EforRed mutant as well as coding gene, expression vector and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to an eforRed mutant as well as a coding gene, an expression vector and application thereof. The amino acid sequence of the eforRed mutant is selected from at least one of SEQ ID NO. 3-10. The chromogenic protein eforRed mutant provided by the invention has good visible light excitation activity, and compared with a wild type eforRed and a K201E mutant thereof, the chromogenic protein eforRed mutant provided by the invention has the advantages of stronger fluorescence intensity in a visible region, more sensitive color development and better structural stability. Besides, compared with a luciferase reporter gene, the eforRed mutant disclosed by the invention has no phototoxicity, does not kill cells, and can develop color without additionally adding an expensive exogenous substrate; as a reporter gene, the gene has wide prospects and application advantages in the fields of visual screening of genetic transformation positive transformants, monitoring of gene expression activity, protein positioning, living cell fluorescence tracing, biological imaging and the like.

Description

Technical Field

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

[0002] Reporter gene is an important tool for studying gene expression and activity detection. Its expression product can be used as a detection marker to indicate certain changes or processes. In genetic transformation, the reporter gene is fused with a gene expression regulatory element to form a chimeric gene, or fused with other target genes for expression. By detecting the expression of the reporter gene, such as reporter protein, mRNA, etc., the expression of the target gene in the cell can be reflected, thereby realizing the visualization of the target gene transformation and expression process. Therefore, reporter genes are usually used to determine whether exogenous genes have been successfully introduced into recipient cells, tissues or organs to efficiently screen positive clones. The expression activity of the target gene can also be monitored by the activity of the reporter gene encoding product, 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 biological imaging.

[0003] Traditional reporter genes such as kanamycin resistance gene, hygromycin marker gene, luciferase gene, β-glucuronidase gene, etc. are gradually replaced by fluorescent protein reporter genes due to the need for expensive exogenous substrate addition, low reaction sensitivity and cytotoxicity. Fluorescent proteins (FP) are a general term for a class of proteins that can emit fluorescence. There are many types of FPs. Different fluorescent proteins will emit different colors of fluorescence under different wavelengths of excitation light. When the target gene carries a fluorescent protein reporter gene, it will express fluorescent protein after transduction into the recipient cell, thereby emitting a visual fluorescent signal. Fluorescent proteins have the advantages of sensitive response, no substrate to excite fluorescence, easy detection of light signals, no cytotoxicity, and in vivo tracing. They have become one of the most widely studied and used reporter tools in biochemistry and cell biology. For example, Zymomonas mobilis is a strain that produces lactic acid, 2-3-butanediol, isobutanol and other products. Some studies have expressed fluorescent proteins such as GFP or mCherry in Zymomonas mobilis, which can achieve visual monitoring in its fermentation production. By monitoring the color value of the fermentation liquid or strain, its growth can be known, which is very helpful for the screening of dominant strains and the control of the fermentation process. However, even though the application of fluorescent proteins has many advantages, the detection still requires instruments such as flow cytometers and fluorescent microplate readers to excite fluorescence and then pass the detection sensor through the instrument data processing, and the inspection process is relatively complicated.

[0004] As a homolog of fluorescent protein, chromogenic protein can be observed in obvious color under natural light due to its strong absorption of visible light, which eliminates the complicated fluorescence detection process and has the potential advantage of being a reporter gene. In the previous study of our research group, it was confirmed that a chromogenic protein eforRed (chromo-redfluorescent GFP-like protein, NBCI accession number: ACD13196.1) derived from Echinoporaforskaliana and its K201E mutant have the characteristics of visible light color development. The visualized cell factory obtained by its transfer into Zymomonas mobilis is more conducive to the detection of the content of various intracellular and extracellular target products and the rapid judgment of whether the strain meets the growth expectations. However, its application bottleneck is mainly due to the low fluorescence signal value, light color under visible light and insufficient protein stability. Therefore, it is necessary to optimize the sequence to obtain a chromogenic protein reporter gene with darker color and stronger fluorescence. Summary of the invention

[0005] In response to the problems in the prior art such as low fluorescence intensity and light color of the chromogenic protein eforRed under visible light, the present invention provides an eforRed mutant and a nucleic acid molecule and an expression vector containing its encoding gene, and further provides the use of the nucleic acid molecule and the 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 NO.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; 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 promoter 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, which comprises 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 the application as a reporter gene in constructing a visualized cell factory, and the visualized 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 a recipient cell.

[0017] Further, the receptor cell is selected from Escherichia coli or Zymomonas mobilis. 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 thalline of the genetically modified engineering bacteria is bright in color and obvious in color development under visible light, and obvious pink changes can be observed only by the naked eye, with the advantage of more intuitive and convenient monitoring. In addition, the eforRed mutant of the present invention has no phototoxicity compared to the luciferase reporter gene, will not kill cells, and can develop color without the need to add expensive exogenous substrates. As a reporter gene, it has broad prospects and application advantages in the fields of visual screening of positive transformants of genetic transformation, monitoring gene expression activity, protein localization, fluorescent tracing of living cells, 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 creative work.

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

[0022] Figure 2 This is a fluorescence intensity graph of Escherichia coli transformed with wild-type eforRed and 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 in 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 embodiments 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 scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the examples. The equipment and reagents used in each embodiment and test example can be obtained from commercial sources unless otherwise specified. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0027] Based on the information contained in this application, it is easy for those skilled in the art to make various changes 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 process, properties or components, because these embodiments and other descriptions are only for illustrating specific aspects of the present invention. In fact, various changes that the technicians in this field or related fields can obviously make to the embodiments of the present invention are all covered within the scope of the appended claims.

[0028] In order to better understand the present invention rather than limit the scope of the present invention, all the numerals and other numerical values ​​used in this application indicating dosage, percentage, etc. should be understood as 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 being obtained according to the reported significant figures and by conventional rounding methods. In addition, the meaning of the terms "include", "comprise", "contain", "have" 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 understandable, 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 has high visible light absorption characteristics. In the previous study of the present invention (see patent "CN116426448A Visualization of Zymomonas mobilis, construction method and application (publication date: 2023-07-14)"), Zymomonas mobilis was used as a recipient cell for genetic transformation to study the luminescence properties of the protein and its K201E mutant. The results showed that the recombinant strain showed a pink color visible to the naked eye after fermentation culture, and no exogenous substrate was required for color development and no cytotoxicity was required, which realized the visual monitoring of the fermentation process of Zymomonas mobilis under visible light. In addition, under natural light observation, the protein color of the K201E mutant was stronger than that of the wild-type eforRed, which was consistent with the result of detecting the fluorescence signal value 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 genetically modified bacteria gene function verification and metabolic engineering transformation.

[0031] In order to further improve the visible light color development function and fluorescence characteristics of the chromogenic protein eforRed, the present invention performs site-directed saturation mutagenesis on the 201st amino acid (K201) site based on the wild-type eforRed and the K201E mutant, connects different mutants to the downstream of the promoter, genetically transforms Escherichia coli and Zymomonas mobilis, and by comparing the fluorescence intensity of the bacteria, it is found that the K201A, K201D, K201G, and K201H mutants have stronger fluorescence in the visible light range, which can improve the fluorescence intensity of the transformed chromogenic protein eforRed. In order to further improve the structural stability of the protein, the 24th amino acid site was mutated from threonine (T) to valine (V) to reduce the Gibbs free energy of the protein, and combined optimization was performed based on K201A, K201D, K201G, K201H mutations and T24V mutations. It was found that the fluorescence intensity of Escherichia coli transformed with T24V-K201E mutants and Zymomonas mobilis transformed with T24V-K201A, T24V-K201G, T24V-K201H mutants was significantly enhanced. Moreover, the overall Gibbs free energy of these mutants is lower and the structural stability is better.

[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, and has stronger fluorescence intensity in the visible light region, more sensitive color development, and better structural stability than the wild-type eforRed and its K201E mutant. 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 by the eforRed mutant of the present invention through flow detection is higher, and the bacteria are bright in color and obvious in color development under visible light. Obvious pink changes can be observed only by the naked eye, which has the advantage of more intuitive and convenient observation, and is more conducive to realizing the visualization monitoring of genetic transformation operations and transgenic engineering bacteria.

[0034] In addition, the eforRed mutant of the present invention is a fluorescent protein homologous protein, which has no phototoxicity compared to the luciferase reporter gene, will not kill cells, and can be colored without the addition of expensive exogenous substrates. 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, fluorescent tracing of living cells, and biological imaging. In practical applications, the eforRed mutant encoding gene of the present invention is fused with the target gene for expression, and the gene sequence of the fusion protein is inserted into a suitable expression vector and transformed into a receptor cell. The expression of the chromogenic protein can indicate whether the target gene is successfully transformed, and then the transgenic and non-transgenic cells can be easily distinguished; by detecting the level of chromogenic protein expression, the expression activity of the target gene 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. Or the eforRed mutant is labeled with other target proteins, and the location study of the target protein is realized 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 the embodiments of the present invention, the chromogenic protein eforRed mutant is used in the construction of a visualized cell factory. Specifically, the eforRed mutant of the present invention is connected to the downstream of a strong promoter, and the recipient bacteria are transformed. After the transgenic engineered bacteria express the eforRed mutant, the bacterial cells show pink color, and the pink color directly reflects the bacterial concentration, which can realize the visualization of the bacterial 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 an obvious visual difference with the color of the environment. For example, the bacteria form colonies with special colors on the plate, or its culture fluid forms a color that is proportional to the growth concentration. Of course, since the eforRed mutant of the present invention has the characteristic of visible light excitation fluorescence, visualization can also be manifested as obvious changes in fluorescence intensity under a fluorescence microscope or under detection by instruments such as flow cytometers.

[0037] Preferably, 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; more preferably, selected from at least one of SEQ ID NO.8-10; further preferably, selected from at least one of SEQ ID NO.9-10. Among them, the eforRed mutants shown in SEQ ID NO.9-10 have obvious fluorescence enhancement effects in Escherichia coli and Zymomonas mobilis, have wide adaptability to different strains, high fluorescence intensity, are more conducive to visible light monitoring, and have good 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 the advantages 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), and the molecules 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 its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis.

[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 according to 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 sequences of Pgap-6M or Pgap-4S promoter are shown in 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 the advantages 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, adenovirus). Correspondingly, the recipient cells transformed or transfected by the expression vector can be prokaryotic cells and eukaryotic cells, which are selected according to the type of expression vector. For example, when it is a prokaryotic expression vector, the recipient cell is a prokaryotic cell, and examples of commonly used prokaryotic cells include Escherichia coli, Bacillus subtilis, and Zymomonas mobilis, etc.; when it is a eukaryotic expression vector, the recipient cell is a eukaryotic cell, and examples of commonly used eukaryotic cells include Saccharomyces cerevisiae and Saccharomyces cerevisiae, etc.

[0046] Typical vectors include plasmids (such as pBR322, pUC series, pET series, pGEX series, pEZ15A), viral vectors, bacteriophages (such as λgt4λB, λ-Charon, λΔz1 and M13), cosmids and minichromosomes. Plasmids are the most commonly used vectors, 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. The pEZ15A vector connected with the nucleic acid molecule of the present invention is used to transform prokaryotic cells (such as Escherichia coli and Zymomonas mobilis), and the chromogenic protein can be expressed in the cells by culturing them by conventional methods.

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

[0049] Specifically, the visualized cell factory is a transgenic engineering 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, is 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 total gene synthesis or by PCR amplification. When obtained by PCR amplification, preferably pEZ-Pgap-6M-eforRed plasmid is used as a template, and PCR amplification is performed by 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 by 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, and each eforRed mutant is connected to the linearized pEZ15A vector by the Gibson assembly method; and the eforRedK201A, K201D, K201G or K201H mutant recombinant expression vector is constructed. When constructing a combined mutant containing a T24V mutation site, the eforRed K201A, K201D, K201G or K201H mutant recombinant expression vector is used as a template, and PCR amplification is performed by the upstream primer shown in SEQ ID NO.40 and the downstream primer shown in SEQ ID NO.41, and the amplified product is cyclized by the Gibson assembly method to obtain a recombinant expression vector with a T24V mutation site. The construction method of the starting pEZ-Pgap-6M-eforRed plasmid and pEZ-Pgap-6M-K201E can be found in patent "CN116426448A".

[0053] The recombinant expression vector can be transformed or transfected into the recipient cell 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 cell can also be introduced by gene gun bombardment.

[0054] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as the conditions described in the Molecular Cloning Laboratory Guide (Fourth Edition) published by Cold Spring Harbor Laboratory, or usually under the conditions recommended by the manufacturer.

[0055] 1. Preparation of chromogenic protein eforRed mutant

[0056] The present invention has previously studied "CN116426448A Visualized Zymomonas mobilis, construction method and application (publication date: 2023-07-14)" and confirmed that the chromogenic protein eforRed and its K201E mutant have visible light absorption characteristics, have obvious color development reaction under visible light, and have application value as a reporter gene. In order to further improve the color performance of the chromogenic protein eforRed, the present invention, on the basis of the wild-type eforRed and the K201E mutant, performs site-directed saturation mutation 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, connects different mutants to promoters and transforms Escherichia coli and Zymomonas mobilis, observes the fluorescence signal value of the bacteria, and screens out K201A, K201D, K201G, and K201H mutants with enhanced fluorescence signals. In addition, through the analysis of protein energy and protein structure, the 24th amino acid site was mutated from threonine (T) to valine (V), and the mutants K201A, K201D, K201G, K201H and T24V obtained by optimizing the site-directed saturation mutation of K201E were combined and mutated, and 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 mutants newly screened by the present invention and promoters for their expression

[0058]

[0059]

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

[0061] The pEZ15A expression plasmid and Escherichia coli and Zymomonas mobilis were selected as recipient cells to construct a prokaryotic expression system. The coding gene sequences of the chromogenic protein eforRed and each mutant were respectively integrated into the expression vector pEZ15A, and the gene expression amount was controlled by a constitutive strong promoter to obtain a recombinant vector, which was transferred into the recipient cell to obtain a visualization strain capable of synthesizing chromogenic proteins. The recombinant expression vector constructed in this example is shown in Table 2.

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

[0063]

[0064]

[0065] The eforRed gene and promoter gene of the above-mentioned expression vector and the primer sequence used are shown in Table 3, F represents the upstream primer (the shadow bold shows the mutation site codon), R represents the downstream primer, and the underlined part in the primer is the vector homology arm sequence. It should be noted that Table 3 exemplarily gives the eforRed gene sequence after the codon optimization for Zymomonas mobilis and the 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 embodiments of the present invention

[0067]

[0068]

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

[0070] Plasmid 1 construction: eforRed (universal puc plasmid vector for bacteria preservation) synthesized by Beijing Qingke Company and Pgap-6M promoter were used as DNA amplification templates, eforRed gene was amplified by eforRed-F and eforRed-R primer pairs, and Pgap-6M promoter was amplified by Pgap-F and Pgap-R primer pairs. Overlap PCR was used to connect the two DNA fragments of Pgap-6M and eforRed into a long fusion gene. At this time, both ends of the fusion gene carried homology arm sequences homologous to the pEZ15A vector. The fusion gene can be connected to the linearized pEZ15A vector by the Gibson assembly method to form a circularized pEZ-Pgap-6M-eforRed vector. Specifically, the fusion gene and the linearized pEZ15A vector were co-incubated with Escherichia coli DH5α competent cells to achieve simultaneous transformation and assembly. The positive clones on the plate were verified by colony PCR, and plasmid 1 was extracted after overnight culture. The linearized pEZ15A vector was generated by PCR amplification using primer pairs 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, K201C-F, etc. with mutant base sequences and universal downstream primer TB201-R to obtain various eforRed mutants. In addition, using plasmid 1 as a template, PCR amplification was performed using primer pairs BH201ZT-F and BH201ZT-R to obtain a linear plasmid. The DNA fragment of the eforRed mutant was reconnected to the linear plasmid through the Gibson assembly method to form a circular plasmid with a K201 saturated mutation site.

[0072] Construction of plasmids 21-26: Plasmids 1, 2, 3, 5, 7 and 8 were used as templates to amplify the full-length plasmids by PCR using bidirectional primers T24V-F and T24V-R with T24V mutation base sequences to obtain linear plasmids with mutation sites. The linear plasmids with mutation sites were circularized by the Gibson assembly method to obtain circular plasmids with T24V mutation sites.

[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, the Pgap-4S promoter was amplified by the Pgap-F and Pgap-R primer pairs, and the eforRed-F and Pgap-FK-R primer pairs were used to amplify the rest of plasmid 1, plasmid 2, and plasmid 26 except Pgap-6M, respectively. At this time, both 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 rest of the linearized vector by the Gibson assembly method to form a circularized plasmid 27-29.

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

[0075] Transformation of E. coli DH5α: When constructing the plasmid, the eforRed gene fragment and the pEZ15A vector were mixed in a ratio of 3:1. The reaction system included: 0.12pM of the eforRed gene fragment, 0.04pM of the pEZ15A vector, 0.5μL of 10×Buffer 4 (Thermo) and 0.5U of nuclease (T5 Exonuclease), and ddH2O was added to 5μL; it was allowed to stand on ice for 5 minutes, and then the DH5α competent medium was added for chemical transformation. The positive clones were screened using LB plates containing 60μg / mL kanamycin resistance, and a single colony was picked and the transformation was verified to be successful by sequencing.

[0076] Transformation of Zymomonas mobilis ZMNP: Take Zymomonas mobilis ZMNP competent cells on ice, take 50 μL after the competent cells melt and add them to the electroporation cup, and add 1 μg of the plasmid to be transferred to the electroporation cup. The electroporation conditions are 1800V, 25 μF and 200Ω. After the electroporation is completed, the transformed strain is inoculated into RMG5 liquid culture medium and revived in a 30°C incubator for 4-6 hours. After that, the culture is centrifuged at 6000 rpm / min for 1 minute, and part of the supernatant is removed. Take 100 μL of the suspended bacteria and spread it on a resistance plate containing 300 μg / mL kanamycin and culture at 30°C for 2 days. Single colonies were selected for sequencing to verify the successful transformation of the plasmid and obtain the target strain.

[0077] IV. Determination of fluorescence intensity of receptor cells

[0078] The target strains successfully transformed with wild-type eforRed and each mutant were inoculated into cryopreservation tubes containing 1 mL of RMG5 medium (with 3 μL of kanamycin) and placed in a 30°C incubator until turbid. Then, they were transferred to 10 mL of RMG5 medium (with kanamycin) to control the initial OD600.nm The concentration of the culture medium was 0.1, and the cells were cultured in a constant temperature shaker at 30°C and 100 rpm. After 48 hours, 200 μL of bacterial solution was taken from each well, centrifuged at 12000 rpm for 1 min, the supernatant was removed, 500 μL of PBS buffer was added to the precipitate and the precipitate was resuspended after shaking. Then the cells were centrifuged at 12000 rpm for 1 min, the supernatant was removed, 500 μL of PBS buffer was added to the precipitate and the precipitate was resuspended after shaking to complete the sample processing. After the processing, the fluorescence intensity of the bacterial cells was detected using a flow cytometer.

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

[0080] The 201st amino acid position of eforRed was subjected to site-directed saturation mutagenesis, 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, wherein the horizontal axis is the mutation mode and the vertical axis is the fluorescence intensity. As a result, four mutants with higher fluorescence intensity than K201E were observed, namely K201A, K201D, K201G and K201H mutations.

[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 with Pgap-6M as promoter to obtain the mutant strains Eco-Pgap-6M-T24V and Eco-Pgap-6M-T24V-K201E.

[0084] The fluorescence intensity of wild-type eforRed and mutants T24V, T24V-K201E, and K201E is shown in 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 the 24th and 201st amino acid combination mutants

[0086] Based on the combined mutation of mutants K201A, K201D, K201G, 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 with the Pgap-6M promoter and then transferred into Zymomonas mobilis ZMNP to obtain the strain ZM-Pgap-6M-eforRed and mutant strains ZM-Pgap-6M-K201E, Z201E-eforRed and Z201H-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 FoldX5.0 software, where the energy calculations for the site-directed saturation mutation at K201 and the T24V mutation were based on eforRed, and the PositionScan function in FoldX5.0 was used to predict all amino acid mutations. After the calculations were completed, the energy data was exported for plotting. The energy calculations for the site-directed saturation mutation at K201 and the combined mutation of T24V were based on the mutation site 24, with T24V-K201A as an example, and the T24V mutant as the starting point. The energy scan site was the energy change data for the K201A amino acid at site 201 in the 201 observation data. After the calculations were completed, the energy data was exported for plotting.

[0090] Figure 4 The protein energy of different mutants is shown, where the horizontal axis is the mutation mode and the vertical axis is the 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] In order to overexpress the chromogenic protein mutant and make its color change more obvious, the Pgap-6M promoter in the expression vector was replaced with the Pgap-4S promoter to obtain 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 transformed into Zymomonas mobilis ZMNP to obtain 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. It can be seen that the strain is pink after cultivation, and the color of strain ZM-Pgap-4S-T24V-K201H is brighter and more obvious than that of ZM-Pgap-4S-eforRed and ZM-Pgap-4S-K201E, which is consistent with the results of fluorescence signal detection by flow cytometry. Moreover, by replacing the Pgap-4S promoter and achieving overexpression of eforRed protein, the color change can be 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

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

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.3-6 and SEQ ID NO.8-10.

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

4. A nucleic acid molecule, characterized in that It comprises a gene sequence encoding the eforRed mutant as described in any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, 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 promoter are shown in SEQ ID NO.12 and SEQ ID NO.13 respectively.

6. An expression vector, characterized in that: Comprising the nucleic acid molecule according to any one of claims 4-5.

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

8. Use of the nucleic acid molecule according to any one of claims 4 to 5 or the expression vector according to claim 6 as a reporter gene.

9. Use of the nucleic acid molecule or expression vector according to claim 8 as a reporter gene, characterized in that: The application is the application as a reporter gene in constructing a visualized cell factory, and the visualized cell factory is a transgenic engineered bacterium obtained by transferring the nucleic acid molecule according to any one of claims 4-5 or the expression vector according to claim 6 into a recipient cell.

10. Use of the nucleic acid molecule or expression vector according to claim 9 as a reporter gene, 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 a recipient cell; Wherein, the receptor cell is selected from Escherichia coli or Zymomonas mobilis.

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