Method for improving expression efficiency and catalytic efficiency of FAP protein and FAP mutant

Abstract

The invention discloses a method for improving FAP protein expression efficiency and catalytic efficiency and an FAP mutant, and belongs to the technical field of molecular biology. The method specifically comprises the following steps: deleting a plurality of non-key redundant peptide fragments which do not influence the activity of a protein core in wild-type FAP protein, and then mutating the C terminal of the wild-type FAP protein to enhance the hydrophobic property of the wild-type FAP protein. The invention develops a brand new FAP protein structure optimization method, innovatively starts from the non-key peptide fragment of the protein, and explores a possible path for improving the industrial adaptability of the protein through accurate structure deletion and recombination design. Meanwhile, the C-terminal region of the FAP protein is modified, the hydrophobicity of the FAP protein is enhanced, the lipophilic property of the FAP protein is improved, and therefore the binding capacity and stability of enzyme and a substrate are improved, and an innovative solution is provided for industrial application of the FAP.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biotechnology, and particularly relates to a method for improving the expression efficiency and catalytic efficiency of FAP protein and a FAP mutant. Background Art

[0002] Fatty acid photodecarboxylase (FAP) is a light-driven biocatalyst that efficiently decarboxylates fatty acids to alkanes or alkenes under illumination (particularly blue light), promising broad applications. Due to its environmentally friendly and efficient properties, FAP exhibits significant potential in fields such as bioenergy, biomaterials, and environmental protection. However, the practical application of FAP still faces several technical bottlenecks, particularly in terms of protein expression, stability, and catalytic efficiency.

[0003] First, the low expression efficiency of FAP is one of the main bottlenecks in current research. Although scientists have tried to improve its expression level through strategies such as optimizing expression vectors and using efficient expression hosts, due to the complex molecular structure of FAP itself, the increase in its expression level is often accompanied by problems of protein aggregation or misfolding, which makes it difficult to significantly increase the yield of active enzyme. Low expression not only increases production costs, but also makes subsequent purification and application more complicated. For example, in the Escherichia coli expression system, the expression of many exogenous enzymes is limited by solubility, resulting in most proteins appearing in the form of inclusion bodies, further increasing the difficulty of extraction and purification. In order to increase the expression level of the protein, it is usually necessary to perform gene optimization, adjust the expression conditions, and even improve the solubility and expression level of the protein by engineering the expression host. However, these methods have not fundamentally solved the problem of low efficiency of fatty acid photodecarboxylase in industrial production.

[0004] Secondly, the stability problem of FAP also limits the scope of its industrial application. Under light conditions, FAP is easily affected by the external environment, including temperature fluctuations, changes in solution pH, and photooxidation. In most common expression systems, the stability of the protein shows low tolerance, especially under harsh environmental conditions, and its catalytic activity often decreases significantly. Although some studies have improved by adding stabilizers and optimizing buffer formulations, due to the characteristics of the protein itself and the inherent problems of the dissolution system, it is still unable to meet the operational requirements of industrial high-efficiency and continuous catalysis in practical applications.

[0005] In addition, research on optimizing the catalytic activity of FAP is still in its early stages. Existing structural modification strategies, such as directed evolution and point mutation methods, although they have demonstrated local improvements under laboratory conditions, often lack systematicity and have difficulty balancing the expression level, stability, and catalytic efficiency of the enzyme. In some experiments, the catalytic efficiency can be slightly improved by optimizing the catalytic conditions or changing the concentration of the substrate, but the catalytic activity is still difficult to reach the ideal level. These shortcomings have led to a large technical gap in the industrial application of fatty acid photodecarboxylase.

[0006] Overall, despite recent progress in FAP research, challenges such as low expression, poor stability, and low catalytic efficiency remain pressing scientific challenges. Further exploring and optimizing the structure-function relationship of FAP and developing novel enzyme engineering methods with enhanced performance are crucial for advancing this field. Summary of the Invention

[0007] To address the aforementioned deficiencies in the prior art, the present invention provides a method for improving the expression and catalytic efficiency of FAP protein and a FAP mutant. This invention aims to develop a novel FAP protein structure optimization method. This innovative approach, starting from non-critical peptide segments of the protein, explores potential pathways for enhancing the protein's industrial adaptability through precise structural deletion and recombinant design. Simultaneously, the C-terminal region of the FAP protein is modified to enhance its hydrophobicity and improve its lipophilicity, thereby improving the enzyme's binding ability and stability to the substrate, providing an innovative solution for the industrial application of FAP.

[0008] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0009] The purpose of the present invention is to provide a method for improving the expression efficiency of FAP protein, specifically by deleting several non-critical redundant peptide segments that do not affect the core activity of the protein.

[0010] Furthermore, redundant peptides include the following peptides:

[0011] Amino acids 1 to 82, amino acids 96 to 104, amino acids 145 to 154, amino acids 176 to 223, amino acids 277 to 285, amino acids 355 to 373, amino acids 412 to 423, amino acids 493 to 517, and amino acids 580 to 600.

[0012] Based on the method constructed by the present invention, the above-mentioned fragments are deleted at the same time, which minimizes the non-critical domains in the FAP protein structure that have little effect on the catalytic function and reduces structural redundancy; improves the folding efficiency of the FAP protein, reduces the risk of misfolding during expression, and avoids possible interference with protein folding and stability; helps to improve the expression efficiency and overall stability of FAP, and provides a more stable enzyme source for subsequent catalytic reactions. Through the above-mentioned transformation, the expression efficiency of the modified FAP protein is significantly improved. Although the catalytic stability and catalytic activity are not significantly improved, the photodecarboxylation activity of long-chain fatty acids is still retained, ensuring functional integrity. The simplification of protein structure and the improvement of expression efficiency provide a good foundation for the next step of research.

[0013] Furthermore, the amino acid sequence of the modified FAP protein is shown in SEQ ID NO.1.

[0014] Another object of the present invention is to provide a FAP protein with high expression efficiency, the amino acid sequence of which is shown in SEQ ID NO.1.

[0015] KYDYILVGGGTAAADGSKRVLVLEAGPDNTSRDVKIPAAITRLFRSPLDWNLFSQIYMARGRLLGGSSATLYHNPRYTNKQLHTAFFKAAEEVGLTPNSDFND WSHDHAGYGTFQVMQDKGTRADMGRRNLQVLTGAAVTKVNIDQAAGKAQALGVEFSTDGPTGERLSAELAPGGEVIMCAGAVHTPFLLKHSGGVGQNLQDQPACLT AAPVKEKYDGIAISDHIYNEKGQIRGGLTSTGCDRGAFVRTAGQALPDLQVRFVPGMALDPDGVSTYVRFAKFQSQGLKWPSGITMQLIACRPDKDGADLATLRKG IHWARDVARSSALSEYLDGELFPGSGVVSDDQIDEYIRRSIHSSNAITGGVEGLRVVDASVVPKIPGGQTGAPVVMIAERAAALLTGKATIGASAAAPATVAA(SEQ ID NO.1)

[0016] The amino acid sequence of the wild-type FAP protein is as follows:

[0017] MASITSRASARASCSQANTRAGRVALSGGALLRPARPARSFVPARKQQQ

[0018] GAVRRGGALSARASAVEDIRKVLSDSSSPVAGQKYDYILVGGGTAACVLANR

[0019] LSADGSKRVLVLEAGPDNTSRDVKIPAAITRLFRSPLDWNLFSELQEQLAERQ

[0020] IYMARGRLLGGSSATNATLYHRGAAGDYDAWGVEGWSSEDVLSWFVQAET

[0021] NADFGPGAYHGSGGPMRVENPRYTNKQLHTAFFKAAEEVGLTPNSDFNDWS

[0022] HDHAGYGTFQVMQDKGTRADMYRQYLKPVLGRRNLQVLTGAAVTKVNID

[0023] QAAGKAQALGVEFSTDGPTGERLSAELAPGGEVIMCAGAVHTPFLLKHSGV

[0024] GPSAELKEFGIPVVSNLAGVGQNLQDQPACLTAAPVKEKYDGIAISDHIYNEK

[0025] GQIRKRAIASYLLGGRGGLTSTGCDRGAFVRTAGQALPDLQVRFVPGMALD

[0026] PDGVSTYVRFAKFQSQGLKWPSGITMQLIACRPQSTGSVGLKSADPFAPPKL

[0027] SPGYLTDKDGADLATLRKGIHWARDVARSSALSEYLDGELFPGSGVVSDDQI

[0028] DEYIRRSIHSSNAITGTCKMGNAGDSSSVVDNQLRVHGVEGLRVVDASVVP

[0029] KIPGGQTGAPVVMIAERAAALLTGKATIGASAAAPATVAA. (SEQ ID NO.5)

[0030] Another object of the present invention is to provide a nucleic acid molecule encoding the above-mentioned FAP protein, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0031] Another object of the present invention is to provide a method for improving the catalytic efficiency of FAP protein, specifically: performing site-directed mutagenesis on the C-terminus of the above-mentioned FAP protein to improve its hydrophobicity.

[0032] Furthermore, the amino acids M, E, R, K in the peptide segment MIAERAAALLTGKATIGASAAAPATVAA consisting of 28 amino acids at the C-terminus of the above-mentioned FAP protein, and the amino acids T, S, and T after K were mutated to V, V, V, L, V, A, and V, respectively, to obtain the mutated fragment VIAVVAAALLTGLAVIGAAAAAPAVVAA. The amino acid sequence of the mutated FAP protein is shown in SEQ ID NO.3.

[0033] KYDYILVGGGTAAADGSKRVLVLEAGPDNTSRDVKIPAAITRLFRSPLDWNLFSQIYMARGRLLGGSSATLYHNPRYTNKQLHTAFFKAAEEVGLTPNSDFND WSHDHAGYGTFQVMQDKGTRADMGRRNLQVLTGAAVTKVNIDQAAGKAQALGVEFSTDGPTGERLSAELAPGGEVIMCAGAVHTPFLLKHSGGVGQNLQDQPACLT AAPVKEKYDGIAISDHIYNEKGQIRGGLTSTGCDRGAFVRTAGQALPDLQVRFVPGMALDPDGVSTYVRFAKFQSQGLKWPSGITMQLIACRPDKDGADLATLRKG IHWARDVARSSALSEYLDGELFPGSGVVSDDQIDEYIRRSIHSSNAITGGVEGLRVVDASVVPKIPGGQTGAPVVVIAVVAAALLTGLAVIGAAAAAPAVVAA(SEQ ID NO.3).

[0034] Another object of the present invention is to provide a FAP mutant with high expression efficiency and high catalytic efficiency, the amino acid sequence of which is shown in SEQ ID NO.3.

[0035] These mutations significantly increased the hydrophobicity of the mutant's C-terminus, which helps enhance the affinity of the FAP mutant for its substrate (palmitic acid), thereby improving its catalytic efficiency. Although catalytic stability was not significantly improved, the enhanced hydrophobicity provided a stronger driving force for FAP's binding to the substrate, optimizing the initial stage of the enzymatic reaction.

[0036] Another object of the present invention is to provide a nucleic acid molecule encoding the above mutant, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0037] Beneficial effects of the present invention:

[0038] 1. Optimize the substrate binding ability of the enzyme

[0039] The present invention enhances the hydrophobicity of the C-terminal region of FAP by mutation, thereby improving its ability to bind to substrates. Although the improvement in catalytic stability and efficiency is not significant, the present invention effectively improves the lipophilic properties of the enzyme, making it more efficient in the process of binding to the substrate. This modification enhances the affinity of FAP for fatty acid substrates, making it more suitable for efficient and stable catalytic reactions. The enzyme optimization strategy provided by the present invention provides potential for applications in the fields of green chemical production and bioenergy, and provides a possible direction for further improving catalytic effects and industrial applications, especially in the process of industrial production, which can reduce the consumption of enzyme preparations and improve cost-effectiveness.

[0040] 2. Increase enzyme expression

[0041] This study optimized the FAP protein structure by deleting non-critical peptide segments, improving the enzyme's stability in the expression system. The modified protein showed a modest increase in expression and enhanced expression efficiency. The yield in the E. coli expression system was significantly higher than that of wild-type FAP, making it more suitable for large-scale production.

[0042] 3. Provide new ideas for enzyme structure optimization

[0043] By deleting noncritical peptide segments from the FAP protein and enhancing the hydrophobicity of the C-terminal region, this study provides a novel strategy for enzyme structural optimization. While the improvements in catalytic stability and efficiency are limited, this technique provides strong theoretical support and practical insights for the structural modification of other similar enzymes. This approach could open up new research avenues for improving the performance of a wide range of fatty acid decarboxylases. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of the protein structure of wild-type FAP and Trim-FAP;

[0045] Figure 2 Schematic diagram of RMSD analysis of FAP wild type and Trim-FAP proteins;

[0046] Figure 3 Schematic diagram of RMSF analysis of FAP wild type and Trim-FAP proteins;

[0047] Figure 4Schematic diagram of the analysis of the Rg gyration radius of FAP wild type and Trim-FAP protein;

[0048] Figure 5 Schematic diagram of hydrogen bond analysis between wild-type FAP and Trim-FAP protein;

[0049] Figure 6 This is the result of the detection of Trim-FAP protein expression efficiency;

[0050] Figure 7 This is the result of the detection of Trim-FAP protein catalytic efficiency. DETAILED DESCRIPTION

[0051] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0052] Example 1

[0053] Based on the crystal structure model of WT-FAP protein ( Figure 1 a) Identify the inactive core region. The WT-FAP protein structure was redesigned, and the amino acids 1 to 82, 96 to 104, 145 to 154, 176 to 223, 277 to 285, 355 to 373, 412 to 423, 493 to 517, and 580 to 600 in the non-core structure were conservatively deleted to avoid affecting the function of the core catalytic region. Combined with molecular dynamics simulation and molecular docking technology, the peptide segment MIAERAAALLTGKATIGASAAAPATVAA consisting of 28 amino acids at the C-terminus of the deleted protein was mutated to VIAVVAAALLTGLAVIGAAAAAPAVVAA, the deleted protein three-dimensional model was optimized, and the redesigned FAP protein (Trim-FAP) was simulated to obtain the modified FAP protein model (such as Figure 1 b).

[0054] Example 2 Protein kinetics verification of Trim-FAP

[0055] To verify the stability and dynamic properties of the modified Trim-FAP protein structure, molecular dynamics simulation analysis was performed, focusing on the overall stability of the protein and changes in key parameters during the simulation. The specific process is as follows:

[0056] (1) The root mean square deviation (RMSD) was used to evaluate the degree of deviation of the protein backbone atoms during the simulation. Figure 2 , Figure 2 (a) is a schematic diagram of WT-FAP RMSD analysis; (b) is a schematic diagram of WT-FAP binding ligand RMSD analysis; (c) is a schematic diagram of Trim-FAP RMSD analysis; (d) is a schematic diagram of Trim-FAP binding ligand RMSD analysis.

[0057] like Figure 2 As shown in the figure, the RMSD value of the modified Trim-FAP fluctuates within a smaller range during the entire simulation process, indicating that the protein structure remains generally stable.

[0058] (2) The flexibility changes of different amino acid residues in the protein during the simulation were analyzed by root mean square fluctuation (RMSF). The results are shown in Figure 3 , Figure 3 (a) is a schematic diagram of WT-FAP RMSF analysis; (b) is a schematic diagram of Trim-FAP RMSF analysis; (c) is a schematic diagram of Trim-FAP binding to all ligands RMSF analysis.

[0059] like Figure 3 As shown, RMSF analysis showed that the modified Trim-FAP active site region had lower volatility, indicating that its structure was stable and helpful to maintain the enzyme catalytic function.

[0060] (3) The radial distribution function (Rg) was used to analyze whether the protein collapsed during the simulation. The results are shown in Figure 4 , Figure 4 (a) is a schematic diagram of the analysis of the Rg gyration radius of WT-FAP; (b) is a schematic diagram of the analysis of the Rg gyration radius of Trim-FAP; (c) is a schematic diagram of the analysis of the Rg gyration radius of Trim-FAP bound to all ligands.

[0061] like Figure 4 As shown in the figure, the Rg value of Trim-FAP binding to all ligands did not show a significant decrease during the entire simulation process, indicating that the protein structure did not collapse and the overall conformation was compact and stable.

[0062] (4) The number and stability of hydrogen bonds within proteins were studied by hydrogen bond analysis. The results are shown in Figure 5 , Figure 5 (a) is a schematic diagram of hydrogen bond analysis of WT-FAP binding to all ligands; (b) is a schematic diagram of hydrogen bond analysis of Trim-FAP binding to all ligands.

[0063] like Figure 5As shown, the number of hydrogen bonds remained high throughout the simulation, indicating that the internal non-covalent forces were strong, further verifying the stability of the modified structure.

[0064] Example 3 Verification of Trim-FAP protein expression efficiency and catalytic efficiency

[0065] 1. Protein expression and verification

[0066] The modified FAP protein was obtained in E. coli through an optimized expression system. Subsequently, the crude FAP enzyme was obtained through induced expression and its catalytic activity was verified. The specific process is as follows:

[0067] (1) Expression system selection: Using Escherichia coli (such as Rosetta) as the host, construct an optimized expression vector, drive protein expression through an enhanced promoter, and fuse expression tags (such as Flag tags) for subsequent purification.

[0068] (2) Induction expression conditions: 20°C was selected as the candidate temperature. Low temperature is more conducive to the correct folding of proteins. The inducer concentration (such as IPTG, 1 mM) and expression time (such as 20 h) were used to maximize protein yield.

[0069] (3) The expressed crude enzyme solution was ultrasonically disrupted and the supernatant was obtained by centrifugation. The crude enzyme was then tested for catalytic activity. At the same time, M2 Beads were used to purify the Trim-FAP protein with a Flag tag. SDS-PAGE was used to analyze the expression of Trim-FAP in the crude enzyme and its purification effect to ensure that the purified protein showed a clear single band at the target molecular weight, verifying the purification effect. The results are shown in Figure 6 .

[0070] like Figure 6 As shown, by deleting non-critical peptide segments, the present invention optimizes the structure of the FAP protein and improves expression efficiency. The yield in the E. coli expression system is significantly higher than that of wild-type FAP, making it more suitable for large-scale production.

[0071] 2. Catalytic performance evaluation

[0072] The catalytic performance of the modified FAP protein was evaluated by testing its affinity for substrates, catalytic stability, and catalytic activity. Figure 7 .

[0073] like Figure 7 As shown in the results, although the catalytic activity and stability were not significantly improved, the catalytic activity of FAP was still retained on the basis of substantial structural deletion and significantly improved expression efficiency. Moreover, compared with the wild-type FAP protein, its catalytic activity was still enhanced (e.g. Figure 7 ).

[0074] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for improving the expression efficiency of FAP protein, characterized in that: Delete several non-critical redundant peptides that do not affect the core activity of the protein.

2. The method according to claim 1, characterized in that The redundant peptide segments include the following peptide segments: Amino acids 1 to 82, amino acids 96 to 104, amino acids 145 to 154, amino acids 176 to 223, amino acids 277 to 285, amino acids 355 to 373, amino acids 412 to 423, amino acids 493 to 517, and amino acids 580 to 600.

3. The method according to claim 2, characterized in that The amino acid sequence of the modified FAP protein is shown in SEQ ID NO.

1.

4. A FAP protein with high expression efficiency, characterized in that: The amino acid sequence of the FAP protein is shown in SEQ ID NO.

1.

5. A nucleic acid molecule encoding the FAP protein according to claim 4, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

6. A method for improving the catalytic efficiency of FAP protein, characterized in that: The C-terminus of the FAP protein according to claim 4 is subjected to site-directed mutagenesis to improve its hydrophobicity.

7. The method according to claim 5, characterized in that The peptide segment MIAERAAALLTGKATIGASAAAPATVAA consisting of 28 amino acids at the C-terminus of the FAP protein according to claim 4 is mutated to VIAVVAAALLTGLAVIGAAAAAPAVVAA, and the amino acid sequence of the mutated FAP protein is shown in SEQ ID NO.

3.

8. A FAP mutant with high expression efficiency and high catalytic efficiency, characterized in that: The amino acid sequence of the FAP mutant is shown in SEQ ID NO.

3.

9. A nucleic acid molecule encoding the FAP mutant according to claim 8, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.4.

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