NnLuz, NnH3H protein mutants and their use in enhancing the intensity of fungal or plant bioluminescence

Abstract

The application discloses NnLuz and NnH3H protein mutants and application thereof in enhancing the intensity of fungal or plant bioluminescence. The application significantly improves the catalytic efficiency and the light emission intensity of a bioluminescence system by rationally designing and optimizing core enzymes NnLuz and NnH3H of a fungal bioluminescence pathway. Experiments prove that: 1) the light emission intensity of NnLuzAN19_v2 (I103T / L167K double mutant) in yeast and plants is significantly better than that of the reported NnLuz_v4 mutant, reaching 3-4 times that of the NnLuz_v4 mutant; 2) the catalytic efficiency of NnH3H_v3 (K307T mutant) is improved by more than 2 times, and the light emission stability is significantly enhanced; and 3) when NnLuzAN19_v2 and NnH3H_v3 are expressed in cooperation, the system light emission intensity reaches an optimal level, and a self-luminous plant with significantly enhanced light emission is created.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to NnLuz and NnH3H protein mutants and their application in enhancing the bioluminescence intensity of fungi or plants. Background Technology

[0002] Bioluminescence is a light phenomenon produced by organisms through natural chemical reactions. It is widely present in nature and has important ecological functions and potential application value (Kotlobay et al., 2018). The fungal bioluminescence pathway (FBP) is a light-generating system based on the caffeic acid metabolic cycle. Its core enzymes include luciferase (Luz) and milkweed alkaloid-3-hydroxylase (H3H). Luz catalyzes the oxidation of 3-hydroxymilkweed alkaloid (fungal luciferin), releasing photons and generating caffeoylpyruvate (Kotlobay et al., 2018); H3H catalyzes the hydroxylation of milkweed alkaloid to generate 3-hydroxymilkweed alkaloid (Mitiouchkina et al., 2020). However, the catalytic efficiency and stability of natural Luz and H3H are limited, especially in plant heterologous expression systems. Their activity is often limited by substrate concentration, environmental conditions (such as temperature and oxygen) and the plant's endogenous metabolic network (Khakhar et al., 2020), resulting in weak luminescence intensity that is difficult to meet the needs of practical applications.

[0003] Currently, increasing the content of endogenous caffeic acid and milkweed alkaloids in plants through metabolic engineering has been shown to enhance luminescence intensity (Mitiouchkina et al., 2020), but the catalytic efficiency of the enzymes themselves remains a key factor limiting the performance of luminescent systems. Therefore, protein modification of Luz and H3H to improve their catalytic efficiency and stability is an effective way to further optimize bioluminescent systems. In existing technologies, Shakhova et al. disclosed the modification of these two key enzymes, generating the NnLuz_v3 variant containing three amino acid substitutions (T99P, T192S, and A199P) through directed evolution of NnLuz. This variant exhibits higher stability and sustainability, and increases the luminescence intensity of E. coli and HEK293T cells by 1.5 times. Subsequently, random mutagenesis of NnLuz_v3 produced NnLuz_v4, which contained four additional amino acid substitutions (I3S, N4T, F11L, and I63T), exhibiting improved thermal stability and catalytic activity in yeast and increasing the luminescence intensity of Escherichia coli by 3-fold.Simultaneously, the team conducted directed evolution of NnH3H, identifying five amino acid substitutions that enhance brightness (D37E, V181I, A183P, S323M, and M385K). By combining these mutations, they obtained NnH3H_v2 with further enhanced brightness (Shakhova ES, Karataeva TA, Markina NM, Mitiouchkina T, Palkina KA, Perfilov MM, Wood MG, Hoang TT, Hall MP, Fakhranurova LI, Alekberova AE, Malyshevskaia AK, Gorbachev DA, Bugaeva EN, Pletneva LK, Babenko VV, Boldyreva DI, Gorokhovatsky AY, Balakireva AV, Gao F, Choob VV, Encell LP, Wood KV, Yampolsky IV, Sarkisyan KS, Mishin AS). An improved pathway for autonomous bioluminescence imaging in eukaryotes. Nat Methods. 2024 Mar;21(3):406-410. doi: 10.1038 / s41592-023-02152-y. Epub 2024 Jan 22. PMID:38253843; PMCID: PMC10927554.), however, the effects of the modified fungal bioluminescence pathway core enzymes NnLuz and NnH3H in enhancing bioluminescence intensity still need to be further improved. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art. By optimizing the core enzymes NnLuz and NnH3H in the fungal bioluminescence pathway through directed evolution, NnLuz protein mutant NnLuzΔN19_v2 and / or NnH3H protein mutant NnH3H_v3 were obtained. Compared with the reported mutants NnLuz_v4 and NnH3H_v2, their catalytic efficiency and bioluminescence intensity are significantly enhanced.

[0005] A second object of the present invention is to provide the encoding gene of the protein mutant.

[0006] A third object of the present invention is to provide a recombinant vector, expression cassette, or recombinant bacteria containing the said coding gene.

[0007] A fourth object of the present invention is to provide the application of the said protein mutant, recombinant vector, expression cassette or recombinant bacteria in enhancing the bioluminescence intensity of fungi or plants.

[0008] A fifth object of the present invention is to provide a method for enhancing the bioluminescence intensity of fungi or plants.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] Traditional protein engineering methods include rational design and directed evolution. Rational design is based on a deep understanding of the relationship between protein structure and function, optimizing the catalytic site of enzymes through site-directed mutagenesis (Reetz et al., 2007). For example, molecular docking and molecular dynamics simulations can predict the impact of key amino acid residues on substrate binding and catalytic efficiency. Furthermore, the application of artificial intelligence tools such as AlphaFold2 has made accurate prediction of protein structures possible, providing a more reliable theoretical basis for rational design (Jumper et al., 2021).

[0011] This invention utilizes rational design to perform point mutations on the core enzymes in the fungal bioluminescence pathway, namely fungal luciferase (NnLuz) and 3-hydroxyhispidin hydroxylase (NnH3H), to enhance the luminescence intensity of the fungal bioluminescence pathway. Specifically:

[0012] (1) Optimization and modification of NnLuz enzyme:

[0013] Based on our previous research, this invention found that NnLuzΔN19, which has 19 amino acids truncated at the N-terminus, has a stronger luminescence intensity than wild-type NnLuz and the reported best mutant NnLuz_v4 (Shakhova et al., 2024).

[0014] Building upon this foundation, this invention further deepens the research, significantly improving the catalytic efficiency and luminescence intensity of the bioluminescent system. First, the three-dimensional structural model of NnLuz was predicted using AlphaFold2. Molecular docking simulations were performed using AutoDock Vina 1.5.7, precisely determining that its active pocket consists of five key amino acid residues: Glu149, Glu181, His184, His186, and His194. Multiple sequence alignment analysis showed that these catalytic residues are highly conserved during evolution. Based on this structural information, this invention systematically compared Luz homologous protein sequences from different species, screening for eight potential mutation sites (positions 58, 68, 103, 122, 126, 128, 145, and 167). Through site-directed mutagenesis, a single mutant library including V49A, F58I / L, S68A, I103T, T122L, I126K, K128T, T145L, and L167K was constructed. Screening in a yeast expression system revealed that single mutations at the I103T, I126K, and L167K sites significantly increased luminescence intensity. Further validation in tobacco showed that co-infection with *Agrobacterium tumefaciens* FBP-ΔNnLuz lacking the NnLuz gene resulted in better effects from the I103T, I122L, and L167K mutations. Double mutation validation further demonstrated the most significant effect, and this mutation, named NnLuzΔN19_v2 (amino acid sequence shown in SEQ ID No. 1), exhibited a luminescence intensity 3–4 times higher than the previously reported optimal mutant NnLuz_v4 (Shakhova et al., 2024).

[0015] (2) Optimization and modification of NnH3H enzyme:

[0016] This invention, through molecular docking studies, identified 10 key residues in the active region of NnH3H, including Gly18, Asp37, Ser38, Val137, and Asp318, which are highly conserved during evolution. Based on multiple sequence alignment, 12 potential mutation sites (A43T / S, T66V / I, G81A, K210N, A251G, V276F, K307T, Q353D, E379Q, and L395M) were screened. Screening using a yeast expression system revealed that the K307T mutant exhibited the most significant activity enhancement. Further validation in tobacco showed that this mutant (named NnH3H_v3) had a superior luminescence intensity compared to the previously reported NnH3H_v2 (Shakhova et al., 2024) mutant, and its amino acid sequence is shown in SEQ ID No. 2.

[0017] (3) Application of modified NnLuz and NnH3H in improving plant luminescence intensity:

[0018] By co-expressing the modified and optimized NnLuzΔN19_v2 and NnH3H_v3 with the remaining genes (NnHispS, NnCPH, AnNPGA) of the fungal bioluminescence system (FBP), the bioluminescence intensity in the plant was significantly enhanced, creating a self-luminescent plant with significantly enhanced luminescence.

[0019] Therefore, the present invention first provides a protein mutant, comprising the NnLuz protein mutant NnLuzΔN19_v2 and / or the NnH3H protein mutant NnH3H_v3; the amino acid sequence of the NnLuz protein mutant NnLuzΔN19_v2 is shown in SEQ ID No. 1; the amino acid sequence of the NnH3H protein mutant NnH3H_v3 is shown in SEQ ID No. 2. That is, the protein mutant is selected from 1) the NnLuz protein mutant NnLuzΔN19_v2; 2) the NnH3H protein mutant NnH3H_v3; or 3) a combination of the NnLuz protein mutant NnLuzΔN19_v2 and the NnH3H protein mutant NnH3H_v3.

[0020] This invention also provides a coding gene encoding the above-mentioned protein mutant. NnLuzΔN19_v2 and / or NnH3H_v3 .

[0021] Furthermore, the coding gene of the aforementioned protein mutant can be codon-optimized before expression as needed. Preferably, the coding gene of the NnLuz protein mutant NnLuzΔN19_v2... NnLuzΔN19_v2 The nucleotide sequence is shown in SEQ ID No. 3; the gene encoding the NnH3H_v3 mutant NnH3H protein is described. NnH3H_v3 The nucleotide sequence is shown in SEQ ID No. 4.

[0022] The present invention also provides a recombinant vector, expression cassette or recombinant bacteria containing the above-mentioned coding gene.

[0023] This invention also provides the application of the above-mentioned protein mutants, the above-mentioned encoding genes, or the above-mentioned recombinant vectors, expression cassettes, or recombinant bacteria in enhancing the bioluminescence intensity of fungi or plants; wherein the fungal or plant genome integrates... HispS Gene, CPH Gene, H3H Gene, NPGA Gene or HispS Gene, CPH Gene, Luz Gene, NPGA Gene or HispS Gene, CPH Gene, NPGA Gene.

[0024] This invention also provides a method for enhancing the bioluminescence intensity of plants, which involves encoding the gene of the NnLuz protein mutant NnLuzΔN19_v2. NnLuzΔN19_v2 After codon optimization, the remaining genes of the fungal bioluminescence system FBP were compared: HispS Gene, CPH Gene, H3H Gene, NPGA Genes are simultaneously and transiently transformed in plants for co-expression;

[0025] Or the encoding gene of the above-mentioned NnH3H protein mutant NnH3H_v3 NnH3H_v3 After codon optimization, the remaining genes of the fungal bioluminescence system FBP were compared: HispS Gene, CPH Gene, Luz Gene, NPGA Genes are simultaneously and transiently transformed in plants for co-expression;

[0026] Alternatively, the encoding genes of the aforementioned NnLuz protein mutant NnLuzΔN19_v2 and NnH3H protein mutant NnH3H_v3 could be used. NnLuzΔN19_v2 and NnH3H_v3 After codon optimization, the remaining genes of the fungal bioluminescence system FBP were compared: HispS Gene, CPH Gene, NPGA Genes are simultaneously and transiently transformed in plants for co-expression;

[0027] Obtain self-luminescent plants with significantly enhanced bioluminescence intensity.

[0028] Furthermore, the method includes the following steps:

[0029] S1. Construct respectively containing NnLuzΔN19_v2 Gene or NnH3H_v3 Plant binary expression vectors for genes;

[0030] S2. Utilizing multi-genome assembly technology, HispS Gene, CPH Gene, H3H Gene, NPGA Genes were integrated into a receptor vector to construct the multi-gene vector FBP-ΔNnLuz; multi-gene assembly technology was then used to... HispS Gene, CPH Gene, Luz Gene, NPGA Genes are integrated into recipient vectors to construct multi-gene vectors FBP-ΔNnH3H; or multi-gene assembly technology is used to... HispS Gene, CPH Gene, NPGAGenes were integrated into the receptor vector to construct the multi-gene vector FBP-ΔNnLuz-ΔNnH3H;

[0031] S3. Containing NnLuzΔN19_v2 Simultaneous transient transformation of recipient plants using the plant binary expression vector and the multi-gene vector FBP-ΔNnLuz; or the generation of genes containing NnH3H_v3 Simultaneous transient transformation of recipient plants using a plant binary expression vector and a multi-gene vector FBP-ΔNnH3H; or the simultaneous transient transformation of recipient plants containing... NnLuzΔN19_v2 Plant binary expression vectors containing genes NnH3H_v3 The plant binary expression vector and the multi-gene vector FBP-ΔNnLuz-ΔNnH3H simultaneously transiently transformed recipient plants.

[0032] S4. Cultivate and obtain transgenic plants with significantly enhanced bioluminescence intensity.

[0033] Furthermore, the plant binary expression vector is pCAMBIA1300. That is, pCAMBIA1300- NnLuzΔN19_ v2 pCAMBIA1300- NnH3H_v3 .

[0034] Preferably, the target gene in the vector contains a 35S promoter sequence upstream.

[0035] Furthermore, the multi-genome assembly was performed using the TransGene Stacking II system (Zhu et al., 2017) developed by Academician Liu Yaoguang's team at South China Agricultural University. That is, through a multi-round recombination strategy, multiple genomes containing [specific genomes] were assembled according to requirements. HispS Gene, H3H gene, Luz Gene, CPH Gene, H3H Gene, NPGA The gene donor vectors pYL322d1 and pYL322d2 were co-transformed with the recipient vector pYLTAC380GW to obtain the target multi-gene vector. The FBP-ΔNnLuz vector contains... HispS, CPH, NPGA, H3H The gene, contained in the FBP-ΔNnH3H vector. HispS , CPH, NPGA, Luz The gene, FBP-ΔNnLuz-ΔNnH3H vector contains HispS , CPH, NPGA Gene.

[0036] Furthermore, the plant in question is a higher plant.

[0037] Preferably, the higher plant is chrysanthemum, petunia, rapeseed, orchid, or tobacco.

[0038] Furthermore, the fungus is yeast.

[0039] Preferably, the HispS Gene, H3H Gene, Luz Genes and CPH The gene comes from the fungus Ganoderma lucidum ( Neonothopanusnambi ),Right now NnHispS , NnH3H , NnLuz、NnCPH The NPGA The gene comes from Aspergillus nidus ( Aspergillus nidulans ),Right now AnNPG A.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] This invention significantly improves the catalytic efficiency and luminescence intensity of the bioluminescent system by rationally designing and optimizing the core enzymes NnLuz and NnH3H in the fungal bioluminescence pathway. Experiments demonstrate that: 1) the luminescence intensity of NnLuzΔN19_v2 (I103T / L167K double mutant) in yeast and plants is significantly superior to the previously reported NnLuz_v4 mutant, reaching 3-4 times the intensity; 2) the catalytic efficiency of NnH3H_v3 (K307T mutant) is increased by more than 2 times, and its luminescence stability is significantly enhanced; 3) when NnLuzΔN19_v2 and NnH3H_v3 are co-expressed, the system's luminescence intensity reaches the optimal level, creating a self-luminescent plant with significantly enhanced luminescence.

[0042] This invention overcomes the key bottleneck of low catalytic efficiency in fungal bioluminescence systems through a rational design strategy, and has the following outstanding advantages: 1) The luminescence intensity is significantly improved, reaching 3 to 4 times that of the wild-type system; 2) It has a wide range of applications and is suitable for various bioluminescence reporter systems.

[0043] This invention provides an innovative solution for creating efficient bioluminescence systems, which can be widely used in fields such as biosensor development, in vivo imaging, and gene expression analysis, and has significant scientific value and broad application prospects. Attached Figure Description

[0044] Figure 1 Schematic diagrams of the FBP-ΔNnH3H, FBP-ΔNnLuz, and FBP-ΔNnLuz-ΔNnH3H carriers are shown. Figure 1 A is a schematic diagram of the FBP-ΔNnH3H vector; B is a schematic diagram of the FBP-ΔNnLuz vector; C is a schematic diagram of the FBP-ΔNnLuz-ΔNnH3H vector.

[0045] Figure 2The luminescence intensity of NnLuzΔN19 and the previously reported NnLuz_v4 was compared in yeast. Figure 2 In the image, A represents the bioluminescence image; B represents the quantitative analysis results of photon flux. V4 represents NnLuz_v4, V4ΔN19 represents a mutant of NnLuz_v4 with 19 amino acids truncated at the N-terminus, Luz represents wild-type NnLuz, and LuzΔN19 represents NnLuzΔN19.

[0046] Figure 3 Analysis of the catalytic mechanism of NnLuz. Among them, Figure 3 In the diagram, A represents the results of molecular docking simulation; B represents the results of multiple sequence alignment analysis.

[0047] Figure 4 Consensus mutation sites were determined after NnLuz sequence alignment.

[0048] Figure 5 The effect of single-point mutations NnLuz and NnLuzΔN19 on luminescence intensity in yeast.

[0049] Figure 6 The effect of different single-point mutations in NnLuzΔN19 on luminescence intensity in tobacco. Figure 6 Image A shows bioluminescence; image B shows the results of quantitative analysis of photon flux.

[0050] Figure 7 The effect of NnLuz double protrusions on luminescence. Figure 7 Image A shows bioluminescence; image B shows the results of quantitative analysis of photon flux.

[0051] Figure 8 Analysis of the NnH3H catalytic mechanism. Among them, Figure 8 In the diagram, A represents the results of molecular docking simulation; B represents the results of multiple sequence alignment analysis.

[0052] Figure 9 Consensus mutation sites were determined after NnH3H sequence alignment.

[0053] Figure 10 The effect of the NnH3H mutation on luminescence in yeast. Figure 10 Image A shows bioluminescence; image B shows the results of quantitative analysis of photon flux.

[0054] Figure 11 The effect of the NnH3H mutation on luminescence in tobacco. Figure 11 In the image, A represents the bioluminescence image; B represents the results of quantitative analysis of photon flux. H3H_K307T is a dot projection at position 307 based on the original H3H, and NnH3H_v3 is a dot projection at position 307 based on NnH3H_v2.

[0055] Figure 12 This represents the effect of the synergistic expression of NnLuzΔN19_v2 and NnH3H_v3. Among them, Figure 12 Image A shows the bioluminescence image; image B shows the results of quantitative analysis of photon flux.

[0056] Figure 13 NnLuzΔN19_v2 and NnH3H_v3 were used to create enhanced self-luminous plants. Detailed Implementation

[0057] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0059] The plant expression vector was pCAMBIA1300-35S-Flag-NOS, preserved in the laboratory: the backbone vector pCAMBIA1300, the MCS region containing a 35S overexpression promoter, a 3×Flag tag and a NOS terminator, Kan resistance, and hygromycin plant selection markers.

[0060] Example 1: Design and Construction of FBP-ΔNnLuz, FBP-ΔNnH3H and FBP-ΔNnLuz-ΔNnH3H Carriers

[0061] The FBP-ΔNnLuz and FBP-ΔNnH3H vectors were assembled and constructed using the TGSII multi-gene stacking system (Zhu et al., 2017) developed by Academician Liu Yaoguang's team at South China Agricultural University. Using the original donor vectors pYL322d1 and pYL322d2 provided by the team as the backbone, empty vectors pYL322d1-P35S and pYL322d2-P35S were constructed through enzyme digestion and ligation. The 35S promoter and NOS terminator on pYL322d1-P35S were respectively... Xho I / Xba I and Sal I / Hin The two dIII restriction sites are ligated, while the 35S promoter and NOS terminator on pYL322d2-P35S are ligated via... Hin d III / Nco I and Eco RI / XhoI. Construction of two sets of restriction enzyme sites. Derived from *Gymnospermum glabra* (… Neonothopanusnambi )of NnLuz , NnHispS , NnH3H , NnCPH Four gene coding sequences and those derived from Aspergillus nidus ( Aspergillus nidulans )of AnNPGA The gene coding sequence was chemically synthesized at Shanghai Jereh Biotechnology Co., Ltd. after optimization using tobacco codons. NnHispS , NnH3H and NnLuz pass Eco RI and Sal I site was cloned into pYL322d1-P35S, while NnCPH , AnNPGA pass Bam HI and Eco RI is connected to the pYL322d2-P35S vector.

[0062] Referring to the TGSII construction method, the specific steps are as follows:

[0063] 1) The donor vector pYL322d1-NnHispS and the recipient vector pYLTAC380GW were co-transformed into NS3529 competent cells using the heat shock transformation method. The specific operation procedure is as follows: after incubating on ice for 30 minutes, a 90-second heat shock was performed, followed by an ice incubation for 2-3 minutes; 700 μL of antibiotic-free LB liquid medium was added, and the cells were cultured at 37°C and 200 rpm for 2 hours to allow the cells to recover; after centrifugation to collect the bacterial cells, they were evenly spread on LA plates containing 25 mg / L kanamycin and 15 mg / L chloramphenicol; after single colonies were formed, all clones were washed with ddH2O and mixed, and finally the mixed plasmid was extracted.

[0064] 2) Take approximately 50–100 ng of the mixed plasmid for I- Sce For the NEB enzyme digestion reaction, 1 μL of enzyme was added to a 10 μL reaction system and digested at 37°C for 4–5 hours. The digestion product was transformed into *E. coli* NEB10-β competent cells from Biomed. The transformation product was plated on LA plates containing kanamycin and incubated at 37°C for 15 hours. Single colonies were picked and amplified in LB broth containing 25 mg / L kanamycin and 0.5 mM IPTG. Positive clones were initially screened by bacterial PCR, and plasmids were extracted from strains showing specific amplification bands. 200 ng of the extracted plasmid was used for… Not I. Enzyme digestion verification: 0.2 μL of enzyme was added to a 20 μL reaction system. Electrophoresis detection showed that the clone containing four bands and a 6243 bp characteristic band was the target recombinant plasmid pYLTAC380GW-HispS.

[0065] 3) The donor vector pYL322d2-CPH and the recipient vector pYLTAC380GW-HispS obtained in step (2) were mixed and co-transformed into NS3529 competent cells. The transformation method was the same as described in step (2), and the transformation products were evenly spread on LB agar plates containing 25 mg / L kanamycin and 70 mg / L ampicillin. After single colonies formed, all clones were washed with sterile double-distilled water and collected for subsequent extraction of mixed plasmids.

[0066] 4) Take 40-90 ng of the mixed plasmid for PI- Sce I. NEB digestion reaction: 10 μL reaction system contains 1 μL enzyme and 0.5 μL BSA, digested at 37℃ for 4-5 hours. The digestion product is transformed and verified according to step (2). Electrophoresis detection shows five characteristic bands. The clone with both 1757 bp (CPH gene) and 6243 bp (HispS gene) present is the target recombinant plasmid pYLTAC380GW-HispS-CPH.

[0067] 5) Through a multi-round recombination strategy, co-transformation was performed using d1 and d2 series donor vectors carrying different genes alternately with the previously constructed recipient vector, ultimately successfully constructing the FBP-ΔNnLuz and FBP-ΔNnH3H vectors. The FBP-ΔNnLuz vector contains... NnHispS , NnCPH, AnNPGA, NnH3H Gene( Figure 1 (B), FBP-ΔNnH3H contains NnHispS , NnCPH, AnNPGA NnLuz Gene( Figure 1 (A), FBP-ΔNnLuz-ΔNnH3H contains NnHispS , NnCPH, AnNPGA Gene( Figure 1 (C) The final carrier undergoes [process] before use. Not I enzyme digestion and next-generation sequencing verification ensured sequence accuracy.

[0068] Example 2 NnLuz and NnH3H Design and construction of related mutants

[0069] Based on the sequence information of the vector pCAMBIA1300-35S-Flag-NOS, and referring to the instructions for the one-step rapid cloning kit, a design was created. NnLuz and NnH3H The relevant primers were used for amplification with KOD One high-fidelity DNA polymerase. NnLuz and NnH3HThe gene coding region sequence was ligated into the pCAMBIA1300-35S-Flag-NOS vector, which already contained a 3×FLAG tag, using homologous recombination to construct the pCAMBIA1300-NnLuz, pCAMBIA1300-NnH3H, and pCAMBIA1300-NnLuzΔN19 vectors.

[0070] Point mutations were performed on the pCAMBIA1300-NnLuz, pCAMBIA1300-NnH3H, and pCAMBIA1300-NnLuzΔN19 vectors, and the plasmid templates were amplified using KOD One high-fidelity DNA polymerase. Dpn I enzyme removes methylated template DNA (original plasmid), retains the mutant product, transforms the product into competent cells (such as DH5α), plates positive clones are screened, and sent for sequencing to confirm the mutation site (the mutation region must be covered).

[0071] Example 3: Verification of the transient expression effect of related vectors in tobacco

[0072] The aforementioned mutant vectors were transformed into *Agrobacterium tumefaciens* EHA105. After verification, they were transiently expressed in tobacco. This was achieved by simultaneously and transiently transforming tobacco with the mutant vectors constructed in Example 1 (FBP-ΔNnLuz, FBP-ΔNnH3H, FBP-ΔNnLuz-ΔNnH3H) and Example 2. The specific methods are as follows:

[0073] 1) Activate in LB medium containing Kana+Rif and incubate for 1 day at 200 rpm in a shaker at 28°C;

[0074] 2) Take 1 mL of bacterial culture and add it to 20 mL of LB medium containing Kana + Rif + 15 μM acetylsyringone for expansion culture. Incubate at 28℃ and 200 rpm until OD reaches 100%. 600 =0.8-1.0;

[0075] 3) Centrifuge at 5000 rpm for 10 min to collect bacteria. Wash Agrobacterium with infiltration buffer (10 mM MES, 10 mM MgCl2, 150 μM AS) and resuspend at OD. 600 =0.8-1.0, let stand at room temperature for 2-3 hours;

[0076] 4) Use a 1 mL syringe with the needle removed to draw up the bacterial solution. Gently poke a small hole (without piercing the leaf) on the lower epidermis of a tender ZY100 tobacco leaf that is about 3 weeks old. Press the back with your thumb and slowly inject Agrobacterium into the tobacco leaf with a syringe. Mark the water-soaked area of ​​the tobacco leaf.

[0077] 5) The plants were cultured at 25℃ under normal growth conditions for approximately 72 hours. Subsequently, the bioluminescence signals of the plants were acquired and the photon flux was quantitatively analyzed using the NIGHTSHADE LB985 system manufactured by Berthold GmbH, Germany. Bioluminescence images were captured with a 20-second exposure, and then the region used for photon calculation was selected. Finally, the data was exported for analysis.

[0078] Example 4 Optimization and Modification of NnLuz Enzyme

[0079] (1) Optimization and modification of NnLuz enzyme:

[0080] Our laboratory has successfully preserved and validated NnLuzΔN19 (amino acid sequence as shown in SEQ ID No. 6), which has truncated 19 amino acids at the N-terminus. By removing the redundant N-terminal sequence, the efficiency of the fungal bioluminescence system has been significantly improved, exhibiting stronger luminescent signals in both Saccharomyces cerevisiae and plants (Zhong Jingling. Research on optimization strategies of fungal bioluminescence systems and their application in plants [D]. Zhejiang University, 2023. DOI:10.27461 / d.cnki.gzjdx.2023.002148.), providing an important strategy for optimizing bioluminescent reporter systems. This invention first further validated wild-type NnLuz (amino acid sequence as SEQ ID No. 5), NnLuzΔN19, NnLuz_v4 (Shakhova et al., 2024), and NnLuz_v4ΔN19 in yeast. Similar to previous results, the luminescence intensity of NnLuzΔN19 after truncating the N-terminal redundant sequence was significantly higher than that of wild-type NnLuz. Similarly, after truncating the N-terminal 19 redundant amino acid sequence of the published NnLuz_v4, its luminescence intensity was also significantly higher than that of NnLuz_v4. In the yeast study, NnLuzΔN19 had the highest luminescence intensity, significantly higher than that of the published NnLuz_v4. Figure 2 (AB). Therefore, this invention further conducts in-depth research based on NnLuzΔN19 in order to further improve the catalytic efficiency and luminescence intensity of the bioluminescent system.

[0081] First, the three-dimensional structural model of NnLuz was predicted using AlphaFold2. Molecular docking simulation was then performed using AutoDock Vina 1.5.7, which precisely determined that its active pocket is composed of five key amino acid residues: Glu149, Glu181, His184, His186, and His194. Figure 3 (A). Multiple sequence alignment analysis showed that these catalytic residues are highly conserved during evolution. Figure 3Based on this structural information, this invention systematically compared Luz homologous protein sequences from different species and screened out 8 potential mutation sites (positions 58, 68, 103, 122, 126, 128, 145, and 167). Figure 4 ).

[0082] Following the method described in Example 2, based on the pCAMBIA1300-NnLuz and pCAMBIA1300-NnLuzΔN19 vectors, the primers shown in Table 1 were used.

[0083] Table 1 Luz dot-protrusion primers

[0084]

[0085] By constructing a self-luminescent Saccharomyces cerevisiae YCA113-FBP-N (Zhong Jingling. Research on optimization strategies of fungal bioluminescence systems and their application in plants [D]. Zhejiang University, 2023.DOI:10.27461 / d.cnki.gzjdx.2023.002148.), and then knocking out NnLuz in the self-luminescent Saccharomyces cerevisiae, the sgRNA sequence of NnLuz was designed using the Benchling CRISPR tool. The N19 sequence targeting NnLuz was screened by balancing the On-Target Score and Off-Target Score, and constructed into the p423-SpSgH plasmid to become p423-SpSgH-△NnLuz. p423-SpSgH-△NnLuz was then transferred into YCA113-FBP-N to knock out NnLuz to become YCA113-FBP(Luz△N19). Using site-directed mutagenesis, a single mutant library was constructed, including wild-type NnLuz and NnLuzΔN19 at the following eleven sites: V49A / G, F58I / L, S68A, I103T, T122L, I126K, K128T, T145L, and L167K. Point mutations were then performed on the pESC-URA (for yeast-related validation) and pCAMBIA1300-35S-NOS (for plant-related validation) vector backbones. In the yeast expression system, by reintroducing the relevant NnLuz mutants into YCA113-FBP(LuzΔN19), screening revealed that single mutations at the I103T, I126K, and L167K sites significantly increased luminescence intensity. Figure 5 ).

[0086] Using the method described in Example 3, further verification in tobacco revealed that co-infection with FBP-ΔNnLuz Agrobacterium lacking the NnLuz gene showed that I103T, T122L, and L167K exhibited better results. Figure 6Further validation with dual mutations was conducted, and the dual mutations of I103T and L167K showed the most significant effect. This mutation was named NnLuzΔN19_v2, and its amino acid sequence is shown in SEQ ID No. 1. The encoding gene... NnLuzΔN19_v2 The nucleotide sequence is shown in SEQ ID No. 3, and its luminescence intensity is 3 to 4 times that of the previously reported optimal mutant NnLuz_v4 (Shakhova et al., 2024). Figure 7 ).

[0087] Example 5: Optimization and Modification of NnH3H Enzyme

[0088] Molecular docking studies have identified 10 key residues in the active region of NnH3H, including Gly18, Asp37, Ser38, Val137, and Asp318. Figure 8 These sites (A) are also highly conserved during evolution. Figure 8 Based on multiple sequence alignment, 12 potential mutation sites were screened (A43T / S, T66V / I, G81A, K210N, A251G, V276F, K307T, Q353D, E379Q, L395M). Figure 9 Following the method described in Example 2, using the primers shown in Table 2 on the pCAMBIA1300-NnH3H vector,

[0089] Table 2 H3H dot projection primers

[0090]

[0091] Using site-directed mutagenesis, a single mutant library was constructed at the following twelve sites: A43T / S, T66V / I, G81A, K210N, A251G, V276F, K307T, Q353D, E379Q, and L395M. Referring to the method in Example 4, screening using a yeast expression system revealed that the K307T mutant exhibited the most significant activity enhancement. Figure 10 (AB).

[0092] Further verification in tobacco using the method described in Example 3 showed that this mutant (named NnH3H_v3, amino acid sequence as shown in SEQ ID No. 2, encoding gene) is effective. NnH3H_v3 The luminescence intensity of the nucleotide sequence shown in SEQ ID No. 4 was superior to that of the previously reported NnH3H_v2 (Shakhova et al., 2024) mutant. Figure 11 (AB).

[0093] Example 6: Co-expression of NnLuzΔN19_v2 and NnH3H_v3

[0094] Referring to the method described in Example 3, the FBP-ΔNnLuz-ΔNnH3H vector was combined with the mutant vector pCAMBIA1300- constructed above. NnLuzΔN19_v2 pCAMBIA1300- NnH3H_v3 Co-expression. The results showed that the system luminescence intensity reached the optimal level when NnLuzΔN19_v2 and NnH3H_v3 were co-expressed, and was stronger than that of NnLuzΔN19_v2 and NnH3H_v3 expressed alone. Figure 12 (AB), created a self-luminescent plant with significantly enhanced luminescence ( Figure 13 ).

Claims

1. A protein mutant, characterized in that, NnLuz protein mutant NnLuzAN19_v2 and / or NnH3H protein mutant NnH3H_v3; the amino acid sequence of the NnLuz protein mutant NnLuzAN19_v2 is shown as SEQ ID No. 1; the amino acid sequence of the NnH3H protein mutant NnH3H_v3 is shown as SEQ ID No.

2.

2. A gene encoding the protein mutant of claim 1 NnLuzAN19_v2 and / or NnH3H_v3 .

3. The genetic code according to claim 2, wherein The gene encoding the NnLuz protein mutant NnLuzΔN19_v2 NnLuzAN19_v2 The nucleotide sequence is shown in SEQ ID No. 3; the encoding gene of the NnH3H protein mutant NnH3H_v3. NnH3H_v3 The nucleotide sequence is shown in SEQ ID No.

4.

4. A recombinant vector, expression cassette or recombinant bacteria containing the coding gene of claim 2 or 3.

5. Use of the protein mutant of claim 1, the encoding gene of claim 2 or 3, or the recombinant vector, expression cassette or recombinant bacteria of claim 4 for enhancing the intensity of bioluminescence of fungi or plants, characterized in that, The fungal or plant genome has integrated therein HispS a gene, CPH a gene, H3H a gene, NPGA a gene; or HispS genes, CPH genes, Luz genes, NPGA genes; or HispS genes, CPH genes, NPGA genes.

6. A method of enhancing the bioluminescence intensity of a plant, characterized by, To construct the coding gene of NnLuz protein mutant NnLuzΔN19_v2 of claim 1 NnLuzAN19_v2 After codon optimization, the rest of the genes of the fungal bioluminescence system FBP are: HispS Gene, CPH Gene, H3H Gene, NPGA Gene, or the coding gene of the NnH3H protein mutant NnH3H_v3 of claim 1 NnH3H_v3 After codon optimization, the rest of the genes of the fungal bioluminescence system FBP: HispS gene, CPH gene, Luz gene, NPGA genes are co-expressed by transiently transforming plants; or the genes encoding the NnLuz protein mutant NnLuzAN19_v2 and the NnH3H protein mutant NnH3H_v3 of claim 1 NnLuzAN19_v2 and NnH3H_v3 after codon optimization with the rest of the fungal bioluminescent system FBP genes: HispS gene, CPH gene, NPGA gene were co-expressed simultaneously in transiently transformed plants; A self-luminous plant with significantly enhanced bioluminescence intensity is obtained.

7. The method of claim 6, wherein, comprising the following steps: S1. Constructing plant binary expression vectors containing NnLuzAN19_v2 genes or NnH3H_v3 genes, respectively; S2. Using multi-gene assembly technology, the genes of HispS gene, CPH gene, H3H gene, NPGA are integrated into a recipient vector to construct a multi-gene vector FBP-ΔNnLuz; or using multi-gene assembly technology, the genes of HispS gene, CPH gene, Luz gene, NPGA are integrated into a recipient vector to construct a multi-gene vector FBP-ΔNnH3H; or using multi-gene assembly technology, the genes of HispS gene, CPH gene, NPGA are integrated into a recipient vector to construct a multi-gene vector FBP-ΔNnLuz-ΔNnH3H; S3. Transiently transforming recipient plants with a plant binary expression vector containing NnLuzAN19_v2 the genes of the FBP-ΔNnLuz and FBP-ΔNnH3H multiple gene vectors; or NnH3H_v3 transiently transforming recipient plants with a plant binary expression vector containing NnLuzAN19_v2 the genes of the FBP-ΔNnLuz and FBP-ΔNnH3H multiple gene vectors; or NnH3H_v3 transiently transforming recipient plants with a plant binary expression vector containing S4. Cultivation to obtain transgenic plants with significantly enhanced bioluminescence intensity.

8. The method of claim 7, wherein, The plant binary expression vector is pCAMBIA1300.

9. The method of claim 7, wherein, The multi-gene assembly is performed by using the TransGene Stacking II system.

10. The method of claim 6, wherein, The plant is a higher plant. The plant is a higher plant.

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