A SEPHCHC synthase mutant derived from bacillus subtilis

Abstract

The application provides a kind of aromatic isopentenyl transferase mutant, to be derived from Bacillus subtilis ( Bacillus subtilis )SEPHCHC synthase gene MenD is based, six aromatic isopentenyl transferase mutants MenD-1, MenD-2, MenD-3, MenD-4, MenD-5 and MenD-6 are obtained by site-directed mutagenesis PCR technology, and the recombinant bacteria Bacillus subtilis expressing the above mutants is respectively constructed, and the yield of heptamethylnaphthoquinone fermentation is 70.77 mg / L, 75.48 mg / L, 81.72 mg / L, 88.41 mg / L, 99.88 mg / L and 133.40 mg / L respectively, which is 116%, 123%, 134%, 144%, 162% and 218% of the yield of heptamethylnaphthoquinone of Bacillus subtilis expressing wild-type SEPHCHC synthase MenD respectively.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and protein engineering, and specifically relates to a novel SEPHCHC synthase mutant protein. Background Technology

[0002] 2-Succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid (SEPHCHC) synthase, also known as MenD (EC 2.2.1.9), is a thiamine diphosphate (ThDP)-dependent decarboxylase that catalyzes the conversion of α-ketoglutarate and isobromate to SEPHCHC. Its catalytic reaction is characterized by high chemo and regioselectivity. Thiamine diphosphate (ThDP)-dependent enzymes typically convert electron-deficient carbonyl substrates into nucleophiles during catalysis. MenD is essential for the biosynthesis of methylnaphthoquinone and phylloquinone and is present in the genomes of various bacteria. It has wide applications in consumer chemicals, food processing, and pharmaceutical synthesis, and is a promising target for biotechnology and medical applications.

[0003] Site-directed mutagenesis refers to the introduction of desired changes (usually changes indicating a favorable direction) into DNA fragments encoding a target protein (which can be in the genome or plasmids) using methods such as polymerase chain reaction (PCR). These changes include base addition, deletion, and point mutations. Site-directed mutagenesis can rapidly and efficiently improve the traits and characterization of the target protein expressed by DNA, making it a very useful tool in gene research. In vitro site-directed mutagenesis is an important experimental technique in various fields of biology and medicine, a convenient method for modifying and optimizing genes, and a powerful tool for studying the complex relationship between protein structure and function. By selectively altering, deleting, or inserting specific bases in a known gene, the corresponding amino acid sequence and protein structure can be changed. Studying the expression products of mutated genes helps us understand the relationship between protein structure and function and explore protein structure / domains. The potential applications of site-directed mutagenesis are wide-ranging, such as studying the structure of protein-protein interaction sites, modifying the different activities or kinetic properties of enzymes, improving protein stability and activity, studying protein crystal structures, and gene therapy. Site-directed mutagenesis of enzymes has opened up new avenues for understanding the structure and function of enzymes, achieving great success in fields such as industry, agriculture, food processing, and the environment. Summary of the Invention

[0004] This invention provides a SEPHCHC synthase mutant with significantly enhanced enzyme activity to address the problems of existing technologies. The mutant is derived from Bacillus subtilis (…). Bacillus subtilis The SEPHCHC synthase gene MenD was obtained using site-directed mutagenesis and expressed in Bacillus subtilis.

[0005] This invention provides a SEPHCHC synthase mutant, the amino acid sequence of which is the 117th amino acid of the aromatic isopentenyltransferase MenD in SEQ ID NO:1 is changed from Gln to Ala, and the amino acid sequence of the mutant is SEQ ID NO:2;

[0006] Alternatively, the amino acid sequence of the aromatic isopentenyltransferase MenD with the amino acid sequence SEQ ID NO:1 is changed from Leu to Asn, and the mutant amino acid sequence is SEQ ID NO:3.

[0007] Alternatively, the amino acid sequence of the aromatic isopentenyltransferase MenD with the amino acid sequence SEQ ID NO:1 is changed from Ala to Ile, and the mutant amino acid sequence is SEQ ID NO:4.

[0008] Alternatively, the amino acid sequence of the aromatic isopentenyltransferase MenD with the amino acid sequence SEQ ID NO:1 is changed from Glu to Pro, and the mutant amino acid sequence is SEQ ID NO:5.

[0009] Alternatively, the amino acid sequence of the aromatic isopentenyltransferase MenD with the amino acid sequence SEQ ID NO:1 is changed from Leu to Arg, and the mutant amino acid sequence is SEQ ID NO:6.

[0010] Alternatively, the amino acid sequence of the aromatic isopentenyltransferase MenD with the amino acid sequence SEQ ID NO:1 is changed from Ala to Tyr at position 115, and the mutant amino acid sequence is SEQ ID NO:7.

[0011] The present invention also provides a gene encoding a SEPHCHC synthase mutant, which encodes the aforementioned SEPHCHC synthase mutant.

[0012] The present invention further provides a recombinant expression vector, wherein the recombinant expression vector carries the encoding gene of the SEPHCHC synthase mutant as described in claim 1.

[0013] Preferably, the starting expression vector is PDH1730, and the coding gene is generated by the promoter P. glv Or P cspd Or P cspB Initiation. More preferably, the integration site of the coding gene is amyE, ldh, or aprE.

[0014] Most preferably, the encoding gene is the promoter P. cspd It is initiated, and the integration site is ldh.

[0015] This invention provides a recombinant host cell, wherein the recombinant host cell is a host cell transformed / transfected with the recombinant expression vector according to any one of claims 3-6, preferably the host cell being Bacillus subtilis (…). Bacillus subtilis ).

[0016] This invention also provides the application of the aforementioned SEPHCHC synthase mutant or its encoding gene in the preparation of heptaene-menaquinone.

[0017] The present invention also provides a method for preparing heptaene-menaquinone by culturing the recombinant host cells to produce heptaene-menaquinone.

[0018] Preferably, the method further includes the steps of collecting and purifying heptaene-menaquinone; more preferably, the fermentation medium used for cultivation consists of: 3% glycerol, 6% soybean peptone, 0.5% yeast extract, 0.3% K2HPO4, 0.5% MgSO4 •7H2O, 0.1% L-Tryptophan, and 3% maltose; the cultivation conditions are 40 °C and 200 rpm shaking culture for 96 h.

[0019] This invention is based on the SEPHCHC synthase MenD derived from Bacillus subtilis. Six SEPHCHC synthase mutants, MenD-1, MenD-2, MenD-3, MenD-4, MenD-5, and MenD-6, were obtained through site-directed mutagenesis. These mutants were then fermented in Bacillus subtilis, and the yields of heptaene menaquinone produced were 70.77 mg / L, 75.48 mg / L, 81.72 mg / L, 88.41 mg / L, 99.88 mg / L, and 133.40 mg / L, respectively. These yields represent 116%, 123%, 134%, 144%, 162%, and 218% of the heptaene menaquinone yield of Bacillus subtilis expressing the wild-type SEPHCHC synthase MenD. Attached Figure Description

[0020] Figure 1 : Plasmid map of the SEPHCHC synthase mutant expression vector;

[0021] Figure 2 Bar graph showing the production of heptaene-naphthoquinone by the SEPHCHC synthase mutant Bacillus subtilis strain. Detailed Implementation

[0022] The method of the present invention will be further illustrated below with reference to examples. Experimental methods not specified in the examples can generally be performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual* by J. Sambrook et al., or according to the manufacturer's recommendations. Those skilled in the art can better understand and master the present invention with the help of these examples. However, the methods for implementing the present invention should not be limited to the specific method steps described in the embodiments of the present invention.

[0023] The terminology and related measurement methods involved in this invention are explained as follows:

[0024] The SEPHCHC synthase mutant is identified by the amino acid at the original amino acid position being replaced. For example, Ser33Ile indicates that the amino acid at position 33 has been replaced by Ile from the parental SEPHCHC synthase, with the position number corresponding to the number in SEQ ID NO:1 of the appendix sequence listing. Ala115Tyr indicates that the amino acid at position 115 has been mutated.

[0025] Example 1: Construction of SEPHCHC synthase MenD expression vector and recombinant strain

[0026] Using PDH1730 plasmid as a template, the vector DNA sequence was amplified by PCR to obtain the vector backbone sequence to be cloned. Using Bacillus subtilis genomic DNA as a template, the complete MenD gene was amplified by PCR to obtain the SEPHCHC synthase MenD sequence, whose encoded amino acid sequence is SEQ ID NO: 1. The PCR amplification conditions were: 98℃ for 2 min; 98℃ for 10 s, 60℃ for 20 s, 72℃ for 3 min 10 s, 30 cycles, 72℃ for 5 min, and incubation at 4℃. The PCR amplification products were recovered using the EZNA Gel Extraction Kit. The recovered enzyme gene sequence and vector backbone sequence were then processed using ClonExpress. ®The recombinant plasmid was ligated using the OneStep Cloning Kit to assemble the vector and fragment. The assembly system was as follows: 4 μL of 5×CEⅡ Buffer, 2 μL of ExnaseⅡ, gene fragment (MenD fragment) (0.04× number of insert bases) ng / concentration (ng / μL), vector backbone fragment (PDH1730) (0.02× number of cloning vector bases) ng / concentration (ng / μL), and ddH2O to a final volume of 20 μL. After adding the appropriate components as described above, the mixture was incubated in a 37 ℃ water bath for 30 min to complete the assembly. The assembly product was then transformed into E. coli competent cells DH5α. After obtaining positive transformants, the plasmid was extracted using the EZNA Plasmid Extraction Kit and named PDH-MenD. Its plasmid map is attached. Figure 1 As shown. The plasmid PDH-MenD was transformed. Bacillus subtilis168 Host bacteria were used to screen for positive transformants, resulting in a recombinant strain expressing wild-type SEPHCHC synthase MenD, which was named Bacillus subtilis MenD. Bacillus subtle MenD).

[0027] The above-mentioned Bacillus subtilis ( Bacillus subtilis )168 was transformed using the competent state method. BS1 competent states were prepared via the Spizizen two-step method, see the article (Spizizen J. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate[J].Proc Natl Acad Sci USA, 1958, 44: 1072-1075.). The specific transformation process is as follows: freshly activated... Bacillus subtilis 168The culture was inoculated from LB agar plates (1% tryptone, 0.5% yeast extract, 1% NaCl) into 5 mL GMⅠ (GMI solution prepared as follows: 0.5 mL 10×Spizizen salt solution, 0.1 mL 2% acid-hydrolyzed casein, 0.1 mL 40% glucose, 0.1 mL 5% yeast extract, 0.005 mL 20% MgSO4·7H2O, 0.05 mL 0.5% L-Tryptophan, 3.145 mL ddH2O); the 10×Spizizen salt solution was prepared as follows: 18.3 g / L K2HPO4, 6 g / L KH2PO4, 2 g / L (NH4)2SO4, 1.2 g / L trisodium citrate, ddH2O, dissolved sequentially in distilled water. The culture was incubated overnight at 37 °C with shaking at 220 rpm. The next day, 0.5 mL was transferred to a freshly prepared 4.5 mL culture medium. In sterile GMⅠ medium, culture at 37 ℃ and 220 rpm for 4.5 h. Then, transfer 0.75 mL of the culture medium from the previous step to freshly prepared 4.25 mL of sterile GMⅡ medium (GMⅡ is prepared as follows: 0.5 mL 10×Spizizen salt solution, 0.05 mL 2% acid-hydrolyzed casein, 0.1 mL 40% glucose, 0.035 mL 20% MgSO4·7H2O, 0.005 mL 0.5% L-Tryptophan, 3.310 mL ddH2O, culture at 37 ℃ and 220 rpm for 90 min, then centrifuge at 5000 g for 10 min to collect the cells. Gently resuspend the cells in 10 mL of the original culture supernatant; the resuspended cells are the competent cells. Add an appropriate amount of DNA to 1 mL of competent cells, culture at 37 ℃ and 220 rpm with shaking for 1.5 h, centrifuge, plate, and then incubate at 37 ℃. Incubate overnight at ℃ with the inverted incubator, and check and verify the transformants the next day.

[0028] Example 2: Construction of the SEPHCHC synthase MenD mutant using point mutation technology

[0029] Phylogenetic analysis of the MenD gene of SEPHCHC synthase from Bacillus subtilis was performed, comparing its amino acid sequence with that of other highly homologous SEPHCHC synthases to identify potential amino acid mutation sites that could improve its enzymatic properties. Following a series of ClustalX sequence alignments, amino acid residues at positions 33, 48, 126, 115, 493, and 521 were selected for mutation. Nucleotide mutations were introduced into the MenD gene of the SEPHCHC synthase from Bacillus subtilis using point mutagenesis technology. The amplification system for the Ser33Ile site mutation PCR was as follows: 25 μL of 2×PimerStarMax, 2.5 μL of upper primer (10 μM), 2.5 μL of lower primer (10 μM), 2 μL of template DNA PDG-MenA plasmid (100 ng / μL), 18 μL of ddH2O, upstream mutation primer 5'-atcgtttgtgtgggtgctgctcggaaccgttttg-3', and downstream mutation primer 5'-cgagcagcacccacacaaacgatgcggttaacgtatgag-3'. The reaction conditions were: 98℃ for 2 min; 98℃ for 10 s, 60℃ for 20 s, 72℃ for 3 min 10 s, 30 cycles; 72℃ for 5 min; and incubation at 4℃. The PCR product was transformed into E. coli DH5α competent cells. After obtaining positive transformants, the plasmid was extracted using the EZNA Plasmid Extraction Kit and named PDH-MenD-1. The amplification system for the Val48Tyr site mutation PCR was as follows: 2×PimerStarMax 25 μL, upper primer (10 μM) 2.5 μL, lower primer (10 μM) 2.5 μL, template DNA PDH-MenD plasmid (100 ng / μL) 2 μL, ddH2O 18 μL, upstream mutation primer 5'-gtttttcatttatacagataagatcaatatgcagagcattttgatttcc-3', and downstream mutation primer 5'-gatcttatctgtataaatgaaaaacgaaatcagcacaaacaccg-3'. The reaction conditions were: 98℃ for 2 min; 98℃ for 10 s, 60℃ for 20 s, 72℃ for 3 min 10 s, 30 cycles; 72℃ for 5 min; and incubation at 4℃. The PCR product was transformed into *E. coli* DH5α competent cells. Positive transformants were obtained, and the plasmid was extracted using the EZNAPlasmid Extraction Kit and named PDH-MenD-2.The amplification system for Tyr521Trp site mutation PCR was as follows: 25 μL of 2×PimerStarMax, 2.5 μL of upper primer (10 μM), 2.5 μL of lower primer (10 μM), 2 μL of template DNA PDH-MenD plasmid (100 ng / μL), 18 μL of ddH2O, upstream mutation primer 5'-gtttttcatttatacagataagatcaatatgcagagcattttgatttcc-3', and downstream mutation primer 5'-gatcttatctgtataaatgaaaaacgaaatcagcaaacaccg-3'. The reaction conditions were: 98℃ for 2 min; 98℃ for 10 s, 60℃ for 20 s, 72℃ for 3 min 10 s, 30 cycles; 72℃ for 5 min; and incubation at 4℃. The PCR product was transformed into E. coli DH5α competent cells. After obtaining positive transformants, the plasmid was extracted using the EZNA Plasmid Extraction Kit and named PDH-MenD-3. The amplification system for the Gly126His site mutation PCR was as follows: 2×PimerStarMax 25 μL, upper primer (10 μM) 2.5 μL, lower primer (10 μM) 2.5 μL, template DNA PDH-MenD plasmid (100 ng / μL) 2 μL, ddH2O 18 μL, upstream mutation primer 5'-gtttttcatttatacagataagatcaatatgcagagcattttgatttcc-3', and downstream mutation primer 5'-gatcttatctgtataaatgaaaaacgaaatcagcacaaacaccg-3'. The reaction conditions were: 98℃ for 2 min; 98℃ for 10 s, 60℃ for 20 s, 72℃ for 3 min 10 s, 30 cycles; 72℃ for 5 min; and incubation at 4℃. The PCR product was transformed into *E. coli* DH5α competent cells. Positive transformants were obtained, and the plasmid was extracted using the EZNA Plasmid Extraction Kit and named PDH-MenD-4. The amplification system for the Leu493Arg site mutation PCR was as follows: 2×PimerStarMax 25 μL, upper primer (10 μM) 2.5 μL, lower primer (10 μM) 2.5 μL, template DNA PDH-MenD plasmid (100 ng / μL) 2 μL, ddH2O 18 μL, upstream mutation primer 5'-cggattcctgatttcgatcggattattgatcagctat-3', and downstream mutation primer 5'-aatccgatcgaaatcaggaatccgaaaaatgtatttgt-3'.The reaction conditions were: 98℃ for 2 min; 98℃ for 10 s, 60℃ for 20 s, 72℃ for 3 min 10 s, 30 cycles; 72℃ for 5 min; and incubation at 4℃. The PCR product was transformed into *E. coli* DH5α competent cells. Positive transformants were obtained, and the plasmid was extracted using the EZNA Plasmid Extraction Kit and named PDH-MenD-5. The amplification system for the Ala115Tyr site mutation PCR was as follows: 25 μL of 2×PimerStarMax, 2.5 μL of upper primer (10 μM), 2.5 μL of lower primer (10 μM), 2 μL of template DNA PDH-MenD plasmid (100 ng / μL), 18 μL of ddH2O, upstream mutation primer 5'-gtttttcatttatacagataagatcaatatgcagagcattttgatttcc-3', and downstream mutation primer 5'-gatcttatctgtataaatgaaaaacgaaatcagcaaacaccg-3'. The reaction conditions were: 98℃ for 2 min; 98℃ for 10 s, 60℃ for 20 s, 72℃ for 3 min 10 s, 30 cycles; 72℃ for 5 min; and incubation at 4℃. The PCR product was transformed into E. coli DH5α competent cells. After obtaining positive transformants, the plasmid was extracted using the EZNA Plasmid Extraction Kit and named PDH-MenD-6. Plasmids PDH-MenD-1, PDH-MenD-2, PDH-MenD-3, PDH-MenD-4, PDH-MenD-5, and PDH-MenD-6 were then transformed. Bacillus subtilis 168 Host bacteria were used to screen for positive transformants, and recombinant strains expressing the wild-type SEPHCHC synthase MenD mutant were obtained, which were named Bacillus subtilis MenD-1. Bacillus subtle MenD-1), MenD-2 Bacillus subtilis MenD-2), MenD-3 Bacillus subtle MenD-3), MenD-4 Bacillus subtilis MenD-4), MenD-5 Bacillus subtle MenD-5) and MenD-6 Bacillus subtilis MenD-6).

[0030] Example 3: Screening of SEPHCHC synthase MenD mutants

[0031] 3.1 Shake-flask fermentation

[0032] The six recombinant SEPHCHC synthase mutant strains (MenD-1, MenD-2, MenD-3, MenD-4, MenD-5, and MenD-6) and the control strain Bacillus subtilis MenD were inoculated into 5 mL of seed culture medium (0.5% yeast extract, 1% tryptone, 1% NaCl, 25 μg / mL kanamycin) and cultured at 37 °C with shaking at 200 rpm for 12 h. Then, the seed culture was inoculated into 30 mL of fermentation medium (3% glycerol, 6% soybean peptone, 0.5% yeast extract, 0.3% K2HPO4, 0.5% MgSO4·7H2O, 0.1% L-Tryptophan, 3% maltose) at an inoculation rate of OD600 = 0.1 and cultured at 40 °C with shaking at 200 rpm for 96 h.

[0033] 3.2 Collect bacterial cells and detect heptaene-naphthoquinone production

[0034] Add 4 times the volume of extraction buffer (isopropanol: n-hexane = 1:2) to the fermentation broth, shake for 20 min, and perform all operations in the dark. After standing and separating the layers, aspirate the supernatant into a centrifuge tube. Add the chromatographic detection solution to the centrifuge tube after vacuum freeze concentration for reconstitution, and filter the dissolved solution through a 0.22 μm organic filter membrane to obtain the sample detection solution. The yield of heptaene-menaquinone was detected by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS). The detection conditions were as follows: Waters XBridge @ BEH C18 column (2.5 µm, 3.0 × 100 mm), column temperature 40 ℃, mobile phase 100% pure methanol, flow rate 0.8 mL / min, detection wavelength 270 nm, and single injection volume 3 µL. Meanwhile, before the production of heptaene-menaquinone, five standard samples with different concentration gradients were prepared. The production of heptaene-menaquinone in the fermentation broth was measured simultaneously with the standard samples and the fermentation samples. The production of heptaene-menaquinone in the fermentation broth was obtained by calculating the results using the standard curve.

[0035] Specific results: The yield of heptaenoquinone from Bacillus subtilis expressing wild-type SEPHCHC synthase was 61.18 mg / L. The yields of heptaenoquinone after fermentation of the SEPHCHC synthase mutants MenD-1 to MenD-6 were 70.77 mg / L, 75.48 mg / L, 81.72 mg / L, 88.41 mg / L, 99.88 mg / L, and 133.40 mg / L, respectively, which were 116%, 123%, 134%, 144%, 162%, and 218% of the yield of heptaenoquinone from Bacillus subtilis expressing wild-type SEPHCHC synthase MenD.

Claims

1. A SEPHCHC synthase mutant, the amino acid sequence of which is encoded by nucleotides as shown in SEQ ID NO:

7.

2. A gene encoding a SEPHCHC synthase mutant, wherein the gene encodes the SEPHCHC synthase mutant of claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector carries the encoding gene of the SEPHCHC synthase mutant as described in claim 1.

4. The recombinant expression vector as described in claim 3, characterized in that, The encoded gene is generated by promoter P. glv Or P cspd Or P cspB start up.

5. The recombinant expression vector as described in claim 4, characterized in that, The integration site of the coding gene is amyE, ldh, or aprE.

6. The recombinant expression vector as described in claim 4, characterized in that, The encoding gene is promoter P. cspd It is initiated, and the integration site is ldh.

7. A recombinant host cell, characterized in that, The recombinant host cell is a host cell transformed / transfected with the recombinant expression vector according to any one of claims 3-6.

8. The recombinant host cell as described in claim 7, characterized in that, The host cell is Bacillus subtilis.

9. The use of the SEPHCHC synthase mutant of claim 1 or its encoding gene in the preparation of heptaene-menaquinone.

10. A method for preparing heptaene-menaquinone, characterized in that, Culturing the recombinant host cells as described in claim 7 or 8 to produce heptaene-menaquinone.

11. The method as described in claim 10, characterized in that, It also includes the steps of collecting and purifying heptaene-menaquinone.

12. The method as described in claim 10, characterized in that, The fermentation medium used for cultivation consisted of: 3% glycerol, 6% soybean peptone, 0.5% yeast extract, 0.3% K2HPO4, 0.5% MgSO4·7H2O, 0.1% L-Tryptophan, and 3% maltose. The cultivation conditions were 40 ℃ and 200 rpm shaking for 96 h.

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