5-aminolevulinic acid synthetase mutant and application thereof

Abstract

The invention relates to the technical field of synthetic biology and microbial fermentation, in particular to a 5-aminolevulinic acid synthetase mutant and application thereof. According to the invention, 5-aminolevulinic acid synthetase derived from rhodobacter capsulatus is expressed in recombinant Escherichia coli, point mutation is carried out on non-conservative amino acid of wild-type ALAS to obtain I86C mutants, the enzymatic activity of the 5-aminolevulinic acid synthetase is improved, and the ability of the 5-aminolevulinic acid synthetase in synthesis of 5-aminolevulinic acid is improved. The method can be used for solving the problems that the enzyme activity of the ALAS is low and the activity of the ALAS is easily inhibited by high-concentration substrate glycine and the like at present, and the yield of the 5-ALA is increased.

Description

Technical Field

[0001] The present invention relates to the field of synthetic biology and microbial fermentation technology, and in particular to a 5-aminolevulinic acid synthase mutant and application thereof. Background Art

[0002] 5-aminolevulinic acid (5-ALA, molecular formula NH2CH2COCH2CH2COOH) is widely present in the cells of bacteria, fungi, animals and plants. It is a synthetic porphyrin, heme, chlorophyll, vitamin B 12 Metabolic intermediates of tetrapyrrole compounds such as 5-aminolevulinic acid are widely used in agricultural production. They can not only be used as herbicides and pesticides, but also improve the salt and freezing resistance of plants. This has important practical significance for alleviating the deterioration of the ecological environment and improving the level of agricultural production. In addition, 5-aminolevulinic acid is also widely used in the diagnosis of tumors and the treatment of cancer.

[0003] Many microorganisms, plants and animals in nature can synthesize 5-ALA naturally. There are two pathways for synthesis: C4 pathway and C5 pathway. C5 pathway mainly exists in higher plants, algae and bacteria (such as Escherichia coli). Escherichia coli and Corynebacterium glutamicum Corynebacterium glutamicum ), the C4 pathway is mainly found in animals, fungi, and non-sulfur photosynthetic bacteria (such as Rhodobacter sphaeroides Rhodobacter spherical and Rhodopseudomonas palustris Rhodopseudomonas palustris ), the key enzyme in the C4 pathway is hemA The gene encodes 5-aminolevulinic acid synthase (ALAS), glycine and succinyl-CoA generate 5-ALA under the catalysis of ALAS. ALAS is the rate-limiting enzyme in 5-ALA synthesis. Currently, the main method for obtaining 5-ALA high-yield engineered strains is to enhance the expression of key enzymes for 5-ALA synthesis, thereby obtaining a higher extracellular 5-ALA yield, but there are relatively few reports on the modification of the ALAS enzyme itself. Summary of the invention

[0004] The present invention expresses Rhodobacter capsulatum in recombinant Escherichia coli ( Rhodobacillus capsulatum) from 5-aminolevulinic acid synthase (ALAS), and improve its enzyme activity and its ability to synthesize 5-aminolevulinic acid (5-ALA) by making point mutations in non-conservative amino acids of wild-type ALAS. This can be used to solve the current problems of low ALAS enzyme activity and susceptibility to inhibition of activity by high concentrations of the substrate glycine, and increase the production of 5-ALA.

[0005] In order to solve the above problems, the technical route adopted by the present invention is as follows: One of the technical solutions provided by the present invention is a 5-aminolevulinic acid synthetase mutant, which is obtained by mutating the 86th amino acid from isoleucine I to cysteine ​​C on the basis of the wild-type 5-aminolevulinic acid synthetase shown in SEQ ID NO.1, and the amino acid sequence of the 5-aminolevulinic acid synthetase mutant is shown in SEQ ID NO.3; The present invention also provides a gene encoding the 5-aminolevulinic acid synthetase mutant described in one of the technical solutions, and the nucleotide sequence of the gene encoding is shown in SEQ ID NO.4.

[0006] The second technical solution provided by the present invention is a recombinant vector or recombinant strain comprising the above mutant encoding gene; Furthermore, the expression plasmid used in the recombinant vector is pET28a; Furthermore, the host cell used by the recombinant strain is Escherichia coli BL21 (DE3).

[0007] The third technical solution provided by the present invention is the application of the recombinant vector or recombinant strain described in the second technical solution, especially in the production of the 5-aminolevulinic acid synthetase mutant described in the first technical solution.

[0008] The fourth technical solution provided by the present invention is the application of the 5-aminolevulinic acid synthetase mutant described in the first technical solution, especially the application in the synthesis of 5-aminolevulinic acid, and further the application in catalyzing glycine and succinyl-CoA to generate 5-aminolevulinic acid.

[0009] Beneficial effects: The present invention improves the enzyme activity of ALAS by mutating the non-conservative amino acid I86 of ALAS, reduces the inhibition of the substrate glycine on the enzyme activity of ALAS, and is beneficial to increasing the yield of 5-ALA. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is the standard curve of 5-ALA.

[0011] Figure 2 Graph showing 5-ALA production by wild-type 5-aminolevulinic acid synthase and the I86C mutant at different glycine concentrations. DETAILED DESCRIPTION

[0012] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0013] 1. The following definitions are used in the present invention: Amino acid and DNA sequences are referred to using the generally accepted IUPAC nomenclature for amino acid residues, using the three-letter / one-letter code format. DNA sequences use the generally accepted IUPAC nomenclature.

[0014] In the present invention, ALAS means Rhodobacter capsulatus ( Rhodobacillus capsulatum ) Source: wild-type 5-aminolevulinic acid synthase, hemA The gene encodes the wild-type 5-aminolevulinic acid synthase (ALAS), I86C represents the 5-aminolevulinic acid synthase mutant, here It is the coding gene of the I86C mutant. The specific information is as follows:

[0015] 2. Some of the culture media or solutions used in the present invention are as follows: (1) Solid culture medium (g / L): peptone 10, sodium chloride 10, yeast powder 5, agar 15, pH 7.0; (2) LB medium (g / L): peptone 10, sodium chloride 10, yeast powder 5, pH 7.0. 3. Some of the culture conditions used in the present invention are as follows: (1) Seed culture: Inoculate a single clone into 4 mL of LB medium, add ampicillin (100 μg / mL) according to the resistance of the transferred plasmid, and culture at 37°C, 220 rpm for 8-10 hours.

[0016] (2) Fermentation culture: Add 20 mL of LB medium to a 100 mL shake flask, add kanamycin (50 μg / mL) according to the resistance of the transferred plasmid, and inoculate the cells to an initial OD of 600 When the value reaches 0.1, culture at 37 ℃ and 200 r / min.600 When the pH reached 0.6-0.8, 0.2 mM IPTG was added to induce gene expression, and the culture was continued at 28°C for 8-9 h. Then the absorbance of the cells at 600 nm was measured, and a certain amount of cells were collected and centrifuged to remove the supernatant. Then, the cells were resuspended with a certain amount of phosphate buffer solution (pH = 7.4) and transferred to a 50 mL centrifuge tube for ultrasonic disruption.

[0017] 4. 5-aminolevulinic acid synthase activity assay (1) Assay method and system: ALAS enzyme activity assay reaction system (200 μL): 80 μL purified ALAS (wild type or mutant, enzyme solution prepared in Example 2), 80 μL succinyl-CoA (initial concentration 0.01 mg / mL), 4 μL PLP (initial concentration 10 mg / mL), 40 μL glycine (final concentration in the system is 5 mM), react at 37°C for 1 h (all components in the reaction system are dissolved in 50 mM pH=7.0 PBS solution).

[0018] Take 20 μL of reaction solution or 5-ALA standard sample solution of different concentrations (25 mg / L-500 mg / L) to prepare 5-ALA standard curve, such as Figure 1 200 μL 1M sodium acetate and 20 μL acetylacetone were added to the mixture (as shown), and the mixture was reacted in a boiling water bath for 15 min. 100 μL Ehrlich's developer (42 mL glacial acetic acid, 8 mL 70% perchloric acid, 1 g p-diaminobenzaldehyde) was added to the mixed solution, and the absorbance was detected at a wavelength of 554 nm by an enzyme reader to determine the production of 5-ALA.

[0019] (2) Definition of enzyme activity: The amount of enzyme required to generate 1 μmol of 5-ALA in 1 min under certain conditions (37°C, pH 7.0 unless otherwise specified) is defined as one enzyme activity unit (U).

[0020] (3) Enzyme activity calculation formula: Enzyme activity (U / mg) = C × V / t / m, where: C: 5-ALA concentration (μmol / L); V: reaction volume (L); t: reaction time (min); m: protein mass (mg).

[0021] In the present invention, the amino acid sequence of the wild-type 5-aminolevulinic acid synthase is shown in SEQ ID NO.1: MDYNLALDKAIQKLHDEGRYRTFIDIEREKGAFPKAQWNRPDGGKQDITVWCGNDYLGMGQHPVVLAAMHEALEAVGAGSGGTRNISGTTAYHRRLEAEIAGLHQKEAALVFSSAYNANDATLSTLRVLFPGLIIYSDSLNHASMIEGIKRNAGPKRIFRHNDVAHLRELIAADDPAAPKLIAFESVYSMDGDFGPIKEICDI AEEFGALTYIDEVHAVGMYGPRGAGVAERDGLMHRIDIFNGTLAKAYGVFGGYIAASARMVDAVRSYAPGFIFSTSLPPAIAAGAQASIAFLKTAEGQKLRDAQQMHAKVLKMRLKALGMPIIDHGSHIVPVVIGDPVHTKAVSDMLLSDYGVYVQPINFPTVPRGTERLRFTPSPVHDLKQIDGLVHAMDLLWARCAHHHHHH In the present invention, the amino acid sequence of the 5-aminolevulinic acid synthase mutant I86C is shown in SEQ ID NO.3: MDYNLALDKAIQKLHDEGRYRTFIDIEREKGAFPKAQWNRPDGGKQDITVWCGNDYLGMGQHPVVLAAMHEALEAVGAGSGGTRNCSGTTAYHRRLEAEIADLHGKEAALVFSSAYIANDATLSTLRLLFPGLIIYSDSLNHASMIEGIKRNAGPKRIFRHNDVAHLRELIAADDPAAPKLIAFESVYSMDGDFGPIKEICDI ADEFGALTYIDEVHAVGMYGPRGAGVAERDGLMHRIDIFNGTLAKAYGVFGGYIAASAKMVDAVRSYAPGFIFSTSLPPAIAAGAQASIAFLKTAEGQKLRDAQQMHAKVLKMRLKALGMPIIDHGSHIVPVVIGDPVHTKAVSDMLLSDYGVYVQPINFPTVPRGTERLRFTPSPVHDLKQIDGLVHAMDLLWARCAHHHHHH The present invention is further explained below by means of specific examples.

[0022] Example 1 Obtaining the I86C mutant From Rhodobacter capsulatus ( Rhodobacillus capsulatum) hemA (SEQ ID NO.2) was integrated into the pET28a plasmid, and the obtained recombinant plasmid was named hemA -pET28a, design primers hemA The codon encoding the 86th amino acid isoleucine I mutated to the codon encoding cysteine ​​C. The designed mutation site is located in the middle of the primer. The forward and reverse primers have an overlap region of 15-20 bp. The primer sequences are as follows: Forward primer I86C-F: CCCGTAACTGCTCTGGTACCACCGCTTACCACCGTCG; Reverse primer I86C-R: TACCAGAGCAGTTACGGGTACCACCAGAACCAGCACCAACAGC.

[0023] The reaction system is as follows:

[0024] The reaction procedure is as follows: 98℃, 10s; 56℃, 15s; 72℃, 3min; 35 cycles.

[0025] Through the above PCR site-directed mutagenesis procedure, the coding gene of 5-aminolevulinic acid synthase mutant I86C was obtained. here , use ligase to connect the PCR fragments into a new plasmid, transform it into DH5α, spread it on an ampicillin-resistant plate, and pick a single clone for sequencing. The correctly sequenced plasmid is named here -pET28a plasmid.

[0026] Example 2: Purification of ALAS wild-type and mutant proteins The expression vector hemA -pET28a or here -pET28a transformed into Escherichia coli expression host E.coli From BL21(DE3), a single clone was picked and cultured in LB medium at 37°C for 8 h, then transferred to 20 mL LB medium until OD 600When the pH reaches 0.6-0.8, 0.2 mM IPTG is added to induce gene expression, cultured at 28 °C for 10 h, and the cells are collected by centrifugation. The collected cells are resuspended and broken with buffer (50 mM PBS, pH=7.4), and the supernatant is obtained by centrifugation. The protein is purified by AKta protein purifier, the column is balanced with loading buffer (20 mM PBS, pH=7.4; 100 mM NaCl; 10 mM imidazole), the target protein is eluted with elution buffer (20 mM PBS, pH=7.4; 100 mM NaCl; 500 mM imidazole), and the enzyme solution is obtained by centrifugation and desalting with a 10kDa ultrafiltration tube.

[0027] Example 3: ALAS activity assay ALAS reaction system (200 μL): 80 μL of purified ALAS (wild type or mutant, enzyme solution prepared in Example 2) with a final concentration of 0.28 mg / L, 80 μL of succinyl-CoA (initial concentration 0.01 mg / mL), 4 μL of PLP (initial concentration 10 mg / mL), 40 μL of glycine with a final concentration of 5 mM (each component was dissolved in 50 mM pH=7.0 PBS solution), and reacted at 37°C for 1 h. Take 20 μL of the reaction solution, add 200 μL of 1M sodium acetate and 20 μL of acetylacetone, react in a boiling water bath for 15 min, add 100 μL of Ehrlich's colorimetric reagent (42 mL of glacial acetic acid, 8 mL of 70% perchloric acid, 1 g of p-diaminobenzaldehyde) to the mixed solution, and use a microplate reader to detect the absorbance at a wavelength of 554 nm to determine the yield of 5-ALA and calculate the enzyme activity.

[0028] The results showed that when the glycine concentration was 5 mM, the enzyme activity of wild-type ALAS was 3.53 U / mg and that of mutant I86C was 6.66 U / mg.

[0029] Example 4: 5-ALA generated by glycine at different concentrations under ALAS catalysis By changing the concentration of glycine in the reaction system of Example 3, the yield of 5-ALA in the glycine reaction catalyzed by ALAS at different glycine reaction concentrations was measured.

[0030] The final concentration of glycine in the reaction system was set to 2.5, 5, 10, 15, 20, 25, and 30 mM, respectively. Figure 2 As shown, it can be seen that the ability of ALAS to catalyze the synthesis of 5-ALA from glycine is improved by mutating the ALAS non-conservative amino acid I86 to cysteine, and a high catalytic ability can be maintained under high concentrations of glycine. Combined with the enzyme activity determination results of Example 2, it is speculated that the I86C mutant improves the enzyme activity while also improving the tolerance to the substrate glycine.

[0031] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A 5-aminolevulinic acid synthase mutant, characterized in that: The amino acid sequence of the 5-aminolevulinic acid synthetase mutant is shown in SEQ ID NO.

3.

2. A gene encoding the 5-aminolevulinic acid synthetase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence is shown in SEQ ID NO.

4.

4. A recombinant vector or recombinant strain comprising the coding gene according to claim 2.

5. The recombinant vector according to claim 4, characterized in that The expression plasmid used was pET28a.

6. The recombinant strain according to claim 4, characterized in that The host cell used was Escherichia coli BL21 (DE3).

7. Use of the recombinant vector or recombinant strain according to claim 4 in producing the 5-aminolevulinic acid synthetase mutant according to claim 1.

8. Use of the recombinant vector or recombinant strain according to claim 4 in producing 5-aminolevulinic acid.

9. Use of the 5-aminolevulinic acid synthetase mutant according to claim 1 in the synthesis of 5-aminolevulinic acid.

10. The use according to claim 9, characterized in that Application in catalyzing the production of 5-aminolevulinic acid from glycine and succinyl-CoA.

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