TE enzyme mutant with improved activity and application thereof

Abstract

The invention discloses a TE enzyme mutant with improved activity and application thereof, and belongs to the technical field of biology. Schrodinger molecular docking is combined with fixed-point saturation mutation to establish a mutant library, Rosetta is used for screening the mutant library, searching a target point and carrying out single-point mutation and combined mutation, the activity of thioesterase is improved, the synthesis efficiency of Surfactin is further improved, and a production strain with better fermentation performance in industrial production is obtained. Through verification, mutations comprise A113W, A113G, K122H, K192D and a combination of the A113W, the A113G, the K122H and the K192D, fermentation verification shows that the yield of the Surfactin is increased by 41.2% to the maximum compared with that of a wild type and reaches 9.6 g / L, thioesterase with higher activity and a production strain with better fermentation performance in industrial production are obtained, and powerful support is provided for efficient production of the Surfactin.

Description

Technical Field

[0001] This invention relates to a TE enzyme mutant with enhanced activity and its applications, belonging to the field of biotechnology. Background Technology

[0002] Surfactin is a class of cyclic lipopeptide biosurfactants synthesized by microorganisms such as Bacillus subtilis, and it has shown great application potential in many fields. Its unique molecular structure consists of a peptide ring containing seven amino acids linked to a hydrophobic fatty acid chain of thirteen to fifteen carbon atoms. This unique structure endows it with excellent surface activity, such as an extremely low critical micelle concentration (typically 0.001-0.1 mg / mL) and a significant ability to reduce interfacial tension (reducing the surface tension of water from 72 mN / m to below 27 mN / m). It possesses good surface activity, antibacterial properties, antitumor activity, and other biological activities. However, the current yield of surfactin produced by natural strains is low, limiting its large-scale industrial application, and there is an urgent need to increase its yield through technological means.

[0003] TE thioesterases, belonging to the α / β hydrolase superfamily, play a crucial role in biosynthesis and metabolism. Members of this family all possess a secondary structure containing eight β-sheets and six α-helices, exhibiting a conserved catalytic trielement (nucleophilic group-histidine-acidic amino acid). Some amino acid residues can form "oxygen ion holes" that stabilize intermediates. For example, subtilis lipopeptide thioesterase (SrfTE), containing 235 amino acid residues, is a globular molecule with a small nest of active sites. Its catalytic trielement is Ser80-His207-Asp107. The active element Ser80 is located between the β5-sheet and the αC-helix, while Ala81 and the amide group of the Val27 backbone form an oxygen ion hole, stabilizing the formation and decomposition of peptide / acyl-O-TE tetrahedral intermediates. In the synthesis of nonribosomal peptides (NRPs), TE enzymes are responsible for the final cyclization of NRPs, exhibiting regio-, chemo-, and stereo-oriented properties. They play a decisive role in the formation of the final cyclic peptide, terminating the reaction and releasing the product. Targeted modification of the TE enzyme may further increase the yield of Surfactin, hence this invention. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides a TE enzyme mutant with enhanced activity, aiming to improve the synthesis efficiency of Surfactin and enhance its fermentation performance, with the potential to further improve the fermentation performance of Surfactin in industrial production.

[0005] The first object of the present invention is to provide a thioesterase (TE enzyme) mutant having at least one of the following mutations relative to the amino acid sequence shown in SEQ ID NO.1:

[0006] The alanine at position 113 is replaced with tryptophan;

[0007] The alanine at position 113 is replaced with glycine;

[0008] The lysine at position 122 is replaced with histidine;

[0009] The lysine at position 192 is replaced with aspartic acid.

[0010] Furthermore, the mutation has one or a combination of the above mutations, preferably any one of the following:

[0011] The alanine at position 113 is replaced with tryptophan;

[0012] The alanine at position 113 is replaced with glycine;

[0013] The lysine at position 122 is replaced with histidine;

[0014] The lysine at position 192 is replaced with aspartic acid;

[0015] The alanine at position 113 is replaced with tryptophan and the lysine at position 122 is replaced with histidine;

[0016] The alanine at position 113 is replaced with glycine and the lysine at position 122 is replaced with histidine.

[0017] A second object of the present invention is to provide a nucleic acid molecule encoding the thioesterase mutant. The DNA molecule encoding the thioesterase mutant is obtained by altering the coding sequence of the wild-type enzyme-encoding DNA molecule according to the changes in the amino acid residues of the mutant.

[0018] A third objective of the present invention is to provide a gene expression cassette or recombinant plasmid carrying the nucleic acid molecule.

[0019] Furthermore, the backbone vector of the recombinant plasmid includes, but is not limited to, the pHT01 plasmid.

[0020] Furthermore, the gene expression cassette or recombinant plasmid contains a promoter, including but not limited to the T7 promoter.

[0021] A fourth objective of this invention is to provide recombinant cells expressing the thioesterase mutant.

[0022] Furthermore, the host of the recombinant cells is a non-plant cell, including bacterial, fungal, or animal cells.

[0023] Furthermore, the host of the recombinant cells is preferably Bacillus subtilis.

[0024] A fifth objective of this invention is to provide the application of the aforementioned thioesterase mutant, nucleic acid molecule, gene expression cassette, recombinant plasmid, or recombinant cell in the preparation of Surfactin. By utilizing thioesterase mutants or host cells, the Surfactin synthesis pathway is enhanced, promoting the final phagocytosis of Surfactin and product efflux, thereby further promoting Surfactin synthesis.

[0025] A sixth objective of this invention is to provide a recombinant Bacillus subtilis that overexpresses the encoding gene of the thioesterase mutant.

[0026] Furthermore, the host bacteria of the recombinant Bacillus subtilis includes modified or unmodified Bacillus subtilis BS168 (such as B. subtilis Q1).

[0027] A seventh object of the present invention is to provide a method for producing Surfactin, comprising the step of fermentation production using the recombinant Bacillus subtilis.

[0028] The beneficial effects of this invention are:

[0029] This invention provides a thioesterase (TE) mutant and its application in increasing surfactin yield. The binding mode between the TE enzyme and substrate was analyzed using Schrödinger molecular docking technology to identify key active sites. These key sites were then modified using site-directed saturation mutagenesis, and high-activity mutants were obtained through fermentation screening to further increase surfactin fermentation yield. After expression of the screened TE enzyme mutant in Bacillus subtilis, the surfactin yield was significantly increased by 41.2% compared to the wild type, reaching a final yield of 9.6 g / L. This invention solves the problems of low surfactin yield and insufficient enzyme catalytic efficiency in existing technologies, providing a new approach and method for the industrial production of surfactin. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the structure of thioesterase.

[0031] Figure 2 The results are from single-point mutation fermentation screening.

[0032] Figure 3 The results are from the fermentation screening of two-point combination mutants. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0034] The solution involved in this invention is as follows:

[0035] A method for discovering TE enzyme mutants with enhanced activity includes the following steps:

[0036] (a) Identifying key active sites through Schrödinger molecular docking;

[0037] (b) Constructing mutant libraries through site-directed saturation mutagenesis;

[0038] (c) The optimal mutant was determined by fermentation screening.

[0039] The culture media involved in the following examples are as follows:

[0040] LB medium (g / L): peptone 10, sodium chloride 10, yeast extract 5, sterilized at 121℃ for 20 min.

[0041] Shake-flask fermentation medium (g / L): sucrose 60 g / L, yeast extract 8 g / L, NaNO3 17 g / L, MgSO4·7H2O 0.2 g / L, FeSO4·7H2O 0.1 g / L, MnSO4·H2O 0.1 g / L, Na2HPO4 25 g / L, citric acid monohydrate 4 g / L; sterilized at 121℃ for 20 min.

[0042] GM I medium: 10×Tbase 10mL, 100g / L glucose 5mL, 50g / L yeast extract 2mL, 10g / L acid hydrolyzed casein 2mL, 2.5g / L tryptophan 2mL, add water to 100mL;

[0043] GMII medium: 10×Tbase 10mL, 100g / L glucose 5mL, 50g / L yeast extract 0.04mL, 10g / L acid hydrolyzed casein 0.5mL, 0.5mol / L MgCl2 0.5mL, 0.05mol / L CaCl2 1mL, add water to 100mL;

[0044] IPTG (1 mol / L): IPTG is isopropyl thio-β-D-galactoside (molecular weight 238.3). Dissolve 2.38 g of IPTG in 8 mL of distilled water, then bring the volume to 10 mL with distilled water. Filter the solution through a 0.22 μm filter to remove bacteria, aliquot into 1 mL portions, and store at -20 °C. Its main function is to induce the expression of Surfactin in Bacillus subtilis.

[0045] The starting strains involved in the following examples are as follows:

[0046] BS168-Q1: It is described in the literature Regulation mechanism and bioactivity characteristic of Surfactin homologues with C14 and C15 fatty acid chains, where it is named B. subtilis Q1.

[0047] The amino acid sequence (SEQ ID NO.1) of the wild-type thioesterase involved in this invention is as follows:

[0048] MSQLFKSFDASEKTQLICFPFAGGYSASFRPLHAFLQGECEMLAAEPPGGTNQTSAIEDLEELTDLYKQELNLRPDRPFVLFGHSMGGMITFRLAQKLEREGIFPQAVIISAIQPPHIQR KKVSHLPDDQFLDHIIQLGGMPAELVENKEVMSFFLPSFRSDYRALEQFELYDLAQIQSPVHVFNGLDDKKCIRDAEGWKKWAKDITFHQFDGGHMFLLSQTEEVAERIFAILNQHPIIQP

[0049] The methods for preparing and transforming competent lysimachia cells involved in the following examples are as follows:

[0050] (1) Streak Bacillus subtilis strain onto LB agar plates;

[0051] (2) Take a single colony and inoculate it into 5 mL of GMI medium, and incubate overnight at 30°C and 100 rpm.

[0052] (3) Take 1 mL of culture medium and transfer it to 10 mL of GMII medium. Incubate at 37°C and 200 rpm for 3.5 h.

[0053] (4) Take 2 mL of culture medium and transfer it to 18 mL of GMII medium. Incubate at 37°C and 100 rpm for 1.5 h.

[0054] (5) Centrifuge at 4℃, 5000g for 10 min and collect the bacterial cells;

[0055] (6) The bacterial cells were suspended in 2 mL of the original culture supernatant. The suspended bacterial cells are competent cells.

[0056] (7) Add 1 μg / ml of the constructed expression vector to a centrifuge tube containing 200 μl of competent bacterial culture, and incubate at 37°C and 80 rpm for 1 h. Add 1 μg / ml of the constructed knockout vector to a centrifuge tube containing 200 μl of competent bacterial culture, and incubate at 30°C and 100 rpm for 1 h.

[0057] (8) Competent strains containing the expression vector were plated on chloramphenicol resistance plates and incubated at 37°C for 12 h; competent strains containing the knockout vector were plated on kanamycin resistance plates and incubated at 30°C for 12-16 h. Transformants were then verified by colony PCR. PCR products with band sizes matching the theoretical values ​​were sent for sequencing. Transformants were then verified by colony PCR. PCR products with band sizes matching the theoretical values ​​were sent for sequencing. Strains with correct sequencing were amplified and stored at -80°C.

[0058] The Surfactin detection scheme involved in the following embodiments is as follows:

[0059] HPLC was performed using an Agilent C18 column with an elution column of 4.6 mm × 250 mm × 5 μm. The eluent consisted of 1‰ trifluoroacetic acid ultrapure water and 1‰ trifluoroacetic acid acetonitrile. The elution gradient conditions were: 0–9 min, 60% volume B phase, 40% volume A phase; 9–30 min, 93% volume B phase, 7% volume A phase, with a flow rate of 0.800 mL / min. The injection volume was 20 μL, and the detection wavelength was 210 nm. Specifically, phase B consisted of 1‰ trifluoroacetic acid acetonitrile, and phase A consisted of 1‰ trifluoroacetic acid ultrapure water.

[0060] Example 1: Construction of engineered thioesterase bacteria (using wild type as an example)

[0061] (1) Using the pHT01 vector as a template, PCR amplification was performed using primers pHT01-F:gatcctctagagtcgacgtcc and pHT01-R:tcctcctttaattgggaattgttatcc. The PCR product was recovered to obtain the linearized vector.

[0062] (2) Using the encoding nucleotide sequence of wild-type thioesterase (amino acid sequence see SEQ ID NO:1) as a template, PCR amplification was performed using primers TE-F:caattaaaggaggaaggatccATGAGCCAACTCTTCAAATCATTTG and TE-R:gacgtcgactctagaggatccTCACGGTTGAATGATCGGATG. The amplified product was recovered to obtain the target fragment of the TE gene.

[0063] (3)Use The two target fragments were ligated using the one-step homologous recombination kit to obtain a recombinant plasmid, which was named pHT01-TE. Sequencing confirmed its correctness, and the recombinant plasmid was successfully constructed.

[0064] (4) The pHT01-TE plasmid was transformed into Bacillus subtilis expression host strain 168 by chemical method and spread on LB solid medium containing ampicillin. The LB plates were cultured at 37°C until transformants grew. Positive transformants were picked to obtain wild-type thioesterase engineered bacteria (WT).

[0065] Example 2 Selection of mutation sites

[0066] This invention uses the predicted structure of wild-type thioesterase AlphaFold (e.g.) Figure 1 Using Schrödinger molecular docking as the initial model, the active sites at the binding pocket were analyzed. Through simulation of key regions, eight amino acid sites were identified: 81V, 86S, 113A, 122K, 134D, 135H, 190D, and 192K. Subsequently, saturation mutagenesis was used to mutate these eight amino acid sites, resulting in a mutant library with 160 mutants. Then, multiple algorithms, including FoldX, I-Mutant, dDFIRE, and Rosetta Cartesian_ddG, were used to predict the mutant library. Finally, Rosetta was used for structure optimization, flexible docking, and catalytic residue optimization, ultimately yielding 14 single-point mutation sites. They are S86E, S86D, A113W, A113G, K122H, K112R, D134R, D134K, H135W, H135K, D190E, D190F, K192D, and K192F, respectively.

[0067] Example 3: Fermentation Screening of Single-Point Mutants

[0068] The mutant recombinant plasmid was constructed according to the method in Example 1 and transformed into Bacillus subtilis to obtain a wild-type thioesterase mutant engineered strain. The optimal mutant was then determined by shake-flask yield screening. The strain was diluted and plated, incubated overnight at 37°C with inverted incubation. Newly activated single colonies were picked and cultured in LB medium at 37°C with shaking at 200 rpm for 12-16 h. The culture was then transferred at an inoculum of 3-6% to a baffle-type shake flask containing 50 mL of fermentation medium and incubated at 37°C with shaking at 200 rpm for 18-24 h. Each experiment had three biological replicates. After fermentation, samples were taken, centrifuged at 12000 rpm for 3 min, and the supernatant was collected, appropriately diluted, and the surfactant yield was measured. The experiment used strain BS168-Q1 as a base for modification, and this strain was used as the experimental control (CV).

[0069] Depend on Figure 2The experimental results showed that four mutants with superior fermentation performance were obtained: A113W, A113G, K122H, and K192D. The fermentation yield of all four mutants was better than the control group, with the K122H mutant exhibiting the highest yield, reaching 8.5 g / L, a 25% increase compared to the control. This demonstrates that we have obtained fermentation strains with stronger activity and better suited for production.

[0070] Example 4: Two-point combination mutation screening

[0071] The four optimal mutants were combined in pairs, and after successful mutation, the mutant recombinant plasmids were constructed according to Example 1 above and transformed into Bacillus subtilis to obtain wild-type thioesterase mutant engineered strains. The optimal mutant was then determined by shake-flask yield screening. The strain was diluted and plated, incubated overnight at 37°C with inverted incubation, and newly activated single colonies were picked and cultured in LB medium at 37°C with shaking at 200 rpm for 12-16 h. Then, 3-6% of the inoculum was transferred to 50 mL shake-flasks containing fermentation medium and incubated at 37°C with shaking at 200 rpm for 18-24 h. Each experiment had three biological replicates. After fermentation, samples were taken, centrifuged at 12000 rpm for 3 min, and the supernatant was collected, appropriately diluted, and the surfactant yield was measured. The experiment used strain BS168-Q1 as a base for modification, and this strain was used as an experimental control.

[0072] Depend on Figure 3 The experimental results show that the A133W / K122H double-point mutant improved by 41.2% compared with the control strain, and by 17.1% and 12.9% compared with the single-point mutant, respectively, reaching 9.6 g / L. The double-point mutant A113G / K122H also showed a significant improvement, further enhancing the application of fermentation strains in industrial production.

[0073] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A thioesterase mutant, characterized in that, The thioesterase mutant has at least one of the following mutations relative to the amino acid sequence shown in SEQ ID NO.1: The alanine at position 113 is replaced with tryptophan; The alanine at position 113 is replaced with glycine; The lysine at position 122 is replaced with histidine; The lysine at position 192 is replaced with aspartic acid.

2. The thioesterase mutant according to claim 1, characterized in that, Having any of the following mutations: The alanine at position 113 is replaced with tryptophan; The alanine at position 113 is replaced with glycine; The lysine at position 122 is replaced with histidine; The lysine at position 192 is replaced with aspartic acid; The alanine at position 113 is replaced with tryptophan and the lysine at position 122 is replaced with histidine; The alanine at position 113 is replaced with glycine and the lysine at position 122 is replaced with histidine.

3. A nucleic acid molecule encoding the thioesterase mutant of claim 1 or 2.

4. A gene expression cassette or recombinant plasmid carrying the nucleic acid molecule of claim 3.

5. The gene expression cassette or recombinant plasmid according to claim 4, characterized in that, The backbone vector of the recombinant plasmid includes the pHT01 plasmid; And / or the gene expression cassette or recombinant plasmid contains a promoter, including the T7 promoter.

6. A recombinant cell expressing the thioesterase mutant of claim 1 or 2, characterized in that, The host of the recombinant cells is a non-plant cell.

7. The use of the thioesterase mutant of claim 1 or 2, the nucleic acid molecule of claim 3, the gene expression cassette or recombinant plasmid of claim 4 or 5, or the recombinant cell of claim 6 in the preparation of Surfactin.

8. A recombinant Bacillus subtilis, characterized in that, The recombinant Bacillus subtilis overexpresses the encoding gene of the thioesterase mutant of claim 1 or 2.

9. The recombinant Bacillus subtilis according to claim 8, characterized in that, The host bacteria of the recombinant Bacillus subtilis include modified or unmodified Bacillus subtilis BS168.

10. A method for producing Surfactin, characterized in that, The step includes fermentation production using the recombinant Bacillus subtilis as described in claim 8 or 9.

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