Screening method of protein mutant with improved stability and protein mutant

Abstract

The invention discloses a screening method of a protein mutant with improved stability and the protein mutant. The screening method comprises the following steps: (1) obtaining a sequence with relatively high heterogeneity, a sequence with relatively high homogeneity or a sequence of a sequenced plant gene of a protein; (2) respectively carrying out positive selection analysis on various sequences and combining with an informatics means to obtain a potential mutation site with the lowest free energy; and (3) carrying out combinatorial mutation on the potential mutation sites of various sequences to construct a mutant library, and calculating the free energy of each combinatorial mutation to obtain a combined mutant of which the free energy is maximally lower than that of the initial enzyme, so as to obtain the zymoprotein mutant of which the stability is improved. The acetolactate synthase mutant with high stability and obviously improved inhibitor resistance efficiency is obtained through positive selection analysis, mutation site screening, combination with bioinformatics means to construct a mutant library, and single-site or multi-site mutation, and the acetolactate synthase mutant has the advantages of high stability, prolonged half-life period, strong acid-base tolerance and strong inhibitor resistance, and significantly broadens the application field.

Description

Technical Field

[0001] The present invention relates to a method for screening protein mutants, in particular to a method for screening protein mutants with improved stability and the protein mutants with improved stability obtained by the screening method, and belongs to the field of protein mutant screening. Background Art

[0002] Protein stability research has important practical applications in medicine, industry, and agriculture. The application of protein physicochemical properties and the catalytic activity of enzymes require mechanisms that support their stability. Protein stability determines the economic viability of such proteins in social production. While many proteins involved in important cellular regulatory processes are inherently disordered, the majority of functional proteins are in a highly ordered, folded state.

[0003] As natural biocatalysts, enzymes possess high catalytic capacity, strong specificity, and relatively mild reaction conditions, offering enormous potential for application in industrial production. Enzymes play a major role in protein research. However, due to their relatively mild reaction conditions, enzymes face limitations in their ability to function properly, such as excessive acidity, alkalinity, high salt concentrations, and high temperatures. Due to long-term natural selection, most enzymes originate from mesophilic bacteria and have adapted to environmental changes. Consequently, these enzymes generally suffer from poor heat resistance. However, high temperatures are frequently used in industrial production processes, for example to reduce the risk of microbial contamination. Therefore, improving the heat resistance of enzymes is of great value for the application of biocatalysts in industrial production.

[0004] Acetolactate synthase (ALS) is a FAD and ThDP-Mg 2+ ALS is a key enzyme that catalyzes the first step in the biosynthesis of branched-chain amino acids (BCAAs). Acetolactate synthase was first discovered in Escherichia coli. In 1997, Chang et al. first purified ALS from Arabidopsis thaliana. Studies have shown that ALS is found only in plants and microorganisms, not animals. This characteristic makes ALS a common target enzyme for herbicides and is now also being considered as a target enzyme for emerging antimicrobial agents.

[0005] In plants, Arabidopsis thaliana acetolactate synthase (AtALS) is a tetramer composed of four identical subunits. ALS typically exists as a tetramer and monomer in plants, consisting of a catalytic subunit (CSU) (≈65 kDa) and a regulatory subunit (RSU) (11-60 kDa), with sizes varying depending on species.

[0006] In the evolutionary analysis of protein families, some key positive selection sites will appear. During the evolutionary process, changes in nucleotides will lead to synonymous or non-synonymous substitutions at amino acid sites. Non-synonymous substitutions are more conducive to environmental selection than synonymous substitutions and are thus fixed, resulting in amino acid sites that have undergone positive selection.

[0007] Random mutations in natural proteins are often neutral or deleterious, potentially rendering them unstable and causing many proteins to malfunction. Furthermore, natural proteins have adapted to their environment through long periods of natural selection, leaving most proteins on the brink of stability. Many natural proteins are not stable enough for research, pharmaceuticals, or industrial applications, and many diseases are caused by single amino acid mutations that destabilize proteins. Mutations affect not only the overall stability of proteins but also their local structure. This low stability is a significant limitation to protein research and application, as proteins are also susceptible to pathologically destabilizing mutations. The instability of natural proteins and their variants makes them difficult to purify and process. Therefore, research to improve protein stability is of great significance. Summary of the Invention

[0008] One of the objects of the present invention is to provide a method for screening protein mutants with improved stability;

[0009] A second object of the present invention is to provide a mutant of acetolactate synthase with improved stability obtained by the screening method.

[0010] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0011] A method for screening protein mutants with improved stability comprises: (1) obtaining a protein sequence with high heterogeneity, a protein sequence with high homogeneity, or a sequence of a sequenced plant gene; (2) subjecting each sequence to positive selection analysis and combining it with informatics to obtain potential mutation sites with the lowest free energy; (3) performing combined mutations on the potential mutation sites of each sequence to construct a mutant library, calculating the free energy of each combined mutation, obtaining a combined mutant with the most reduced free energy compared to the starting enzyme, and obtaining an enzyme protein mutant with improved stability.

[0012] In a preferred embodiment of the present invention, the protein includes but is not limited to enzyme proteins, hormones, antibodies, transport proteins or contractile proteins, and is preferably ALS enzyme (acetolactate synthase). The original amino acid sequence of the ALS enzyme is AT3G48560 in the GenBank sequence number on NCBI, and its amino acid sequence is shown in SEQ ID No. 1. The reference ALS protein sequence is reverse-translated and optimized according to the codon preference of the host Escherichia coli, and the nucleotide sequence information after codon optimization is shown in SEQ ID No. 2.

[0013] In a preferred embodiment of the present invention, the protein sequence with higher heterogeneity is obtained by artificially selecting 80 acetolactate synthase sequences from 80 species of dicotyledons, monocotyledons, lower plants and microorganisms using the BLASTP search method.

[0014] In a preferred embodiment of the present invention, the protein sequence with higher homogeneity is obtained by obtaining a sequence with higher homogeneity of acetolactate synthase, wherein the public database NCBI is searched using BLASTP to obtain the first 500 sequences as the sequences with higher homogeneity.

[0015] In a preferred embodiment of the present invention, the sequence of the sequenced plant gene for obtaining the protein is preferably the sequence of the sequenced plant gene for obtaining acetolactate synthase, wherein a total of 82 homologous sequences of acetolactate synthase were identified from the plant database Phytozome as the sequence of the sequenced plant gene.

[0016] In a preferred embodiment of the present invention, the sequences of acetolactate synthase with high heterogeneity obtained in step (2) are respectively subjected to positive selection analysis and combined with informatics means to obtain the potential amino acid mutation sites with the lowest free energy, namely Q184I, K339L, L350F, G353S, R356M and Q364K.

[0017] In a preferred embodiment of the present invention, the highly homogeneous sequences of acetolactate synthase obtained in step (2) are respectively subjected to positive selection analysis and combined with informatics means to obtain the potential amino acid mutation sites with the lowest free energy, namely D9Q, E144H, H278Y, T321Q, L350F, S568A and N573D.

[0018] In a preferred embodiment of the present invention, the sequence of the sequenced plant gene of acetolactate synthase obtained in step (2) is subjected to positive selection analysis and combined with informatics means to obtain the potential various amino acid mutation sites with the lowest free energy, which are A7L, D9Q, R114K, E183S, Q184R, Q184I, E200P, H203L and N360K.

[0019] In a preferred embodiment of the present invention, step (2) further comprises comparing the potential mutation sites of various sequences to find overlapping mutation sites, combining these overlapping mutation sites with the important potential mutation sites obtained by screening to construct a mutant library, calculating the free energy of each combined mutation, obtaining the combined mutant with the largest free energy reduction compared to the starting enzyme, and obtaining an enzyme protein mutant with improved stability; preferably, when the protein is acetolactate synthase, the potential mutation sites of various sequences of acetolactate synthase are compared to find that the overlapping mutation sites are Q184I, L350F and D9Q, and the important potential mutation sites are S568A and N360K.

[0020] The "Q184I" single-site mutation in the present invention refers to the mutation of asparagine at position 184 to isoleucine, and the meanings of the remaining single-site mutations are similar.

[0021] Another aspect of the present invention is to provide an acetolactate synthase mutant with improved stability obtained by the screening method, selected from any one of the acetolactate synthase mutants described in (1) to (4) below:

[0022] (1) A multi-site mutant obtained by subjecting the acetolactate synthase having the amino acid sequence shown in SEQ ID NO. 1 to any one of the amino acid multi-site mutations K339L-G353S-R356M, Q184I-R356M-Q364K, G353S-R356M-Q364K, Q184I-K339L-R356M-Q364K, Q184I-G353S-R356M-Q364K, or Q184I-K339L-G353S-R356M-Q364K, wherein the multi-site mutants G353S-R356M-Q364K and Q184I-G353S-R356M-Q364K are preferred;

[0023] (2) The amino acid sequence is SEQ ID A multi-site mutant of the acetolactate synthase shown in NO.1 obtained by subjecting the acetolactate synthase to any one of the amino acid multi-site mutations E144H-H278Y-T321Q, E144H-T321Q-N573D, D9Q-E144H-H278Y-T321Q, E144H-H278Y-T321Q-N573D, E144H-H287Y-L350F-S568A, E144H-H278Y-L350F-N573D, or D9Q-E144H-H278Y-T321Q-N573D, wherein the two multi-site mutants E144H-H278Y-L350F-N573D and D9Q-E144H-H278Y-T321Q-N573D are preferred;

[0024] (3) The acetolactate synthase having the amino acid sequence shown in SEQ ID NO. 1 was subjected to D9Q-R114K-E183S-Q184I-E200P-H203L-N360K, A7L-R114K-E183S-Q184R-E200P-H203L-N360K, A7L-R114K-E183S-Q184I-E200P-H203L-N360K, A7L-R114K-E183S-Q184I-E200P-H203L-N360K, , A7L-D9Q-E183S-E200P-H203L-N360K, D9Q-R114K-E183S-Q184I-E200P-N360K, A7L-D9Q-R114K-E183S-Q184R-H203L-N360K or A7L-D9Q-R114K-Q184R-H203L-N360K, wherein preferably the mutants obtained by the two multi-site mutations of A7L-D9Q-E183S-E200P-H203L-N360K or D9Q-R114K-E183S-Q184I-E200P-N360K;

[0025] (4) A plurality of multi-site mutants obtained by subjecting the acetolactate synthase having the amino acid sequence shown in SEQ ID NO.1 to any one of the amino acid multi-site mutations Q184I-L350F-D9Q, Q184I-L350F-N360K, D9Q-S568A-N360K, S568A-Q184I-L350F, L350F-S568A-N360K or D9Q-N360K-L350F, wherein the two multi-site mutants L350F-S568A-N360K or D9Q-N360K-L350F are preferred.

[0026] The "K339L-G353S-R356M" multi-site mutation described in the present invention means that the lysine at position 339 is mutated to leucine, the glycine at position 353 is mutated to serine, and the arginine at position 356 is changed to methionine. The meanings of the remaining multi-site mutations are similar.

[0027] The present invention further provides a coding gene for the acetolactate synthase mutant and a chimeric gene, expression cassette or recombinant expression vector containing the coding gene.

[0028] The present invention also provides a recombinant host cell containing the chimeric gene, expression cassette or recombinant expression vector.

[0029] The present invention expressed these single-site mutants or multi-site mutants in Escherichia coli. The expression results showed that the stability of the mutants screened by positive selection was significantly higher than that of the original enzyme, reflecting the advantage of positive selection analysis in screening mutation sites.

[0030] Another aspect of the present invention is to provide a method for preparing a mutant, comprising:

[0031] (1) constructing a prokaryotic expression vector of the coding gene of the ALS enzyme mutant; (2) transforming the constructed prokaryotic expression vector into corresponding host cells, expressing the fusion protein in the host cells, and purifying the fusion protein.

[0032] The present invention expresses single- and multi-point mutants of the ALS enzyme in prokaryotes, uses molecular dynamics simulations to preliminarily predict the stability of the designed enzymes with combined mutants, and further combines experimental methods to determine the changes in residual enzyme activity of the designed enzymes under different conditions. The mutants expressed by the present invention have higher stability than the original enzyme and also possess excellent herbicide resistance. Therefore, screening for positively selected sites can effectively identify sites that are beneficial to protein stability, improve the stability of the ALS enzyme, extend the half-life of the ALS enzyme, and increase the enzyme protein's inhibitor resistance.

[0033] Compared with the prior art, the present invention has the following advantages and effects:

[0034] 1. The present invention provides a new approach to protein improvement by using positive selection analysis to screen enzyme protein mutation sites. Combined with molecular dynamics simulation, the stability of the designed enzyme with combinatorial mutations can be preliminarily predicted, making it easier and more accurate to obtain mutation sites that are beneficial to protein stability.

[0035] 2. The method for analyzing and screening enzyme protein mutation sites by positive selection has a wide range of applications and can be applied to a variety of different proteins. By means of bioinformatics, a virtual mutant library is constructed to screen the mutant combination with the largest reduction in free energy. In this process, there is no need for continuous repetition and screening, which is time-consuming and laborious. Therefore, compared with the traditional method for improving enzyme proteins, it has a shorter operation time, is simpler, has a lower cost, and is suitable for rapid and accurate protein improvement.

[0036] 3. The screened mutation sites are highly accurate. The created enzyme protein mutants have high stability, strong acid and alkali resistance, and can be used in some environments with strong acids, strong alkalis or high temperatures. They have high inhibitor resistance. The present invention uses the prokaryotic expression system, the Escherichia coli expression system, to express ALS enzyme mutants, and the preparation process is simple.

[0037] Definitions of terms used in this invention

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0039] The term "positive selection" is a process in which advantageous mutations driven by natural selection are fixed. The genetic differences between species that we observe today are the products of this complex process. In molecular evolution analysis, the analysis of selection pressure is a commonly used method. For the analysis of gene family evolution, researchers have proposed the maximum likelihood method for detecting functional differences at single codon sites, which can be used to measure different selection pressures and identify amino acid sites affected by different selections, and use the ratio ω (ω = dN / dS) of the non-synonymous substitution rate (dN) and the synonymous substitution rate (dS) as the pressure of natural selection acting on proteins, especially for identifying the adaptive evolution of proteins. When dN > dS, it is positive selection; when dN < dS, it is negative selection, also known as purifying selection; when dN = dS, it is neutral evolution.

[0040] The term "free energy" refers to that the stability of a protein is usually represented by the change in Gibbs free energy (ΔΔG) before and after folding, which calculates the difference in free energy between the folded state and the extended state of the protein. The larger the absolute value of the negative number, the higher the stability. Substitution of a single amino acid in a protein sequence can cause a significant change in protein stability (ΔΔG). A positive value of ΔΔG indicates an unstable mutation, while a negative value of ΔΔG indicates a stable mutation.

[0041] The terms "mutation" and "mutant" have their common meanings herein, referring to genetic, naturally occurring or introduced changes in nucleic acid or polypeptide sequences, and their meanings are the same as those commonly known to those skilled in the art.

[0042] The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector, such as a plasmid, or may be integrated into the host genome. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Phylogenetic analysis and motif analysis diagram of ALS 80 family members were performed based on heterogeneous sequence information, where a: Bayesian tree, b: Motif analysis.

[0044] Figure 2 For the design based on heterogeneous sequence information, the phylogenetic analysis diagram of ALS 80 family members was performed, where a: NJ tree; b: ML tree.

[0045] Figure 3 For design based on heterogeneous sequence information, a line graph is plotted based on the average B-factor of each amino acid residue in the ALS protein, clearly showing the 3D structure of the ALS protein. (a) Line graph; b) 3D structure diagram.

[0046] Figure 4 For the design based on heterogeneous sequence information, a virtual mutant library was constructed according to the four methods of FoldX, Rosetta, PSSM, and B-factor, and the intersection was taken to obtain 6 mutable amino acid sites Q184, K339, L350, G353, R356, and Q364.

[0047] Figure 5 When designing based on heterogeneous sequence information, molecular dynamics simulations were performed on the combined mutants. The PMSD values of S1, S2, S3, and S4 had small fluctuations overall, and it was inferred that the stability was better than that of the original enzyme.

[0048] Figure 6 For design based on heterogeneous sequence information, the thermal stability analysis and acid-base analysis of the combined mutant designed enzyme are shown. (a) The temperature at which the enzyme activity of the combined mutant and the original enzyme is reduced by 50% when treated at different temperatures. (b) The time it takes for the enzyme activity of the combined mutant and the original enzyme to be reduced by half. (c) The acid-base resistance analysis of the combined mutant designed enzyme.

[0049] Figure 7 For the design based on heterogeneous sequence information, the resistance of the combined mutant designed enzyme and the original enzyme to three herbicides was analyzed, among which a: chlorsulfuron-resistant group; b: bispyribac-sodium-resistant group; c: imidacloprid-resistant group.

[0050] Figure 8For design based on heterogeneous sequence information, salt bridge and surface electrostatic potential analysis of the combined mutant designed enzyme and the original enzyme are performed. Where A: salt bridge; B: surface electrostatic potential.

[0051] Figure 9 For the design based on homogeneous sequence information, the system evolution analysis diagram is as follows: a: Bayesian tree; b: NJ tree; c: ML tree.

[0052] Figure 10 For the design based on homogeneous sequence information, the intersection of the four results of FoldX, Rosetta, PSSM, and B-factor was taken to finally obtain 7 candidate mutation amino acid sites, namely D9, E144, H278, T321, L350, S568, and N573.

[0053] Figure 11 Figure 3. Molecular dynamics simulations of combined mutants for design based on homogeneous sequence information.

[0054] Figure 12 For design based on homogeneous sequence information, the thermostability analysis and acid-base analysis of the combined mutant designed enzyme are shown. (a) The temperature at which the enzyme activity of the combined mutant and the original enzyme is reduced by 50% when treated at different temperatures. (b) The time it takes for the enzyme activity of the combined mutant and the original enzyme to be reduced by half. (c) The acid-base resistance analysis of the combined mutant designed enzyme.

[0055] Figure 13 For the design based on homogeneous sequence information, the resistance of the combined mutant designed enzyme and the original enzyme to three herbicides was analyzed, among which a: chlorsulfuron-resistant group; b: bispyribac-sodium-resistant group; c: imidacloprid-resistant group.

[0056] Figure 14 For design based on homogeneous sequence information, salt bridge and surface electrostatic potential analysis of the combined mutant designed enzyme and the original enzyme. Where A: salt bridge; B: surface electrostatic potential.

[0057] Figure 15 The topology of the Bayesian tree and molecular dynamics simulations of combined mutants for the design of ALSs in sequenced plant genes. A: Bayesian tree topology; B: molecular dynamics simulation.

[0058] Figure 16Thermostability analysis, acidity, alkalinity, and herbicide resistance analysis of the combined mutant designed enzymes based on sequenced ALS in plant genes. (a) The temperature at which the enzyme activity of the combined mutants and the original enzyme decreased by 50% when treated at different temperatures. (b) The time it took for the enzyme activity of the combined mutants and the original enzyme to decrease by half. (c) Analysis of the acidity and alkalinity resistance of the combined mutant designed enzymes. (d) Analysis of chlorsulfuron resistance.

[0059] Figure 17 When designing ALS based on sequenced plant genes, surface electrostatic potential analysis of the combined mutant designed enzyme and the original enzyme was performed. DETAILED DESCRIPTION

[0060] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0061] 1. Experimental Materials and Reagents

[0062] Strains and vectors: The prokaryotic expression vector pET-28a(+), Escherichia coli competent cells Trans5α, and BL21(DE3) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0063] ALS enzyme sequence: Provide the nucleotide sequence to be synthesized to Beijing Shengyuan Kemeng Gene Biotechnology Co., Ltd. for gene synthesis and clone the gene into the vector pET-28a.

[0064] Enzymes and reagents: Restriction endonucleases and T4 DNA ligase were purchased from Promega. LA Taq DNA polymerase and other related reagents used in the PCR reaction were purchased from TakaRa. The preparation of solutions and culture media was based on reference texts (Joseph et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, 2002; Osber et al., A Compendium of Molecular Biology, 1998). Unless otherwise noted, percentages and parts are calculated by weight.

[0065] 2. Positive Selection Analysis

[0066] Null hypothesis models (M0 / M3) and alternative hypothesis models (M7 / M8) were selected to detect the occurrence of positively selected sites. M0, known as the single-rate evolution model, assumes that all amino acid sites evolve at the same rate. M3, known as the discrete model, assumes that each site evolves at a different rate and calculates the probability of each site being subject to three types of evolutionary selection: p0 (purifying selection), p1 (neutral selection), and p2 (positive selection). The M7 model uses a Beta distribution, assuming ω lies between 0 and 1, and serves as a null test for positive selection. The M8 model also uses a Beta distribution (0 < ω < 1) but adds a category of sites with ω > 1 to detect sites under positive selection. Comparing the M0 and M3 models allows for differences in ω values across sites and for detecting whether they are subject to different selective pressures. Based on the M0 and M3 models, the M7 and M8 models were used to examine whether the ALS protein family has been subject to positive selection during evolution and to identify sites under positive selection. In the positive selection results, the 2△lnL values (twice the difference in the logarithm of the likelihood values) of the two groups of models are first compared, and then the χ 2 The likelihood ratio (LRT) distribution test was performed, and the resulting p-value was used to determine the validity of the alternative hypothesis model. When the likelihood ratio test confirmed that a codon site had ω > 1, the posterior probability of each codon site was calculated using the BEB (Bayes empirical Bayes) method. A posterior probability > 0.95 was considered a significant positively selected site. A p < 0.05 indicated a significantly positively selected amino acid site, and a p < 0.01 indicated a very significantly positively selected amino acid site.

[0067] 3. Construction of mutant library based on free energy

[0068] Using the B-FITTER computer-aided tool, load the 5K6Q_A.pdb file and execute B-FITTER.exe. This program automatically generates an output file containing the B-factor for each amino acid residue and calculates the average B-factor for all amino acid residues in a given protein, excluding hydrogen. A higher B-factor indicates a less rigid amino acid site, which is more unstable and thus a more flexible site.

[0069] Position-Specific Scoring Matrix (PSSM), derived from multiple sequence alignments, has been widely used to extract protein sequence features. This paper constructs a local database based on retrieved ALS protein family sequences and generates a PSSM output file. A matrix is generated within the output file, assigning each amino acid position a weight. A position with a value ≥ 0 is considered permissible for mutation.

[0070] Rosetta is used to calculate the ΔΔG (ΔG is the free energy of folding) after replacing amino acids at positively selected sites with different amino acids to assess changes in protein stability. Depending on the selectivity of the replacement, including single amino acid substitutions or multiple combinations, different files are edited. Based on the high-resolution protocol, the specified tasks for calculating free energy and the output files are set. Finally, the result files are reviewed to collect and analyze ΔΔG data. Detailed steps and command program writing are available on the official Rosetta website.

[0071] The present invention uses FoldX5.0 and uses the command line call software that comes with Windows. The PositionScan module can be used to mutate an amino acid to a selected amino acid and repair adjacent residues. The BuildModel function module introduces mutations and optimizes them to calculate ΔΔG. The present invention replaces the amino acid at the positive selection site with an amino acid that has appeared at the multiple sequence alignment position, and statistics the change in free energy after the amino acid site replacement. For the FoldX5.0 version, the result with a ΔΔG value greater than zero is an unstable mutation, and the ΔΔG result less than zero is a stable mutation. For software download and detailed command content, please refer to the FoldX official website.

[0072] 4. Molecular dynamics simulation steps

[0073] In this study, NAMD was selected as the molecular dynamics simulation software. Developed by the Theoretical and Computational Biophysics Research Group at the University of Illinois, NAMD is primarily used to calculate various numerical values for biological macromolecules. Molecular dynamics simulations were performed using the designed mutant amino acid sequences at the selected sites. Simulation conditions were constant pressure, a temperature of 310 K, and an all-atom CHARMM force field. The simulation time was set to 1 ns, and the specific steps were as follows:

[0074] (1) Generate protein PSF file

[0075] The PSF file is a file containing protein structural information. In this step, the water molecules in the protein PDB file need to be removed to generate a PSF file that only stores protein structural information and contains hydrogen atoms, so that the optimized protein conformation can be obtained.

[0076] (2) Solubilization of proteins

[0077] The goal of this step is to simulate a more realistic biological environment for proteins. The protein is placed in a water environment, either spherical or cubic. In this study, a spherical water environment was used, and VMD was used to visualize the protein molecules within the spherical water environment.

[0078] (3) Perform dynamic simulation

[0079] The temperature was set to 310°C, and the molecular dynamics simulation run time was set to 500,000 timesteps. The results were visualized using VMD visualization software. Scripts were run within the VMD TK console to calculate detailed parameters such as RMSD, RMSF, and salt bridges. Perl was used to analyze the changes in salt bridges within the protein molecules during the simulation time.

[0080] Experimental Example 1 Expression and Activity Assay of ALS Mutants Designed Based on Heterogeneous Sequence Information in Escherichia coli 1. Experimental Methods

[0081] 1.1 Construction of phylogenetic tree and positive selection analysis

[0082] Multiple sequence alignments of ALS protein family members were performed using MUSCLE software. To investigate the evolutionary relationships among ALS protein family members, a Bayesian phylogenetic tree was constructed using MrBayes 3.2.5. Neighbor-joining and maximum likelihood trees were constructed using MEGA 7.0 software, respectively. The bootstrap value was set to 1000, and all other settings were kept as default. The protein multiple sequence alignment files were first converted into codon alignment files using PAL2NAL. Then, the CODEML program was used to perform computational analysis of the ALS gene family using a site-specific model. Null hypotheses M0 (one-ratio) and M3 (discrete) were used, and alternative hypotheses M7 (beta) and M8 (beta & ω > 1) were used. The difference in the logarithm of the maximum likelihood values between the two models was tested using a likelihood ratio test. Posterior probabilities were calculated using a chi-squared test of significance to identify amino acid sites potentially under positive selection.

[0083] 1.2 Screening of mutation sites and construction of virtual mutant library

[0084] In this experiment, flexible sites and non-conserved sites were first screened as candidate mutation sites based on positive selection sites. At the same time, a potential mutant library was constructed by combining the calculation results of Rosetta and FoldX. Amino acid sites with B-factor > 115 were used as potential sites for subsequent protein design. Amino acid sites with 0 < PSSM < 0.4 were used as potential sites for subsequent protein design. The free energy of amino acid substitution was calculated using Rosetta, and the results with △△G < -1 Kcal / mol were selected to construct a potential mutant library. The free energy of amino acid substitution was calculated using FoldX, and the results with △△G < -0.2 Kcal / mol were selected to construct a potential mutant library. The intersection of the four results was taken to determine the mutant amino acid sites, and then the types of amino acids after substitution were determined according to the △△G results. The obtained single sites were combined into multiple sites, including two sites, three sites, four sites, etc. The △△G of the combined results was calculated using Rosetta, and the combined mutants with lower △△G values were selected for subsequent experimental verification.

[0085] 1.3 Construction of 3D model and molecular dynamics simulation

[0086] Using the ALS protein reference sequence AT3G48560 as a template, according to the designed combined mutants, the mutant protein sequence was determined, and the protein sequence was submitted to the I-TASSER website (https: / / zhanggroup.org / / I-TASSER / ) to predict the 3D structure of the protein. The PyMol software (https: / / pymol.org / ep) was used to display the three-dimensional structure of the protein and label the amino acid sites. Molecular dynamics simulation was performed using NAMD.

[0087] 1.4 Synthesis of designed ALS mutant genes based on heterogeneous sequence information

[0088] The original amino acid sequence of the ALS enzyme has the GenBank accession number AT3G48560 on NCBI, and its amino acid sequence is shown as SEQ ID No.1. The ALS enzyme with the amino acid sequence shown as SEQ ID NO.1 was subjected to any one of the multi-site mutation methods of S1 (K339L-G353S-R356M), S2 (Q184I-R356M-Q364K), S3 (G353S-R356M-Q364K), S4 (Q184I-K339L-R356M-Q364K), S5 (Q184I-G353S-R356M-Q364K), S6 (Q184I-K339L-G353S-R356M-Q364K) to obtain multiple multi-site mutants. According to the codon preference of the host Escherichia coli, the reference ALS protein sequence was reverse-translated and optimized, and the above optimized designed gene sequence was artificially synthesized.

[0089] 1.5 Conversion

[0090] Take 2 μL of ALS protein plasmid and add it to the competent E. coli, mix gently, and let it stand on ice for 30 minutes; heat shock in a 42°C water bath for 45 to 90 seconds, let it stand on ice for 2 minutes; add 1 mL of LB liquid culture medium without resistance to the tube and culture it in a shaker at 37°C at 200 rpm for 45 minutes to 1 hour; take out the centrifuge tube and centrifuge at 2000 rpm for 3 minutes to discard the supernatant, mix the precipitate and residual liquid retained in the tube gently with a pipette, take the mixed bacterial liquid and place it on LB solid culture medium containing kanamycin resistance, spread it evenly, place it upside down in a 37°C incubator, and culture it for 14 hours.

[0091] 1.6 Expression and purification of recombinant proteins

[0092] Pick a single colony from the LB medium and place it in 1 mL of LB liquid medium containing kanamycin resistance. Culture at 37°C, 220 rpm for 14 hours, and preserve the strain. Take 200 μL of the bacterial solution and inoculate it into 100 mL of LB culture medium containing resistance. Incubate at 37°C, 200 rpm, and shake for 3 hours to 3.5 hours until the absorbance at 600 nm reaches 0.6-0.8. Add IPTG and shake and culture at 16°C, 160 rpm for 16 hours (save the bacterial solution for SDS-PAGE identification). Centrifuge the above bacterial solution at 4°C, 8000 rpm for 12 minutes, discard the supernatant, collect the bacteria, and wash the bacteria with sterilized deionized water. Repeat twice, and finally retain the precipitate.

[0093] 1.7 Enrichment and concentration determination of target protein

[0094] Add MilliQ H2O to a new, dry, 15 mL ultrafiltration tube with a molecular weight of 10 kDa, pre-chilled. Discard the water before use and add the enzyme solution to concentrate the protein. Place the ultrafiltration tube in a centrifuge with the membrane perpendicular to the spindle to prevent breakage. Incubate at 4°C, 4000 rpm, and an acceleration of 3-4. Add potassium phosphate buffer at a volume of approximately 10 times the volume of the enzyme concentrate. Change the buffer three times to ensure a volume of at least 1000 times. The final volume of concentrated protein is generally between 200 and 500 μL. Transfer the concentrated protein to a new centrifuge tube. Gently remove the residual liquid at the bottom of the ultrafiltration membrane with a pipette and store at -80°C for enzyme activity determination. Protein concentration is determined using a total protein quantification kit (with BCA standard; colorimetric method). See the kit instructions for detailed procedures. Protein concentration is calculated based on the absorbance value.

[0095] 1.8 Acetolactate synthase activity assay

[0096] The ALS protein sample to be tested was added to a substrate reaction buffer solution containing sodium pyruvate, incubated at 37°C for 1 hour, and 100 mL of 3M concentrated sulfuric acid was added to terminate the reaction. After mixing evenly, the reaction was incubated at 60°C for 15 minutes. 0.5% creatine and 5% α-naphthol were added and the reaction was carried out at 60°C for 15 minutes. After the color development reaction was completed, the solution was centrifuged at 10,000 rpm for 8 minutes. The supernatant was added to a 96-well ELISA plate, and the absorbance at 525 nm was read using an ELISA reader.

[0097] 1.8.1 Kinetic stability determination

[0098] Determination of ALS T 50

[0099] T 50 The temperature at which enzyme activity is reduced to 50% when treated at different temperatures. In this study, the temperature gradient was set at 35, 40, 45, 50, 55, and 60°C. Enzyme solutions of equal concentration were treated at different temperatures for 10 minutes. The treated enzyme solution was then added to the substrate reaction buffer and incubated at 37°C for 1 hour. Enzyme activity at 35°C was used as a reference, representing 100% enzyme activity. Enzyme activity was determined according to the method in 1.8.

[0100] Determination of ALS inactivation half-life

[0101] Half-life refers to the time it takes for enzyme activity to decrease by half. First, dilute the mutant and starting enzyme to the same protein concentration and incubate at 45°C for three replicates at 10-min intervals: 0, 10, 20, 30, 40, 50, 60, and 70 min. Add the incubated enzyme to the substrate reaction buffer. In this experiment, 0 min of incubation was used as a reference for 100% enzyme activity. Enzyme activity was determined according to the method in 1.8.

[0102] 1.8.2 Determination of acid and alkali resistance of acetolactate synthase

[0103] Prepare a series of substrate reaction buffers with a pH gradient (pH = 4, 5, 6, 7, 8, 9, 10, 11), add enzyme solutions of equal concentrations to react, and determine the enzyme activity according to the method in 1.8, taking the reaction condition of pH = 7 as the reference enzyme activity of 100%.

[0104] 1.8.3 Determination of acetolactate synthase resistance to three types of herbicides

[0105] Chlorsulfuron was tested at different concentrations (0.000, 0.003, 0.006, 0.009, 0.012, and 0.015 μg / μL). The enzyme activity was determined using the above method, assuming the enzyme activity of ALS protein without inhibitors was 100%. Bispyribac-sodium and chlorsulfuron have the same inhibitory constants for ALS, so the same inhibitor concentration was used for the experiments. Imidacloprid was tested at different concentrations (0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 μg / μL). The enzyme activity was determined using the above method, assuming the enzyme activity of ALS protein without inhibitors was 100%.

[0106] 2. Experimental Results

[0107] 2.1 Phylogenetic analysis and positive selection site analysis results

[0108] Based on the Arabidopsis ALS protein sequence (AT3G48560), a total of 80 sequences from different species (including monocots, dicots, lower plants and microorganisms) were obtained after screening in the Phytozome database and the NCBI database. A phylogenetic analysis was performed on the ALS 80 family members, and Bayesian trees were constructed according to three different methods ( Figure 1 a), NJ tree ( Figure 2 a) and ML tree ( Figure 2 b), red, yellow, and blue represent Group I, II, and III subfamilies, respectively. Based on the phylogenetic tree, ALS genes from multiple species were divided into three branches, corresponding to three subfamilies. Motif analysis was further performed on ALS 80 family members ( Figure 1 b) The motif structure in the same subfamily is conserved, the types and numbers of motifs are basically similar, and the number and classification of motifs in different subfamilies are different, which proves the reliability of the evolutionary tree branch.

[0109] To detect whether there is a variable ω ratio at amino acid sites, two pairs of models (M0 / M3 and M7 / M8) were used for comparison. The results showed that for M0 and M3, 2ΔlnL = 4962.47 (p < 0.01, d.f. = 4, χ2 value = 18.467), and the likelihood ratio test result was extremely significant, rejecting the single-rate model. Comparing models M7 and M8, 2ΔlnL = 31270.596 (p < 0.01, d.f. = 2, χ2 value = 13.815), and the likelihood ratio test was very significant. Moreover, the ω estimated by the M8 model was 2.09710, far greater than 1, indicating that there were amino acid sites under positive selection. Combining with the empirical Bayes method for evaluation, 76 positive selection sites were detected, with a posterior probability greater than 95%. Among them, 66 positive selection sites reached an extremely significant level (p < 0.01), and 10 positive selection sites were at a significant level (p < 0.05).

[0110] 2.2 Obtaining Mutation Sites and Screening of Combinatorial Mutants

[0111] Based on the average B-factor of each amino acid residue, a line graph was plotted ( Figure 3 a), and the B-factor values were shown on the 3D structure of the ALS protein ( Figure 3 b). Amino acid sites with B-factor > 115 were selected as flexible candidate sites for subsequent protein sequence modification, and a total of 47 amino acid sites were screened. Using 80 ALS homologous sequences from different species as a database to calculate the PSSM values, amino acid sites with 0 < PSSM < 0.4 were selected, and 89 amino acid sites were screened. Rosetta was used to calculate the ΔΔG value after amino acid substitution, and results with ΔΔG < -1 Kcal / mol were selected. FoldX was used to calculate the ΔΔG after amino acid substitution at positive selection sites, and amino acid sites with ΔΔG < -0.2 Kcal / mol were selected to construct a potential mutant library. The intersection of the above four methods yielded 6 mutable amino acid sites, Q184, K339, L350, G353, R356, and Q364 ( Figure 4 ). Multiple-site combinations were performed on the six screened mutant amino acid sites, including combinations of two, three, four, five, and six sites, resulting in a total of 57 combinatorial mutation results. Moreover, Rosetta was used to calculate the ΔΔG of the combinatorial mutations, and combinatorial mutants with ΔΔG < -11 Kcal / mol were named S1, S2, S3, S4, S5, and S6 (Table 1).

[0112] Table 1 Results of 6 Combinatorial Mutants

[0113]

[0114] 2.3 Molecular Dynamics Simulation of Combinatorial Mutants

[0115] The change in the overall stability of the protein was determined based on the change trend of the RMSD value of the molecular dynamics simulation within 1000ps. Compared with the starting enzyme, the average RMSD of the combined mutant designed enzyme S2 was less than The RMSD values of S1, S3, and S4 are arrive The overall fluctuation range was lower than that of the starting enzyme ( Figure 5 ), it is speculated that the stability of S1, S2, S3, and S4 is improved compared to the starting enzyme. For S5 and S6, the RMSD values are lower than that of the starting enzyme before 500 ps, and their fluctuation amplitudes tend to be smaller than that of the starting enzyme. However, after 500 ps, the RMSD fluctuation amplitude is slightly higher than that of the starting enzyme.

[0116] 2.4 Thermal stability analysis of enzymes designed by combinatorial mutation

[0117] To test the thermal stability of the designed enzyme, the enzyme was kept at a series of temperature gradients (35, 40, 45, 50, 55, 60°C) for 10 min, and then the residual activity of the enzyme was measured, as shown in ( Figure 6 a). Starting enzyme T 50 The T values of enzymes S3, S5, and S6 were 49.08°C. 50 They were 50.56℃, 50.10℃, and 49.32℃, respectively, which were 1.48℃ higher than those of the starting enzyme.

[0118] 1.02°C, 0.24°C. When the temperature was between 40 and 50°C, the activity of the combined mutant enzymes, except for S4, was higher than that of the original enzyme. The combined mutant enzymes exhibited good thermal stability.

[0119] Treat with a warm bath at 45°C, set a series of time gradients at intervals of 10 min, including treatments of 0, 10, 20, 30, 40, 50, 60, and 70 min, and set three sets of repetitions. Add the enzyme after warm bath treatment to the substrate reaction buffer, as shown in ( Figure 6 As shown in b), the half-life of the starting enzyme is 30 minutes, while the half-life of the combined designed mutant enzyme S3 is greater than 70 minutes, more than double that of the starting enzyme, significantly improving its half-life. The half-lives of the combined designed mutant enzymes S1 and S5 are 37.58 minutes and 57.79 minutes, respectively, increasing their half-lives by 7.58 minutes and 27.79 minutes, respectively, compared to the starting enzyme.

[0120] 2.5 Analysis of acid and alkali resistance of enzymes designed by combinatorial mutation

[0121] Using sodium pyruvate as substrate, the residual activity of ALS starting enzyme and combined mutant designed enzyme was measured at different pH. The results are shown in ( Figure 6As shown in Figure c), the starting enzyme and all the combined mutant designed enzymes had the highest enzyme activity at pH 7. Compared to the starting enzyme, the combined mutant designed enzyme S6 maintained a residual activity of over 50% within the pH range of 5.5 to 10.5, indicating enhanced acid-base tolerance and stability within this range. S2 exhibited enhanced acid resistance, maintaining 5% residual activity at pH 5, while the starting enzyme was nearly inactivated. S1 exhibited enhanced alkaline resistance, maintaining 59% residual activity at pH 9, a 14% increase over the starting enzyme.

[0122] 2.6 Analysis of resistance of the designed enzymes to three herbicides

[0123] Chlorsulfuron-resistant group ( Figure 7 a), compared to the initial enzyme I 50 =0.197 μg / mL, combined mutation design enzyme S3 I 50 =0.518 μg / mL, which is 162.9% higher than that of the starting enzyme. 50 The resistance of S3 to chlorsulfuron was significantly improved. The resistance of the other five mutants, S1, S2, S4, S5, and S6, was significantly improved. 50 The concentrations of the enzymes S2, S5 and S6 were 0.199 μg / mL, 0.257 μg / mL, 0.205 μg / mL, 0.329 μg / mL and 0.265 μg / mL, respectively, which were 1%, 30.5%, 4%, 67% and 34.5% higher than those of the starting enzyme. Compared with the starting enzyme, the ability of the combined mutant designed enzymes S2, S5 and S6 to resist chlorsulfuron herbicide was enhanced.

[0124] Anti-bispyribac group ( Figure 7 b), compared to the initial enzyme I 50 =0.182μg / mL, S3 showed the greatest resistance enhancement, and its I 50 =0.393μg / mL, an increase of 0.211μg / mL, an increase of 115.9%. At the same time, with the increase of bispyribac-sodium concentration, the residual activity of S3 was still higher than that of the starting enzyme, and S3 showed a strong tolerance to bispyribac-sodium. 50 The residual activities of S5 and S6 were 0.242 μg / mL and 0.191 μg / mL, respectively, which were 0.06 μg / mL and 0.009 μg / mL higher than those of the starting enzyme, representing increases of 32.9% and 4.9%, respectively. Within the bispyribac-sodium concentration range of 0 to 0.6 μg / mL, the residual activities of S5 and S6 were higher than those of the starting enzyme, indicating a higher resistance compared to the starting enzyme.

[0125] Anti-imidazole group ( Figure 7 c), I of the starting enzyme 50 =48.491μg / mL, combined mutation design enzymes S3 and S5 I50 The activity of S3 was 94μg / mL and 56.504μg / mL respectively. Compared with the starting enzyme, S3 increased by 45.509μg / mL, an increase of 93.85%, and S5 increased by 8.013μg / mL, an increase of 16.5%. The overall residual activity of S3 was greater than that of the starting enzyme, indicating that S3 and S5 had enhanced resistance to imidacloprid. 50 =50.437μg / mL, which is 1.946μg / mL higher than the starting enzyme. At the same time, according to the results, it can be found that when the concentration of imidacloprid is lower than 50μg / mL, the residual activity of S1 is higher than that of the starting enzyme, indicating that within this concentration range, the resistance of S1 to imidacloprid is enhanced. 50 The activity of S2 and S4 was 36.244 μg / mL and 46.671 μg / mL respectively, which were both lower than that of the starting enzyme. The residual activity of the enzyme as a whole was lower than that of the starting enzyme, indicating that the resistance of S2 and S4 to imidacloprid was not enhanced. 50 =48 μg / mL, which is less than the starting enzyme, but its residual enzyme activity is higher than the starting enzyme in the range of 48-100 μg / mL, indicating that it is resistant to imazethapyr at high concentrations.

[0126] 2.7 Analysis of Salt Bridges and Surface Electrostatic Potential of Combinatorial Mutation Designed Enzymes (Only Preferred Mutant Combinations are Listed)

[0127] The starting enzyme formed a total of 27 salt bridges, of which four salt bridges persisted stably, namely Glu315-Lys318, Glu231-Lys331, Glu40-Lys13, and Asp553-Arg529. Only the preferred S3 and S5 are shown here. Compared with the starting enzyme, the total number of salt bridges of the two combined mutant designed enzymes increased ( Figure 8A) The number of salt bridges formed by the combined mutation design enzyme S3 is 35. It shares the persistent and stable salt bridges Glu231-Lys331 and Glu40-Lys13 with the starting enzyme. On this basis, S3 adds 5 salt bridges that are persistent and stable during the simulation time, namely Glu578-Lys371, Glu298-Lys296, Glu200-Lys197, Asp290-Arg288, Asp256-Lys414, and Glu258-Lys363. S5 has 37 salt bridges, of which the same persistent and stable salt bridges as the starting enzyme are Glu40-Lys13, Glu315-Lys318, and Glu231-Lys331. On this basis, six more persistent and stable salt bridges are added, namely Glu578-Lys371, Glu346-Lys349, Glu258-Lys363, Asp256-Lys414, Asp16-Arg47, and Asp580-Lys371. Among them, the Glu231-Lys331 salt bridge shows persistent stability in the starting enzyme and all combinatorial mutant designed enzymes, indicating that this salt bridge is crucial for the stability of the protein structure. ALS-inhibiting herbicides are non-competitive inhibitors. The role of herbicides in inhibiting acetolactate synthase is mainly through hindering the binding of the enzyme activity channel to the substrate. The combinatorial mutant designed enzyme S3, which has the best herbicide resistance characteristics, shows that the blue around the substrate binding channel deepens ( Figure 8 B) It is speculated that the increased positive electrostatic potential on the protein surface here leads to increased conformational rigidity, which facilitates enzyme-substrate binding and thus conferring a certain degree of tolerance to herbicides. Compared to the starting enzyme, the worst combination of mutants, designed enzyme S1, showed no significant increase in positive charge around the substrate-binding channel, maintaining a relatively neutral potential. This may be one of the reasons why the combination of mutants designed enzymes did not exhibit significant herbicide resistance.

[0128] Experimental Example 2 Expression and activity determination of ALS mutants designed based on homogeneous sequence information in Escherichia coli

[0129] 1. Experimental Methods

[0130] The specific experimental method is shown in Experimental Example 1.

[0131] 2. Experimental Results

[0132] 2.1 Phylogenetic analysis and positive selection site analysis results

[0133] Using the Bayesian method ( Figure 9 a) NJ method ( Figure 9 b) and ML method ( Figure 9c) Phylogenetic tree construction revealed that the ALS family is divided into three subfamilies: Sub I, encompassing 245 dicots, Sub II, encompassing 97 monocots, and Sub III, encompassing 5 lower plants. Likelihood ratio analysis using the M0 and M3 models yielded a significant 2ΔlnL value of 8629.998 (p < 0.01, df = 4, χ² = 18.467), indicating that each amino acid site evolved at a different rate. Analysis using the M7 and M8 models revealed a 2ΔlnL value of 16.642 (p < 0.01, df = 2, χ² = 13.815), which was not significantly greater than 13.815. Furthermore, the ω value did not show a trend greater than 1, indicating that no significant positively selected sites were detected. Due to the high similarity and lack of heterogeneity among the sequences, it is difficult to detect sites of positive selection at the protein level. For the subsequent process of designing protein mutants, a total of 132 amino acid sites were selected as positive selection sites based on the sites with relatively large ω values in the results.

[0134] 2.2 Obtaining mutation sites and screening of combined mutants

[0135] 116 flexible sites were screened out based on the results of B-factor, and 313 non-conserved amino acid sites were screened using the results of PSSM. Secondly, two different software, Rosetta and FoldX, were used to construct a potential mutant library. The intersection of these amino acid sites was taken, and a total of 25 amino acid sites were obtained. The intersection of the four results finally obtained 7 candidate mutation amino acid sites, D9, E144, H278, T321, L350, S568, N573 ( Figure 10 These seven single mutation sites were combined with two, three, four, five, six, and seven mutation sites to obtain 119 combined mutation results. The combined mutation design enzymes with ΔΔG values <-9 Kcal / mol after Rosetta calculation were named D1, D2, D3, D4, D5, D6, and D7.

[0136] Table 2 Results of 7 combined mutants

[0137]

[0138]

[0139] 2.3 Molecular dynamics simulation of combined mutants

[0140] Compared with the starting enzyme, the average fluctuation of the three mutants D2, D6, and D7 is smaller, and the average RMSD is less than It is speculated that the stability of D2, D6, and D7 is significantly improved compared to the starting enzyme. The overall fluctuation range of D3, D4, and D5 is smaller than that of the starting enzyme, but compared with D2, D6, and D7, they show a larger fluctuation range. It is speculated that the stability of D3, D4, and D5 is also improved, but the improvement is smaller than that of D2, D6, and D7. Figure 11 ).

[0141] 2.4 Thermal stability analysis of enzymes designed by combinatorial mutation

[0142] T of seven combinatorial mutant designed enzymes D1-D7 50 The results showed that ( Figure 12 a) T of the other combined mutant designed enzymes except the two combined mutant designed enzymes D4 and D5 50 All of them were improved, and the T of the combined mutant designed enzymes D1, D2, D3, D6, and D7 50 They were 50.20℃, 50.58℃, 50.49℃, 50.68℃ and 51.27℃ respectively, which were 1.07℃, 1.45℃, 1.36℃, 1.55℃ and 2.14℃ higher than the starting enzyme respectively. Among them, the thermal stability of the combined mutation designed enzyme D7 was significantly improved.

[0143] The residual enzyme activity of the starting enzyme and the combined mutant designed enzyme treated at 45°C for different time periods was analyzed ( Figure 12 b), the half-lives of D2, D3, D5, D6, and D7 were 44.85 min, 57.44 min, 47.74 min, 68.6 min, and 57.6 min, respectively, which were 14.85 min, 27.44 min, 17.74 min, 38.6 min, and 27.6 min longer than those of the starting enzyme, and increased by 49.5%, 91.5%, 57.1%, 128.7%, and 92%, respectively, compared with the starting enzyme. Among them, the best performance was the D6 mutant, with an increase in half-life of more than 1 times that of the starting enzyme.

[0144] 2.5 Analysis of acid and alkali resistance of enzymes designed by combinatorial mutation

[0145] Analysis of the tolerance of the combined mutant designed enzymes D1-D7 in acidic and alkaline environments ( Figure 12c) Compared with the starting enzyme, the six mutant enzymes D1, D3, D4, D5, D6, and D7 all showed improved acid resistance or alkaline resistance. Among them, D1, D4, and D6 all showed higher residual activity than the starting enzyme in the pH range of 5 to 11, showing better tolerance to alkaline environments. In an acidic environment, the residual activities of D3 and D5 were 9.6% and 21% respectively at pH = 4. At pH = 9, the residual activity of the starting enzyme was 46.3%, less than 50%, while the residual activities of the mutants D3 and D5 were both greater than 50%. Therefore, the acid resistance and alkaline resistance of D3 and D5 were both increased. In addition, the residual activity of D7 in a strongly acidic environment at pH = 4 was 15%, and in a strongly alkaline environment at pH = 11, its residual activity was 13%. The stability of the combined mutant D7 under acidic and alkaline conditions was significantly increased, and its tolerance to acidic and alkaline environments was improved.

[0146] 2.6 Analysis of herbicide resistance by combinatorial mutation-designed enzymes

[0147] Chlorsulfuron-resistant group ( Figure 13 a), compared with the initial enzyme I 50 =0.197 μg / mL, I of combined mutant D6 50 =0.316μg / mL, an increase of 60.4%, I 50 They were 0.216μg / mL, 0.31μg / mL, 0.206μg / mL, 0.263μg / mL and 0.267μg / mL, respectively, which were increased by 9.6%, 57.3%, 4.6%, 33.5% and 35.5% compared with the starting enzyme.

[0148] Anti-bispyribac group ( Figure 13 b), compared to the starting enzyme I 50 =0.182 μg / mL, I of combined mutant D5 50 =0.586μg / mL, an increase of 0.404μg / mL, an increase of 221.9%, indicating that its resistance to the inhibitor bispyribac-sodium was greatly increased compared with the starting enzyme. 50 =0.24μg / mL, which increased by 0.058μg / mL compared with the starting enzyme, an increase of 31.9%, and the overall residual activity of D2 was greater than that of the starting enzyme, indicating that in the range of 0-1μg / mL, under the action of the same concentration of bispyribac-sodium, D2 retained higher activity and had a higher resistance to herbicide inhibition. In addition, for D7, its I 50=0.194 μg / mL, which is 0.012 μg / mL higher than the starting enzyme, an increase of 6.6%. When the concentration of the herbicide bispyribac-sodium is less than 0.6 μg / mL, the residual activity of the enzyme is higher than that of the starting enzyme. When the herbicide concentration is greater than 0.6 μg / mL, the residual activity of the enzyme is lower than that of the starting enzyme, indicating that when the concentration of bispyribac-sodium is in the range of 0 to 0.6 μg / mL, the resistance of D7 to the herbicide increases.

[0149] Anti-imidazole group ( Figure 13 c), compared to the initial enzyme I 50 =48.491 μg / mL, I of combined mutant D5 50 =100μg / mL, an increase of 43.108μg / mL, an increase of 75.8%, D5's ability to resist the herbicide imazethapyr is significantly better than the starting enzyme. 50 =57.037 μg / mL, which increased by 0.145 μg / mL and 0.3% compared with the starting enzyme. In terms of the residual activity of the enzyme, when the concentration of imidacloprid was greater than 20 μg / mL, the residual activity of the combined mutant designed enzyme D2 was higher than that of the starting enzyme, and its ability to resist imidacloprid herbicide was slightly improved compared with the starting enzyme.

[0150] 2.7 Analysis of Salt Bridges and Surface Electrostatic Potential of Combinatorial Mutation Designed Enzymes (Only Preferred Mutant Combinations are Listed)

[0151] The analysis of the stable salt bridge situation within the simulation time of 1000ps is carried out ( Figure 14A) The number of salt bridges in the combined mutant-designed enzyme increased compared to the starting enzyme. The starting enzyme and combined mutant-designed enzymes D6 and D7 exhibited persistently stable salt bridges during the simulation, including Glu315-Lys318 and Glu231-Lys331, indicating that these two salt bridges are crucial for protein stability. At 310 K, the starting enzyme exhibited 27 salt bridges, while combined mutant-designed enzyme D6 exhibited 40, an increase of 13 compared to the starting enzyme. Combined mutant-designed enzyme D6 also exhibited five additional persistently stable salt bridges: Glu560-Arg582, Glu346-Lys349, Glu298-Lys296, Asp391-Lys393, and Asp145-Arg187. The combined mutant designed enzyme D7 has 31 salt bridges. Although the overall number is only 4 more than that of the starting enzyme, the number of salt bridges that remain stable during the simulation time is 8 more than that of the starting enzyme, namely Glu375-Lys532, Glu358-Lys216, Glu277-Arg303, Glu40-Lys171, Glu23-Arg5, Asp580-Lys371, Asp535-Lys532 and Asp290-Arg288. Based on the mechanism of action of ALS as a herbicide-specific target enzyme, i.e., a non-competitive inhibitor, the combined mutant designed enzyme D5, which showed outstanding resistance to all three herbicides, and the combined mutant designed enzyme D1, which performed poorly, were selected from the seven combined mutant designed enzymes for surface electrostatic potential analysis ( Figure 14 B) Comparison of the starting enzyme with the combined mutant designed enzymes D5 and D1 reveals enhanced surface electrostatic potential around the substrate binding site of D5, while decreased surface electrostatic potential in this region of D1. Based on this, it is speculated that the rigidity of the D5 protein conformation is enhanced, thereby conferring resistance to different herbicides. Experimental Example 3: Expression and Activity Assay of Designed ALS Mutants in E. coli Based on Sequenced Plant Genes

[0152] 1. Experimental Methods

[0153] The specific experimental method is shown in Experimental Example 1.

[0154] 2. Experimental Results

[0155] 2.1 Phylogenetic and positive selection site analysis results

[0156] A total of 82 homologous sequences were identified from the plant database Phytozome and phylogenetic analysis was performed. Based on the topological structure of the Bayesian tree ( Figure 15A) The ALS protein family can be divided into three subfamilies: Group I (cyan), Group II (red), and Group III (blue), representing lower plants, monocots, and dicots, respectively. Each subfamily contains 10, 14, and 58 members, respectively. Intron-exon analysis and protein motif analysis of genes show that members of the same subfamily share similar structures. Positive selection analysis revealed that 85 amino acid sites were strongly positively selected, with 51 sites showing extremely significant effects at p < 0.01 and 12 sites showing significant effects at p < 0.05.

[0157] 2.2 Obtaining mutation sites and screening of combined mutants

[0158] Root mean square deviation (RMSD), temperature factor (B-factor), and position-specific scoring matrix (PSSM) methods were used to screen for stability-improving amino acid sites, resulting in 66, 116, and 85 potential mutation sites, respectively. The intersection of all these methods ultimately identified nine potential mutation sites: A7L, D9Q, R114K, E183S, Q184R, Q184I, E200P, H203L, and N360K. Based on these nine mutations, all possible multi-site combinations were designed and their free energies were calculated. The top eight combinations with the greatest free energy reduction were selected and designated B1, B2, B3, B4, B5, B6, B7, and B8.

[0159] Table 3 Results of 8 combined mutants

[0160]

[0161] 2.3 Molecular dynamics simulation of combined mutants

[0162] Except for B1 and B5, whose RMSD values of the combined mutants are higher than those of WT, the RMSD values of the designed enzymes are lower than those of the original enzymes, that is, the rest of the designed enzymes are theoretically more stable than the original enzymes ( Figure 15 B) Simulation results show that B8 is the most stable mutation among the combined mutations. Molecular dynamics simulations suggest that the overall conformation of these designed enzymes may be more stable than the original enzyme, potentially surpassing its stability.

[0163] 2.4 Thermal stability analysis of enzymes designed by combinatorial mutation

[0164] T of eight combined mutation design enzymes B1-B8 50 The results showed that ( Figure 16 a) After the enzyme was treated under the same conditions, it was compared with the original enzyme T 50Compared with 49.0℃, the temperatures of B5 and B6 were 51.1℃ and 49.2℃, respectively, which were increased by 2.1℃ and 0.1℃.

[0165] The residual enzyme activity of the starting enzyme and the combined mutant designed enzyme treated at 45°C for different time periods was analyzed ( Figure 16 b), the half-life of B4 in the combined mutation was 55.0 min, which was 1.4 min longer than that of the original enzyme.

[0166] 2.5 Analysis of acid and alkali resistance of enzymes designed by combinatorial mutation

[0167] Analysis of the tolerance of the combined mutant designed enzymes B1-B8 in acidic and alkaline environments ( Figure 16c) Both the original enzyme and the designed enzyme retained activity more effectively under alkaline conditions than under acidic conditions. Under acidic conditions, the residual enzyme activities of B1, B5, B6, and B8 were relatively higher than those of the original enzyme. After treatment with a pH 4 buffer, the original enzyme had a residual activity of 11.5%, while B1 had a residual activity of 21.0%, representing a 9.6% increase in activity compared to the original enzyme at pH 4. B5 had a residual activity of 18.7%, a 7.3% increase compared to the original enzyme. B6 had a residual activity of 43.6%, a 32.1% increase compared to the original enzyme. B8 had a residual activity of 28.7%, a 17.2% increase compared to the original enzyme, all of which were superior to the original enzyme, with B6 showing the greatest improvement in activity. Furthermore, at pH 6, the original enzyme had a residual activity of 48.3%, while B7, after the same treatment conditions, had a residual activity of 52.0%, a 3.8% increase compared to the original enzyme, also superior to the original enzyme. Under alkaline conditions, B1, B5, B6, and B8 all showed superior alkaline resistance to the original enzyme. For example, after treatment with a pH 11 buffer, the original enzyme's residual enzyme activity was 11.0%, while B1's residual enzyme activity after treatment under the same conditions was 18.0%, a 7.0% increase compared to the original enzyme. B5's residual enzyme activity after treatment under the same conditions was 20.4%, a 9.3% increase compared to the original enzyme. B6's residual enzyme activity after treatment under the same conditions was 40.2%, a 29.2% increase compared to the original enzyme. B8's residual enzyme activity after treatment under the same conditions was 27.3%, a 16.3% increase compared to the original enzyme. All of these were superior to the original enzyme, with B6 showing the greatest increase in activity. Therefore, B6 exhibits higher enzyme activity than the original enzyme under both acidic and alkaline conditions. In addition, except for the condition of pH = 11, the enzyme activity of B7 is lower than that of the original enzyme. In other alkaline environments, it is higher than that of the original enzyme. For example, at pH = 10, the residual enzyme activity of the original enzyme is 14.6%, while the residual enzyme activity of B7 is 35.6%, which is 21.1% higher than that of the original enzyme.

[0168] 2.6 Analysis of herbicide resistance by combinatorial mutation-designed enzymes

[0169] Chlorsulfuron-resistant group ( Figure 16d) After treatment with the same concentration of chlorsulfuron, the residual enzyme activities of B1, B5, B6, B7, and B8 were all higher than those of the original enzyme. For example, after treatment with chlorsulfuron at a concentration of 0.012 μg / μL, the residual enzyme activity of the original enzyme was 32.2%. Among the five designed enzymes with the best performance in the combined mutations, B1, B5, B6, B7, and B8 had residual enzyme activities of 43.5%, 48.1%, 57.0%, 46.4%, and 44.4%, respectively, after treatment with chlorsulfuron at a concentration of 0.012 μg / μL. These increases were 11.4%, 16.0%, 24.8%, 14.2%, and 12.2%, respectively, compared to the original enzyme. Among these eight inhibitor-resistant mutations, the B6 designed enzyme performed best. After being treated with 0.012 μg / μL chlorsulfuron, its enzyme activity still remained at 57.0%, which was greater than 50%. Its enzyme activity was increased by 24.8% compared with the original enzyme. After being treated with 0.015 μg / μL chlorsulfuron, the original enzyme had 31.1% remaining enzyme activity, while B6 still had 41.6% enzyme activity.

[0170] 2.7 Analysis of enzyme surface electrostatic potential by combined mutation design (only preferred mutant combinations are listed)

[0171] Compared with the original enzyme, B6 has increased positive electrostatic potential near sites 184 and 183, and B7 has increased positive electrostatic potential near sites 183, 184, and 360 to varying degrees ( Figure 17 This change in the designed enzyme may play an important role in maintaining the stability of the ALS enzyme and exerting its catalytic activity.

[0172] Experimental Example 4: Expression and activity determination of ALS mutants in Escherichia coli based on amino acids commonly pointed to by three sequence sources

[0173] 1. Experimental Methods

[0174] The specific experimental method is shown in Experimental Example 1.

[0175] 2. Experimental Results

[0176] 2.1 Screening of combinatorial mutants

[0177] Taking the intersection of the amino acid sites in Examples 1, 2, and 3 above, three potential mutation sites were ultimately obtained: Q184I, L350F, and D9Q, as well as S568A and N360K sites selected based on previous experience. All possible multi-site combination mutation designs were performed and the above-mentioned important parameters were calculated to obtain a mutant combinatorial library. From this library, the following six compound mutants were preferentially obtained through expression experiments. Their specific sequences are shown in (Table 4) and are named C1, C2, C3, C4, C5, and C6.

[0178] Table 4 Results of 6 combined mutants

[0179]

[0180]

[0181] 2.3 Molecular dynamics simulation of combined mutants

[0182] Compared to the starting enzyme, the combined mutants C5 and C6 exhibited smaller average fluctuations, suggesting a significant improvement in the stability of C5 and C6 compared to the starting enzyme. The overall fluctuations of C1, C2, C3, and C4 were smaller than those of the starting enzyme, but they exhibited larger fluctuations compared to C5 and C6, suggesting that the stability of C1, C2, C3, C4, C5, and C6 could be improved to varying degrees.

[0183] 2.4 Analysis of thermal stability experimental results of combined mutation-designed enzymes

[0184] The six combined mutant designed enzymes C1-C6 were expressed and thermodynamically analyzed according to the method of the above embodiment. The corresponding T 50 The results showed that the T of the combined mutation designed enzymes C1, C2, C3, and C5 50 They were 50.81℃, 50.58℃, 50.99℃ and 51.63℃ respectively, which were increased by 1.68℃, 1.85℃, 1.86℃ and 2.50℃ respectively compared with the starting enzyme. Among them, the thermal stability of the combined mutation designed enzyme C5 was significantly improved.

[0185] The residual enzyme activity results of the starting enzyme and the combined mutant designed enzyme were analyzed after treatment at 45°C for different time periods. The half-lives of C2, C3, C5, and C6 were 75.75min, 86.12min, 96.71min, and 98.36min, respectively, which were 45.75min, 56.12min, 66.71min, and 68.36min longer than those of the starting enzyme. Among them, the best performing mutant was the C6 mutant, with a half-life greater than 90min.

[0186] 2.5 Analysis of acid and alkali resistance of the designed enzymes by combined mutation

[0187] Analysis of the tolerance of the combined mutant designed enzymes C1-C6 in acidic and alkaline environments showed that the six mutant enzymes, C1, C2, C3, C4, C5, and C6, showed improved acid and alkaline resistance compared to the starting enzyme. Among them, the combined mutant C5 showed significantly increased stability under both acidic and alkaline conditions, demonstrating improved tolerance to both acidic and alkaline environments.

[0188] 2.6 Analysis of experimental results of herbicide resistance of designed enzymes using combined mutations

[0189] Chlorsulfuron-resistant group, combined mutants C1, C2, C3, C4, C5, C6 I 50 They were 0.523μg / mL, 0.531μg / mL, 0.525μg / mL, 0.529μg / mL, 0.544μg / mL and 0.551μg / mL, respectively, which were 165.5%, 169.5%, 166.5%, 168.5%, 176.1% and 179.7% higher than the starting enzyme.

[0190] The bispyribac-resistant group, combined mutants C1, C2, C3, C4, C5, C6 I 50 They were 0.397μg / mL, 0.411μg / mL, 0.402μg / mL, 0.451μg / mL, 0.513μg / mL and 0.504μg / mL, respectively, which increased by 118.1%, 125.8%, 120.9%, 147.8%, 181.9% and 176.9% respectively compared with the starting enzyme.

[0191] Imidazole-resistant group, combined mutants C1, C2, C3, C4, C5, C6 I 50 They were 95.3μg / mL, 97.2μg / mL, 94.4μg / mL, 95.7μg / mL, 116.1μg / mL and 127.5μg / mL, respectively, which were increased by 96.5%, 100.5%, 94.7%, 97.4%, 139.4% and 162.9% respectively compared with the starting enzyme.

[0192] From the above experimental results, it was found that the comprehensive performance of the composite mutants C5 and C6 was significantly improved.

Claims

1. A method for screening protein mutants with improved stability, characterized in that: include: (1) Obtaining a protein sequence with high heterogeneity, a protein sequence with high homogeneity, or a sequenced plant gene; (2) Various sequences are subjected to positive selection analysis and combined with informatics to obtain potential mutation sites with the lowest free energy; (3) Potential mutation sites of various sequences are subjected to combined mutation to construct a mutant library, and the free energy of each combined mutation is calculated to obtain the combined mutant with the largest free energy reduction compared to the starting enzyme, thereby obtaining enzyme protein mutants with improved stability.

2. The screening method according to claim 1, wherein The protein includes but is not limited to enzyme proteins, hormones, antibodies, transport proteins or contractile proteins; preferably, acetolactate synthase, the amino acid sequence of which is shown in SEQ ID No. 1, and the codon-optimized nucleotide sequence is shown in SEQ ID No.

2.

3. The screening method according to claim 1, wherein The method of obtaining a protein sequence with high heterogeneity is to obtain a sequence with high heterogeneity of acetolactate synthase, wherein 80 acetolactate synthase sequences from 80 species of dicotyledons, monocotyledons, lower plants and microorganisms are obtained through manual selection as sequences with high heterogeneity by using a BLASTP search method; The method of obtaining a protein sequence with high homogeneity is to obtain a sequence with high homogeneity of acetolactate synthase, wherein the public database NCBI is searched using BLASTP to obtain the first 500 sequences as the sequence with high homogeneity; The sequence of the sequenced plant gene for obtaining the protein is preferably the sequence of the sequenced plant gene for obtaining acetolactate synthase, wherein a total of 82 homologous sequences of acetolactate synthase were identified from the plant database Phytozome as the sequence of the sequenced plant gene.

4. The screening method according to claim 1, wherein In step (2), the obtained acetolactate synthase sequences with high heterogeneity were subjected to positive selection analysis and combined with informatics methods to obtain the potential amino acid mutation sites with the lowest free energy, namely Q184I, K339L, L350F, G353S, R356M and Q364K; In step (2), the obtained acetolactate synthase sequences with high homogeneity were subjected to positive selection analysis and combined with informatics methods to obtain the potential amino acid mutation sites with the lowest free energy, namely D9Q, E144H, H278Y, T321Q, L350F, S568A and N573D; In step (2), the sequence of the sequenced plant gene of acetolactate synthase obtained is subjected to positive selection analysis and combined with informatics to obtain the various potential amino acid mutation sites with the lowest free energy, namely A7L, D9Q, R114K, E183S, Q184R, Q184I, E200P, H203L and N360K.

5. The screening method according to claim 1, wherein Step (2) also includes comparing the potential mutation sites of various sequences to find overlapping mutation sites, combining these overlapping mutation sites with the important potential mutation sites obtained by screening to construct a mutant library, calculating the free energy of each combined mutation, obtaining the combined mutant with the largest free energy reduction compared to the starting enzyme, and obtaining an enzyme protein mutant with improved stability; preferably, when the protein is acetolactate synthase, the potential mutation sites of various sequences of acetolactate synthase are compared to find that the overlapping mutation sites are Q184I, L350F and D9Q, and the important potential mutation sites are S568A and N360K.

6. An acetolactate synthase mutant with improved stability obtained by screening according to any one of the screening methods of claims 1 to 5, characterized in that: The amino acid sequence of the acetolactate synthase mutant is selected from any one of the following (1)-(4): (1) A multi-site mutant obtained by subjecting the acetolactate synthase having the amino acid sequence shown in SEQ ID NO. 1 to any one of the amino acid multi-site mutations K339L-G353S-R356M, Q184I-R356M-Q364K, G353S-R356M-Q364K, Q184I-K339L-R356M-Q364K, Q184I-G353S-R356M-Q364K, or Q184I-K339L-G353S-R356M-Q364K; (2) a multi-site mutant obtained by subjecting the acetolactate synthase having the amino acid sequence shown in SEQ ID NO. 1 to any one of the amino acid multi-site mutations E144H-H278Y-T321Q, E144H-H278Y-T321Q-N573D, D9Q-E144H-H278Y-T321Q, E144H-H278Y-T321Q-N573D, E144H-H287Y-L350F-S568A, E144H-H278Y-L350F-N573D, or D9Q-E144H-H278Y-T321Q-N573D; (3) The acetolactate synthase having the amino acid sequence shown in SEQ ID NO. 1 was subjected to D9Q-R114K-E183S-Q184I-E200P-H203L-N360K, A7L-R114K-E183S-Q184R-E200P-H203L-N360K, A7L-R114K-E183S-Q184I-E200P-H203L-N360K, A7L-R114K-E183S-Q184I-E200P-H203L-N360K, Multiple multi-site mutants obtained by multi-site mutation of any one of L-D9Q-E183S-E200P-H203L-N360K, D9Q-R114K-E183S-Q184I-E200P-N360K, A7L-D9Q-R114K-E183S-Q184R-H203L-N360K, or A7L-D9Q-R114K-Q184R-H203L-N360K; (4) A multi-site mutant obtained by subjecting the acetolactate synthase having the amino acid sequence shown in SEQ ID NO.1 to any one of the amino acid multi-site mutations Q184I-L350F-D9Q, Q184I-L350F-N360K, D9Q-S568A-N360K, S568A-Q184I-L350F, L350F-S568A-N360K or D9Q-N360K-L350F.

7. A gene encoding the acetolactate synthase mutant according to claim 6.

8. A chimeric gene, expression cassette or recombinant expression vector comprising the coding gene of claim 7.

9. A recombinant host cell containing the chimeric gene, expression cassette or recombinant expression vector according to claim 8.

10. A method for preparing the acetolactate synthase mutant according to claim 6, characterized in that: include: (1) constructing a prokaryotic expression vector of the coding gene of the acetolactate synthase mutant; (2) transforming the constructed prokaryotic expression vector into corresponding host cells, expressing the fusion protein in the host cells, and purifying the fusion protein.

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