LcfA enzyme mutant and application thereof in synthesis of Surfactin by bacillus subtilis

Abstract

The invention discloses an LcfA enzyme mutant and application of the LcfA enzyme mutant in synthesis of Surfactin from bacillus subtilis, and belongs to the technical field of biology. According to the invention, bacillus subtilis LcfA enzyme is modified, Schrodinger molecular docking is combined with site-specific saturation mutation to establish a mutant library, Rosetta is used for screening the mutant library, target sites are searched, single-point mutation and combined mutation are carried out, the activity of long-chain fatty acid coenzyme A ligase is improved, and the synthesis efficiency of Surfactin is further improved. The obtained mutations comprise T217H, S332R, R452W, K538R and a combination of any two of T217H, S332R, R452W and K538R, fermentation verification shows that the yield of Surfactin is increased by 35.3% and reaches 9.2 g / L, a production strain with higher activity of long-chain fatty acid coenzyme A ligase and better fermentation performance in industrial production is obtained, and powerful support is provided for efficient production of Surfactin.

Description

Technical Field

[0001] This invention relates to an LcfA enzyme mutant and its application in the synthesis of Surfactin by Bacillus subtilis, 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. In applications, in the oil extraction industry, surfactin can be used as a highly efficient oil displacement agent to reduce oil-water interfacial tension and improve crude oil recovery; in the pharmaceutical industry, its antibacterial, antiviral, and antitumor properties offer possibilities for the development of new drugs; in the food industry, it can act as a natural emulsifier and preservative; and in environmental remediation, its solubilizing effect on hydrophobic pollutants is beneficial for the treatment of soil and water pollution. However, the current yield of Surfactin produced by natural strains is low, which limits its large-scale industrial application. There is an urgent need to increase the yield through technological means.

[0003] LcfA enzyme, short for long-chain fatty acid-coagulant ligase, plays a central role in lipid metabolism in organisms. It primarily catalyzes the linkage reaction between long-chain fatty acids (typically straight-chain fatty acids with a carbon chain length of 12 to 22 atoms) and coenzyme A (CoA) to generate the more reactive long-chain fatty acyl-CoA (LCFA-CoA).

[0004] Structurally, LcfA enzymes typically consist of multiple domains. Their N-terminal region often possesses substrate recognition and binding functions, enabling precise recognition of long-chain fatty acids with varying carbon chain lengths and saturation levels. The C-terminal region is frequently associated with ATP binding and catalytic activity, achieving efficient hydrolysis and catalytic reaction of ATP through specific amino acid residue arrangements. LcfA enzymes from different species exhibit variations in amino acid sequences and three-dimensional conformations, affecting their substrate affinity and catalytic efficiency. For example, LcfA enzymes from Bacillus subtilis exhibit a unique folding pattern in their crystal structure, allowing their active site to effectively accommodate long-chain fatty acids while strategically positioning ATP and coenzyme A molecules to promote the reaction. In the surfactin synthesis pathway, long-chain fatty acids can be linked to polypeptide chains to promote surfactin synthesis; however, their lower activity limits their applications. Summary of the Invention

[0005] To address the aforementioned issues, this invention aims to modify the LcfA enzyme of Bacillus subtilis to enhance its activity, thereby further improving the synthesis efficiency of Surfactin, enhancing the fermentation performance of Surfactin, and ultimately improving the fermentation performance of Surfactin in industrial production.

[0006] The first object of the present invention is to provide an LcfA enzyme mutant having at least one of the following mutations relative to the amino acid sequence shown in SEQ ID NO. 1:

[0007] Replace the threonine at position 217 with histidine;

[0008] Replace serine at position 332 with arginine;

[0009] Replace arginine at position 452 with tryptophan;

[0010] Replace lysine at position 538 with arginine.

[0011] Preferably, it has one or a combination of the above mutations.

[0012] A second object of the present invention is to provide a nucleic acid molecule encoding the LcfA enzyme mutant. The DNA molecule encoding the long-chain fatty acid coenzyme A ligase 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.

[0013] A third objective of the present invention is to provide a gene expression cassette or recombinant plasmid carrying the nucleic acid molecule.

[0014] Furthermore, the backbone vector of the recombinant plasmid includes, but is not limited to, the pHT01 plasmid.

[0015] Furthermore, the gene expression cassette or recombinant plasmid contains a promoter, including but not limited to the T7 promoter.

[0016] A fourth objective of this invention is to provide recombinant cells expressing the LcfA enzyme mutant.

[0017] Furthermore, the host of the recombinant cells is a non-plant cell, including bacterial, fungal, or animal cells.

[0018] Furthermore, the host of the recombinant cells is preferably Bacillus subtilis.

[0019] A fifth objective of this invention is to provide the application of the LcfA enzyme mutant, nucleic acid molecule, gene expression cassette, recombinant plasmid, or recombinant cell in the preparation of Surfactin. By utilizing long-chain fatty acid coenzyme A ligase mutants or host cells, the Surfactin synthesis pathway is enhanced, and the fatty acid chain is strengthened to link the polypeptide chain, thereby further promoting Surfactin synthesis.

[0020] The sixth objective of this invention is to provide a recombinant Bacillus subtilis that overexpresses the encoding gene of the LcfA enzyme mutant.

[0021] Furthermore, the host bacteria of the recombinant Bacillus subtilis includes modified or unmodified Bacillus subtilis BS168 (such as B. subtilis Q1).

[0022] 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.

[0023] The beneficial effects of this invention are:

[0024] This invention provides a long-chain fatty acid coenzyme A ligase (LcfA) mutant and its application in increasing surfactin yield. The binding mode of the LcfA enzyme to the 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 LcfA enzyme mutant in Bacillus subtilis, the surfactin yield was significantly increased compared to the control strain, with a maximum increase of 35.3% and a maximum yield of 9.2 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

[0025] Figure 1 This is a schematic diagram of the structure of a long-chain fatty acid coenzyme A ligase.

[0026] Figure 2 The results are from single-point mutation fermentation screening.

[0027] Figure 3 The results are from the fermentation screening of two-point combination mutants. Detailed Implementation

[0028] 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.

[0029] The solution involved in this invention is as follows:

[0030] A method for discovering LcfA enzyme mutants with enhanced activity includes the following steps:

[0031] (a) Identifying key active sites through Schrödinger molecular docking;

[0032] (b) Constructing mutant libraries through site-directed saturation mutagenesis;

[0033] (c) The optimal mutant was determined by fermentation screening.

[0034] The culture media involved in the following examples are as follows:

[0035] LB medium (g / L): peptone 10, sodium chloride 10, yeast extract 5, sterilized at 121℃ for 20 min.

[0036] 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

[0037] 25g / L, citric acid monohydrate 4g / L; sterilize at 121℃ for 20min.

[0038] GM I medium: 10×T base 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;

[0039] 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;

[0040] 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.

[0041] The starting strains involved in the following examples are as follows:

[0042] 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.

[0043] The amino acid sequence (SEQ ID NO.1) of the wild-type long-chain fatty acid coenzyme A synthase involved in this invention is as follows:

[0044] MQSQKPWLAEYPNDIPHELPLPNKTLQSILTDSAARFPDKTAISFYGKKLTFHDILTDALKLAAFLQCNGLQKGDRVAVMLPNCPQTVISYYGVLFAGGIVVQTNPLYTEHELEYQLRDAQVSVIITLDLLFPKAIKMKT LSIVDQILITSVKDYLPFPKNILYPLTQKQKVHIDFDKTANIHTFASCMKQEKTELLTIPKIDPEHDIAVLQYTGGTTGAPKGVMLTHQNILANTEMCAAWMYDVKEGAEKVLGIVPFFHVYGLTAVMNYSIKLGFEMIL LPKFDPLETLKIIDKHKPTLFPGAPTIYIGLLHHPELQHYDLSSIKSCLSGSAALPVEVKQKFEKVTGGKLVEGYGLSEASPVTHANFIWGKNKPGSIGCPWPSTDAAIYSEETGELAAPYEHGEIIVKGPQVMKGYWNK PEETAAVLRDGWLFTGDMGYMDEEGFFYIADRKKDIIIAGGYNIYPREVEEALYEHEAIQEIVVAGVPDSYRGETVKAFVVLKKGAKADTEELDAFARSRLAPYKVPKAYEFRKELPKTAVGKILRRRLLEEETENHHIK

[0045] The methods for preparing and transforming competent lysimachia cells involved in the following examples are as follows:

[0046] (1) Streak Bacillus subtilis strain onto LB agar plates;

[0047] (2) Take a single colony and inoculate it into 5 mL of GMI medium, and incubate overnight at 30°C and 100 rpm.

[0048] (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.

[0049] (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.

[0050] (5) Centrifuge at 4℃, 5000g for 10 min and collect the bacterial cells;

[0051] (6) The bacterial cells were suspended in 2 mL of the original culture supernatant. The suspended bacterial cells are competent cells.

[0052] (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.

[0053] (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.

[0054] The Surfactin detection scheme involved in the following embodiments is as follows:

[0055] 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.

[0056] Example 1: Construction of engineered bacteria for long-chain fatty acid coenzyme A ligase (using wild type as an example)

[0057] (1) Using the pHT01 vector as a template, PCR amplification was performed using primers pHT01-F: gatcctctagagtcgacgtcc and pHT01-R: ttcctcctttaattgggaattgttatcc. The PCR product was recovered to obtain the linearized vector.

[0058] (2) Using the encoding nucleotide sequence of wild-type long-chain fatty acid coenzyme A ligase (amino acid sequence see SEQ ID NO.1) as a template, PCR amplification was performed using primers LA-F: caattaaaggaggaaggatccATGCAGTCTCAAAAGCCGTGG and LA-R: gacgtcgactctagaggatccTTATTTGATATGATGATTTTCGGTTTC. The amplification product was recovered to obtain the target fragment of the LA gene.

[0059] (3)Use The two target fragments were ligated using the one-step homologous recombination kit to obtain a recombinant plasmid, which was named pHT01-LA. Sequencing confirmed its correctness, and the recombinant plasmid was successfully constructed.

[0060] (4) The pHT01-LA 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 long-chain fatty acid coenzyme A ligase engineered bacteria (WT).

[0061] Example 2 Selection of mutation sites

[0062] This invention uses the predicted structure of AlphaFold, a wild-type long-chain fatty acid coenzyme A ligase (file: AF-P94547-F1; e.g.) Figure 1 Using Schrödinger molecular docking as the initial model, active sites at the binding pocket were analyzed. Ten amino acid sites were identified through simulation of key regions: 105N, 215G, 217T, 260H, 332S, 353E, 534G, 359E, 452R, and 538K. Subsequently, saturation mutagenesis was used to mutate these 10 amino acid sites, resulting in a mutant library with 200 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 16 single-point mutation sites. They are N105M, G215E, T217W, T217G, T217H, H260E, H260D, S332R, S332H, S332W, E359Q, E353D, E359R, R452W, K538R, and K538N, respectively.

[0063] Example 3: Fermentation Screening of Single-Point Mutants

[0064] The mutant recombinant plasmid was constructed according to the method in Example 1 and transformed into Bacillus subtilis to obtain a long-chain fatty acid coenzyme A ligase 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 inoculum was then transferred at a rate of 3-6% to a 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).

[0065] Depend on Figure 2 The experimental results showed that four mutants with superior fermentation performance were obtained: T217H, S332R, R452W, and K538R. The fermentation yield of all four mutants was better than the control group, with the T217H mutant exhibiting the highest yield, reaching 8.3 g / L, a 22.1% increase compared to the control. Fermentation strains with stronger activity and better suited for production were obtained.

[0066] Example 4: Combination Mutation of the Optimal Mutant

[0067] The four optimal mutants were combined in pairs, and after successful mutation, recombinant plasmids were constructed according to Example 1 above. These plasmids were then transformed into Bacillus subtilis to obtain a long-chain fatty acid coenzyme A ligase mutant 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. Then, 3-6% of the inoculum was transferred to a 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 a control.

[0068] Depend on Figure 3 The experimental results showed that the combined mutants were superior to the single-point mutants, and significantly improved compared to the control strain. The optimal combined mutant was T217H / S332R, with a yield of 9.2 g / L, which was 35.3% higher than the control strain BS168-Q1, and 10.8% and 13.5% higher than the single-point mutants, respectively. This further enhances the application of the fermentation strain in industrial production.

[0069] 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. An LcfA enzyme mutant, characterized in that, The LcfA enzyme mutant has at least one of the following mutations relative to the amino acid sequence shown in SEQ ID NO.1: Replace the threonine at position 217 with histidine; Replace serine at position 332 with arginine; Replace arginine at position 452 with tryptophan; Replace lysine at position 538 with arginine.

2. A nucleic acid molecule encoding the LcfA enzyme mutant of claim 1.

3. A gene expression cassette or recombinant plasmid carrying the nucleic acid molecule of claim 2.

4. The gene expression cassette or recombinant plasmid according to claim 3, 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.

5. A recombinant cell expressing the LcfA enzyme mutant of claim 1, characterized in that, The host of the recombinant cells is a non-plant cell.

6. The recombinant cell according to claim 5, characterized in that, The host of the recombinant cells is bacteria, fungi, or animal cells; The host of the recombinant cells includes Bacillus subtilis.

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

8. A recombinant Bacillus subtilis, characterized in that, The recombinant Bacillus subtilis overexpressed the encoding gene of the LcfA enzyme mutant of claim 1.

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.

Images