CN118086267A - Fumaric acid enzyme mutant and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of bioengineering, and discloses a fumaric acid enzyme mutant and application thereof. The fumaric acid enzyme mutant is obtained by single mutation of 148 th site in fumaric acid enzyme with an amino acid sequence shown as SEQ ID NO. 1. The specific enzyme activity of the fumaric acid enzyme mutant is 1.45 times that of the wild type, and the kcat/Km value of the malic acid is 2.02 times that of the wild type, so that the utilization efficiency of a substrate is increased, and the yield of a target product fumaric acid is improved. By heterologously expressing the fumaric acid enzyme mutant in Saccharomyces cerevisiae, the yield of fumaric acid is improved by 6.25 times.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a fumaric acid enzyme mutant and application thereof.
Background
Fumaric acid is an important four-carbon organic acid and has wide application as a potential platform chemical in foods, pharmaceuticals, biopolymers, paints, green solvents and plasticizers. The process reported in the prior art for producing fumaric acid is the maleic anhydride (also known as maleic anhydride) isomerisation process. In addition, the process for producing fumaric acid also comprises a furfural oxidation method, a malease catalytic isomerism method, a candida fermentation method, a biomass raw material fermentation method and the like, wherein the maximum yield of fumaric acid cannot be realized by the above methods, and the reduction TCA pathway is reported by relevant documents to be the realization pathway of the maximum yield of fumaric acid, and the maximum enzyme activity of fumaric acid enzyme (FumR) is the key for ensuring the efficient synthesis of fumaric acid. The maximum enzyme activity of the rate-limiting reaction in the whole TCA cytoplasmic circulation reaction plays an important role in promoting the generation of succinic acid. However, the yield of fumaric acid enzyme is low, and the high production cost is one of the main factors which hamper the wide production thereof. In order to better adapt to the industrialized demand, the continuous supply of the malic acid precursor of the fumaric acid by the pyruvate carboxylase and the malate dehydrogenase is realized, and the fumaric acid enzyme has to have higher yield and enzyme activity under the same condition.
In the prior art, rhizopus oryzae is mutagenized by ultraviolet and Y rays of Kang, a mutant strain with the yield of fumaric acid increased by 1.9 times is obtained by screening, and the level of a 5L fermentation tank is 32.1g/L. Wen Jian the strain is equally bred by using the femtosecond laser mutagenesis technology, the positive mutation rate of the mutant strain is 8% -21%, and the fermentation unit is improved by 15% -50% compared with the original strain. However, due to the complex growth morphology of rhizopus fungi, the controllability and industrial scale of the fermentation process is limited; chen Xiulai and the like take Torulopsis glabrata as a host, carry out different amino acid substitutions on four amino acid residues close to the B site of the enzyme surface, finally overexpress the P160A mutant, obtain engineering strain T.G-PMS-P160A, and can generate 5.2g/L fumarate. Genetic engineering means are still difficult in the aspect of strain transformation, and further improvement of the fumaric acid enzyme activity is limited.
Therefore, there is a need to develop a fumaric acid enzyme with high yield so as to exert the catalytic performance of the natural enzyme to the maximum extent, thereby making it more suitable for industrial application.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a fumaric acid enzyme mutant and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a fumaric acid enzyme mutant, which is obtained by single mutation of 148 th site in fumaric acid enzyme with an amino acid sequence shown as SEQ ID NO. 1.
The specific enzyme activity of the fumaric acid enzyme mutant is 1.45 times that of the wild type, and the kcat/Km value of the malic acid is 2.02 times that of the wild type, so that the utilization efficiency of a substrate is increased, and the yield of a target product fumaric acid is improved. By heterologous expression of the fumaric acid enzyme mutant in CEN.PK2SE 04 of the fumaric acid producing Saccharomyces cerevisiae, the yield of fumaric acid was improved by 6.25 times.
As a preferred embodiment of the fumaric acid enzyme mutant, the amino acid sequence is shown as SEQ ID NO. 2. Namely, serine at position 148 is mutated to arginine (S148R).
In a second aspect, the invention provides a nucleic acid encoding the fumarase mutant.
As a preferred embodiment of the nucleic acid of the present invention, the nucleotide sequence is shown in SEQ ID NO. 2.
In a third aspect, the invention provides a recombinant plasmid comprising said nucleic acid.
In a fourth aspect, the invention provides a recombinant bacterium comprising the recombinant plasmid.
As a preferred embodiment of the recombinant bacterium of the present invention, the host cell is any one of Saccharomyces cerevisiae, pichia pastoris, yarrowia lipolytica and Escherichia coli.
In a fifth aspect, the present invention provides a method for high yield of fumaric acid enzyme, comprising the steps of:
culturing the recombinant bacteria, inducing, separating thalli, breaking walls and collecting supernatant; purifying the obtained fumaric acid enzyme.
As a preferred embodiment of the method of the invention, the induction is: when the recombinant bacteria are cultured until the OD 600 is 0.5-0.7, the added IPTG (isopropyl-beta-D-thiogalactoside) is added.
The method comprises the following steps: culturing the recombinant escherichia coli until the OD 600 is within the range of 0.5-0.7, adding 0.4mmol/L IPTG, and inducing for 12h at 25 ℃ and 200 r/min.
In a sixth aspect, the invention applies the fumaric acid enzyme mutant, the nucleic acid, the recombinant plasmid and the recombinant bacterium to fumaric acid production or catalysis of L-malic acid.
Compared with the prior art, the invention has the beneficial effects that:
The specific enzyme activity of the fumaric acid enzyme mutant is 1.45 times that of the wild type, and the kcat/Km value of the malic acid is 2.02 times that of the wild type, so that the utilization efficiency of a substrate is increased, and the yield of a target product fumaric acid is improved. By heterologous expression of the fumaric acid enzyme mutant in CEN.PK2SE 04 of the fumaric acid producing Saccharomyces cerevisiae, the yield of fumaric acid was improved by 6.25 times.
Drawings
FIG. 1 is a map of the molecular docking sites of fumaric and malic acid in example 1 of the present invention;
FIG. 2 is a schematic diagram showing the mutation site of fumaric acid enzyme in example 1 of the present invention;
FIG. 3 shows the effect of fumaric acid mutants (SE 051) on fumaric acid production in example 5 of the invention.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting. In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
In the following examples and comparative examples, the test methods used, unless otherwise specified, were conventional; the materials, reagents and the like used, unless otherwise specified, are all commercially available. The pET28a (+) -WT was purchased from Optimago Corp. The pGL0_16 is purchased from Addgene.
Example 1: homology modeling and molecular docking simulation
And carrying out homologous protein modeling and evaluation on the protein which is not subjected to tertiary structure analysis by using computer simulation software such as pymol, autoDock and the like to obtain the required mutant enzyme. The substrate L-malic acid was molecularly docked with the fumaric acid enzyme, and the RMSD threshold was set at 1.0 angstrom to ensure as diverse docking conformations as possible, and the docking mode with the highest scoring function was selected.
The fumaric acid enzyme (RoFumR) from rhizopus oryzae Rhizopus oryzae ATCC and 10260 (GenBank: P55250.1, namely SEQ ID NO: 1) is subjected to virtual mutation, molecular simulation software is utilized to carry out rational design on the fumaric acid enzyme of Rhizopus oryzae ATCC and 10260, and mutation sites are selected, so that the screening time of the mutation sites can be effectively saved, and the mutation efficiency is improved.
And (3) carrying out data analysis on the butt joint result to determine the spatial distance and the action relation of each amino acid in the active region. Based on the interaction diagram analysis shown in FIG. 1, the site where the single mutation was performed was selected as S148 (see FIG. 2, where serine at position 148 was mutated to arginine). The amino acid sequence of the obtained mutant is shown as SEQ ID NO.2, and the nucleotide sequence is shown as SEQ ID NO. 3.
Example 2: construction and expression of mutation sites
The candidate mutation site S148 of example 1 was mutated by a method of introducing site-directed mutation using whole plasmid PCR. Aiming at the selected mutation site, a primer is designed, a rhizopus oryzae fumaric acid enzyme gene (GenBank: P55250.1, namely SEQ ID NO: 1) is used as a template for site-directed PCR amplification, a mutant with single point mutation is obtained, and the mutation with improved enzyme activity is screened.
The site-directed mutagenesis primer is as follows:
upstream primer (S148R): 5'-AATATGTCTCAGTCTAGAAACGACACCTTTCCG-3' the process of the preparation of the pharmaceutical composition,
Downstream primer (S148R): 5'-CGGAAAGGTGTCGTTAGAAGACTGAGACATATT-3'.
The PCR reaction system is as follows:
2X pfx mix 25. Mu.L, forward primer (10. Mu. Mol. L -1) 1. Mu.L, reverse primer (10. Mu. Mol. L -1) 1. Mu.L, template DNA 1. Mu.L, distilled water to 50. Mu.L; wherein the template is pET28+ (a) -WT, and the mutant plasmid is constructed according to the primer sequences shown above.
The PCR amplification procedure was set as follows:
firstly, pre-denaturation at 98 ℃ for 4min; then enter 30 cycles: denaturation at 98℃for 30s, annealing at 55℃for 30s, and extension at 72℃for 8min; finally, the mixture is extended for 10min at 72 ℃ and is kept at 4 ℃. The PCR products were detected by 1% agarose gel electrophoresis.
Dpn I is added into the PCR product for verification, the temperature is 37 ℃, the water bath is carried out for 2 hours, the template is degraded, then E.coli JM109 competent cells are transformed, the transformed product is coated on LB solid culture medium containing 50 mg.mL -1 kanagacillin, the culture is carried out for 10 hours to 12 hours at the temperature of 37 ℃, positive clones are picked, and the LB liquid culture medium is cultured for 8 hours to 10 hours.
Wild-type and inoculated with the correctly sequenced mutant S148R was grown in LB broth (containing 30 mg. ML -1 kanapigenin) for 8h and seeds were inoculated in 5% inoculum size into TB broth (containing 30 mg. ML -1 kanapigenin). E.coli BL21 is cultivated for 2 hours at 37 ℃, IPTG is added, and the mixture is transferred into a shaking table at 25 ℃ to continue cultivation and fermentation for 24 hours, the fermentation liquor is centrifuged for 10 minutes at 4 ℃ and 12000rpm, the supernatant is discarded, bacterial cells are collected, 50mL 25mM pH 7.0PPS buffer solution is added into the bacterial cells, after the bacterial cells are fully resuspended, the wall breaking is carried out by using an ultrasonic wall breaking machine, and after centrifugation for 5 minutes at 10000rpm, the wall breaking supernatant is collected.
Example 3: wild-type and mutant S148R purification
Breaking the cell walls of the recombinant bacteria of the wild type and the mutant S148R obtained in the example 2 respectively, and adding 175g of solid ammonium sulfate into the supernatant to carry out salting-out for 12 hours; centrifuging the salted crude enzyme solution at 4 ℃ and 10000rpm for 20min, dissolving the precipitate in a buffer solution A containing 50mM sodium phosphate, 0.5M sodium chloride, 20mM imidazole and pH 7.4, dialyzing in the buffer solution A for 10h, and filtering through a 0.22 μm membrane to prepare a sample;
After the Ni affinity column is equilibrated by the buffer A, sucking the sample into the Ni column to make the sample completely adsorbed, respectively eluting by using 100mL of the buffer A, 100mL of the buffer A containing 25mM imidazole, 100mL of the buffer A containing 50mM imidazole, 100mL of the buffer A containing 75mM imidazole and 100mL of the buffer A containing 500mM imidazole in sequence at a flow rate of 1mL min -1, and collecting the part of the eluent; the active component (buffer A containing 500mM imidazole) was dialyzed against 50mM phosphate buffer, pH5.0, for 10 hours to obtain a purified enzyme preparation.
Example 4: enzymatic reaction kinetics of wild-type and mutant enzymes
The enzyme kinetics of the enzymatic reaction was determined using the wild-type fumaric acid enzyme obtained by fermentation in example 3 as a control, and the fumaric acid enzyme mutants obtained in this example as a test example, using L-malic acid as a substrate, using different concentrations of L-malic acid to react with the wild-type and different mutant purified enzymes at their optimal temperatures for 10min, measuring their absorbance at 290nm, and calculating their enzyme activities. K m and V max were obtained by nonlinear fitting curve analysis using Origin software, and the K cat values were calculated by combining the results of the fitting analysis with the protein molecular weight.
The measurement results are shown in Table 1:
TABLE 1 kinetics of enzymatic reactions
| Enzymes | Km(mM) | kcat(s-1) | kcat/Km(mM-1·s-1) |
| WT | 52.2±0.6 | 245.3±11.0 | 4.7±0.7 |
| S148R | 25.4±0.2 | 210.8±3.2 | 8.3±0.1 |
Compared with the wild type fumaric acid enzyme, the k cat/Km of the S148R is obviously increased, the catalytic efficiency is improved by 2.02 times from 4.7 of the wild type fumaric acid enzyme to 8.3.
Example 5: application of mutant enzyme in producing fumaric acid
The construction method of the saccharomyces cerevisiae engineering bacteria comprises the following steps:
(1) PCR amplification or chemical synthesis of RoFumR fragment with nucleotide sequence shown as SEQ ID NO.1 and mutation RoFumR-S148R fragment;
(2) The RoFumR fragment and the mutant RoFumR-S148R fragment were ligated to plasmid pGL0_16 to obtain two recombinant plasmids pGL0_16-RofumR, pGL0_16-RofumR-S148R, respectively, and verified;
(3) The recombinant plasmids obtained in the last step are respectively transformed into recipient bacteria S.cerevisiae CEN.PK2SE 04, and Ura flat plates are coated for screening;
(4) And verifying correct recombinant bacteria, namely the saccharomyces cerevisiae engineering bacteria for producing fumaric acid.
The obtained fumaric acid enzyme mutant S148R is introduced into Saccharomyces cerevisiae CEN.PK2SE04 to obtain Saccharomyces cerevisiae experimental strain CEN.PK2SE 051, and simultaneously, wild type fumaric acid enzyme is also introduced into Saccharomyces cerevisiae CEN.PK2SE04 to obtain Saccharomyces cerevisiae control strain CEN.PK2SE 052. That is, the experimental strain cen.pk2se 051 and the control strain cen.pk2se 052 (containing RoFumR wild type) differ only in that the experimental strain is capable of expressing RoFumR _s148R and the control strain expresses RoFumR wild type.
Selecting Saccharomyces cerevisiae CEN.PK2SE 051 and Saccharomyces cerevisiae CEN.PK2SE 052 single colony from the plate, inoculating into a test tube containing 5mL YPD culture medium, and shake culturing at 30deg.C and 220rpm for about 24 hr; after the bacterial liquid OD 600 reaches 5-8, transferring the bacterial liquid OD 600 into a 500mL conical flask containing 100mL YPD culture medium, continuing to perform fermentation experiments in a shaking table at 30 ℃ and 220rpm for 72h, sampling and detecting the yield of fumaric acid at intervals.
The method for detecting the yield of fumaric acid comprises the following steps:
Determining the amount of reaction product in the sample by high performance liquid chromatography (high performance liquid chromatography, HPLC); the detection conditions are as follows: agilent-C18 column (4.6 mm. Times.250 mm,5 um), mobile phase 0.02mol/L sodium phosphate solution (pH 7.0) -acetonitrile (80:20); the detection wavelength is 210mm, the flow rate is 1.0mL/min, and the sample injection amount is 10 mu L.
As shown in FIG. 3, the yield of fumaric acid was increased by 6.25-fold by the heterologous expression of the fumaric acid enzyme mutant in CEN.PK2SE 04 of Saccharomyces cerevisiae compared to the control strain.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A fumarase mutant is characterized by being obtained by single mutation of 148 th site in fumarase with an amino acid sequence shown as SEQ ID NO. 1.
2. The fumaric acid enzyme mutant according to claim 1, wherein the amino acid sequence is shown in SEQ ID NO. 2.
3. A nucleic acid encoding the fumarase mutant of claim 1.
4. The nucleic acid of claim 3, wherein the nucleotide sequence is set forth in SEQ ID NO. 2.
5. A recombinant plasmid comprising the nucleic acid of claim 3.
6. A recombinant bacterium comprising the recombinant plasmid according to claim 5.
7. The recombinant bacterium according to claim 6, wherein the host cell is any one of Saccharomyces cerevisiae, pichia pastoris, yarrowia lipolytica, and Escherichia coli.
8. A method for producing fumaric acid enzyme in high yield, comprising the steps of:
culturing the recombinant bacterium according to claim 6 or 7, inducing, separating thalli, breaking walls and collecting supernatant; purifying the obtained fumaric acid enzyme.
9. The method of claim 8, wherein the inducing is: and culturing the recombinant strain until the OD 600 is 0.5-0.7, and adding IPTG.
10. Use of the fumarate enzyme mutant of claim 1 or 2, the nucleic acid of claim 3 or 4, the recombinant plasmid of claim 5, the recombinant bacterium of claim 6 or 7 in fumaric acid production or catalysis of L-malic acid.
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