A method of co-engineering a multimeric fluorinase

Abstract

The application relates to the technical field of bioengineering, and particularly relates to a synergistic engineering modification method of a multimeric fluorinase, which comprises step-by-step synergistic modification of an N-terminal sequence, a distal region and a core region of a target enzyme. The N-terminal sequence is optimized through ancestral sequence reconstruction or hydrophobicity analysis, the distal mutation site is screened by using multiple sequence alignment and free energy calculation, and the catalytic activity of the core region is improved by combining conservativeness analysis and saturation mutation, so as to finally construct a multi-site combined mutant. The application can significantly improve the overall structural stability and local catalytic efficiency of the multimeric fluorinase, and meanwhile, the thermal stability is enhanced, a universal methodology is provided for modification of other highly conservative multimeric enzymes, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and specifically relates to a synergistic engineering modification method of a multimeric fluorinase. BACKGROUND

[0002] Multimeric enzymes play an important role in biological catalysis, and their functions depend on the precise assembly and interaction between subunits. For highly conserved multimeric enzymes (such as fluorinase), traditional enzyme engineering methods usually focus on single-point mutation or local modification of the active site, but this approach can easily disrupt the balance of the multimeric structure, leading to reduced enzyme activity or stability, and it is difficult to significantly improve catalytic efficiency. Therefore, developing an engineering strategy that can coordinate the overall structural stability and local catalytic activity of multimeric enzymes has become a technical problem to be solved.

[0003] Patent CN114502715A discloses a method and device for optimizing biotechnological production, which optimizes the composition of the culture medium and the feeding strategy through digital twin technology to improve product concentration, productivity, biomass concentration and product quality. This method uses model prediction and online optimization to achieve process improvement, but it mainly focuses on the optimization of macro production process and fails to deeply involve the molecular level modification of multimeric enzymes, especially the lack of synergistic regulation ability for the interaction between multimeric enzyme subunits.

[0004] Patent CN108830046A discloses a method for estimating the DNA content of biotechnology products, which detects the DNA copy number of exogenous inserted genes and endogenous genes through two independent tests, and then estimates the relative percentage of DNA. This method provides an accurate mathematical model for quantitative analysis of the gene content of biotechnology products, but its application scenario is limited to the estimation of DNA content, and it does not involve the optimization of structure and function of multimeric enzymes, and lacks systematic research on the dynamic interaction between multimeric enzyme subunits.

[0005] In summary, the existing technology has obvious deficiencies in the engineering modification of multimeric enzymes, especially in the regulation of synergistic interaction between subunits and the maintenance of overall structural stability. SUMMARY

[0006] The present application provides a synergistic engineering modification method of a multimeric fluorinase, which aims to sequentially modify the N-terminal sequence, distal region and core region of the target enzyme through a "from outside to inside" step-by-step strategy, thereby improving the overall structural stability and local catalytic activity of the multimeric enzyme.

[0007] In a first aspect, the present application provides a method for synergistic engineering modification of a multimeric fluorinase, comprising the following steps:

[0008] S10: Obtain an optimized N-terminal short peptide sequence for the N-terminal sequence of the target enzyme using ancestral sequence reconstruction (ASR) technology or rational design based on hydrophobicity analysis, and construct a short peptide library at the N-terminal to replace the wild-type N-terminal;

[0009] S20: For the distal region away from the active center, analyze sequence diversity by multiple sequence alignment (MSA), and select sites with sequence diversity of 50% to 70% as candidate distal sites; predict the effect of mutations on protein stability (ΔΔG) by combining molecular dynamics simulation and free energy calculation, and select mutations that can reduce free energy for experimental verification;

[0010] S30: For the conserved core region around the active center, select all residues within 5Å of the active center substrate molecule as the origin for amino acid conservation analysis; perform saturation mutation screening on residues with lower conservation, and predict the free energy change (ΔΔG) of the mutant by combining calculation tools, and select mutations that significantly reduce free energy and improve activity;

[0011] S40: Combine the N-terminal modification, beneficial mutations in the distal region, and beneficial mutations in the core region to construct the final multi-site combined mutant.

[0012] According to the present application, the optimized N-terminal short peptide sequence is obtained by ancestral sequence reconstruction (ASR) technology or rational design based on hydrophobicity analysis, and a short peptide library is constructed for replacing the wild-type N-terminal, which enhances the efficiency of multimer assembly and the overall structural stability, and lays a foundation for subsequent modification.

[0013] Preferably, in step S10, at least 20 homologous fluorination enzyme sequences are collected for multiple sequence alignment; the ancestral N-terminal sequence is inferred using ancestral sequence reconstruction (ASR) software (such as GRASP); the hydrophobicity of the candidate N-terminal sequence is analyzed, and the sequence with significantly higher hydrophobicity than the wild-type MA37 N-terminal is selected; the selected optimized N-terminal sequence is replaced with the original N-terminal of MA37 by molecular cloning technology, an N-terminal modification library is constructed, and the optimal N-terminal variant is screened by activity determination.

[0014] Preferably, in step S20, global multiple sequence alignment (MSA) is performed on homologous fluorination enzyme sequences; the active center residues directly involved in substrate binding or catalysis and the key structural residues with sequence conservation higher than 75% are excluded, and sites with sequence diversity of 50% to 70% are selected as candidate distal sites; virtual saturation mutation is performed on the candidate sites using molecular modeling and molecular dynamics simulation, and the free energy change (ΔΔG) of each mutant is calculated using FoldX software; mutations with significant negative ΔΔG (such as R92G, ΔΔG = -1.13 kcal / mol) are selected for experimental verification to confirm their effectiveness.

[0015] Preferably, in the step S30, the C5 atom of the substrate SAM is taken as the center of the sphere, and 5Å is taken as the radius to define the core region range; all residues in the range are subjected to amino acid conservation analysis; the residues with lower conservation (such as A264) are subjected to saturation mutation to construct a mutation library; combined with crude enzyme activity screening and pure enzyme accurate activity determination, and assisted by FoldX and other tools to calculate ΔΔG, the mutations (such as A264V) that significantly improve the activity and are stable are identified.

[0016] Preferably, in the step S40, the optimal N-terminal sequence screened in the step S10, the distal mutation (such as R92G) verified in the step S20, and the core region mutation (such as A264V) identified in the step S30 are combined, and a combined mutant plasmid is constructed by molecular biology methods; the plasmid is transformed into an expression host (such as Escherichia coli BL21 (DE3)), induced to express, and purified to obtain a combined mutant protein (named V_G_N_Hbhob).

[0017] Preferably, in the step S10, the software used by the ancestral sequence reconstruction (ASR) technology includes but is not limited to GRASP, PAML or MEGA; the rational design is based on hydrophobicity analysis, and an N-terminal short peptide with significantly higher hydrophobicity than the wild type sequence is selected.

[0018] Preferably, in the step S20, the tool used by the multiple sequence alignment (MSA) includes but is not limited to ClustalOmega, MAFFT or T-Coffee; the software used by the molecular dynamics simulation includes but is not limited to GROMACS, AMBER or CHARMM; and the tool used by the free energy calculation includes but is not limited to FoldX, Rosetta or Discovery Studio.

[0019] Preferably, in the step S30, the tool used by the amino acid conservation analysis includes but is not limited to ConSurf, Rate4Site or Jalview; the mutation library constructed by the saturation mutation screening is realized by a site-directed mutagenesis technology, and the tool used includes but is not limited to QuikChange Lightning Site-Directed Mutagenesis Kit.

[0020] Preferably, in the step S40, the expression host is Escherichia coli BL21 (DE3), and the induction expression condition is that after culturing at 37 degrees Celsius to OD600 reaches 0.6 to 0.8, IPTG with a final concentration of 0.1 millimole per liter to 1 millimole per liter is added to induce expression.

[0021] In a second aspect, the application provides a high-performance multimeric fluorinase mutant, which is obtained by the method according to any one of the first aspect.

[0022] According to the application, the high-performance multimeric fluorinase mutant is obtained by the method, and the catalytic efficiency is improved by 387% compared with the wild type MA37, and the thermal stability is significantly enhanced.

[0023] Compared with the prior art, the application has the following technical effects:

[0024] Through the "from outside to inside" partitioning and step-by-step modification, the synergistic optimization of the overall structural stability and local catalytic activity of the multimeric enzyme is realized, and the limitation problem of the traditional single-point or local mutation strategy for the modification of the multimeric enzyme is solved.

[0025] The N-terminal sequence is optimized by the ancestral sequence reconstruction (ASR) technology or rational design, the multimer assembly efficiency and the overall structural stability are enhanced, the mutation sites in the distal region are screened by multiple sequence alignment (MSA), molecular dynamics simulation and free energy calculation, the enzyme molecular conformation dynamics is optimized, the mutation sites in the core region are screened by amino acid conservation analysis and saturation mutation, and the substrate binding and catalytic efficiency are improved. The above-mentioned method has the characteristics of synergistic effect, and can be widely applied to other highly conserved and difficult to engineer multimeric enzymes. BRIEF DESCRIPTION OF DRAWINGS

[0026] ATTACHMENT Figure 1 A flowchart of the synergistic engineering modification method is shown, which sequentially includes four key steps of N-terminal modification, distal region modification, core region modification and combination optimization, and clearly presents the step-by-step strategy from "outside to inside". DETAILED DESCRIPTION

[0027] In the description of this specification, the description of the terms "one embodiment", "some embodiments", "illustrative embodiment", "example", "specific example" or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the application. In the description of this specification, the illustrative description of the above terms does not necessarily mean the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples with appropriate means.

[0028] In addition, the terms "first", "second", etc. are used only for descriptive purposes and are not to be construed as indicating or implying relative importance or an ordered ranking of the indicated technical features. Thus, features defined with "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality" is at least two, for example, two, three, etc., unless otherwise explicitly and specifically limited.

[0029] As described in the background section above, the current modification of multimeric fluorinase mainly relies on single region mutation strategy, but this strategy often has difficulty in achieving the synergistic optimization of overall structural stability and local catalytic activity. Based on this, the present application provides a synergistic engineering modification method of multimeric fluorinase, which aims to sequentially modify the N-terminal sequence, the distal region and the core region of the target enzyme by a "from outside to inside" step-by-step strategy, so as to improve the overall structural stability and local catalytic activity of the multimeric enzyme.

[0030] In a first aspect, the present application provides a method for synergistic engineering modification of multimeric fluorinase, comprising the following steps: S10, for the N-terminal sequence of the target enzyme, using ancestral sequence reconstruction (ASR) technology or rational design based on hydrophobicity analysis, obtaining an optimized N-terminal short peptide sequence, and constructing a short peptide library at the N-terminal to replace the wild-type N-terminal; S20, for the distal region away from the active center, analyzing sequence diversity by multiple sequence alignment (MSA), and selecting sites with sequence diversity of 50% to 70% as candidate distal sites; combining molecular dynamics simulation and free energy calculation to predict the influence (ΔΔG) of mutation on protein stability, and selecting mutations that can reduce free energy for experimental verification; S30, for the conserved core region around the active center, selecting all residues within 5Å range of the active center substrate molecule for amino acid conservation analysis; performing saturation mutation screening on residues with lower conservation, and combining calculation tools to predict the free energy change (ΔΔG) of the mutant, and selecting mutations that significantly reduce free energy and improve activity; S40, combining the N-terminal modification, distal region beneficial mutation and core region beneficial mutation to construct the final multi-site combination mutant.

[0031] According to the present application, in step S10, the software used by the ancestral sequence reconstruction (ASR) technology includes but is not limited to GRASP, PAML or MEGA; the rational design based on hydrophobicity analysis selects N-terminal short peptides with significantly higher hydrophobicity than the wild-type sequence.

[0032] In step S10, at least 20 homologous fluorinase sequences are collected for multiple sequence alignment; the ancestral sequence reconstruction (ASR) software is used to infer the possible ancestral N-terminal sequence; the hydrophobicity of the candidate N-terminal sequence is analyzed, and the sequence with significantly higher hydrophobicity than the wild-type MA37 N-terminal is selected; the selected optimized N-terminal sequence is replaced with the original N-terminal of MA37 through molecular cloning technology to construct an N-terminal modification library, and the optimal N-terminal variant is screened through activity determination.

[0033] In step S20, the tools used for multiple sequence alignment (MSA) include but are not limited to Clustal Omega, MAFFT or T-Coffee; the software used for molecular dynamics simulation includes but is not limited to GROMACS, AMBER or CHARMM; the tools used for free energy calculation include but are not limited to FoldX, Rosetta or Discovery Studio.

[0034] In step S20, global multiple sequence alignment (MSA) is performed on homologous fluorinase sequences; the active center residues directly involved in substrate binding or catalysis and the key structural residues with sequence conservation higher than 75% are excluded, and the sites with sequence diversity of 50% to 70% are selected as candidate distal sites; virtual saturation mutation is performed on the candidate sites by molecular modeling and molecular dynamics simulation, and the free energy change (ΔΔG) of each mutant is calculated by FoldX software; the mutations with significant negative ΔΔG are selected for experimental verification to confirm their effectiveness.

[0035] In step S30, the tools used for amino acid conservation analysis include but are not limited to ConSurf, Rate4Site or Jalview; the mutant library constructed by saturation mutation screening is realized by site-directed mutagenesis technology, and the tools used include but are not limited to QuikChange Lightning Site-Directed Mutagenesis Kit.

[0036] In step S30, the C5 atom of substrate SAM is taken as the center of the sphere, and 5Å is taken as the radius to define the core region range; amino acid conservation analysis is performed on all residues within this range; saturation mutation is performed on residues with lower conservation to construct a mutant library; combined with crude enzyme activity screening and pure enzyme accurate activity determination, and assisted by FoldX and other tools to calculate ΔΔG, mutations that significantly improve activity and stability are identified.

[0037] In step S40, the optimal N-terminal sequence screened in step S10, the distal mutation verified in step S20 and the core region mutation identified in step S30 are combined, and a plasmid of the combined mutant is constructed by molecular biology methods; the plasmid is transformed into an expression host, induced to express and purified to obtain the combined mutant protein.

[0038] Wherein, the expression host is Escherichia coli BL21 (DE3), and the inducing expression condition is that after culturing at 37 degrees Celsius until OD600 reaches 0.6 to 0.8, IPTG is added to induce expression at a final concentration of 0.1 millimole per liter to 1 millimole per liter.

[0039] In a second aspect, the application provides a high-performance multimeric fluorinase mutant, which is obtained by the method according to any one of the embodiments of the first aspect. The high-performance multimeric fluorinase mutant is obtained by the method described above, and the catalytic efficiency is improved by 387% compared with the wild type MA37, and the thermal stability is significantly enhanced.

[0040] The examples described below are exemplary and are intended to explain the present application, but cannot be understood as a limitation of the present application. In the examples, the specific techniques or conditions not mentioned are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be obtained by purchase. Examples

[0041] The present embodiment provides a method for the cooperative engineering of multimeric fluorinase, comprising the following steps:

[0042] Step S10: Optimization of N-terminal sequence

[0043] Collect 25 homologous fluorinase sequences, and perform multiple sequence alignment using Clustal Omega;

[0044] Infer the possible ancestral N-terminal sequence using GRASP software;

[0045] Analyze the hydrophobicity of the candidate N-terminal sequence, and select the sequence with significantly higher hydrophobicity than the wild type MA37 N-terminal sequence;

[0046] Replace the original N-terminal sequence of MA37 with the selected optimized N-terminal sequence through molecular cloning technology, construct an N-terminal modification library, and screen the optimal N-terminal variant through activity determination.

[0047] Step S20: Screening of distal region mutation sites

[0048] Perform global multiple sequence alignment (MSA) on 25 homologous fluorinase sequences;

[0049] Exclude active center residues and structural core residues with a conservation higher than 75%, and screen sites with sequence diversity of 50% to 70% as candidate distal sites;

[0050] Molecular modeling and molecular dynamics simulation were performed by GROMACS, virtual saturation mutation was performed on candidate sites, and the free energy change (ΔΔG) of each mutant was calculated by FoldX software;

[0051] Mutations with significantly negative ΔΔG were selected for experimental verification to confirm their effectiveness.

[0052] Step S30: Screening of core region mutation sites

[0053] The C5 atom of the substrate SAM was taken as the center of the sphere, and a radius of 5 Å was defined as the range of the core region;

[0054] Amino acid conservation analysis was performed on all residues in this range;

[0055] Low-conservation residues were subjected to saturation mutation to construct a mutation library;

[0056] Combining crude enzyme activity screening and pure enzyme accurate activity determination, and assisted by FoldX and other tools to calculate ΔΔG, mutations that significantly improve activity and stability were identified.

[0057] Step S40: Construction and characterization of combined mutants

[0058] The optimal N-terminal sequence screened in step S10, the effective distal mutation verified in step S20, and the core region mutation identified in step S30 were combined, and a combined mutant plasmid was constructed by molecular biology methods;

[0059] The plasmid was transformed into E. coli BL21 (DE3), induced for expression, and the combined mutant protein was obtained by purification.

[0060] Comparative Example 1:

[0061] This comparative example only optimizes the N-terminal sequence without modifying the distal region and the core region. The optimization process is shown in step S10.

[0062] Comparative Example 2:

[0063] This comparative example only modifies the distal region without optimizing the N-terminal sequence and modifying the core region. The optimization process is shown in step S20.

[0064] Comparative Example 3:

[0065] This comparative example only modifies the core region without optimizing the N-terminal sequence and modifying the distal region. The optimization process is shown in step S30.

[0066] The performance of the multimeric fluorinase mutants obtained in the above examples and comparative examples was tested, and the results are shown in the following table:

[0067]

[0068] According to the results of the above table, the multimeric fluorinase mutant obtained in the present application has higher catalytic efficiency and thermal stability than the comparative examples. In comparative example 1, only the N-terminal sequence is optimized, which improves some performance, but the overall effect is limited; in comparative example 2, only the distal region is modified, which fails to significantly improve the catalytic efficiency; in comparative example 3, only the core region is modified, which improves the catalytic efficiency, but the thermal stability is still insufficient. In the present example, the overall performance is maximized by combined optimization.

Claims

1. A method for the co-engineering of multimeric fluorinated enzymes, characterized in that, The polymeric fluorinase is wild-type MA37 fluorinase; the method includes the following steps: S10: For the N-terminal sequence of MA37, at least 25 homologous fluoridease sequences were collected for multiple sequence alignment. After inferring the possible ancestral N-terminal sequence using ancestral sequence reconstruction technology, the hydrophobicity of the candidate N-terminal sequences was analyzed, and the sequence with significantly higher hydrophobicity than that of the wild-type MA37 N-terminus was selected. The original N-terminus of MA37 was replaced by the selected optimized N-terminal sequence using molecular cloning technology to construct an N-terminal modification library, and the optimal N-terminal variant was screened by activity assay. S20: For the distal region far from the MA37 active site, global multiple sequence alignment was performed on 25 homologous fluoridease sequences to exclude active site residues and structural core residues with conservation higher than 75%, and sites with sequence diversity between 50% and 70% were screened as candidate distal sites; molecular modeling and molecular dynamics simulation were performed using GROMACS software, virtual saturation mutations were performed on the candidate sites, and the free energy change of each mutant was calculated using FoldX software. Mutations with significantly negative free energy changes were selected for experimental verification. S30: For the conserved core region surrounding the active site of MA37, the core region is defined with the C5 atom of substrate SAM as the center and a radius of 5 Å. The amino acid conservation of all residues within this region is analyzed. Saturation mutations are performed on residues with low conservation to construct a mutant library. Combining crude enzyme activity screening and precise pure enzyme activity determination, and using FoldX tool to calculate the free energy change, mutations that significantly reduce free energy and increase activity are selected. S40: Combine the optimal N-terminal sequence selected in step S10, the effective distal mutation verified in step S20, and the core region mutation identified in step S30 to construct a combined mutant plasmid using molecular biology methods; transform the plasmid into an expression host, induce expression, and purify to obtain the combined mutant protein.

2. The method of claim 1, wherein, In step S10, the software used for ancestral sequence reconstruction technology includes GRASP, PAML, or MEGA; after constructing an N-terminal modified library using molecular cloning technology, the optimal N-terminal variant is screened using an activity assay, and the hydrophobicity of the optimal N-terminal variant is significantly higher than that of the wild-type MA37 N-terminus.

3. The method of claim 1, wherein, In step S20, the tools used for multiple sequence alignment include Clustal Omega, MAFFT, or T-Coffee; the tools used for free energy calculation include Rosetta or Discovery Studio; and the experimentally verified effective distant mutations include R92G, where ΔΔG = -1.13 kcal / mol.

4. The method of claim 1, wherein, In step S30, the tools used for amino acid conservation analysis include ConSurf, Rate4Site, or Jalview; the mutant library constructed by saturation mutation screening is achieved through site-directed mutagenesis, using the QuikChange Lightning Site-Directed Mutagenesis Kit; residues with low conservation include A264, and the corresponding activity-enhancing mutation includes A264V.

5. The method of claim 1, wherein, In step S40, the expression host is Escherichia coli BL21 (DE3), and the induction expression conditions are: culture at 37 degrees Celsius until the OD600 reaches 0.6 to 0.8, and then add IPTG at a final concentration of 0.1 mmol / L to 1 mmol / L for induction expression; the combined mutant protein is named V_G_N_Hbhob.

6. The method of claim 2, wherein, In step S10, the number of homologous fluoridease sequences collected shall not be less than 25, and the results of multiple sequence alignment shall be used to assist in the inference of the ancestral N-terminal sequence and hydrophobicity analysis.

7. The method of claim 3, wherein, In step S20, the software used for molecular dynamics simulation also includes AMBER or CHARMM; the scope of virtual saturation mutations is limited to the screened candidate distal sites and does not involve active site residues or highly conserved structural residues.

8. The method of claim 4, wherein, In step S30, residues with low conservation refer to residues with a conservation rate of less than 30%; crude enzyme activity screening is used to preliminarily screen for positive mutants, and pure enzyme precise activity determination is used to verify the catalytic activity enhancement effect of the mutants.

9. The method of claim 5, wherein, In step S40, the molecular biology methods include conventional techniques for plasmid construction, transformation, induced expression, and protein purification; the combinatorial mutant plasmid contains coding sequences for the optimal N-terminal sequence, effective distal mutation, and active mutation in the core region.

10. A high-performance multimeric fluoridease mutant, characterized in that, The mutant obtained by the method according to any one of claims 1 to 9 comprises an optimized MA37 N-terminal sequence, a beneficial mutation R92G in the distal region, and a beneficial mutation A264V in the core region, with a catalytic efficiency that is more than 300% higher than that of wild-type MA37 and significantly enhanced thermal stability.

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