CN116875571A - P450 enzyme mutant, single plasmid three-enzyme co-expression system and application thereof in calcitol synthesis - Google Patents

P450 enzyme mutant, single plasmid three-enzyme co-expression system and application thereof in calcitol synthesis Download PDF

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CN116875571A
CN116875571A CN202310642328.2A CN202310642328A CN116875571A CN 116875571 A CN116875571 A CN 116875571A CN 202310642328 A CN202310642328 A CN 202310642328A CN 116875571 A CN116875571 A CN 116875571A
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王华磊
柳沁怡
沈亚领
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East China University of Science and Technology
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Abstract

The invention discloses a P450 enzyme mutant, a single plasmid three-enzyme co-expression system and application thereof in calcitol synthesis. The P450 enzyme mutants include: the wild P450 enzyme CYP109E1 shown in SEQ ID NO.1 is used as a template, and mutants T78A, T78V, T L and T78I, G81V and G81M formed by the T mutation at the 78 th position or the G mutation at the 81 th position and mutants T78L/G81M formed by the G mutation at the 78 th position and the G mutation at the 81 th position are formed. The invention also constructs a single plasmid three-enzyme co-expression system of the P450 enzyme mutant and the oxidation-reduction partner, improves the electron transfer efficiency in the hydroxylation reaction of the P450 enzyme, can synthesize the calcitol with higher efficiency under mild conditions, solves the problems of low catalytic efficiency and low activity of the catalytic synthesis of the calcitol by the P450 enzyme, and has important industrial application value.

Description

P450 enzyme mutant, single plasmid three-enzyme co-expression system and application thereof in calcitol synthesis
Technical Field
The invention belongs to the technical field of biocatalysis, and in particular relates to catalytic vitamin D with improved activity 3 P450 enzyme mutant for synthesizing calcitonin, single plasmid three enzyme coexpression system constructed by the same and application of the single plasmid three enzyme coexpression system in calcitonin synthesis.
Background
Calcitonin is vitamin D 3 An active form produced in vivo by cytochrome P450 mediated hydroxylation at position 25, also vitamin D in vivo 3 Is a primary storage form of (c). Activated vitamin D 3 Calcium homeostasis in the body is achieved by regulating the absorption of calcium and phosphorus in the intestine, the reabsorption of phosphate in the kidneys and the release of calcium and phosphate in the bones by binding to vitamin D receptors, which contributes invaluably to the prevention or treatment of various chronic diseases such as cancer, cardiovascular diseases and immune diseases. The calcitriol can be further hydroxylated to form calcitriol, and can be used for treating diseases such as osteomalacia and hypothyroidism, and preventing and treating liver cirrhosis.
The synthesis method of the calcitol mainly comprises a chemical synthesis method and a biocatalysis method. Chemical synthesis converts cholesterol to calcitol in about 20 steps, but the synthesis is cumbersome and environmentally hazardous, and the final yield is not optimistic and difficult to use on a large scale industrially. The biocatalysis method for preparing the calcite glycol has the advantages of high regioselectivity, simple reaction steps and less environmental pollution. Wherein the P450 enzyme-catalyzed hydroxylation reaction is capable of introducing one of the oxygen atoms of the oxygen molecule into an inactive C-H bond under relatively mild conditions, the other being reduced to water. The reaction has both regioselectivity and stereoselectivity, so that the P450 enzyme becomes the most potential enzyme for synthesizing the calcitol, but most of the P450 enzymes are difficult to express in vitro at present and have extremely low catalytic activity.
Cytochrome P450 enzymes are a superfamily of proteins containing type B heme that are widely found in different living bodies in nature, and have chemical catalysts that are incomparableCan catalyze more than 20 reactions including toxin metabolism, hormone synthesis and the like. Co-participation of redox chaperones is required in the P450 catalytic cycle, and P450 enzyme catalytic systems can be divided into three main types depending on the mode of interaction of the redox chaperones and the P450 enzyme: the first is a three-component system that exists independently of each other, including P450 enzymes, ferredoxin reductase (FdR) and ferredoxin (Fdx), and is a class of catalytic systems that are commonly found in prokaryotic sources. The second is a two-component system, commonly found in P450 enzymes of eukaryotic origin, whose redox partners are Cytochrome P450 Reductase (CPR); the third class is the monocomponent system, where the P450 enzyme at the N-terminus and the FAD/FMN-containing redox partner domain at the C-terminus are fused in nature, also known as "self-sufficient" P450 enzymes. Because the P450 enzyme of prokaryotic source can realize low-level soluble expression in vitro and is more suitable for synthesis application, a vitamin D pair is developed 3 Three enzyme system with C-25 hydroxylation activity and method for improving vitamin D of CYP109E1 to unnatural substrate through molecular modification 3 Has great potential for industrial application.
Disclosure of Invention
The invention aims to provide a P450 enzyme mutant, a single plasmid three-enzyme co-expression system and application thereof in calcitol synthesis, thereby solving the problem that the P450 enzyme in the prior art is used for vitamin D which is a non-natural substrate 3 The low catalytic activity causes the problem that the calcitol is low in yield and difficult to be used for industrial production.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to a first aspect of the present invention there is provided a catalytic vitamin D with improved activity 3 The P450 enzyme mutant for synthesizing the calcitol comprises a P450 enzyme mutant which takes a wild P450 enzyme CYP109E1 (derived from bacillus megatherium Bacillus megaterium DSM 319) shown in SEQ ID NO.1 as a template, and is formed by mutating the 78 th amino acid residue or the 81 st amino acid residue as follows: the 78 th threonine T is mutated into a P450 enzyme mutant T78A of alanine A, and the amino acid sequence is shown in SEQ ID NO. 2; p45 with threonine T mutation at position 78 to valine VThe amino acid sequence of the 0 enzyme mutant T78V is shown as SEQ ID NO. 3; the threonine T at position 78 is mutated into leucine L, and the amino acid sequence of the P450 enzyme mutant T78L is shown as SEQ ID NO. 4; and a P450 enzyme mutant T78I with threonine T at position 78 mutated into isoleucine I, the amino acid sequence of the mutant is shown as SEQ ID NO. 5; the 81 st glycine G is mutated into a P450 enzyme mutant G81V of valine V, and the amino acid sequence is shown in SEQ ID NO. 6; and the 81 st glycine G is mutated into a P450 enzyme mutant G81M of methionine M, and the amino acid sequence is shown as SEQ ID NO. 7; and P450 enzyme mutant T78L/G81M in which glycine G at position 81 is mutated into methionine M by using mutant P450 enzyme CYP109E1-T78L shown in SEQ ID NO.4 as a template.
According to a second aspect of the present invention there is provided a gene encoding said P450 enzyme mutant.
According to a third aspect of the present invention, there is provided a recombinant vector comprising the coding gene.
According to a fourth aspect of the present invention, there is provided a method for constructing a single plasmid three enzyme co-expression system, comprising the steps of: 1) Taking a wild P450 enzyme CYP109E1 shown in SEQ ID NO.1 as a template, respectively carrying out T mutation at the 78 th site or G mutation at the 81 st site and G combined mutation at the 78 th site and the 81 st site to form P450 enzyme mutants T78A, T78V, T78L, T78I, G81V, G M and T78L/G81M, and inserting an expression plasmid pET28a (+) -CYP109E1 to obtain various mutant plasmids pET28a (+) -CYP109E1; 2) Respectively amplifying gene sequences of ferredoxin reductase PdR and ferredoxin Pdx from pseudomonas aeruginosa, inserting into expression plasmid pET28a (+), and respectively obtaining plasmid pET28a (+) -PdR and plasmid pET28a (+) -Pdx; 3) And respectively carrying out enzyme digestion on the plasmid pET28a (+) -PdR, the plasmid pET28a (+) -Pdx and any one of the mutant plasmids pET28a (+) -CYP109E1 by using endonucleases, and sequentially connecting the plasmids to transform host escherichia coli BL21 (DE 3), so as to construct a single plasmid three-enzyme co-expression system.
According to the construction method of the single plasmid three-enzyme co-expression system provided by the invention, threonine T at the 78 th site is mutated into a P450 enzyme mutant T78A of alanine A, and the amino acid sequence is shown as SEQ ID NO. 2; the threonine T at the 78 th position is mutated into a P450 enzyme mutant T78V of valine V, and the amino acid sequence is shown as SEQ ID NO. 3; the threonine T at position 78 is mutated into leucine L, and the amino acid sequence of the P450 enzyme mutant T78L is shown as SEQ ID NO. 4; the threonine T at the 78 th position is mutated into a P450 enzyme mutant T78I of isoleucine I, and the amino acid sequence is shown as SEQ ID NO. 5; the 81 st glycine G is mutated into a P450 enzyme mutant G81V of valine V, and the amino acid sequence is shown in SEQ ID NO. 6; the 81 st glycine G is mutated into a P450 enzyme mutant G81M of methionine M, and the amino acid sequence is shown in SEQ ID NO. 7; the mutant T78L/G81M of P450 enzyme with threonine T at 78 position mutated into leucine L and glycine G at 81 position mutated into methionine M has an amino acid sequence shown in SEQ ID NO. 8.
The ferredoxin reductase PdR and the ferredoxin Pdx are both derived from pseudomonas aeruginosa (Pseudomonas putida), the amino acid sequence of the ferredoxin reductase PdR is shown as SEQ ID NO.9, and the amino acid sequence of the ferredoxin Pdx is shown as SEQ ID NO. 10.
According to a fifth aspect of the present invention, there is provided a single plasmid three enzyme co-expression system constructed by the construction method.
Preferably, the single plasmid three enzyme co-expression system adopts a P450 enzyme double-point combined mutant T78L/G81M.
According to a sixth aspect of the present invention there is provided the use of a single plasmid three enzyme co-expression system in the synthesis of calcitonin.
According to a preferred embodiment of the invention, the application comprises the following steps: s1: culturing the single plasmid three-enzyme co-expression system in a TB culture medium, and carrying out induced expression to obtain crude enzyme liquid; s2: mixing the solubilizer hydroxypropyl-beta-cyclodextrin and Tris-HCl buffer solution in a mass-volume ratio of 45%, vortex oscillating, and adding a substrate vitamin D 3 Vortex thoroughly to clear, prepare 4mM mother liquor, add substrate vitamin D 3 Adding the crude enzyme solution and a certain amount of cofactor NAD (P) H into the mixture with the final concentration of 0.3-0.5 mM, putting the mixture into a constant temperature shaking table at 30 ℃ and 200-250 rpm for shaking for 20-30 hours, and carrying out substrate vitamin D 3 Is converted into the calcitolAnd (3) forming the finished product.
The invention firstly uses vitamin D 3 The P450 enzyme CYP109E1 (PDB ID:5L92, resolution) derived from Bacillus megaterium (Bacillus megaterium DSM 319) was docked:) In (C) by administering vitamin D 3 Analysis of the binding pocket for the P450 enzyme and simulation of binding molecular dynamics confirm the 78 th amino acid residue and 81 th amino acid residue which play a key role in the substrate specificity of the P450 enzyme CYP109E 1. After subjecting them to saturation mutation, the activity of each mutant in catalyzing the synthesis of calcitol was determined. To analyze the synergistic effect between them, mutants having greatly improved activity were combined, and the optimal double point mutant CYP109E1-T78L/G81M, i.e., the amino acid residue at position 78 and the amino acid residue at position 81 were simultaneously mutated, was determined. Through the protein engineering strategies, a plurality of mutants with improved P450 enzyme activity are obtained, and the obtained mutants have great application potential for industrial production.
Thus, the present invention uses the wild type P450 enzyme shown in SEQ ID NO.1 as a template, and the amino acid site which plays a key role in activity is determined through analysis, so that the following various P450 enzyme mutants are obtained, wherein the following P450 enzyme mutants comprise: the mutation of T at position 78 to A, V, L and I forms mutants T78A, T78V, T L and T78I; g at position 81 is mutated to V and M to form mutants G81V and G81M; the combined mutation at position 78 and position 81 forms a mutant T78L/G81M.
Furthermore, the invention applies the P450 enzyme mutant to the synthesis of calcitol. The wild P450 enzyme CYP109E1 single plasmid three enzyme co-expression system constructed by the invention converts 0.4mM vitamin D in 24h 3 With only 2.8% conversion, multiple mutants with improved P450 enzyme catalysis were obtained by engineering the binding pocket and saturation mutagenesis strategy, with the two mutants with the greatest degree of improvement CYP109E1-T78L and CYP109E1-G81M at 0.4mM vitamin D 3 The 12h conversion at the concentration increased from 2.8% to 16.9% and 17.1% respectively for wild-type CYP109E1, with the double point combination mutant CYP109E1-T78L/G81M at 0.4mM vitamin D 3 At a concentration ofThe conversion rate is as high as 36.5% after 12 hours.
In summary, the beneficial effects of the invention are as follows: the invention carries out molecular modification on the P450 enzyme CYP109E1 through semi-rational design, obtains a plurality of P450 enzyme mutants with enhanced catalytic activity, and improves vitamin D 3 Is a conversion rate of (a). The invention also constructs a single plasmid three-enzyme co-expression system of the P450 enzyme mutant and the oxidation-reduction partner, improves the electron transfer efficiency in the hydroxylation reaction of the P450 enzyme, can synthesize the calcitol with higher efficiency under mild conditions, and does not find other hydroxylation byproducts, thus having great application value.
Drawings
FIG. 1 is an SDS-PAGE protein gel electrophoresis of a single plasmid three enzyme co-expression system of a wild type P450 enzyme CYP109E1 and redox partners PdR and Pdx, wherein M is a protein molecular weight standard reagent, and A is a cell disruption supernatant of the single plasmid three enzyme co-expression system.
Detailed Description
The invention is further described below in conjunction with specific embodiments. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The technical means used in the examples are, unless specified otherwise, conventional in the art or are in accordance with the experimental methods recommended by the manufacturers of the kits and instruments.
Example 1 Strain construction
The sequences of P450 enzyme CYP109E1 from bacillus megaterium (Bacillus megaterium DSM 319) and redox partners PdR and Pdx from pseudomonas aeruginosa (Pseudomonas putida) are cloned respectively, and inserted into expression plasmid pET28a (+) to obtain pET28a (+) -CYP109E1, pET28a (+) -PdR and pET28a (+) -Pdx. The amino acid sequence of CYP109E1 is shown in SEQ ID NO.1, the amino acid sequence of PdR is shown in SEQ ID NO.9, and the amino acid sequence of Pdx is shown in SEQ ID NO. 10. After sequencing, the three plasmids are transferred into an expression host escherichia coli BL21 (DE 3) respectively for the expression of the subsequent recombinant enzyme.
Example 2 selection of mutation sites
Selecting a target protein crystal structure (PDB ID:5L92, resoluti)on:) As receptor proteins, they are molecularly docked with a substrate ligand. After the butt joint is finished, the results are analyzed and screened according to the order of binding energy and ideal butt joint compound is stored.
Two potential hot spots T78 and G81 located on the substrate binding pocket and possibly affecting the hydrophobic interaction of the substrate and enzyme binding are screened out by analyzing the docking structure, then saturation mutation is carried out on the two amino acid residues, a mutant library is constructed, and the optimal mutants on the two sites are subjected to joint mutation.
EXAMPLE 3 construction of CYP109E1 mutant
E.coli with pET28a (+) -CYP109E1 recombinant plasmid is cultivated in an LB liquid culture medium test tube for 10-12h, the plasmid is extracted as a template for constructing subsequent mutants, and primers (SEQ ID NO. 11-24) for mutation are shown in Table 1.
TABLE 1 mutant primer information
The PCR reaction system is shown in Table 2.
TABLE 2 Point mutation PCR reaction System
PCR reaction conditions: pre-denaturation at 95℃for 3min; denaturation at 98℃for 5s, annealing at 58℃for 15s, extension at 72℃for 6min, 30 cycles of this stage; finally, final extension is carried out for 5min at 72 ℃; preserving at 4 ℃.
After the completion of PCR amplification and positive detection by agarose gel electrophoresis, 0.5. Mu.LDpnI enzyme was added to specifically cleave off methylated template DNA, and the mixture was left in a metal bath at 37℃for 3 hours. Transformation, picking up bacteria and sequencing can be performed subsequently.
EXAMPLE 4 construction of Single plasmid Trienzyme Co-expression System
Plasmid pRSFDuet-1 was cut with NdeI and XhoI, and pET28a (+) -Pdx was PCR amplified by primer pair (listed in Table 3) RSP-Pdx-MCS2-F and RSP-Pdx-MCS2-R, followed by ligation of the digested product and the obtained PCR product with a seamless cloning kit, and transferred into E.coli BL21 (DE 3) cells to obtain pRSFDuet-1-Fdx recombinant strain. The plasmid was further cultured and extracted, the plasmid was cut with NdeI, pET28a (+) -PdR was amplified by PCR with primer pairs RSP-PdR-F and RSP-PdR-R, and then the digested product and the obtained PCR product were transferred into E.coli BL21 (DE 3) cells again by ligation, to obtain pRSFDuet-1-PdR/Pdx recombinant strain. The plasmid was further cultured and extracted, cut open with BamHI and HindIII, PCR amplified with primer pairs RSP-E1-F and RSP-E1-R on pET28a (+) -CYP109E1, and the above ligation and transformation steps were repeated to finally obtain pRSFDuet-1-CYP109E1/PdR/Pdx single plasmid recombinant strain.
The mutants sequenced correctly in example 3 were cultivated and plasmids were extracted for construction of a single plasmid three enzyme co-expression system for the mutants. After pRSFDuet-1-PdR/Pdx plasmid was obtained, it was cut with BamHI and HindIII, PCR amplification was performed on mutant plasmid pET28a (+) -CYP109E1 with primer pairs RSP-E1-F and RSP-E1-R, respectively, and the above ligation and transformation steps were repeated, finally obtaining pRSFDuet-1-mutant CYP109E1/PdR/Pdx single plasmid recombinant strain. The primers used in the co-expression system (SEQ ID NOS.25-30) are shown in Table 3.
TABLE 3 Co-expression System construction of primers required
Example 5 Induction and expression of a Single plasmid Trienzyme Co-expression System
The recombinant cells are coated on a solid flat plate, cultured for 12 hours in a constant temperature incubator at 37 ℃, single colonies are picked up into a 5mL LB culture medium test tube, kanamycin solution with one thousandth concentration is added, and the test tube is placed in a constant temperature shaker at 37 ℃ for shake culture for 12 hours. Transferring 500 μl of bacterial liquid into 50mL of TB liquid culture medium, adding kanamycin solution with one thousandth concentration, and culturing for 2-2.5 hr until cell OD 600 Reaching between 0.6 and 0.8. The cultured bacterial liquid is added with one ten thousandth concentration of inducer IPTG (final concentration is 0.1 mM) for induction, and then the shake flask is placed in a constant temperature incubator at 20 ℃ and 200rpm for incubation for 18 hours. After balancing the bacterial solutions, centrifuging (4 ℃,800 rpm,10 min), discarding the supernatant, adding 15mL of physiological saline for washing for 2 times, taking the washed bacterial bodies, adding 15mL of Tris-HCl buffer (pH 7.4) according to the mass ratio of 1:12, fully vibrating and re-suspending, crushing by an ultrasonic cell crusher, centrifuging the suspension bacterial solutions after crushing (4 ℃,800 rpm,10 min), pouring the supernatant on ice, and obtaining the crude enzyme solution. Wherein, the SDS-PAGE protein gel electrophoresis diagram of the single plasmid three enzyme co-expression system of the wild P450 enzyme CYP109E1 is shown in figure 1.
EXAMPLE 6 screening of mutants
The substrate vitamin D was carried out using the crude enzyme solution prepared in example 5 as a catalyst 3 To screen for mutants with increased conversion.
The reaction system comprises the following components: mixing the solubilizer hydroxypropyl-beta-cyclodextrin and Tris-HCl buffer (20 mM pH 7.4) at 45% mass (g) to volume (mL), vortexing, adding substrate vitamin D 3 Vortex well to clear and prepare 4mM stock solution. mu.L of substrate vitamin D was first added to each reaction tube 3 The mother liquor (final concentration 0.4 mM) was added, then the three enzymes were added to co-express the wet cells, and finally, the reaction was started by adding a certain amount of cofactor NAD (P) H (final concentration 0.4 mM), and the mixture was placed in a constant temperature shaker at 30℃and 250rpm and shaken for 24 hours. The reaction system is shown in Table 4.
TABLE 4 reaction system
The extraction method comprises the following steps: after the reaction, 750. Mu.L of ethyl acetate is added to terminate the reaction and extract, after shaking the mixture at a high speed for 10min, the mixture is put into a high-speed centrifuge to be centrifuged (14000 rpm,10 min), the upper organic phase is collected, the steps are repeated once, the organic phases are combined and placed into a fume hood to be volatilized overnight, 200. Mu.L of chromatographic grade methanol is added, shaking is violently carried out for 5min, the centrifugation is continued in the high-speed centrifuge (14000 rpm,10 min), 150. Mu.L of supernatant is filtered by an organic filter membrane of 0.22 μm, and the sample is loaded in HPLC for detection.
Analysis of vitamin D in the reaction solution by HPLC 3 And the concentration of calcitonin: agilentC18 column (5 μm. Times.4.6 mm. Times.250 mm), mobile phase methanol and water, gradient elution conditions are shown in Table 5, UV detection wavelength: 265nm, column temperature: 30 ℃, flow rate: 1.0mL/min, the sample injection amount is 10 mu L, and the detection time is 25min.
TABLE 5 gradient elution conditions
Vitamin D through the substrate 3 The catalytic activity of the above-constructed mutant single plasmid three-enzyme co-expression system was evaluated, and mutants having higher calcitol production ability than the wild type were obtained as shown in Table 6. As can be seen from the data in the table, the long side chain nonpolar mutations at the T78 and G81 sites have a significant boosting effect on the conversion of calcitol, with the best single point mutants T78L and G81M at 0.4mM vitamin D 3 The conversion rate of 12h under the concentration can reach 16.9 percent and 17.1 percent respectively, and the double-point combined mutant CYP109E1-T78L/G81M is 0.4mM vitamin D 3 The conversion rate is as high as 36.5% at 12h at the concentration.
Table 6P450 enzyme and reaction conversion rate of mutant single plasmid three enzyme co-expression system
The above description is only of the preferred embodiments of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can be changed in various ways, namely, any simple, equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the scope of the claims of the present patent. The present invention is not described in detail in the conventional art.

Claims (10)

1. Catalytic vitamin D with improved activity 3 The P450 enzyme mutant for synthesizing the calcitol is characterized by comprising a P450 enzyme mutant formed by taking a wild P450 enzyme CYP109E1 shown by SEQ ID NO.1 as a template and carrying out the following mutation on the 78 th amino acid residue or the 81 st amino acid residue:
the 78 th threonine T is mutated into a P450 enzyme mutant T78A of alanine A, and the amino acid sequence is shown in SEQ ID NO. 2;
the threonine T at the 78 th position is mutated into a P450 enzyme mutant T78V of valine V, and the amino acid sequence is shown as SEQ ID NO. 3;
the threonine T at position 78 is mutated into leucine L, and the amino acid sequence of the P450 enzyme mutant T78L is shown as SEQ ID NO. 4; and
the threonine T at the 78 th position is mutated into a P450 enzyme mutant T78I of isoleucine I, and the amino acid sequence is shown as SEQ ID NO. 5;
the 81 st glycine G is mutated into a P450 enzyme mutant G81V of valine V, and the amino acid sequence is shown in SEQ ID NO. 6; and
the 81 st glycine G is mutated into a P450 enzyme mutant G81M of methionine M, and the amino acid sequence is shown in SEQ ID NO. 7.
2. Catalytic vitamin D with improved activity 3 The P450 enzyme mutant for synthesizing the calcitonin is characterized by taking mutant P450 enzyme mutant T78L shown in SEQ ID NO.4 as a template, and the amino acid sequence of the P450 enzyme mutant T78L/G81M after the 81 st glycine G is mutated into methionine M is shown in SEQ ID NO. 8.
3. A gene encoding the P450 enzyme mutant according to any one of claims 1-2.
4. A recombinant vector comprising the coding gene of claim 3.
5. The construction method of the single plasmid three enzyme co-expression system is characterized by comprising the following steps:
1) Taking a wild P450 enzyme CYP109E1 shown in SEQ ID NO.1 as a template, respectively carrying out T mutation at the 78 th site or G mutation at the 81 st site and G combined mutation at the 78 th site and the 81 st site to form P450 enzyme mutants T78A, T78V, T78L, T78I, G81V, G M and T78L/G81M, and inserting an expression plasmid pET28a (+) -CYP109E1 to obtain various mutant plasmids pET28a (+) -CYP109E1;
2) Respectively amplifying gene sequences of ferredoxin reductase PdR and ferredoxin Pdx from pseudomonas aeruginosa, inserting into expression plasmid pET28a (+), and respectively obtaining plasmid pET28a (+) -PdR and plasmid pET28a (+) -Pdx;
3) And respectively carrying out enzyme digestion on the plasmid pET28a (+) -PdR, the plasmid pET28a (+) -Pdx and any one of the mutant plasmids pET28a (+) -CYP109E1 by using endonucleases, and sequentially connecting the plasmids to transform host escherichia coli BL21 (DE 3), so as to construct a single plasmid three-enzyme co-expression system.
6. The method according to claim 5, wherein in step 1):
the 78 th threonine T is mutated into a P450 enzyme mutant T78A of alanine A, and the amino acid sequence is shown in SEQ ID NO. 2;
the threonine T at the 78 th position is mutated into a P450 enzyme mutant T78V of valine V, and the amino acid sequence is shown as SEQ ID NO. 3;
the threonine T at position 78 is mutated into leucine L, and the amino acid sequence of the P450 enzyme mutant T78L is shown as SEQ ID NO. 4;
the threonine T at the 78 th position is mutated into a P450 enzyme mutant T78I of isoleucine I, and the amino acid sequence is shown as SEQ ID NO. 5;
the 81 st glycine G is mutated into a P450 enzyme mutant G81V of valine V, and the amino acid sequence is shown in SEQ ID NO. 6;
the 81 st glycine G is mutated into a P450 enzyme mutant G81M of methionine M, and the amino acid sequence is shown in SEQ ID NO. 7;
the threonine T at the 78 th position is mutated into leucine L, the glycine G at the 81 th position is mutated into P450 enzyme mutant T78L/G81M of methionine M, and the amino acid sequence is shown as SEQ ID NO. 8.
7. The construction method according to claim 5, wherein the amino acid sequence of the ferredoxin reductase PdR is shown in SEQ ID NO.9, and the amino acid sequence of the ferredoxin Pdx is shown in SEQ ID NO. 10.
8. A single plasmid three enzyme co-expression system constructed according to the construction method of any one of claims 5 to 7.
9. Use of the single plasmid three enzyme co-expression system of claim 8 in the synthesis of calcitonin.
10. The use according to claim 9, characterized by the steps of:
s1: culturing the single plasmid three-enzyme co-expression system in a TB culture medium, and carrying out induced expression to obtain crude enzyme liquid;
s2: mixing the solubilizer hydroxypropyl-beta-cyclodextrin and Tris-HCl buffer solution in a mass-volume ratio of 45%, vortex oscillating, and adding a substrate vitamin D 3 Vortex thoroughly to clear, prepare 4mM mother liquor, add substrate vitamin D 3 Adding the crude enzyme solution and a certain amount of cofactor NAD (P) H into the mixture with the final concentration of 0.3-0.5 mM, putting the mixture into a constant temperature shaking table at 30 ℃ and 200-250 rpm for shaking for 20-30 hours, and carrying out substrate vitamin D 3 The high-efficiency synthesis of the calcitol can be realized through the conversion reaction of the calcitol.
CN202310642328.2A 2023-05-29 2023-06-01 P450 enzyme mutant, single plasmid three-enzyme co-expression system and application thereof in calcitol synthesis Pending CN116875571A (en)

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