as reporter genes in gene function verification and metabolic engineering transformation of transgenic bacteria.

[0031] To further improve the visible light color development function and fluorescence properties of the chromogenic protein eforRed, the present invention performed site-directed saturation mutagenesis on the 201st amino acid (K201) site based on the wild-type eforRed and the K201E mutant. Different mutants were connected to the downstream of the promoter and genetically transformed into Escherichia coli and Zymomonas mobilis. By comparing the fluorescence intensity of the bacteria, it was found that the K201A, K201D, K201G, and K201H mutants had stronger fluorescence in the visible light range, which can improve the fluorescence intensity of the transformed chromogenic protein eforRed. To further enhance the protein's structural stability, the 24th amino acid position was mutated from threonine (T) to valine (V) to reduce the protein's Gibbs free energy. Combinatorial optimization was performed based on the K201A, K201D, K201G, K201H, and T24V mutations. The researchers found that the fluorescence intensity of Escherichia coli transformed with the T24V-K201E mutant and Zymomonas mobilis transformed with the T24V-K201A, T24V-K201G, and T24V-K201H mutants was significantly enhanced. Furthermore, these mutants exhibited lower overall Gibbs free energy and improved structural stability.

[0032] Based on this, an embodiment of the present invention provides an eforRed mutant, whose amino acid sequence is selected from at least one of SEQ ID NO.3-10, wherein the sequences shown in SEQ ID NO.3-10 correspond to eforRed K201A, K201D, K201G, K201H, T24V-K201E, T24V-K201A, T24V-K201G, and T24V-K201H mutants, respectively.

[0033] The chromogenic protein eforRed mutant provided by the present invention has good visible light excitation activity. Compared with the wild-type eforRed and its K201E mutant, the fluorescence intensity in the visible light region is stronger, the color development is more sensitive, and the structural stability is also better. It is fused with a constitutive strong promoter and expressed in a prokaryotic expression system (Escherichia coli and Zymomonas mobilis). Under the same culture system and culture conditions, the fluorescence intensity of the bacteria transformed with the eforRed mutant of the present invention is higher by flow cytometry. The bacteria are brightly colored and have obvious color development under visible light. A clear pink change can be observed only by the naked eye, which has the advantage of more intuitive and convenient observation and is more conducive to the visualization monitoring of genetic transformation operations and genetically modified engineered bacteria.

[0034] In addition, the eforRed mutant of the present invention is a fluorescent protein homologous protein. Compared with the luciferase reporter gene, it has no phototoxicity, will not kill cells, and does not require the addition of expensive exogenous substrates to develop color. As a reporter gene, it has broad prospects and application advantages in the fields of screening positive transformants for genetic transformation, monitoring gene expression activity, protein localization, live cell fluorescence tracing, and biological imaging. In practical applications, the eforRed mutant encoding gene of the present invention is fused with the target gene for expression, the gene sequence of the fusion protein is inserted into a suitable expression vector, and transformed into the recipient cell. By observing whether the chromogenic protein is expressed, it can be indicated whether the target gene transformation is successful or not, and then genetically modified and non-genetically modified cells can be easily distinguished; by detecting the level of chromogenic protein expression, the target gene expression activity can be indicated; by detecting the position and distribution of the chromogenic protein, the location of the target gene expression protein in living cells and tissues can be observed. Alternatively, the eforRed mutant is labeled with other target proteins, and the location study of the target protein is achieved by fluorescence imaging. In cell culture or in vivo models, specific cells are tracked using chromogenic protein markers, and their migration and distribution and other physiological processes can be observed.

[0035] In an embodiment of the present invention, a mutant of the chromogenic protein eforRed is used to construct a visualized cell factory. Specifically, the eforRed mutant of the present invention is linked downstream of a strong promoter and transformed into a recipient bacterium. The resulting transgenic bacteria, after expressing the eforRed mutant, exhibit a pink color. The pink color directly reflects the cell concentration, enabling visualization of the fermentation process under visible light.

[0036] In the context of the present invention, the term "visualization" should be interpreted in the broadest sense, meaning that the growth of bacteria can show a color visible to the naked eye and form a certain proportional relationship with its growth concentration, and can form a clear visual difference visible to the naked eye with the color of the environment. For example, the bacteria form colonies with a special color on the plate, or its culture medium forms a color that is proportional to the growth concentration. Of course, since the eforRed mutant of the present invention has the characteristic of fluorescence excitation by visible light, visualization can also be manifested as a clear change in fluorescence intensity under a fluorescence microscope or when detected by an instrument such as a flow cytometer.

[0037] Preferably, the amino acid sequence of the eforRed mutant is selected from at least one of SEQ ID NOs. 3-6 and 8-10; more preferably, selected from at least one of SEQ ID NOs. 8-10; and even more preferably, selected from at least one of SEQ ID NOs. 9-10. The eforRed mutants shown in SEQ ID NOs. 9-10 exhibit significant fluorescence enhancement effects in both Escherichia coli and Zymomonas mobilis, exhibit broad adaptability to diverse strains, exhibit high fluorescence intensity, are more conducive to visible light monitoring, and exhibit excellent stability.

[0038] Yet another embodiment of the present invention provides a nucleic acid molecule comprising a gene sequence encoding the eforRed mutant as described above.

[0039] The advantages of the nucleic acid molecule over the prior art are the same as those of the eforRed mutant over the prior art as described above, and will not be repeated here.

[0040] Nucleic acid molecules include DNA molecules (e.g., genomic DNA or cDNA) and / or RNA molecules (e.g., mRNA), which can be single-stranded or double-stranded. The sequence of the nucleic acid molecule can be derived from the amino acid (AA) sequence of the eforRed mutant by conventional means, such as codon coding rules. The full-length sequence of the nucleic acid molecule or fragments thereof can generally be obtained by PCR amplification, recombinant methods, or synthetic methods.

[0041] Optionally, the nucleic acid molecule further comprises a gene sequence encoding a promoter, and the eforRed mutant is located downstream of the promoter. The promoter is used to initiate the transcription and translation process of the eforRed mutant and can be adaptively selected based on the expression of the recipient cell and the eforRed mutant.

[0042] In a preferred embodiment, in order to achieve efficient expression of the eforRed mutant and realize visual monitoring under visible light, the promoter is selected from Pgap-6M or Pgap-4S promoter, wherein the nucleotide sequence of Pgap-6M or Pgap-4S promoter is shown as SEQ ID NO.12 and SEQ ID NO.13, respectively.

[0043] Another embodiment of the present invention provides an expression vector, which includes the nucleic acid molecule described above.

[0044] The advantages of the expression vector over the prior art are the same as those of the eforRed mutant over the prior art as described above, and will not be repeated here.

[0045] Optionally, the expression vector includes a prokaryotic expression vector, a eukaryotic expression vector, or a viral expression vector (such as a lentivirus or adenovirus). Correspondingly, the recipient cells transformed or transfected by the expression vector can be prokaryotic cells or eukaryotic cells, depending on the type of expression vector. For example, when it is a prokaryotic expression vector, the recipient cells are prokaryotic cells, and commonly used prokaryotic cells include Escherichia coli, Bacillus subtilis, and Zymomonas mobilis. When it is a eukaryotic expression vector, the recipient cells are eukaryotic cells, and commonly used eukaryotic cells include Saccharomyces cerevisiae and Saccharomyces cerevisiae.

[0046] Typical vectors include plasmids (e.g., pBR322, pUC series, pET series, pGEX series, pEZ15A), viral vectors, bacteriophages (e.g., λgt4λB, λ-Charon, λΔz1, and M13), cosmids, and minichromosomes. Plasmids are the most commonly used vectors, and therefore, in the context of the present invention, unless otherwise specified, plasmids and vectors can be used interchangeably.

[0047] In a preferred embodiment, the expression vector is a pEZ15A vector, and the nucleic acid molecule is inserted into the multiple cloning site of the pEZ15A vector. Prokaryotic cells (such as Escherichia coli or Zymomonas mobilis) can be transformed with the pEZ15A vector of the present invention to which the nucleic acid molecule is linked, and cultured using conventional methods to express the chromogenic protein within the cells.

[0048] Another embodiment of the present invention provides the use of the nucleic acid molecule or expression vector as described above as a reporter gene, especially as a reporter gene in constructing a visual cell factory, and provides related visual cell factories and preparation methods thereof.

[0049] Specifically, the visualized cell factory is a transgenic engineered bacterium obtained by transferring the above-mentioned nucleic acid molecule or the above-mentioned expression vector into a recipient cell.

[0050] The preparation method of the above-mentioned visualized cell factory comprises the following steps: connecting the promoter encoding gene and the eforRed mutant encoding gene to form a fusion gene, inserting the fusion gene into the pEZ15A vector, constructing a recombinant expression vector, and transferring the recombinant expression vector into a recipient cell.

[0051] In a typical embodiment, the recipient cell is selected from Escherichia coli, such as Escherichia coli DH5α, or selected from Zymomonas mobilis, such as Zymomonas mobilis ZMNP. The construction method of Zymomonas mobilis ZMNP refers to the patent "CN115806922A Genetically engineered strains of Zymomonas mobilis and their applications (publication date: 2023-03-17)".

[0052] The promoter encoding gene and the eforRed mutant encoding gene can be obtained by whole gene synthesis or by PCR amplification. When obtained by PCR amplification, preferably using the pEZ-Pgap-6M-eforRed plasmid as a template, PCR amplification is performed using the upstream primer shown in SEQ ID NO.18 and the downstream primer shown in SEQ ID NO.19 to obtain a linearized pEZ15A vector, and PCR amplification is performed using the upstream primer shown in SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25 or SEQ ID NO.26 and the downstream primer shown in SEQ ID NO.39 to obtain eforRed K201A, K201D, K201G or K201H mutants, respectively, and each eforRed mutant is connected to the linearized pEZ15A vector by the Gibson assembly method; and a recombinant expression vector of the eforRedK201A, K201D, K201G or K201H mutant is constructed. To construct a combination mutant containing the T24V mutation site, a recombinant expression vector containing the eforRed K201A, K201D, K201G, or K201H mutant was used as a template. PCR amplification was performed using the upstream primer shown in SEQ ID NO. 40 and the downstream primer shown in SEQ ID NO. 41. The amplified product was circularized by Gibson assembly to obtain a recombinant expression vector containing the T24V mutation site. The construction methods of the starting pEZ-Pgap-6M-eforRed plasmid and pEZ-Pgap-6M-K201E are described in patent CN116426448A.

[0053] The recombinant expression vector can be transformed or transfected into the recipient cells by various methods known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome-mediated transfection, liposome fusion, lipofection and protoplast fusion. In addition, the recipient cells can also be introduced by gene gun bombardment.

[0054] The present invention will be further described below with reference to specific examples. Experimental methods in the following examples where specific conditions are not specified were generally performed under conventional conditions, such as those described in the Molecular Cloning Laboratory Manual (4th Edition) published by Cold Spring Harbor Laboratory, or under conditions recommended by the manufacturer.

[0055] 1. Preparation of chromogenic protein eforRed mutants

[0056] The present invention's prior study "CN116426448A Visualization of Zymomonas mobilis, Construction Method and Application (Publication Date: 2023-07-14)" confirmed that the chromogenic protein eforRed and its K201E mutant have visible light absorption characteristics, have a significant color reaction under visible light, and have application value as a reporter gene. To further improve the color-developing performance of the chromogenic protein eforRed, the present invention, based on the wild-type eforRed and the K201E mutant, performs site-directed saturation mutagenesis on the 201st amino acid site of the protein, that is, mutates the lysine (L) at the 201st site of eforRed to the remaining 18 amino acids except E. After connecting the different mutants to a promoter, they are transformed into Escherichia coli and Zymomonas mobilis, and the fluorescence signal value of the bacteria is observed. The K201A, K201D, K201G, and K201H mutants with enhanced fluorescence signals are screened. In addition, through analysis of protein energy and protein structure, the 24th amino acid site was mutated from threonine (T) to valine (V). At the same time, based on the optimization of the K201E site-directed saturation mutation, the mutants K201A, K201D, K201G, K201H and T24V were combined and mutated. It was found that the fluorescence intensity of the T24V-K201E mutant in Escherichia coli and the T24V-K201A, T24V-K201G, and T24V-K201H mutants in Zymomonas mobilis was enhanced. These mutants can improve the color development performance of eforRed. In addition, the Gibson free energy of these mutants was predicted, and their overall energy was reduced, indicating that the protein structure was more stable; these characteristics make the newly screened mutants of the present invention more advantageous in application than the wild-type protein and the K201E mutant. Table 1 shows the amino acid sequences of the promoters expressing the eforRed of the present invention and its advantageous mutants and the aforementioned proteins.

[0057] Table 1 Amino acid sequences of newly screened mutants and their expression promoters in the examples of the present invention

[0058]

[0059]

[0060] 2. Construction of the chromogenic protein eforRed expression vector

[0061] A prokaryotic expression system was constructed using the pEZ15A expression plasmid and Escherichia coli and Zymomonas mobilis as recipient cells. The gene sequences encoding the chromogenic protein eforRed and each mutant were integrated into the expression vector pEZ15A. A strong constitutive promoter was used to control gene expression, generating recombinant vectors. These recombinant vectors were then transformed into recipient cells to produce a visualization strain capable of synthesizing chromogenic proteins. The recombinant expression vectors constructed in this example are shown in Table 2.

[0062] Table 2 Expression vectors constructed and inserted expression elements in the examples of the present invention

[0063]

[0064]

[0065] The eforRed gene and promoter gene of the above-mentioned expression vector and the primer sequences used are shown in Table 3, where F represents an upstream primer (the shadow boldly shows the mutation site codon), and R represents a downstream primer. The underlined portion in the primer is a vector homology arm sequence. It should be noted that Table 3 exemplarily provides the eforRed gene sequence after codon optimization for Zymomonas mobilis and integration of the termination codon TAA at the end of the gene. Each mutant gene sequence can be based on the wild-type eforRed gene, and the amino acid codon corresponding to the mutation site is replaced with the amino acid codon after mutation, so it is not repeated. When constructing the eforRed K201A mutant, the 604th AAA codon of the gene sequence is replaced with GCA, and the coding gene of the K201E mutant is obtained.

[0066] Table 3 Gene and primer sequences involved in constructing expression plasmids in the examples of the present invention

[0067]

[0068]

[0069] The construction process of the expression vector is as follows:

[0070] Plasmid 1 construction: Using eforRed (a universal puc plasmid vector for bacterial protection) synthesized by Beijing Qingke Company and the Pgap-6M promoter as DNA amplification templates, the eforRed gene was amplified using the eforRed-F and eforRed-R primer pairs, and the Pgap-6M promoter was amplified using the Pgap-F and Pgap-R primer pairs. Overlap PCR was used to connect the Pgap-6M and eforRed DNA fragments to form a long fusion gene. This fusion gene now carries homology arms homologous to the pEZ15A vector at both ends. The fusion gene was then ligated to the linearized pEZ15A vector using the Gibson assembly method to form the circularized pEZ-Pgap-6M-eforRed vector. Specifically, the fusion gene and linearized pEZ15A vector were co-incubated with competent Escherichia coli DH5α cells for simultaneous transformation and assembly. Positive clones on the plates were verified by colony PCR, and plasmid 1 was extracted after overnight culture. The linearized pEZ15A vector was generated by PCR amplification using primers Pgap-FK-F and Pgap-FK-R. The aforementioned process can be found in patent "CN116426448A".

[0071] Construction of plasmid 2-20: Using plasmid 1 as a template, PCR amplification was performed using upstream primers K201A-F and K201C-F containing mutant base sequences and the universal downstream primer TB201-R to obtain various eforRed mutants. Separately, using plasmid 1 as a template, PCR amplification was performed using the primer pair BH201ZT-F and BH201ZT-R to obtain a linear plasmid. The DNA fragments of the eforRed mutants were religated with the linear plasmid by Gibson assembly to form a circular plasmid containing a K201 saturation mutation site.

[0072] Construction of plasmids 21-26: Using plasmids 1, 2, 3, 5, 7, and 8 as templates, full-length plasmids were amplified by PCR using bidirectional primers T24V-F and T24V-R, which carry the T24V mutation sequence, to generate linear plasmids harboring the mutation site. These linear plasmids harboring the mutation site were circularized by Gibson assembly to generate circular plasmids harboring the T24V mutation site.

[0073] Construction of plasmid 27-29: In order to overexpress the chromogenic protein and make it have a more obvious color reaction, the Pgap-6M promoter was replaced with the Pgap-4S promoter. The specific steps are as follows: The Pgap-4S promoter synthesized by Beijing Qingke Company was used as a DNA amplification template, and the Pgap-4S promoter was amplified by the Pgap-F and Pgap-R primer pairs. The eforRed-F and Pgap-FK-R primer pairs were used to amplify the remaining parts of plasmid 1, plasmid 2, and plasmid 26 except Pgap-6M, respectively. At this time, the two ends of the Pgap-4S gene carried homology arm sequences homologous to the rest of the vector. The Pgap-4S can be connected to the remaining part of the linearized vector by the Gibson assembly method to form a circularized plasmid 27-29.

[0074] 3. Transformation of Recipient Cells with the Chromogenic Protein eforRed Expression Vector

[0075] Transformation of Escherichia coli DH5α: To construct the plasmid, mix the eforRed gene fragment and the pEZ15A vector at a ratio of 3:1. The reaction system includes: 0.12 pM eforRed gene fragment, 0.04 pM pEZ15A vector, 0.5 μL 10× Buffer 4 (Thermo), and 0.5 U T5 exonuclease. Add ddH2O to 5 μL. Incubate on ice for 5 minutes, then add the DH5α competent cell for chemical transformation. Screen for positive clones using LB plates containing 60 μg / mL kanamycin resistance. Single colonies were picked and verified for successful transformation by sequencing.

[0076] Transformation of Z. mobilis ZMNP: Thaw competent cells of Z. mobilis ZMNP on ice. Add 50 μL of the cells to an electroporation cuvette, along with 1 μg of the plasmid to be transformed. Electroporation conditions are 1800 V, 25 μF, and 200 Ω. After electroporation, inoculate the transformed strain into RMG5 liquid medium and allow it to recover in a 30°C incubator for 4-6 hours. Centrifuge the culture at 6000 rpm / min for 1 minute, and remove the supernatant. Spread 100 μL of the suspension onto a plate containing 300 μg / mL kanamycin and incubate at 30°C for 2 days. Single colonies were sequenced to verify successful transformation and obtain the desired strain.

[0077] IV. Determination of Receptor Cell Fluorescence Intensity

[0078] The target strains successfully transformed with wild-type eforRed and each mutant were inoculated into cryopreservation tubes containing 1 mL of RMG5 medium (added with 3 μL of kanamycin), and after activation in a 30°C incubator until turbidity, they were transferred to 10 mL of RMG5 medium (containing kanamycin) to control the initial OD600.nm The cells were cultured at a constant temperature of 0.1 on a shaker at 30°C and 100 rpm. After 48 hours, 200 μL of bacterial culture was collected from each well and centrifuged at 12,000 rpm for 1 minute. The supernatant was removed, and 500 μL of PBS buffer was added to the pellet and resuspended by shaking. The cells were then centrifuged again at 12,000 rpm for 1 minute, the supernatant was removed, and 500 μL of PBS buffer was added to the pellet and resuspended by shaking. After processing, the fluorescence intensity of the bacterial cells was measured using a flow cytometer.

[0079] 4.1. Fluorescence intensity detection of amino acid 201 saturation mutants

[0080] Site-directed saturation mutagenesis was performed on the 201st amino acid position of eforRed, and the lysine at the 201st amino acid position of eforRed was mutated to the remaining 19 amino acids. Using Pgap-6M as the promoter, the plasmids carrying each mutant were transformed into Escherichia coli DH5α, and the Eco-Pgap-6M-eforRed strain and mutant strains Eco-Pgap-6M-K201A, Eco-Pgap-6M-K201C, Eco-Pgap-6M-K201D, Eco-Pgap-6M-K201E, Eco-Pgap-6M-K201F, and Eco-Pgap-6M-K201G were obtained, respectively. , Eco-Pgap-6M-K201H, Eco-Pgap-6M-K201I, Eco-Pgap-6M-K201L, Eco-Pgap-6M-K201M, Eco-Pgap-6M-K201N, Eco-Pgap-6M-K201P, Eco-Pgap -6M-K201Q, Eco-Pgap-6M-K201R, Eco-Pgap-6M-K201S, Eco-Pgap-6M-K201T, Eco-Pgap-6M-K201V, Eco-Pgap-6M-K201W and Eco-Pgap-6M-K201Y.

[0081] Figure 1 The fluorescence intensity of E. coli transformed with wild-type eforRed and each K201 mutant is shown, with the horizontal axis representing the mutation type and the vertical axis representing the fluorescence intensity. Four mutants with higher fluorescence intensities than K201E were observed: K201A, K201D, K201G, and K201H.

[0082] 4.2. Fluorescence intensity detection of amino acid 24 mutants

[0083] The 24th amino acid of eforRed and its K201E mutant were site-directed mutagenesis and transformed into Escherichia coli DH5α using Pgap-6M as promoter to obtain the mutant strains Eco-Pgap-6M-T24V and Eco-Pgap-6M-T24V-K201E.

[0084] The fluorescence intensities of wild-type eforRed and mutants T24V, T24V-K201E, and K201E are shown in Figure 2. Figure 2 As shown, it can be observed that the fluorescence intensity of T24V is higher than that of eforRed, and the fluorescence intensity of T24V-K201E is higher than that of K201E.

[0085] 4.3 Fluorescence intensity detection of amino acid combination mutants at positions 24 and 201

[0086] Based on the combined mutation of mutants K201A, K201D, K201G, and K201H obtained by site-directed saturation mutagenesis of K201 and the T24 energy-optimized mutant T24V, mutants T24V-K201A, T24V-K201D, T24V-K201G, and T24V-K201H were obtained. The aforementioned mutants were connected to the Pgap-6M promoter and then transformed into Zymomonas mobilis ZMNP to obtain the strain ZM-Pgap-6M-eforRed and the mutant strains ZM-Pgap-6M-K201E and ZM-Pgap-6M-eforRed. M-Pgap-6M-K201A, ZM-Pgap-6M-K201D, ZM-Pgap-6M-K201G, ZM-Pgap-6M-K201H, ZM-Pgap-6M-T24V, ZM-Pgap-6M-T2 4V-K201E, ZM-Pgap-6M-T24V-K201A, ZM-Pgap-6M-T24V-K201D, ZM-Pgap-6M-T24V-K201G and ZM-Pgap-6M-T24V-K201H.

[0087] Comparison of fluorescence intensities of mutants K201A, K201D, K201G, K201H, T24V-K201A, T24V-K201D, T24V-K201G, and T24V-K201H in ZMNPs of Z. mobilis. Figure 3 As shown, it can be seen that the mutants K201A, K201D, K201G, K201H and T24V-K201A have a certain enhancement compared with K201E, and the fluorescence intensity of T24V-K201G and T24V-K201H is significantly enhanced compared with K201E.

[0088] 5. Protein Energy Prediction

[0089] The overall Gibson free energy of the protein was predicted using FoldX 5.0 software. Energy calculations for site-directed saturation mutations at K201 and T24V were performed using eforRed as a starting point and the PositionScan function in FoldX 5.0 for all amino acid mutations. After calculations, the energy data were exported for plotting. Energy calculations for combined site-directed saturation mutations at K201 and T24V were performed by changing the mutation site to 24. Using the T24V-K201A variant as an example, the T24V mutant was used as a starting point, and the energy scan was performed using the K201A amino acid energy change data from the 201 observation data. After calculations, the energy data were exported for plotting.

[0090] Figure 4 The protein energy of different mutants is shown, where the horizontal axis represents the mutation mode and the vertical axis represents the protein capacity (protein energy). The results show that the Gibson free energy of the T24V mutant is lower than that of eforRed, and the energy of the T24V-K201E mutant is lower than that of K201E. After the T24V mutation is integrated into the eforRed and K201E mutants, the fluorescence intensity is enhanced and the overall energy is reduced. The energy of the combined mutants T24V-K201A, T24V-K201D, T24V-K201G and T24V-K201H is also significantly lower than that of K201E, indicating that the overall structure of these proteins is more stable.

[0091] 6. Overexpression optimization of chromogenic protein mutants

[0092] To overexpress the chromogenic protein mutants and enhance their color change, the Pgap-6M promoter in the expression vector was replaced with the Pgap-4S promoter, generating plasmids pEZ-Pgap-4S-eforRed, pEZ-Pgap-4S-K201E, and pEZ-Pgap-4S-T24V-K201H. Plasmids pEZ-Pgap-4S-eforRed, pEZ-Pgap-4S-K201E, and pEZ-Pgap-4S-T24V-K201H were then transformed into Zymomonas mobilis ZMNP to generate strains ZM-Pgap-4S-eforRed, ZM-Pgap-4S-K201E, and ZM-Pgap-4S-T24V-K201H.

[0093] After each strain was applied to RMG5 solid medium, the color change was observed by naked eyes. The results were as follows: Figure 5As shown in the figure, the strains developed a pink color after culture, and the color of strain ZM-Pgap-4S-T24V-K201H was brighter and more pronounced than that of ZM-Pgap-4S-eforRed and ZM-Pgap-4S-K201E, consistent with the fluorescence signal detected by flow cytometry. Furthermore, by replacing the Pgap-4S promoter and overexpressing the eforRed protein, the color change was enhanced.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An eforRed mutant, characterized in that The amino acid sequence of the eforRed mutant is selected from at least one of SEQ ID NO.3-6 and SEQ ID NO.8-10.

2. The eforRed mutant according to claim 1, characterized in that The amino acid sequence of the eforRed mutant is selected from at least one of SEQ ID NO.9-10.

3. A nucleic acid molecule, characterized in that The invention comprises a gene sequence encoding the eforRed mutant according to any one of claims 1 to 2.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleic acid molecule further comprises a gene sequence encoding a promoter, and the eforRed mutant is located downstream of the promoter; The promoter is selected from Pgap-6M or Pgap-4S promoter, and the nucleotide sequences of Pgap-6M and Pgap-4S promoters are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively.

5. An expression vector, characterized in that The method comprises the nucleic acid molecule according to any one of claims 3 to 4.

6. The expression vector according to claim 5, characterized in that The expression vector is pEZ15A vector.

7. Use of the nucleic acid molecule according to any one of claims 3 to 4 or the expression vector according to any one of claims 5 to 6 in constructing a visual cell factory, characterized in that: The visualized cell factory is a genetically modified engineered bacterium obtained by transferring the nucleic acid molecule according to any one of claims 3 to 4 or the expression vector according to any one of claims 5 to 6 into a recipient cell.

8. Use of the nucleic acid molecule or expression vector according to claim 7 in constructing a visual cell factory, characterized in that: The preparation method of the visualized cell factory comprises the following steps: Connecting the promoter encoding gene and the eforRed mutant encoding gene to form a fusion gene, inserting the fusion gene into the pEZ15A vector to construct a recombinant expression vector, and transferring the recombinant expression vector into recipient cells; Wherein, the recipient cell is selected from Escherichia coli or Zymomonas mobilis.

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