High-temperature-resistant flavin mononucleotide-binding fluorescent protein and application thereof

Abstract

The application discloses a high-temperature-resistant flavin mononucleotide-binding fluorescent protein and application thereof, relates to the technical field of biology, and the amino acid sequence is shown as SEQ ID NO. 3; and application of a differential scanning fluorescence method in screening of a directed evolution of a flavin mononucleotide-binding fluorescent protein YNP3Y116F mutant library. The application applies fluorescent labeling to strict anaerobic super-thermophilic archaea for the first time, obtains a mutant with improved thermal stability through directed evolution, constructs an expression vector, and obtains a recombinant strain by using a thermococcus transformation method. The mutant can be successfully applied to fluorescent imaging of the thermococcus with a growth temperature of 85 DEG C. The application introduces the differential fluorescence scanning technology as a screening method of the mutant library, and can realize simultaneous screening of the two characteristics of the fluorescent intensity and the thermal stability.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a heat-resistant flavin mononucleotide-binding fluorescent protein and its applications. Background Technology

[0002] Fluorescent proteins are valuable non-invasive molecular imaging tools, widely used in in vivo imaging, molecular interaction observation, biofactor activity assessment, tumor activity, and more. However, the most widely used fluorescent proteins (such as GFP) are currently limited to aerobic systems because their chromophores require oxygen to form. To expand the application of fluorescent proteins in anaerobic or hypoxic systems, flavin mononucleotide-based fluorescent protein (FbFP) has been developed. FbFP is mainly composed of light, oxygen, and voltage (LOV) domains, and emits fluorescence upon blue light excitation after binding to the ligand flavin mononucleotide. Compared to GFP and its derivatives, FbFP has advantages such as small molecular weight, oxygen independence, rapid fluorescence maturation, and strong stress resistance. These excellent properties indicate that FbFP has the potential to become a superior fluorescent protein compared to GFP and its derivatives, and can be applied to fields such as microbial fermentation, bioremediation, anaerobic wastewater treatment, tumor metastasis, chronic inflammation development, cerebral hypoxia-ischemia, microbial pathogenesis, and biofilm formation.

[0003] High fluorescence intensity and strong resistance to adverse conditions are considered crucial properties of fluorescent proteins. When fluorescent proteins are exposed to unfavorable, unnatural conditions, their three-dimensional structure can easily change, leading to reduced fluorescence emission or loss of fluorescence. The performance of fluorescent proteins can be improved through directed evolution methods, including irrational, semi-rational, and rational design. For example, a high-fluorescence mutant of SB2 from *Pseudomonas putida* was obtained through error-prone PCR. Computer-aided rational design increased the Tm value of YtvA from *Bacillus subtilis* by 31°C. However, for prokaryotic microorganisms living in extreme environments such as high temperature, high hydrostatic pressure, hypertonicity, and strict anaerobic conditions, many biological studies cannot utilize existing fluorescent proteins. This indicates that existing fluorescent proteins need further improvement to broaden their application range.

[0004] Therefore, those skilled in the art are dedicated to developing a fluorescently labeled flavin mononucleotide-binding fluorescent protein with improved thermostability in strictly anaerobic hyperthermophilic bacterial strains, as well as its directed evolution screening method and application. Summary of the Invention

[0005] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is how to develop a flavin mononucleotide-binding fluorescent protein that can be used for fluorescent labeling and improved thermostability in strictly anaerobic hyperthermophilic bacterial strains, and its application method.

[0006] To achieve the above objectives, the present invention provides a mutant A57S of flavin mononucleotide binding to fluorescent protein YNP3Y116F, the amino acid sequence of which is shown in SEQ ID NO.3.

[0007] This invention also provides an application of differential scanning fluorescence in the directed evolutionary screening of a mutant library of flavin mononucleotide-binding fluorescent protein YNP3Y116F.

[0008] Furthermore, the application includes the following steps:

[0009] Step 1: Construct a mutant library of flavin mononucleotide binding fluorescent protein YNP3Y116F to obtain a saturated mutant library at the target amino acid site;

[0010] Step 2: Induced expression of the saturated mutant library obtained in Step 1 in E. coli host to obtain bacterial culture;

[0011] Step 3: Prepare the bacterial culture obtained in Step 2 into a preliminary screening sample. Screen the preliminary screening sample by differential fluorescence scanning method to obtain a single-point mutant with a Tm value that is higher than that of wild type.

[0012] Step 4: Separate and purify the single-point mutant obtained in Step 3 and YNP3Y116F to obtain the purified single-point mutant;

[0013] Step 5: Induce expression of the purified single-point mutant obtained in Step 4 in Thermococcus.

[0014] Furthermore, step 1 also includes selecting saturation mutation sites by combining the Abacus algorithm and B-factor.

[0015] Furthermore, step 1 also includes the following steps:

[0016] Step 1.1: Based on the amino acid sequence of the flavin mononucleotide fluorescent protein YNP3Y116F and the codon preference of Thermococcus, the gene fragment encoding YNP3Y116F was synthesized.

[0017] Step 1.2: Use Discovery Studio to determine the FMN surrounding the YNP3Y116F ligand molecule. The amino acids within the range were selected, and their B-factor values ​​were calculated. The free energy of each amino acid residue was calculated using the statistical energy function Abacus. Taking into account all factors, ten residues with low free energy and relatively high B-factor values ​​were selected for saturation mutation to obtain the YNP3Y116F mutant with improved thermal stability.

[0018] Furthermore, step 1 also includes:

[0019] Step 1.3: The YNP3Y116F coding gene sequence was cloned and recombined into the vector pET-28a to obtain the first recombinant plasmid pET-28a-YNP3Y116F. The first recombinant plasmid pET-28a-YNP3Y116F was transformed into the expression host Escherichia coli BL21(DE3) by heat shock method to establish heterologous expression of YNP3Y116F in Escherichia coli.

[0020] Step 1.4: Perform full plasmid PCR amplification on the recombinant plasmid obtained in Step 1.3 after heterologous expression in DH5α using degenerate primers. Verify the separation and purification of the target fragment by nucleic acid gel electrophoresis. Digest the purified product with DpnI enzyme to remove the template plasmid and obtain the target fragment.

[0021] Step 1.5: The target fragment obtained in Step 1.4 is cloned and recombined into the vector pET-28a to obtain the second recombinant plasmid pET-28a-YNP3Y116F. The second recombinant plasmid pET-28a-YNP3Y116F is transformed into Escherichia coli BL21(DE3) by heat shock method and cultured overnight at 37°C on LB plates containing kanamycin resistance. Subsequently, a portion of single clones are picked for sequencing verification to construct a saturated mutant library at the target amino acid site.

[0022] Furthermore, in step 1.5, the kanamycin content in the LB plate was 50 μg / mL.

[0023] Furthermore, the method for preparing the initial screening sample in step 3 is as follows: The OD value of the induced bacterial solution is measured using a UV spectrophotometer. 600 The OD values ​​of each bacterial culture were determined. 600 The value was standardized to 2.0. The bacterial cells were collected by centrifugation for the first time, and the cells were repeatedly frozen and thawed. Lysozyme was added to resuspend the cells, and the reaction was carried out at 37°C. The supernatant of the reaction solution was collected by centrifugation for the second time and used as the initial screening sample.

[0024] Furthermore, the steps for screening the initial samples using differential fluorescence scanning in step 3 are as follows:

[0025] Step 3.1 Using wild type as the control group, saturated mutant library as the experimental group, and PBS buffer as template-free blank control (NTC), add the initial screening samples to a 96-well qPCR plate, 50 μL per well. Each sample is performed in 3 technical replicates on the same qPCR plate. Seal the qPCR plate with a high-transmittance sealing film.

[0026] Step 3.2 Reaction program settings: Detection Format is set to SYBR Green / HRM Dye by default. Equilibrate at 37℃ for 4 min, then increase the temperature from 38℃ to 90℃ at a rate of 1℃ / min to determine the Tm value of the sample. The Tm value is the temperature at which the fluorescence intensity drops to half of the initial fluorescence.

[0027] Furthermore, the purification method in step 4 is nickel column affinity chromatography.

[0028] This invention also provides an application of the mutant A57S of flavin mononucleotide binding fluorescent protein YNP3Y116F as a fluorescent marker in strictly anaerobic hyperthermophilic archaea.

[0029] In a preferred embodiment 1 of the present invention, the process of constructing a mutant library of flavin mononucleotide binding fluorescent protein YNP3Y116F is described in detail.

[0030] In another preferred embodiment 2 of the present invention, the process of inducing expression of YNP3Y116F and its mutant library in Escherichia coli host is described in detail.

[0031] In another preferred embodiment 3 of the present invention, the screening process of the mutant library is described in detail;

[0032] In another preferred embodiment 4 of the present invention, the separation and purification process of YNP3Y116F and A57S is described in detail.

[0033] In another preferred embodiment 5 of the present invention, the process of inducing expression of A57S in Thermococcus is described in detail.

[0034] In another preferred embodiment 6 of the present invention, the fluorescence microscopy examination process of A57S in Thermococcus is described in detail.

[0035] The beneficial technical effects of this invention are as follows:

[0036] This invention marks the first application of fluorescent labeling in strictly anaerobic hyperthermophilic archaea. Through directed evolution, mutants with improved thermostability were obtained, expression vectors were constructed, and recombinant strains were obtained using a thermococcal transformation method. These mutants can be successfully applied to fluorescence imaging of thermococci growing at 85°C, demonstrating broad application prospects for studying the performance of bio-components, molecular interactions, and biosensors under extreme conditions of ultra-high temperature and high hydrostatic pressure.

[0037] This invention introduces differential fluorescence scanning technology as a screening method for mutant libraries, enabling simultaneous screening of both fluorescence intensity and thermal stability. Differential fluorescence scanning involves slowly heating the sample on a quantitative PCR instrument. During heating, changes in the three-dimensional structure of the fluorescent protein are reflected in the detection of fluorescence signal intensity, thereby evaluating the protein's thermal stability. This screening method is applicable to all proteins exhibiting fluorescence signals that require improved thermal stability.

[0038] This invention combines the Abacus algorithm and B-factor to select saturation mutation sites, overcoming the problem that insufficiently refined mutation libraries in directed evolution can lead to excessively high screening pressure and be time-consuming and labor-intensive processes. Discovery Studio software is used to determine the FMN surrounding the ligand molecule. The study identifies amino acids within a specific range and calculates their B-factor values, while simultaneously calculating their free energies using the Abacus statistical energy function. This reduces the size of the mutant library, significantly improving screening efficiency and enabling the rapid acquisition of positive mutants.

[0039] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the docking analysis of the ligand molecule FMN with the YNP3Y116F molecule and the selected saturation mutation site in a preferred embodiment 1 of the present invention;

[0041] Figure 2 This is a differential fluorescence scanning result of YNP3Y116F and mutant A57S in a preferred embodiment 3 of the present invention;

[0042] Figure 3 This is a diagram showing the purification results of YNP3Y116F and A57S proteins in a preferred embodiment 4 of the present invention;

[0043] Figure 4 This is a differential fluorescence scanning result of YNP3Y116F and mutant A57S verified in a preferred embodiment 5 of the present invention;

[0044] Figure 5 The results of fluorescence microscopy examination of mutant A57S expressed in (a) Thermococcus eurythermalis A101 strain and (b) Thermococcus kodakarensis KOD1 in a preferred embodiment 6 of the present invention. Detailed Implementation

[0045] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0046] Example 1: Construction of a mutant library of flavin mononucleotide binding to fluorescent protein YNP3Y116F

[0047] The amino acid sequence of the flavin mononucleotide fluorescent protein YNP3Y116F was obtained from the literature (Wingen et al., 2017). Based on the codon preference of Thermococcus, the gene fragment encoding YNP3Y116F was synthesized by Sangon Biotech (Shanghai) Co., Ltd. Discovery Studio was used to determine the FMN surrounding the YNP3Y116F ligand molecule (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2). The amino acids within the range were analyzed, and their B-factor values ​​were calculated. The free energy of each amino acid residue was calculated using the statistical energy function Abacus. Ten residues with low free energy and relatively high B-factor values ​​were selected for saturation mutagenesis to obtain a YNP3Y116F mutant with improved thermal stability. A schematic diagram of the docking analysis between the ligand molecule FMN and the YNP3Y116F molecule, and the selected saturation mutagenesis sites, is shown below. Figure 1 As shown.

[0048] The YNP3Y116F encoding gene sequence was cloned and recombined into the vector pET-28a in a one-step process. The recombinant plasmid pET-28a-YNP3Y116F was transformed into the expression host *E. coli* BL21(DE3) using a heat shock method to establish heterologous expression of YNP3Y116F in *E. coli*. In this invention, whole-plasmid PCR was performed on the above recombinant plasmid using degenerate primers (ANK / TNK / GNK / CNK) (Table 1). The target fragment was isolated and purified by nucleic acid gel electrophoresis. The purified product was digested with DpnI enzyme to remove the template plasmid. *E. coli* BL21(DE3) was transformed using the same method and cultured overnight at 37°C on LB plates containing kanamycin resistance (50 μg / mL). Subsequently, a subset of single clones were picked for sequencing verification to construct a saturated mutant library at the target amino acid sites.

[0049] Example 2: Inducible expression of YNP3Y116F and its mutant library in Escherichia coli host

[0050] Escherichia coli carrying the wild-type recombinant plasmid and its mutant library were cultured at 37°C in 5 mL LB (containing 50 μL / mL kanamycin) until OD500. 600 Approximately 0.8, IPTG was added to the culture system to a final concentration of 0.5 mM, and induction was performed at 20°C for 20 hours.

[0051] Example 3: Screening of mutant libraries

[0052] The OD of the induced bacterial culture was measured using a UV spectrophotometer. 600 The OD values ​​of each bacterial culture were determined. 600 The value was unified to 2.0. The cells were collected by centrifugation at 13680g for 5 min. The cells were repeatedly frozen and thawed 3 times. 200 μL of lysozyme was added to resuspend the cells. The cells were reacted at 37℃ for 15 min. The supernatant of the reaction solution was collected by centrifugation at 13680g for 30 min and used as the initial screening sample.

[0053] Screening method: Differential fluorescence scanning. Wild-type samples were used as the control group, saturated mutant libraries as the experimental group, and PBS buffer as the NTC. 50 μL of the initial screening samples were added to each well of a 96-well qPCR plate. Each sample was tested in triplicate on the same qPCR plate. The qPCR plate was sealed with a high-transmittance sealing film. Reaction program settings: Detection Format was set to SYBR Green / HRM Dye by default. Equilibration was performed at 37°C for 4 min, followed by a temperature increase from 38°C to 90°C at a rate of 1°C / min. During the slow heating process, the intrinsic fluorescence of protein-containing samples gradually decreased due to conformational changes. The temperature at which the fluorescence intensity dropped to half of the initial fluorescence can be considered the Tm value of the protein sample. Using saturation mutation primers, saturation mutation libraries were screened for ten amino acid sites (F116, A57, Q32, R74, R58, Q61, L87, V70, A25, V117) of YNP3Y116F (wild-type, WT), resulting in a single-point mutant A57S with a Tm value 3°C higher than that of wild-type YNP3Y116F. Differential fluorescence scanning results of YNP3Y116F and mutant A57S are shown below. Figure 2 As shown in Table 1, the saturation mutation primer sequences for the ten amino acid sites (F116, A57, Q32, R74, R58, Q61, L87, V70, A25, V117) of YNP3Y116F are saturated.

[0054] Table 1 Saturation Mutant Primers

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061] Example 4: Isolation and purification of YNP3 and A57S

[0062] Recombinant *E. coli* containing the YNP3Y116F (wild-type, WT) and A57S (mutant) genes were cultured in a 200 mL LB medium. After overnight induction (using the same induction method as described above), the bacterial cells were collected by centrifugation at 16260 g for 5 min. The LB medium was poured out, and the bacterial cells were resuspended in 10 mL of protein binding buffer (50 mM Tris-HCl, 500 mM NaCl, pH 7.4). The cells were then sonicated on ice for 30 min to disrupt the cell structure. After centrifugation at 16260 g for 30 min, cell debris was removed, and a cell-free extract was obtained. Since the target protein and its mutants were constructed with a His-tag consisting of 6 histidine residues at the N-terminus, the target protein in this invention could be purified using nickel column affinity chromatography. Specifically, the Ni column was first equilibrated with 20 column volumes of protein binding buffer. The cell-free extract was then poured into the Ni column and incubated for 5 to 10 min. The Ni column was washed with an appropriate concentration of imidazole to elute the target protein. The molecular weight and purity of the purified target proteins were analyzed by SDS-PAGE. The purification results of YNP3Y116F and A57S proteins are shown below. Figure 3 As shown, lane M represents the protein marker, lanes 1-4 contain YNP3Y116F washed with different concentrations of imidazole, and lanes 5-8 contain A57S washed with different concentrations of imidazole. Elution buffers containing relatively high purity of the target protein were selected for DSF method rescreening of mutant efficacy (method as described above). Experimental results showed that the screening results of crude enzymes induced using a small system could be scaled up and validated. The differential fluorescence scanning results of validated YNP3Y116F and mutant A57S are shown below. Figure 4 As shown.

[0063] Example 5: Induced expression of A57S in Thermococcus

[0064] Plasmid construction in *E. coli* DH5α: Using the shuttle plasmid pLC, which can replicate in *Thermococcus* and *Escherichia coli*, as a vector, the 15-20 bp terminal of the linearized vector was added as a homologous sequence to the 5' end of the A57S gene-specific amplification primers. The insert fragment containing the vector homologous sequence was amplified using this primer pair. The linearized vector and the insert fragment were ligated via recombination, and the recombination product was transformed into *E. coli* DH5α competent cells using a heat shock method. Successfully ligated transformants were picked and cultured overnight in LB (100 μg / mL), and the plasmid was extracted for later use.

[0065] Transformation of *Thermococcus eurythermalis*: *T. eurythermalis* A101 was cultured in 50 mL TRM medium at 85°C to the logarithmic growth phase (approximately 12 h). The cells were collected by centrifugation at 6000 g for 5 min, and resuspended in 200 μL of pre-chilled CaCl2 solution (under nitrogen protection) and incubated on ice for 30 min. Then, 3 μg of plasmid was added (under nitrogen protection) and incubated on ice for 1 h. The mixture was then heat-shocked at 85°C for 45 s, immediately followed by an ice incubation for 10 min. The mixture was then transferred to 5 mL of TRM liquid culture and revived at 85°C for 4 h. 2 mL of the revived bacterial culture was injected into TRM solid medium (4 μM simvastatin) and cultured in roller tubes for 16 to 24 h. Single clones were picked for gel electrophoresis. Since the A57S gene fragment in the recombinant pLC plasmid was placed after the high hydrostatic inducible promoter, the correctly identified single clones were cultured and induced to express in a high hydrostatic autoclave (85°C, 30 MPa).

[0066] Using the overexpression plasmid pTE with guanidine-labeled guanidine as a vector, the pTE-A57S recombinant plasmid was constructed and transformed into Thermococcus kodakarensis KOD1 in the same manner to obtain the recombinant strain.

[0067] Example 6: Fluorescence microscopic examination of A57S expressed in Thermococcus

[0068] The recombinant strain *T. eurythermalis* A101, cultured overnight (85℃, 30MPa) at 4065g, was collected in 50mL of medium. The culture was washed twice with isotonic NaCl solution to remove sulfur powder from the medium as much as possible to avoid affecting observation. Microscopic examination was performed using a Nikon Ti-2E fully automated thermostatic inverted fluorescence microscope. *T. eurythermalis* A101 strain carrying the pLC empty vector plasmid was used as a negative control. The results were observed under 100x oil immersion light at 475nm excitation. The recombinant strain carrying the pLC-A57S plasmid emitted moderate green fluorescence; the negative control group showed weak fluorescence due to impurity reflection, but significantly weaker than the experimental group. Similarly, the expression of mutant A57S in the recombinant strain *Thermococcus kodakarensis* KOD1 was examined using fluorescence microscopy. The fluorescence microscopy results of mutant A57S expression in *Thermococcus eurythermalis* A101 and *Thermococcus kodakarensis* KOD1 are shown below. Figure 5 As shown.

[0069] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A mutant A57S of flavin mononucleotide binding to fluorescent protein YNP3Y116F, characterized in that, The amino acid sequence of the mutant A57S is shown in SEQ ID NO.

3.

2. The application of the mutant A57S of flavin mononucleotide binding fluorescent protein YNP3Y116F as described in claim 1 as a fluorescent marker in strictly anaerobic hyperthermophilic archaea.

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