5-amino levulinic acid synthetase pbhem a mutants and uses thereof

Abstract

The application discloses a 5-aminoacetylpropionic acid synthetic enzyme PbHemA-I49L-E46G-L407P, and an amino acid sequence of the 5-aminoacetylpropionic acid synthetic enzyme PbHemA-I49L-E46G-L407P is shown as SEQ ID NO. 5. A 5-aminoacetylpropionic acid synthetic enzyme PbHemA-I49L is also disclosed, and an amino acid sequence of the 5-aminoacetylpropionic acid synthetic enzyme PbHemA-I49L is shown as SEQ ID NO. 3. The application firstly carries out saturated mutation on an I49 site of a 5-aminoacetylpropionic acid synthetic enzyme pbHemA, and a mutant I49L is screened, and the 5-ALA production capacity of the mutant I49L is increased by 11.7% compared with that of a wild enzyme; then, mutation modification is continuously carried out, and a mutant I49L-E46G-L407P is screened, and the 5-ALA production capacity of the mutant I49L-E46G-L407P is increased by 68.9% compared with that of the mutant I49L. The research of the application can provide technical support for synthesis and application of 5-ALA and downstream heme and other porphyrin substances.

Description

Technical Field

[0001] This invention relates to the PbHemA mutant of 5-aminolevulinic acid synthase and its applications, belonging to the field of functional enzyme technology. Background Technology

[0002] 5-Aminolevulinic acid (ALA) is a naturally occurring non-protein amino acid and a key precursor in the biosynthesis of tetrapyrrole compounds. Widely found in plants, animals, and microorganisms, 5-ALA is a common precursor in the biosynthesis of important bioactive substances such as heme, chlorophyll, and vitamin B12. Due to its important physiological functions, 5-ALA has broad applications in medicine, agriculture, animal husbandry, aquaculture, and functional foods.

[0003] With the rapid development of green manufacturing and synthetic biology, compared with traditional chemical synthesis methods, the synthesis of 5-ALA by microbial fermentation has become a current research hotspot due to its advantages such as being green, sustainable, low-cost and environmentally friendly.

[0004] In the study of 5-aminolevulinic acid (5-ALA) biosynthesis, the C4 pathway (Shemin pathway) has attracted widespread attention in metabolic engineering and synthetic biology due to its simple reaction steps and short metabolic pathway, and is considered one of the important strategies for achieving efficient 5-ALA biosynthesis. In the C4 pathway, glycine and succinyl-CoA serve as direct precursors, undergoing a condensation reaction under the catalysis of ALAS to generate 5-ALA, while simultaneously releasing CoA and carbon dioxide. ALAS is the key rate-limiting enzyme in this pathway, and its catalytic efficiency directly determines the synthesis rate and final yield of 5-ALA. However, currently reported ALAS have the following limitations: low catalytic activity, limited enzyme expression levels, and insufficient intracellular supply of the substrates glycine and succinyl-CoA. These problems all limit the efficient accumulation of 5-ALA. Summary of the Invention

[0005] To address the aforementioned limitations of existing technologies, this invention provides two PbHemA mutants of 5-aminolevulinic acid synthase and their applications. This invention, through modification, obtains 5-aminolevulinic acid synthase mutants with higher activity, which can effectively improve the synthesis efficiency of 5-ALA.

[0006] This invention is achieved through the following technical solution: A 5-aminolevulinic acid synthase, PbHemA-I49L-E46G-L407P, has the amino acid sequence shown in SEQ ID NO.5.

[0007] The nucleotide sequence of the gene encoding the 5-aminolevulinic acid synthase PbHemA-I49L-E46G-L407P is shown in SEQ ID NO.6.

[0008] A 5-aminolevulinic acid synthase, PbHemA-I49L, has the amino acid sequence shown in SEQ ID NO.3.

[0009] The nucleotide sequence of the gene encoding the 5-aminolevulinic acid synthase PbHemA-I49L is shown in SEQ ID NO.4.

[0010] The above-mentioned 5-aminolevulinic acid synthases PbHemA-I49L-E46G-L407P and PbHemA-I49L are used in the synthesis of 5-ALA and in the synthesis of heme.

[0011] Furthermore, the specific application method is as follows: using Escherichia coli BL21(DE3) as the host bacterium, construct a recombinant engineered bacterium expressing 5-aminolevulinic acid synthase PbHemA-I49L-E46G-L407P or 5-aminolevulinic acid synthase PbHemA-I49L, culture the recombinant engineered bacterium, and under the action of the 5-aminolevulinic acid synthase PbHemA-I49L-E46G-L407P or 5-aminolevulinic acid synthase PbHemA-I49L expressed by the recombinant engineered bacterium, the recombinant engineered bacterium metabolizes and synthesizes 5-ALA.

[0012] Furthermore, during the cultivation process, glycine (final concentration 3–5 g / L), succinic anhydride (final concentration 0.8–1.2 g / L), pyridoxal phosphate (final concentration 25–35 μM), and IPTG (final concentration 0.08–0.12 mM) are added to promote the efficient synthesis of 5-ALA. Preferably, glycine (final concentration 4 g / L), succinic anhydride (final concentration 1 g / L), pyridoxal phosphate (final concentration 30 μM), and IPTG (final concentration 0.1 mM) are added.

[0013] This invention first involves a saturation mutation at the I49 site of the 5-aminolevulinic acid synthase pbHemA, resulting in the mutant I49L with the highest hydrolytic activity, exhibiting an 11.7% increase in 5-ALA production compared to the wild-type enzyme. Then, further mutations were performed on the I49L mutant, resulting in the mutant I49L-E46G-L407P, which shows a 68.9% increase in 5-ALA production compared to the I49L mutant. These two pbHemA mutants of 5-aminolevulinic acid synthase can efficiently synthesize 5-ALA and have potential for industrial application. This research provides technical support for the synthesis and application of 5-ALA and downstream porphyrins such as heme.

[0014] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0015] Figure 1 Results of fermentation of the NNK saturated mutant library at site I49 in well plates.

[0016] Figure 2 Results of the detection of cell concentration and 5-ALA concentration in fermentation broth containing each mutant.

[0017] Figure 3 Results of plate screening of I49L random mutant library.

[0018] Figure 4 Results of cell concentration and 5-ALA concentration detection in the fermentation broth of each I49L mutant.

[0019] Figure 5 Growth curves of mutant B3 and wild-type enzyme. Detailed Implementation

[0020] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0021] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0022] The plasmid pET28a(+) used in this invention was preserved in the inventor's laboratory; this plasmid is a commonly used gene expression vector in the field and can be purchased from the market.

[0023] The Escherichia coli BL21(DE3) competent cells used in this invention were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0024] The LB medium used in this invention consists of the following components: 10 g / L sodium chloride; 10 g / L tryptone; 5 g / L yeast extract; 15 g / L agar powder (added when preparing solid medium); the remainder is water; sterilization conditions: 121℃, 20 min.

[0025] The M9 culture medium used in this invention was purchased from Aili Biotechnology Co., Ltd.

[0026] The anhydrous glucose and glycine used in this invention were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0027] The DNA polymerase 2×Taq Master Mix used in this invention was purchased from Nanjing Jujiang Biotechnology Co., Ltd.

[0028] The high-fidelity DNA polymerase Prime STAR and DNA standard molecular weight marker used in this invention were purchased from Dalian Takara Co., Ltd.

[0029] The 5-aminolevulinate salt standard, ammonium acetate, and acetonitrile (HPLC grade) used in this invention were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0030] Example 1: Detection of 5-ALA content in fermentation broth 5-Aminolevulinic acid synthase PbHemA is a 5-aminolevulinic acid synthase screened by the inventors in previous studies. Its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene (after codon optimization) is shown in SEQ ID NO.2. This gene fragment was artificially synthesized and ligated into the pET28a(+) expression vector to obtain the recombinant expression vector pET28a-pbhemA. Recombinant engineered bacteria were constructed using conventional methods, and the 5-ALA content in the fermentation broth was detected. It was found that the 5-ALA yield was not ideal. Therefore, this invention attempts to mutate the enzyme to obtain a mutant with higher enzyme activity and better suitability for industrial applications.

[0031] The method for detecting the 5-ALA content in fermentation broth is as follows: (a) Construction of 5-ALA liquid chromatography standard curve (1) Preparation of standard: First, accurately weigh 383.0 mg of 5-aminolevulinic acid salt standard, dissolve it in 50 mL of deionized water to prepare a 6 g / L 5-ALA stock solution, and then serially dilute it to 5-ALA standard solutions with final concentrations of 0.25, 0.5, 1.0, 1.5, 2, 3 and 6 g / L.

[0032] (2) Preparation of reaction reagents: Weigh 1.0 g dansyl chloride and dissolve it in 10 mL acetone as a derivatizing reagent to participate in the reaction; dissolve 571.3 mg NaHCO3 and 160.0 mg NaOH in deionized water, and after complete dissolution, make up to 40 mL as a buffer reagent for the derivatization reaction system.

[0033] (3) Derivatization reaction: 248 μL of buffer system, 38.2 μL of standard sample, 20 μL of derivatization reagent and 294 μL of pure acetonitrile were added to a 1.5 mL brown centrifuge tube in sequence, placed in a 38℃ water bath for 90 min, centrifuged at 12000 rpm for 5 min, and 200 μL of supernatant was taken and filtered through a 0.22 μm organic filter membrane and placed in a liquid phase bottle.

[0034] (4) Liquid chromatography detection: 5-aminolevulinic acid was determined using a Shimadzu high performance liquid chromatograph. The injection volume was set to 10 μL, the chromatographic column was 250×4.6 mm, 5 μm SepaChrom™0002 column, the column temperature was 30℃, the mobile phase was 25% acetonitrile / 75% ammonium acetate solution, the flow rate was 0.8 mL / min, the UV detection wavelength was 387 nm, the single sample detection time was 25 min, and the peak time of 5-ALA in the 5-ALA standard was 15.2 min.

[0035] (5) The peak areas corresponding to different concentrations were determined according to the above HPLC detection method. The peak area was plotted as the abscissa and the concentration of the corresponding 5-ALA standard was plotted as the ordinate to form the standard curve.

[0036] (II) Detection of 5-ALA content in fermentation broth Take 1 mL of fermentation broth into a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 5 min, and then filter the supernatant through a 0.22 μm aqueous filter membrane. Use the above method to perform pre-column derivatization of 5-ALA to achieve pretreatment of the fermentation sample before high performance liquid chromatography (HPLC) detection. Obtain the characteristic peak by HPLC detection, and substitute the peak area of ​​the sample to be tested into the standard curve to calculate the concentration of 5-ALA in the fermentation broth.

[0037] Example 2: Construction and initial screening of saturated mutant libraries (1) Construction of a saturated mutant library at site I49: In previous studies, the effects of non-conserved amino acid residues near the active site of 5-aminolevulinic acid synthase PbHemA on the ability to synthesize 5-ALA were evaluated using alanine scanning technology. The results showed that sites 22, 49, 115, 138, 155, 209, 251, and 252 were potential non-conserved amino acid residues. In this invention, site I49 was selected for saturated mutation to generate a saturated mutant library at this site. High-throughput screening was used to evaluate the effect of replacing different amino acids at this site on the 5-ALA synthesis efficiency.

[0038] Specifically, by designing NNK primers for this site, using pET28a-pbhemA as a template, and I49L-F (nucleotide sequence as shown in SEQ ID NO.7) and I49-R (nucleotide sequence as shown in SEQ ID NO.8) as primers, amplification was performed by polymerase chain reaction. After verifying the presence of the correct band by nucleic acid gel electrophoresis, the sample was transferred into the expression host Escherichia coli BL21(DE3) to obtain a single clone.

[0039] (2) 24-well deep plate fermentation: Nearly 100 single clones were inoculated into 1.5 mL of M9 medium containing 50 μg / mL kanamycin sulfate in 24-well deep plates to ensure coverage of the mutant library. The cells were cultured at 37℃ and 600 rpm for 4 hours. Then, glycine, succinic anhydride, pyridoxal phosphate, and IPTG were added to a final concentration of 4 g / L. Expression was induced at 30℃ and 600 rpm for 24 h. The cell concentration and 5-ALA content in the fermentation broth were measured.

[0040] The results of plate fermentation of the I49 site NNK saturated mutant library (including cell concentration and 5-ALA concentration) are as follows: Figure 1 As shown.

[0041] Example 3: Shake-flask fermentation of recombinant strains (1) Identification and acquisition of recombinant plasmids: Plasmids were extracted from the monoclonal bacterial pellet with the highest yield in Example 2 and sequenced to obtain a series of potentially high-efficiency mutant enzymes, which were named I49E, I49A, I49S, I49V, I49T, and I49L, respectively. The recombinant plasmids were transformed into Escherichia coli BL21(DE3) competent cells to obtain recombinant strains.

[0042] (2) Seed culture: single clones were picked from the plate and inoculated into 5 mL LB liquid medium (containing 50 μg / mL kanamycin sulfate) and cultured at 37℃ and 220 rpm for 16 h.

[0043] (3) Shake-flask fermentation: The cultured seed culture was transferred to a 250 mL Erlenmeyer flask containing 30 mL of M9 medium at an inoculation rate of 2% (v / v), and cultured at 37℃ and 220 rpm until OD. 600 The pH was initially set at 0.6–0.8, and then the following inducers (final concentrations) were added: 4 g / L glycine, 1 g / L succinic anhydride, 30 μM pyridoxal phosphate, and 0.1 mM IPTG. Expression was induced at 30 °C and 220 rpm for 24 h. The pH of the fermentation broth was monitored during fermentation, and was maintained at approximately 7.0 by adding 2 M NaOH solution.

[0044] (4) Yield detection: The cell concentration and 5-ALA content in the fermentation broth of the recombinant strain were detected. The results of the cell concentration and 5-ALA concentration detection of the fermentation broth containing each mutant are as follows: Figure 2 As shown in the figure, the mutant I49L had the highest 5-ALA concentration, with a 11.7% increase in 5-ALA yield compared to the wild-type enzyme. Other mutants (I49E, I49A, I49S, I49V, and I49T) generally performed worse than the wild-type enzyme, or even worse. This may be because while plate screening offers high throughput, the small system size and significant differences between culture conditions and actual fermentation mean that the advantages of some mutants may be dependent on specific microenvironments and difficult to maintain stably, resulting in only potentially high-activity candidate strains. In the further shake-flask rescreening, the culture conditions were closer to actual fermentation conditions, placing more comprehensive demands on the growth, metabolism, and product synthesis capabilities of the strains. Ultimately, only one mutant still showed significantly higher activity than the wild-type enzyme in the rescreening, indicating that it possesses a stable and transferable superior phenotype, making it more valuable for further research and application.

[0045] The mutant I49L is named 5-aminolevulinic acid synthase PbHemA-I49L, and its amino acid sequence is shown in SEQ ID NO.3, while the nucleotide sequence of its encoding gene is shown in SEQ ID NO.4.

[0046] Example 4: Construction and initial screening of I49L random mutant library Although the mutant I49L obtained in Example 3 produced 5-ALA more efficiently than the wild-type enzyme, it was still not ideal. Therefore, this invention attempts to further mutate the mutant I49L in order to obtain a mutant with higher enzyme activity.

[0047] (1) Construction of I49L random mutant library The pbhemA-I49L gene fragment was mutated using error-prone PCR, and the mutation was performed using DNA polymerase 2×Taq MasterMix (Dye), while simultaneously inhibiting Mg. 2+ Mn2+ Different concentrations and dosages were experimented with, with the specific dosages as follows: 0 mM Mg 2+ 7 mMMg 2+ ;0 mM Mn 2+ 0.2 mM Mn 2+ 0.4 mM Mn 2+ 0.5 mM Mn 2+ 0.6 mM Mn 2+ Ten orthogonal combinations of PCR reaction systems were performed. After obtaining fragments with the correct band size, the fragments were ligated to pET28a(+) using C115 recombinase, and then transformed. The mixed plasmid library was extracted and transformed into E. coli BL21(DE3) for plate verification.

[0048] (2) Initial screening of I49L random mutant library in well plate Nearly 100 single clones were selected and inoculated into 24-well deep-well plates containing 1.5 mL of M9 medium with 50 μg / mL kanamycin sulfate to construct as many mutant libraries as possible. After culturing at 37℃ and 600 rpm for 4 hours, glycine, succinic anhydride, pyridoxal phosphate, and IPTG were added to a final concentration of 4 g / L. Expression was induced at 30℃ and 600 rpm for 24 h, and cell concentration and 5-ALA concentration were measured.

[0049] The results of the I49L random mutant library plate screening (including cell concentration and 5-ALA concentration) are as follows: Figure 3 As shown.

[0050] Example 5: Shake-flask rescreening of the I49L mutant (1) Identification and acquisition of recombinant plasmids: Plasmids were extracted from the monoclonal bacterial pellet with the highest yield in Example 4 and sequenced to obtain a series of potential high-efficiency mutant enzymes, which were numbered as B15, B3, B14, B5, and D17. The recombinant plasmids were transformed into Escherichia coli BL21(DE3) competent cells to obtain recombinant strains.

[0051] (2) Seed culture: single clones were picked from the plate and inoculated into 5 mL LB liquid medium (containing 50 μg / mL kanamycin sulfate) and cultured at 37℃ and 220 rpm for 16 h.

[0052] (3) Shake-flask fermentation: The cultured seed culture was transferred to a 250 mL Erlenmeyer flask containing 30 mL of M9 medium at an inoculation rate of 2% (v / v), and cultured at 37℃ and 220 rpm until OD. 600The pH value was initially set at 0.6–0.8. Subsequently, the following inducers (final concentrations) were added: 4 g / L glycine, 1 g / L succinic anhydride, 30 μM pyridoxal phosphate, and 0.1 mM IPTG. Expression was induced at 30℃ and 220 rpm for 24 h. The pH of the fermentation broth was monitored during fermentation, and the pH was maintained at approximately 7.0 by adding 2 M NaOH solution.

[0053] (5) Yield detection: The cell concentration and 5-ALA content in the fermentation broth of the recombinant strains were measured. The cell concentration and 5-ALA concentration detection results of the fermentation broth of each I49L mutant are as follows: Figure 4 As shown in the figure, the results indicate that B3 exhibited the highest 5-ALA concentration, with a 68.9% increase in 5-ALA production compared to mutant I49L. Other mutants (B15, B14, B5, D17) generally showed even lower yields than mutant I49L. Therefore, mutant B3 demonstrated the best 5-ALA production performance and possesses potential for industrial application.

[0054] The specific mutation of mutant B3 is I49L-E46G-L407P. In this invention, it is named 5-aminolevulinic acid synthase PbHemA-I49L-E46G-L407P. Its amino acid sequence is shown in SEQ ID NO.5, and its encoding gene nucleotide sequence is shown in SEQ ID NO.6.

[0055] Example 6: Plotting the growth curve of 5-aminolevulinic acid synthase PbHemA-I49L-E46G-L407P Following the shake-flask fermentation conditions of Example 5, the induction time was adjusted to 3 hours. A recombinant engineered bacterial strain containing mutant B3 was cultured, and OD was measured every 6 hours during the induction expression period. 600 The 5-ALA content was determined, and fermentation lasted for 36 hours. Wild-type enzyme (5-aminolevulinic acid synthase PbHemA) was used as a control.

[0056] Growth curves (changes in cell concentration and 5-ALA concentration with fermentation time) of mutant B3 and wild-type enzyme are shown below. Figure 5 As shown, after 30 hours of fermentation, the 5-ALA content in the fermentation broth of mutant B3 can reach 3.7 g / L, which is significantly better than the 2.5 g / L of the wild enzyme.

[0057] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A 5-aminolevulinic acid synthase PbHemA-I49L-E46G-L407P, characterized in that: The amino acid sequence is shown in SEQ ID NO.

5.

2. The encoding gene of 5-aminolevulinic acid synthase PbHemA-I49L-E46G-L407P according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

6.

3. The use of the 5-aminolevulinic acid synthase PbHemA-I49L-E46G-L407P according to claim 1 in the synthesis of 5-ALA or heme.

4. The application according to claim 3, characterized in that, The specific application method is as follows: using Escherichia coli BL21(DE3) as the host bacterium, a recombinant engineered bacterium expressing 5-aminolevulinic acid synthase PbHemA-I49L-E46G-L407P is constructed. The recombinant engineered bacterium is cultured, and under the action of the 5-aminolevulinic acid synthase PbHemA-I49L-E46G-L407P expressed by the recombinant engineered bacterium, the recombinant engineered bacterium metabolizes and synthesizes 5-ALA.

5. The application according to claim 4, characterized in that: During the culture process, glycine (3–5 g / L), succinic anhydride (0.8–1.2 g / L), pyridoxal phosphate (25–35 μM), and IPTG (0.08–0.12 mM) were added to a final concentration.

6. A 5-aminolevulinic acid synthase PbHemA-I49L, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

7. The encoding gene for 5-aminolevulinic acid synthase PbHemA-I49L as described in claim 6, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

4.

8. The use of the 5-aminolevulinic acid synthase PbHemA-I49L according to claim 6 in the synthesis of 5-ALA or heme.

9. The application according to claim 8, characterized in that, The specific application method is as follows: using Escherichia coli BL21(DE3) as the host bacterium, a recombinant engineered bacterium expressing 5-aminolevulinic acid synthase PbHemA-I49L is constructed. The recombinant engineered bacterium is cultured, and under the action of the 5-aminolevulinic acid synthase PbHemA-I49L expressed by the recombinant engineered bacterium, the recombinant engineered bacterium metabolizes and synthesizes 5-ALA.

10. The application according to claim 9, characterized in that: During the culture process, glycine (3–5 g / L), succinic anhydride (0.8–1.2 g / L), pyridoxal phosphate (25–35 μM), and IPTG (0.08–0.12 mM) were added to a final concentration.

Images