Mutants of enzymes involved in folate synthesis and uses thereof

Abstract

The application discloses an enzyme mutant in a folate synthesis pathway and application thereof. The enzyme mutant comprises AtDRTS2M, and the amino acid sequences of the AtDRTS2M are respectively shown as SEQ ID NO:1, and the gene sequences of the AtDRTS2M are respectively shown as SEQ ID NO:2. The application carries out molecular modification on the related enzymes in the folate synthesis pathway, so that the biological activity of the modified AtDRTS2M in a plant body is higher, and technical support is provided for cultivating a high-yield folate crop.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to enzyme mutants in the folic acid synthesis pathway and their applications. Background Technology

[0002] Folic acid fortification is an internationally recognized public health strategy that improves public folate nutrition and prevents birth defects by adding folic acid to foods that are widely consumed in daily life, such as flour and rice.

[0003] Utilizing genetic engineering to increase crop folate content has become an important research direction in recent years for addressing the global problem of "hidden hunger," and significant progress has been made in multiple dimensions, but several key challenges remain. De novo folate synthesis exists only in plants and microorganisms. In the cytoplasm, guanosine triphosphate cyclase I (GTPCHI) catalyzes guanosine triphosphate and subsequent reactions to ultimately produce 6-hydroxymethyldihydropterin; in the plastid, aminodeoxybranched acid synthase (ADCS) catalyzes branched acid and subsequent reactions to ultimately produce p-aminobenzoic acid. In the mitochondria, the plant's dual-function hydroxymethyldihydropterin pyrophosphate kinase / dihydropterin synthase (HPPK / DHPS) catalyzes the production of dihydrofolate; then, dihydrofolate synthase (DHFS) links the first glutamate residue to it. Subsequently, dihydrofolate reductase (DRTS) reduces dihydrofolate to tetrahydrofolate. At the technological breakthrough level, scientists have successfully identified and utilized several key metabolic genes to achieve significant enrichment of folic acid. For example, by introducing two genes from Arabidopsis thaliana, GTPCHI and ADCS, into rice and tomatoes—a "gene stacking" strategy—the folic acid synthesis pathway can be synergistically enhanced, with effects far exceeding those of single gene modification. In lettuce, after introducing the aforementioned two genes, the folic acid content of transgenic lines increased by 3.4 times compared to the wild type, and even by 1.9 times compared to spinach, a well-known folic acid-rich vegetable, and remained stable in field trials for two consecutive years. Furthermore, a research team from the Chinese Academy of Agricultural Sciences discovered the key "valve" gene ZmGFT in maize. Natural variations in its single base can affect the conversion of active folic acid to its inactive form. By knocking out this "valve," the accumulation of active folic acid in maize kernels can be significantly increased, providing a precise target for molecular marker-assisted breeding. The biosynthesis of folate in plants is highly complex, with its biosynthetic pathways occurring in the cytoplasm, chloroplasts, and mitochondria of the cell. This compartmentalized distribution and intricate regulatory network mean that single-gene modifications often have limited effects, necessitating complex multi-gene engineering. Furthermore, overexpression of enzymes involved in the folate synthesis pathway (such as ADCS, HPPK / DHPS, and DRTS2) has limited effect on increasing folate production, requiring molecular modification of these enzymes to further enhance their activity. Summary of the Invention

[0004] The purpose of this invention is to provide mutants of key enzymes in the folic acid synthesis pathway and their applications.

[0005] A mutant of an enzyme related to the folic acid synthesis pathway, including AtDRTS2M, has the amino acid sequence shown in SEQ ID NO: 1 and the gene sequence shown in SEQ ID NO: 2.

[0006] The AtDRTS2M was originally AtDRTS2 before the mutation. The amino acid sequence before the mutation is shown in SEQ ID NO: 3, and the gene sequence is shown in SEQ ID NO: 4.

[0007] A recombinant plasmid carrying the aforementioned gene.

[0008] A recombinant bacterium expressing the AtDRTS2M mutant described above.

[0009] Application of enzyme mutants related to the folic acid synthesis pathway in promoting folic acid synthesis.

[0010] A method for increasing the folic acid content in Arabidopsis thaliana, wherein the folic acid content is increased by... AtDRTS2M The gene was transferred into Arabidopsis thaliana.

[0011] The beneficial effects of this invention are as follows: This invention molecularly modifies the key enzyme gene in the folic acid synthesis pathway, resulting in higher biological activity of the modified AtDRTS2M in plants, providing technical support for cultivating crops rich in folic acid. Attached Figure Description

[0012] Figure 1 The CD spectra of AtDRTS2 and AtDRTS2M after heat treatment for 0 min, 10 min, 20 min, and 30 min are shown in the figure; a in the figure is AtDRTS2; b in the figure is AtDRTS2M.

[0013] Figure 2 Characterization of the enzyme activities of AtDRTS2 and AtDRTS2M; Figure a shows the enzyme kinetics of AtDRTS2 and AtDRTS2M; b shows the determination of the optimal reaction temperature of AtDRTS2 and AtDRTS2M; c shows the change in residual activity of AtDRTS2 and AtDRTS2M over time; d shows the enzyme kinetic parameters of AtDRTS2 and AtDRTS2M. The DHFR enzyme activity data provided are representative results from two independent experiments and the mean ± sd of three biological and three technical replicates.

[0014] Figure 3Figure 1 shows the folic acid content of AtDRTS2 and AtDRTS2M; Figure 2a compares the total folic acid content in wild-type and AtDRTS2 and AtDRTS2M gain-of-function plants (AtDRTS2 and AtDRTS2M); Figure 2b compares the THF content in wild-type and AtDRTS2 and AtDRTS2M gain-of-function plants (AtDRTS2 and AtDRTS2M); Figure 2c compares the DHF content in wild-type and AtDRTS2 and AtDRTS2M gain-of-function plants (AtDRTS2 and AtDRTS2M). Detailed Implementation

[0015] To facilitate understanding of the present invention, a more comprehensive description will be provided below. However, the present invention can be implemented using different techniques and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0016] Example 1: The mutant AtDRTS2M and its activity To explore novel strategies for enhancing plant folate production, we modified and optimized genes in the Arabidopsis thaliana folate synthesis pathway. We selected dihydrofolate reductase (DHFR) from Arabidopsis as the target for modification. DHFR catalyzes the synthesis of NADPH and DHF (C6H2O). 19 H 23 Using N7O6 as a substrate, THF (C) is generated. 19 H 21 N7O6) and NADP. The Arabidopsis genome has three homologous proteins of DHFR-TS, located in the cytoplasm, plastids and mitochondria, respectively. Since the synthesis and assembly of folic acid usually occur in mitochondria, the mitochondrial-localized DHFR-TS2 (abbreviated as AtDRTS2) was selected as the target for modification.

[0017] Homologous proteins were searched in the Non-Redundant Protein Sequence Database. Homologous protein sequences from higher plants were selected, and the position-specific amino acid probability (PSAP) of each position in AtDRTS2 (SEQ ID NO: 3) was calculated to find amino acid sites with modification potential. Residues located in the DHFR protein domain with PASP ≥ 0.5 and higher than that of the wild type at the same site were selected as modification sites for mutation to obtain the modified protein sequence AtDRTS2M (SEQ ID NO: 1).

[0018] To confirm the effectiveness of the modification, the HIS label will be incorporated. AtDRTS2M (SEQ ID NO: 2) and AtDRTS2(SEQ ID NO: 4) was induced to express in E. coli, and the purified protein was detected by circular dichroism spectroscopy.

[0019] Plasmids pET28A and pCAMBIA3301 were used to construct overexpression vectors for the folic acid synthesis gene. To construct plant overexpression vectors with 3*FLAG tags fused to the ends of the target gene, each vector was used to carry the synthesized folic acid. AtDRTS2 , AtDRTS2M Using a vector containing the gene CDS sequence (codon-optimized) and a vector containing the 3*FLAG sequence as templates, PCR amplification was performed using TransStartFastPfu DNA Polymerase to obtain a target gene fragment containing the Bgl II restriction site sequence at the 3' to 5' ends and the 3*FLAG tag and BstEII restriction site sequence fused at the 5' to 3' ends. The Pcambia3301 vector and the purified target gene fragment were double-digested with enzymes, and the linearized vector and the target gene fragment were ligated by incubation with T4 ligase.

[0020] Amplification ends fused with 3*FLAG AtDRTS2 , AtDRTS2 Gene sequence:

[0021] Reaction conditions: 95°C pre-denaturation for 5 min; 95°C, 20 s; 60°C annealing for 20 s; 72°C extension for 20 s, 35 cycles; 72°C final extension for 5 min; incubation at 4°C to obtain insert 1 fragment. PCR product purification and recovery (DNA gel rapid purification kit).

[0022] Double digestion of Pcambia3301 vector and PCR products: using BstE II and Bgl Double digestion with restriction endonuclease II, digestion at 37°C for 15 min, followed by inactivation at 65°C.

[0023]

[0024] The PCR products and the enzyme-digested Pcambia3301 linearized vector fragment were purified and recovered (DNA gel rapid purification kit). A small amount of the recovered solution was electrophoresed to estimate the DNA concentration in the system. The purified Pcambia3301 linearized vector fragment and the purified target fragment were ligated using T4 ligase. The system was incubated at 50°C for 15 min.

[0025]

[0026] After ligation, the system was transformed into E. coli Trans1-T1 competent cells, and single colonies were used for sequencing verification.

[0027] The validated Pcambia3301-AtDRTS2 and Pcambia3301-AtDRTSM vectors were used to transform Agrobacterium GV3101 competent cells.

[0028] Transformation by inflorescence immersion method: Immerse the above-ground parts (inflorescences) of Arabidopsis thaliana in Agrobacterium suspension for about 20 seconds, gently agitating to ensure full contact between the inflorescence and the bacterial solution. After infection, cover the plants with plastic wrap to maintain high humidity and incubate in the dark for 16-24 hours, then restore normal light. Repeat the above operation every 3 days to ensure a sufficient number of flower buds are infected during the reproductive period.

[0029] Plant culture: Culture the plants normally until maturity (about 3-5 weeks), during which time the siliques will gradually turn yellow and dry. Collect the dried siliques to obtain T0 generation seeds.

[0030] To characterize the stability of the protein structure, the sequence encoding the protein was overexpressed in *E. coli* using the PET28A vector to carry the synthesized protein. AtDRTS2 , AtDRTS2 Using a PMV vector containing the gene CDS sequence (codon-optimized) as a template, the DNA was amplified using TransStart FastPfu DNA Polymerase. AtDRTS2 , AtDRTS2 CDS sequence of the gene

[0031] Reaction conditions: 95°C pre-denaturation for 5 min; 95°C for 20 s; 60°C annealing for 20 s; 72°C extension for 20 s, 35 cycles; 72°C final extension for 5 min; 4°C holding.

[0032] The PET28A vector was double-digested with BAMHI and XHOI restriction endonucleases at 37°C for 15 minutes, and then inactivated at 65°C.

[0033] The obtained PET28A linearized vector fragment after enzyme digestion was purified and recovered. The purified PET28A linearized vector fragment and the purified gene fragment were ligated using T4 ligase, and the system was incubated overnight at 16°C.

[0034] The ligation system was then transformed into E. coli BL21 competent cells.

[0035] BL21 bacteria carrying PET28A-AtDRTS2 and PET28A-AtDRTSM were inoculated into kanamycin-resistant LB liquid medium and cultured overnight at 37°C with shaking at 200 rpm. The culture was then transferred to 500 ml of kanamycin-resistant LB medium and cultured at 37°C with shaking for 2-3 hours. When the OD600 reached 1, protein expression was induced using 0.2 mM / L IPTG and induced overnight at 16°C with shaking at 200 rpm. After collection, the bacterial cells were sonicated to disrupt the protein composition. The target protein was purified using Ni-NTA. The supernatant was passed through a column five times at 4°C, followed by washing the column four times at 4°C with contaminating protein wash buffer (10 times column volume each time). Finally, the target protein was eluted with 5 times column volume of the target protein eluent. The purified protein was dialyzed against pure water for 24 hours to remove salt ions. The concentration of the purified protein was determined using the Bradford method, and the protein concentration was adjusted to 0.2 mg / mL for circular dichroism spectroscopy.

[0036] To characterize the stability of the protein structure, the samples were heat-treated at 70℃ for 10 min, 20 min, and 30 min, and their secondary structures were examined. It was found that both AtDRTS2 and AtDRTS2M possessed normal folding ability. Without heat treatment, the secondary structure composition of AtDRTS2M was more stable than that of AtDRTS2 (higher α-helix content). However, after heat treatment, the rigidity of AtDRTS2 was disrupted more quickly; the α-helix content decreased significantly within just 10 min, while the random coil content increased rapidly, indicating severe damage to the secondary structure. Thereafter, the proportions of various secondary structures did not change significantly. These results indicate that AtDRTS2M has stronger thermal stability than AtDRTS2. Figure 1 (Table 1-2)

[0037] Table 1. Percentage analysis of the secondary structure of the AtDRTS2 protein by CDNN.

[0038] Table 2. Percentage analysis of the secondary structure of the AtDRTS2M protein by CDNN.

[0039] The optimal catalytic temperature of AtDRTS2 and AtDRTS2M was determined by characterizing the degree of NADPH oxidation at 310 nm, and parameters such as enzyme turnover number and half-life were further determined. Figure 2The optimal reaction temperature of AtDRTS2M was found to be nearly 10°C higher than that of AtDRTS2, demonstrating an improvement in the enzyme's heat resistance. The results of the remaining enzyme activity over time showed that AtDRTS2 had a half-life of 110 min at 25°C and pH 7.4, while AtDRTS2M had a half-life of 300 min, indicating that the modified AtDRTS2M protein is more stable than AtDRTS2. Based on the Michaelis-Menten equation, the enzyme turnover number (Kcat) and maximum reaction rate (Vmax) of AtDRTS2M were significantly increased (1190.00 ± 15.21) compared to AtDRTS2's (130.00 ± 7.45), demonstrating a significant improvement in the catalytic efficiency of AtDRTS2M.

[0040] Therefore, Arabidopsis thaliana utilizes a 35S promoter-driven, C-terminal fused 3*FLAG tag... AtDRTS2 and AtDRTS2M The Pcambia3301 vector encoding the sequence was stably transformed to obtain... AtDRTS2 and AtDRTS2M The contents of folic acid and folic acid synthesis precursors in the overexpression transgenic lines AtDRTS2, AtDRTS2M and Col-0 were detected (Figure 3). It was found that THF, as a direct product of DHFR, had increased contents in both AtDRTS2 and AtDRTS2M lines. Compared with Col-0, the total folic acid content of the former increased by nearly 0.5 times, while the total folic acid content of the latter increased by nearly 1 time.

[0041] The primers synthesized by Liuhe Huada are listed in Table 3.

[0042] Table 3 Primers

[0043] The embodiments described above are merely examples of several implementations of the present invention and should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A mutant of an enzyme involved in the folic acid synthesis pathway, characterized in that, The mutant is AtDRTS2M, whose amino acid sequence is shown in SEQ ID NO: 1 and gene sequence is shown in SEQ ID NO:

2.

2. The enzyme mutant related to the folic acid synthesis pathway according to claim 1, characterized in that, The AtDRTS2M was originally AtDRTS2 before the mutation. The amino acid sequence before the mutation is shown in SEQ ID NO: 3, and the gene sequence is shown in SEQ ID NO:

4.

3. A recombinant plasmid carrying the gene of claim 1.

4. A recombinant bacterium expressing the AtDRTS2M mutant of claim 1.

5. The application of the enzyme mutant related to the folic acid synthesis pathway described in claim 1 in promoting folic acid synthesis.

6. A method for increasing the folic acid content in Arabidopsis thaliana, characterized in that, The claim 1 AtDRTS2M The gene was transferred into Arabidopsis thaliana.

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