P450 enzyme mutant CYP107D1-MTM

Abstract

The invention provides a P450 enzyme mutant capable of efficiently catalyzing LCA to synthesize UDCA, compared with a reported P450 enzyme mutant CYP107D1, the yield of UDCA is increased by 33%-67.3%, and the yield is the highest yield of UDCA production through Escherichia coli whole-cell catalysis of LCA at present. In addition, the mutant is applied to industrial biotransformation of LCA to synthesize UDCA, reaction conditions are mild, steps are simple, and the mutant has the advantages of being green and environmentally friendly.

Description

Technical field:

[0001] The invention belongs to the field of enzyme mutant construction and application, and specifically relates to a mutant of a P450 enzyme mutant CYP107D1-MTM and application thereof. Background technology:

[0002] Ursodeoxycholic acid (UDCA) has a relatively good therapeutic effect in dissolving cholesterol gallstones. UDCA has a litholytic effect. Its pharmacological action is mainly through inhibiting the reabsorption of cholesterol in the intestine to reduce the saturation of cholesterol in bile, while enhancing the degree of cholesterol transport in bile, and finally making cholesterol gradually dissolve from the surface of the stone. As a hydrophilic endogenous bile acid with multiple biological activities, UDCA can not only dissolve gallstones, but also treat cholestasis, biliary pancreatitis, primary cholangitis, colitis, (non) alcoholic hepatitis and drug-induced hepatitis; and improve the outcome of liver transplantation; and UDCA evolved by intestinal microorganisms also reduces colon inflammation in low birth weight infants by enhancing the polarization of macrophage M2. In recent years, it has been found that UDCA can also mediate and influence the regulation of oncogenic signaling pathways in the tumor genome and be used for cancer treatment; it can specifically regulate the apoptosis threshold, inhibit cancer cell growth, induce autophagy and apoptosis, and it has also been found that it can improve damaged mitochondrial function and play a neuroprotective role in the treatment of progressive neurological diseases.

[0003] At present, the synthesis methods of UDCA include chemical synthesis and biosynthesis. Among them, chemical synthesis faces many problems, such as low yield, expensive and environmentally unfriendly reagents, and complicated steps. With the continuous development of science and technology, a research team has transformed the P450 enzyme that can hydroxylate the C7 position of lithocholic acid (LCA). Producing UDCA through microorganisms is a production method with milder reaction conditions and more environmentally friendly.

[0004] The mutant of cytochrome P450 enzyme CYP107D1 can catalyze the C7β hydroxylation of LCA to produce UDCA (Sascha Grobe et al. Engineering regioselectivity of a P450 monooxygenase enables the synthesis of ursodeoxycholic acid via 7β-hydroxylation of lithocholic acid. Angew. Chem. Int. Ed. 2021, 60, 753-757), but it still has problems such as low activity, poor stability and poor catalytic specificity in the process of catalyzing the hydroxylation reaction of LCA.

[0005] In view of the prominent problem of low catalytic activity of the above enzymes, it is very important for large-scale production of UDCA to improve the catalytic efficiency through rational design and targeted modification of the enzymes. Summary of the invention:

[0006] Based on the above reasons, the present invention provides a P450 enzyme mutant with higher catalytic activity from the perspective of industrial production.

[0007] The technical solution of the present invention is as follows:

[0008] A P450 enzyme mutant CYP107D1-MTM is obtained by using a cytochrome P450 monooxygenase CYP107D1 mutant derived from antibiotic Streptomyces (whose nucleotide sequence and amino acid sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 4, respectively) as a parent, and by mutating the key amino acid residues of its electron transport chain and catalytic active center through site-directed mutagenesis technology. The specific mutation sites are as follows:

[0009] The invention is based on a mutant of cytochrome P450 monooxygenase CYP107D1 of antibiotic Streptomyces, and is obtained by mutating at least one of the 58th, 86th or 197th amino acid residues in its amino acid sequence (shown in SEQ ID NO: 4); wherein: the mutation of the 58th amino acid residue is from Met to Phe, His, Trp or Tyr; the mutation of the 86th amino acid residue is from Thr to Cys, Leu, Val or Trp; and the mutation of the 197th amino acid residue is from Met to Ala or Tyr.

[0010] Furthermore, the present invention provides a DNA molecule comprising a nucleotide sequence encoding the mutant CYP107D1-MTM.

[0011] Furthermore, the present invention provides a recombinant expression vector comprising the above-mentioned DNA molecule.

[0012] Preferably, the recombinant expression vector is a pET28a vector.

[0013] Furthermore, the present invention provides a host cell, wherein the host cell contains the above-mentioned recombinant expression vector.

[0014] Preferably, the host cell is Escherichia coli.

[0015] Furthermore, the present invention provides a method for constructing a recombinant expression vector comprising the above mutant CYP107D1-MTM gene sequence, characterized by comprising the following steps:

[0016] 1) Connecting the gene sequence of the cytochrome P450 monooxygenase CYP107D1 mutant of Streptomyces antibioticus to the pET28a vector to obtain a pET28a recombinant vector containing the gene;

[0017] 2) Using the pET28a recombinant vector as a template, designing site-directed mutagenesis primers to perform site-directed mutagenesis PCR amplification reaction, and the reaction product is digested with Dpn I enzyme to remove the template and then purified and recovered to obtain the target product;

[0018] 3) transforming the target product obtained in step 2) into competent E. coli cells, reviving and culturing them on plates overnight until transformants grow;

[0019] 4) The transformants were picked and placed in LB liquid medium containing kanamycin. After overnight culture for 12 hours, the plasmids were extracted for sequencing verification. The plasmids with correct sequencing were the recombinant expression vectors containing the mutant CYP107D1-MTM gene sequence.

[0020] Preferably, in the above step 2), the site-directed mutagenesis primers: the nucleotide sequences of the forward primer M58W-F and the reverse primer M58W-R used to introduce the mutation of the amino acid residue at position 58 are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively; the nucleotide sequences of the forward primer T86L-F and the reverse primer T86L-R used to introduce the mutation of the amino acid residue at position 86 are shown in SEQ ID NO:9 and SEQ ID NO:10, respectively.

[0021] Furthermore, the present invention provides a method for constructing an engineered strain using the above-mentioned recombinant expression vector containing the mutant CYP107D1-MTM gene sequence, characterized in that: the recombinant expression vector containing the mutant CYP107D1-MTM gene sequence is transformed into Escherichia coli competent cells by heat shock transformation, and after recovery culture, the cells are plated and cultured overnight until transformants grow out; the transformants are picked up in LB liquid culture medium containing kanamycin, shaken and cultured at 37°C and 220rpm / min for 12h, and then cultured in transfer bottles to obtain the engineered strain.

[0022] The present invention mutates and transforms the key amino acid residues of the electron transport chain and catalytic active center of the cytochrome P450 monooxygenase CYP107D1 mutant derived from antibiotic Streptomyces, and the catalytic activity of the obtained mutant is significantly improved. By comparing with the original CYP107D1 mutant, it is found that the UDCA yield is increased by 33% to 67.3%, which is the highest yield of UDCA produced by LCA catalyzed by the whole cell of Escherichia coli known so far. In addition, the mutant of the present invention is applied to the industrial bioconversion of LCA to synthesize UDCA, which not only has mild reaction conditions and simple steps, but also has the advantages of being green and environmentally friendly. Description of the drawings:

[0023] Figure 1 HPLC profile of control whole cells after 24 h of LCA reaction.

[0024] Figure 2 HPLC profile of mutant 1 whole cells after 24 h of LCA reaction.

[0025] Figure 3 HPLC profile of mutant 2 whole cells after 24 h of LCA reaction. Specific implementation method:

[0026] The method of the present invention is described below by way of specific embodiments. Unless otherwise specified, the technical methods (including pharmaceutical reagents) used in the present invention are all methods known to those skilled in the art.

[0027] Example 1

[0028] Construction of CYP107D1-MTM mutant 1:

[0029] 1) Construction of CYP107D1 mutant recombinant vector: The CYP107D1 mutant gene sequence (SEQ ID NO: 1) was synthetically connected to the pET28a vector to obtain a recombinant expression vector containing the CYP107D1 mutant gene. The pET28a vector in this step can also be replaced by other pET vectors.

[0030] 2) Site-directed mutagenesis: Using the recombinant vector containing the CYP107D1 mutant gene as a template, site-directed mutagenesis primers were designed, including forward primer M58W-F and reverse primer M58W-R (nucleotide sequences are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively) for site-directed mutagenesis PCR amplification reaction, and the reaction system is shown in Table 1. The purpose is to change the 58th amino acid residue of the CYP107D1 mutant from Met to Trp. PCR reaction conditions and process: 95℃ pre-denaturation for 30s; 95℃ denaturation for 10s; 66℃ annealing for 20s; 72℃ extension for 7min; 72℃ extension for another 2min after 25 cycles:

[0031] Table 1 Site-directed mutagenesis PCR reaction system

[0032]

[0033] The PCR product after the reaction was digested with Dpn I enzyme to remove the template, and the target product was purified and recovered after 37°C for 30 minutes. The Dpn I digestion system is shown in Table 2.

[0034] Table 2 Dpn I digestion system

[0035]

[0036] 3) Transformation and sequencing verification: Take 10 μL of the target product and transform it into E. coli JM109 competent cells by heat shock method. After recovery culture, plate it and culture it overnight. When the transformants grow out, pick the transformants and place them in LB liquid culture medium containing kanamycin. After culturing for 12 hours at 37°C and 220rpm / min, extract the plasmid for sequencing verification. The plasmid with correct sequencing is the recombinant expression vector containing the gene sequence of CYP107D1-MTM mutant 1. The nucleotide sequence of mutant 1 is shown in SEQ ID NO:2, and the amino acid sequence is shown in SEQ ID NO:5.

[0037] Example 2

[0038] Whole-cell catalytic reaction of mutant 1:

[0039] 1) Transformant induction culture: The recombinant expression vector containing the gene sequence of mutant 1 was transformed into the competent cells of E. coli BL21 (DE3) containing the redox chaperone Pdr / Pdx by heat shock method, and then plated and cultured overnight after recovery culture; after the transformants grew out, the transformants were picked up and placed in a 5 mL LB tube containing 50 μg / mL kanamycin, cultured at 37°C and 220 rpm / min for 12 h, and then expanded by transfer flask with a volume ratio of 2%. When the bacterial solution concentration OD 600 When the pH value reached 0.6-0.8, IPTG with a final concentration of 0.4 mM, ALA with a final concentration of 0.64 mM and FeSO4 with a final concentration of 0.3 mM were added, and the culture was induced at 30°C and 200 rpm / min for 20 h.

[0040] 2) Whole-cell catalysis of LCA to UDCA: After induction culture, substrate LCA was added at a feed rate of 0.1 mg / mL, and the culture was continued in a shaker at 30°C. After 24 hours, 1 mL of the sample was taken out, and the sample was extracted with 1 mL of ethyl acetate and the upper organic phase was taken out. After drying in a fume hood, it was redissolved with 200 μL of methanol, filtered with a 0.22 μm organic filter membrane, and detected by HPLC.

[0041] The same method was used to carry out the LCA reaction catalyzed by the whole cell of CYP107D1 mutant, and the reaction results were used as the control. The liquid phase results of the whole cell catalyzed LCA reaction of the control and mutant 1 after 24 hours were as follows: Figure 1 , 2The yield is shown in Table 5. The yield of mutant 1 was 3.3 times higher than that of the control, and 1.34 times higher than that reported in the literature (Sascha Grobe et al. Engineering regioselectivity of a P450 monooxygenase enables the synthesis of ursodeoxycholic acid via 7β-hydroxylation of lithocholic acid. Angew. Chem. Int. Ed. 2021, 60, 753-757).

[0042] Example 3

[0043] Construction of CYP107D1-MTM mutant 2:

[0044] 1) Site-directed mutagenesis: Using the CYP107D1 mutant recombinant vector constructed in Example 1 as a template, site-directed mutagenesis primers were designed, including forward mutation primer T86L-F and reverse mutation primer T86L-R (nucleotide sequences are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively) for site-directed mutagenesis PCR amplification reaction, and the reaction system is shown in Table 3. The purpose of the reaction is to change the 86th amino acid residue of the CYP107D1 mutant from Thr to Leu. PCR reaction conditions and process: 95°C pre-denaturation for 30s; 95°C denaturation for 10s; 66°C annealing for 20s; 72°C extension for 7min; 72°C extension for another 2min after 25 cycles:

[0045] Table 3 Site-directed mutagenesis PCR reaction system

[0046]

[0047] The PCR product after the reaction was digested with Dpn I enzyme to remove the template, and the target product was purified and recovered after 37°C for 30 minutes. The Dpn I digestion system is shown in Table 4.

[0048] Table 4 Dpn I digestion system

[0049]

[0050] 2) Transformation and sequencing verification: Take 10 μL of the target product and transform it into E. coli JM109 competent cells by heat shock method. After recovery culture, plate it and culture it overnight. When the transformants grow out, pick the transformants and place them in LB liquid culture medium containing kanamycin. After culturing for 12 hours at 37°C and 220rpm / min, extract the plasmid for sequencing verification. The plasmid with correct sequencing is the recombinant expression vector containing the gene sequence of CYP107D1-MTM mutant 2. The nucleotide sequence of mutant 2 is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:6.

[0051] Example 4

[0052] Whole-cell catalytic reaction of mutant 2:

[0053] The transformant containing the gene sequence of mutant 2 was constructed and induced to culture, and after culture, the whole cell catalyzed LCA to synthesize UDCA. The method was as described in Example 2.

[0054] The liquid phase results after 24h of LCA reaction catalyzed by CYP107D1-MTM mutant 2 whole cells are as follows Figure 3 The yield is shown in Table 5. The yield of mutant 2 was 4.1 times higher than that of the control, and 1.69 times higher than that reported in the literature (Sascha Grobe et al. Engineering regioselectivity of a P450 monooxygenase enables the synthesis of ursodeoxycholic acid via 7β-hydroxylation of lithocholic acid. Angew. Chem. Int. Ed. 2021, 60, 753-757).

[0055] Table 5 The yield of UDCA produced by whole cells catalyzed by LCA of mutant 1, mutant 2, control and literature reports

[0056]

Claims

1. A P450 enzyme mutant CYP107D1-MTM, characterized in that: The mutant is obtained after at least one of the 58th, 86th or 197th amino acid residues on the amino acid sequence shown in SEQ ID NO: 4 is mutated; wherein: the mutation of the 58th amino acid residue is from Met to Phe, His, Trp or Tyr; the mutation of the 86th amino acid residue is from Thr to Cys, Leu, Val or Trp; the mutation of the 197th amino acid residue is from Met to Ala or Tyr.

2. A DNA molecule, characterized in that: The DNA molecule comprises a nucleotide sequence encoding the mutant CYP107D1-MTM.

3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the DNA molecule according to claim 2.

4. The recombinant expression vector according to claim 3, characterized in that: The recombinant expression vector is a pET28a vector.

5. A host cell, characterized in that: The host cell contains the recombinant expression vector according to claim 4.

6. A method for constructing a recombinant expression vector comprising the gene sequence of the mutant CYP107D1-MTM as claimed in claim 1, characterized in that The steps include: 1) Connecting the gene sequence of the cytochrome P450 monooxygenase CYP107D1 mutant of Streptomyces antibioticus to the pET28a vector to obtain a pET28a recombinant vector containing the gene; 2) Using the pET28a recombinant vector as a template, designing site-directed mutagenesis primers to perform site-directed mutagenesis PCR amplification reaction, and the reaction product is digested with Dpn I enzyme to remove the template and then purified and recovered to obtain the target product; 3) transforming the target product obtained in step 2) into competent E. coli cells, reviving and culturing them on plates overnight until transformants grow; 4) The transformants were picked and placed in LB liquid culture medium containing kanamycin. After overnight culture for 12 hours, the plasmids were extracted for sequencing verification. The plasmids with correct sequencing were the recombinant expression vectors containing the mutant CYP107D1-MTM gene sequence.

7. The method for constructing a recombinant expression vector according to claim 6, characterized in that: In the step 2), the nucleotide sequences of the site-directed mutagenesis primers: the forward primer M58W-F and the reverse primer M58W-R used to introduce the mutation of the amino acid residue at position 58 are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; the nucleotide sequences of the forward primer T86L-F and the reverse primer T86L-R used to introduce the mutation of the amino acid residue at position 86 are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

8. A method for constructing an engineering strain comprising the recombinant expression vector according to claim 4, characterized in that: The recombinant expression vector was transformed into competent Escherichia coli cells by heat shock transformation method, and after recovery culture, the cells were plated and cultured overnight until transformants grew out; the transformants were picked up and placed in LB liquid culture medium containing kanamycin, and cultured at 37°C and 220 rpm / min for 12 hours, and then cultured in transfer bottles to obtain engineered strains.

9. Use of the mutant CYP107D1-MTM as claimed in claim 1 in biotransformation of lithocholic acid to synthesize ursodeoxycholic acid.

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