Method for producing heme by using callus of transgenic plant
By expressing nine enzymes in the heme synthesis pathway in rice callus, the efficient production of heme is achieved, solving the problems of high cost, low yield and biosafety risks in traditional methods, and providing a simple and economical heme production method.
Patent Information
- Application Number
- CN202411468743.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-21
- Publication Date
- 2025-01-14
AI Technical Summary
Traditional heme production methods are costly and have biosafety risks, and microbial fermentation yield is low and it is difficult to achieve large-scale production. How to reduce cytotoxicity and maximize heme production is a challenge.
By transferring expression vectors containing 9 enzymes in the heme synthesis pathway into rice callus, heme is expressed and isolated and extracted, the simple production process and low equipment requirements of rice callus to achieve stable and large-scale heme Production.
High expression of heme is achieved in transgenic rice callus, the production process is simple and cost-saving, and the heme content reaches 6.1 times that of the control, avoiding the complex conditions and cytotoxicity problems of microbial fermentation.
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Abstract
Description
(I) Technical field
[0001] The invention relates to a method for producing heme by utilizing transgenic plant callus. (II) Background technology
[0002] Heme is composed of Fe and protoporphyrin IX, and is a cofactor for hemoglobin, myoglobin, peroxidase, cytochrome P450, etc. Heme plays an important role in catalysis, transcription, signal transduction, and electron transfer. Due to its unique physical and chemical properties, heme is widely used in food, medicine, and health care industries. In the food industry, heme is mainly used as a colorant in meat processing. In the health care industry, heme has been developed as an iron supplement to improve iron deficiency anemia. In the pharmaceutical field, heme can be used as a raw material for the preparation of some drugs, such as sodium protoporphyrin, which can be prepared for the treatment of liver disease.
[0003] In organisms, the synthesis pathway of heme involves multiple steps and multiple enzymes. 5-Aminolevulinic acid (5-ALA) is a key precursor of heme and can be synthesized through two pathways, C4 and C5. The C4 pathway exists in humans, animals, fungi, and purple non-sulfur photosynthetic bacteria, while the C5 pathway exists in archaea, plants, and other bacteria. In the C4 pathway, ALA synthase directly converts glycine and succinyl-CoA into 5-ALA. In the C5 pathway, glutamyl-tRNA synthase (GluRS) catalyzes glutamate to form glutamyl-tRNA, which then forms glutamate-1-semialdehyde under the action of glutamyl t-RNA reductase (GluTR). Finally, glutamate-1-semialdehyde aminotransferase (GSAM) catalyzes glutamate-1-semialdehyde to form 5-ALA. There are seven more steps from 5-ALA to heme, involving seven enzymes, namely porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD), uroporphyrinogen III synthase (UROS), uroporphyrinogen decarboxylase (UROD), coproporphyrinogen III oxidase (CPO), protoporphyrin oxidase (PPO) and ferrochelatase (FECH).
[0004] Traditional methods of producing heme include chemical synthesis and extraction from plant tissue or animal blood. Chemical synthesis is costly, while extraction from animal blood may pose a biosafety risk. In plants, the more typical hemoglobin comes from the nodules of legumes and non-legumes, but the number of nodules is small, and extracting heme from nodules cannot solve the problem of quantity.
[0005] In recent years, China has gradually carried out relevant research on the synthesis of heme through microbial fermentation, but the low heme yield and the inability to achieve large-scale production are currently huge challenges. Biological fermentation conditions are complex, and heme production is affected by factors such as culture medium composition, temperature, pH, dissolved oxygen, etc. Excessive intracellular heme can cause lipid peroxidation and produce reactive oxygen, which is cytotoxic. Optimizing the heme synthesis pathway and fermentation process and increasing heme production are current problems that need to be solved. How to reduce cytotoxicity while maximizing heme production is extremely challenging. With the rapid development of plant biotechnology, it is becoming increasingly attractive to use plants as bioreactors to produce valuable proteins or compounds. (III) Summary of the invention
[0006] The present invention aims to provide a method for producing heme using transgenic plant callus. The present invention uses rice callus to produce heme. Rice is a staple food crop, which is long-term edible and has good food and nutritional safety. A large amount of callus can be obtained through tissue culture. Compared with microbial fermentation, the production process is simple and the production equipment requirements are low. The method can stably and scale up the production of heme, so it can be applied to the food, medicine and health care products industries.
[0007] The technical solution adopted by the present invention is:
[0008] The invention provides a method for producing heme by using transgenic plant callus. The method comprises the following steps: transferring an expression vector containing expression frames of 9 enzymes in a heme synthesis pathway into rice callus for expression, obtaining callus rich in heme, separating and extracting the enzymes, and obtaining the heme. The 9 enzymes are: glutamyl t-RNA reductase (GluTR), glutamate-1-semialdehyde aminotransferase (GSAM), porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD), uroporphyrinogen III synthase (UROS), uroporphyrinogen decarboxylase (UROD), coproporphyrinogen III oxidase (CPO), protoporphyrin oxidase (PPO) and ferrochelatase (FECH).
[0009] Further, the enzyme expression frame includes a promoter, an enzyme gene, and a terminator. The promoter can initiate high-level expression of the enzyme in callus tissue. The promoter includes, but is not limited to, a constitutive promoter, preferably a maize ubiquitin promoter (pZmUbi, the nucleotide sequence is shown in SEQ ID NO: 1), a rice actin promoter (pActin, the nucleotide sequence is shown in SEQ ID NO: 2) and a cauliflower mosaic virus 35S promoter (pCaMV35S, the nucleotide sequence is shown in SEQ ID NO: 3). Other promoters that can initiate high-efficiency expression of the target gene in rice callus tissue can also be used, and the method of cloning and testing the promoter function is currently a mature method. The terminator includes but is not limited to NOS terminator (T-NOS, nucleotide sequence as shown in SEQ ID NO: 24), cauliflower mosaic virus 35S (CaMV35S) terminator (nucleotide sequence as shown in SEQ ID NO: 25), potato proteinase inhibitor II terminator (T-pinII, nucleotide sequence as shown in SEQ ID NO: 26), these terminators are common terminators, and the same effect can be achieved by replacing them with other common terminators.
[0010] Furthermore, the nine enzymes in the heme synthesis pathway are: glutamyl t-RNA reductase (GluTR, nucleotide sequence as shown in SEQ ID NO: 4, amino acid sequence as shown in SEQ ID NO: 13), glutamate-1-semialdehyde aminotransferase (GSAM, nucleotide sequence as shown in SEQ ID NO: 5, amino acid sequence as shown in SEQ ID NO: 14), porphobilinogen synthase (PBGS, nucleotide sequence as shown in SEQ ID NO: 6, amino acid sequence as shown in SEQ ID NO: 15), porphobilinogen deaminase (PBGD, nucleotide sequence as shown in SEQ ID NO: 7, amino acid sequence as shown in SEQ ID NO: 16), uroporphyrinogen III synthase (UROS, nucleotide sequence as shown in SEQ ID NO: 8, amino acid sequence as shown in SEQ ID NO: 17), uroporphyrinogen decarboxylase (UROD, nucleotide sequence as shown in SEQ ID NO: 9, amino acid sequence as shown in SEQ ID NO: 18), coproporphyrinogen III oxidase (CPO, nucleotide sequence as shown in SEQ ID NO: 10, amino acid sequence as shown in SEQ ID NO: 19), urea-1-dopamine oxidase (ELD ... NO:19), protoporphyrin oxidase (PPO, nucleotide sequence as shown in SEQ ID NO:11, amino acid sequence as shown in SEQ ID NO:20) and ferrochelatase (FECH, nucleotide sequence as shown in SEQ ID NO:12, amino acid sequence as shown in SEQ ID NO:21).
[0011] Furthermore, the enzyme expression frame also includes a connecting peptide; the connecting peptide includes but is not limited to porcine teschovirus 2A peptide segment (P2A, amino acid sequence as shown in SEQ ID NO:22, nucleotide sequence as shown in SEQ ID NO:28) and hand, foot and mouth disease virus 2A peptide segment (F2A, amino acid sequence as shown in SEQ ID NO:23, nucleotide sequence as shown in SEQ ID NO:29), which is used to connect different genes so that different genes share a promoter and terminator, thereby reducing the size of the inserted sequence.
[0012] Furthermore, the expression vector further comprises a selection marker gene expression cassette; the selection marker gene expression cassette is preferably composed of a promoter, a herbicide-resistant gene, and a terminator, wherein the promoter and the terminator are both promoters and terminators of cauliflower mosaic virus 35S, and the same effect can be achieved by replacing them with other common promoters and terminators. The herbicide-resistant gene comprises a glyphosate-resistant gene, a glufosinate-resistant gene, and a flazol-methyl-resistant gene, preferably a glyphosate-resistant gene g10, the nucleotide sequence of which is shown in SEQ ID NO: 27, the promoter is a CaMV35S promoter, and the terminator is a CaMV35S terminator.
[0013] Furthermore, the basic vector of the expression vector of the present invention is pCambia1300 (NCBI sequence number AF234296).
[0014] Furthermore, the present invention also relates to an expression vector constructed by the method, wherein the expression vector is constructed as follows: (1) the hygromycin resistance gene hptII (located between two XhoI sites) on pCambia1300 is replaced with a herbicide resistance gene (preferably a glyphosate resistance gene g10 (nucleotide sequence as shown in SEQ ID NO:27), so that the glyphosate resistance gene g10 is placed under the regulation of the CaMV35S promoter and terminator, and the transformed vector is named p1300-g10); (2) the vector of step (1) is double-digested with HindIII / KpnI; the expression frames of the 9 enzyme genes in the heme synthesis pathway are connected to the vector after HindIII / KpnI digestion by a seamless cloning method, the seamless cloning product is transferred into the Escherichia coli TG1 strain, and the expression vector is obtained after enzyme digestion and identification.
[0015] The method of introducing the expression vector into rice callus tissue of the present invention includes Agrobacterium-mediated method (Hiei Y, Komari T. Agrobacterium-mediated transformation of rice using immature embryosor calliinduced from mature seed. Nat Protoc, 2008, 3 (5): 824-34.), gene gun method (Tang K, Tinjuangjun P, et al. Particle-bombardment-mediated co-transformation of elite Chinese rice cultivars with genes conferring resistance to bacterial blight and sap-sucking insect pests. Planta, 1999, 208 (4), 552-563.), etc. These methods are mature methods, and those skilled in the art can use these methods to obtain transgenic rice. These different methods can achieve the purpose of obtaining transgenic rice expressing heme of the present invention. The more commonly used transformation method is Agrobacterium-mediated method. This method has the advantages of simple operation and low equipment cost.
[0016] The invention also relates to transgenic rice callus and cells constructed by an expression vector.
[0017] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: (1) the present invention achieves high expression of hemoglobin in transgenic rice callus, the callus with high hemoglobin expression is obviously red, and the hemoglobin expressed is up to 6.1 times that of the control callus; (2) the hemoglobin production method provided by the present invention is simple to operate and convenient for purification; (3) compared with the microbial fermentation method, the method provided by the present invention saves more energy and reduces production costs. (IV) Description of the drawings
[0018] Figure 1 , rice callus expressing heme.
[0019] Figure 2 , relative levels of heme content in callus transformed with vector A. CK, callus of control group (transformed with p1300-g10); A1-10 are callus of different transgenic lines.
[0020] Figure 3, relative levels of heme content in callus transformed with vector B. CK, control group (transformed with p1300-g10) callus; B1-9 are callus of different transgenic lines.
[0021] Figure 4 , Relative levels of heme content in callus transformed with vector C. CK, control group (transformed with p1300-g10) callus; C1-10 are callus of different transgenic lines. (V) Specific implementation methods
[0022] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:
[0023] Example 1. Synthesis of 9 enzyme genes in the heme synthesis pathway
[0024] The NCBI database was used to search for rice-derived glutamyl t-RNA reductase, glutamate-1-semialdehyde aminotransferase, porphobilinogen synthase, porphobilinogen deaminase, uroporphyrinogen III synthase, uroporphyrinogen decarboxylase, coproporphyrinogen III oxidase, protoporphyrin oxidase, and ferrochelatase. The nucleotide and amino acid sequences of the above nine enzymes are as follows: (1) the nucleotide sequence of glutamyl t-RNA reductase is shown in SEQ ID NO:4, and the amino acid sequence is shown in SEQ ID NO:13; (2) the nucleotide sequence of glutamate-1-semialdehyde aminotransferase is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:14; (3) the nucleotide sequence of porphobilinogen synthase is shown in SEQ ID NO:6, and the amino acid sequence is shown in SEQ ID NO:15; (4) the nucleotide sequence of porphobilinogen deaminase is shown in SEQ ID NO:7, and the amino acid sequence is shown in SEQ ID NO:16; (5) the nucleotide sequence of uroporphyrinogen III synthase is shown in SEQ ID NO:8, and the amino acid sequence is shown in SEQ ID NO:17; (6) the nucleotide sequence of uroporphyrinogen decarboxylase is shown in SEQ ID NO:9, and the amino acid sequence is shown in SEQ ID NO:18; (7) the nucleotide sequence of coproporphyrinogen III oxidase is shown in SEQ ID NO:10, and the amino acid sequence is shown in SEQ ID NO:19. NO:19; (8) protoporphyrin oxidase (PPO, the nucleotide sequence is shown in SEQ ID NO:11, and the amino acid sequence is shown in SEQ ID NO:20; (9) ferrochelatase, the nucleotide sequence is shown in SEQ ID NO:12, and the amino acid sequence is shown in SEQ ID NO:21.
[0025] In order to reduce the length of the T-DNA insertion sequence, a 2A connecting peptide strategy was adopted. Specifically, the above nine enzymes were divided into groups of three, each group of enzymes shared a promoter and terminator, and the enzymes in the same group were connected by connecting peptides P2A (nucleotide sequence such as SEQ ID NO: 28) or F2A (nucleotide sequence such as SEQ ID NO: 29). The nucleotide sequence containing the enzyme and the connecting peptide was synthesized by Shanghai Shenggong Company. The grouping of enzymes and the connecting peptides used are shown in Table 1.
[0026] Table 1 Enzyme grouping and connecting peptides used
[0027] serial number Sequentially linked enzymes and linker peptides Group 1 GluTR, F2A, GSAM, F2A, PBGS Group 2 PBGD, F2A, UROS, F2A, UROD Group 3 CPO, F2A, PPO, F2A, FECH Group 4 GluTR, P2A, GSAM, P2A, PBGS Group 5 PBGD, P2A, UROS, P2A, CPO Group 6 UROD, P2A, PPO, P2A, FECH Group 7 GluTR, P2A, GSAM, F2A, FECH Group 8 UROD, P2A, CPO, F2A, PPO Group 9 PBGS, P2A, PBGD, F2A, UROS
[0028] Example 2: Synthesis of promoter and terminator
[0029] Artificially synthesized maize ubiquitin promoter (pZmUbi, nucleotide sequence as shown in SEQ ID NO: 1), rice actin promoter (pActin, nucleotide sequence as shown in SEQ ID NO: 2) and cauliflower mosaic virus 35S promoter (pCaMV35S, nucleotide sequence as shown in SEQ ID NO: 3).
[0030] Artificially synthesized NOS terminator (T-NOS, nucleotide sequence as shown in SEQ ID NO: 24), cauliflower mosaic virus 35S (CaMV35S) terminator (T-35S, nucleotide sequence as shown in SEQ ID NO: 25) and potato protease inhibitor II terminator (T-pinII, nucleotide sequence as shown in SEQ ID NO: 26).
[0031] Example 3: Construction of Rice Genetic Transformation Vector
[0032] Through the seamless cloning method, a rice transformation vector containing the gene expression frames of nine enzymes in the heme synthesis pathway and the glyphosate-tolerant gene expression frame was constructed. Seamless cloning technology can easily achieve multi-fragment recombination, which is a very mature method known in the industry.
[0033] (1) Basic carrier
[0034] The rice genetic transformation vector of the present invention is constructed based on pCambia1300 (NCBI sequence number AF234296). The hygromycin resistance gene hptII (located between two XhoI sites) on pCambia1300 is replaced with the glyphosate resistance gene g10 (nucleotide sequence as shown in SEQ ID NO: 27), so that the glyphosate resistance gene g10 is placed under the regulation of the CaMV35S promoter and terminator, and the transformed vector is named p1300-g10. The vector p1300-g10 is double-digested with HindIII / KpnI.
[0035] (2) Rice transformation vector
[0036] Using seamless cloning technology, according to Table 2, the nucleotide sequence containing enzymes and connecting peptides synthesized in Example 1 was combined with the promoter and terminator artificially synthesized in Example 2, and finally the 9 enzyme gene expression frames in the heme synthesis pathway were connected to the vector p1300-g10 after HindIII / KpnI digestion, and the seamless cloning products (vectors A, B, C) were transferred into the Escherichia coli TG1 strain. After enzyme digestion identification and sequencing, the Escherichia coli plasmid was electroporated into the competent cells of Agrobacterium LBA4404. After enzyme digestion identification was correct, it was preserved in glycerol bacteria and used for rice transformation. The combination of the promoter, terminator, enzyme and connecting peptide used is shown in Table 2.
[0037] Table 2 Combinations of promoters, terminators, enzymes and linker peptides used
[0038]
[0039] Example 4: Rice genetic transformation
[0040] The rice genetic transformation was carried out by Agrobacterium-mediated method (Hiei Y, Komari T. Agrobacterium-mediated transformation of rice using immature embryos or calliinduced from mature seeds. Nat Protoc, 2008, 3(5): 824-34.) The rice variety Xiushui 134 was used as the receptor material, and the specific steps were as follows:
[0041] (1) Callus induction
[0042] The hulled mature rice seeds were treated with 70% alcohol for 90 seconds and then with 20% sodium hypochlorite for 25 minutes; the seeds were sown on 2N6 medium after being washed with sterile water five times and cultured in the dark at 30°C for 7 days; the scutellum was cut from the seeds, transferred to 2N6 medium, and cultured in the dark at 30°C for 10 days; the induced callus was cut into small pieces and used for Agrobacterium infection.
[0043] (2) Agrobacterium infection and screening of resistant calli
[0044] Rice callus was infected with Agrobacterium solution (OD660nm was 1.0) containing the transformation vector and cultured in the dark at 25°C on 2N6-As co-culture medium for 3 days; then cultured in the dark at 28°C on a screening medium (2NBK medium + 200 mg / L of Timentin) containing 400 mg / L of glyphosate. After 4 weeks, the calli were transferred to a new screening medium and subcultured for four weeks, during which the growth of resistant calli was observed.
[0045] The rice callus expressing heme in vector A showed different degrees of red ( Figure 1 A), while the callus of the control group (transformed by p1300-g10) was light yellow ( Figure 1 (B). Figure 1 In A, 1, 2, 3 and 4 are calli of different transgenic lines, which are in red. Figure 1 B is the control group rice callus, which is light yellow. By observing the color, callus with higher hemoglobin content can be screened, such as Figure 1 Calli of A 3 and 4 transgenic lines.
[0046] Both vector B and vector C can produce red callus containing heme.
[0047] (3) Resuspension culture of resistant callus
[0048] The resistant callus with high heme expression was used for cell resuspension culture. The callus tissue was transferred to 2N6 liquid medium and cultured in the dark at 28°C in a shaker at 110 rpm, and subcultured every 7 days until sufficient callus tissue was obtained.
[0049] Example 5: Determination of heme content in callus
[0050] The hemoglobin content in the callus tissue was determined according to the method of Liu et al. (Liu L, Martínez J, Liu Z, et al. Balanced globin protein expression and heme biosynthesis improve production of human hemoglobin in Saccharomyces cerevisiae. Metab Eng. 2014 Jan: 21: 9-16.). The results are shown in Figure 2-4 As shown, Figure 2 , Figure 3 and Figure 4These are the results of heme content determination in callus tissues of different transgenic lines transformed with transformation vectors A, B and C in Example 3. The results show that there are certain differences in heme content in callus tissues of different transgenic lines from the same transformation vector. The maximum heme content in transgenic callus tissues obtained by transformation vectors A, B and C is relatively similar, which are 5.8, 5.9 and 6.1 times that of the control, respectively.
[0051] The results showed that both the connecting peptides P2A and F2A can successfully express multiple enzymes sharing the same promoter and terminator, and the promoters pCaMV35S, pActin and pZmUbi can have multiple combinations with the terminators T-35S, T-NOS and T-pinII, and the maximum heme content among different combinations is relatively similar. The promoters, terminators and connecting peptides known in the art that can express the target gene in Example 1 can all be used for the synthesis of heme in rice callus.
[0052] Example 6: Observation of heme in transgenic rice seeds
[0053] The differentiation and rooting steps of resistant callus were carried out according to the method in the literature (Hiei Y, Komari T. Agrobacterium-mediated transformation of rice using immature embryos or calliinduced from mature seed. Nat Protoc, 2008, 3 (5): 824-34.). The resistant callus obtained by the three vectors in Example 4 and screened by color observation was cultured for differentiation, specifically: the resistant callus was transferred to the differentiation medium (NBKRH40 medium, without hygromycin) and cultured under light at 26°C, and after differentiation to form seedlings, it was transferred to the rooting medium (NBKFH40 medium, hygromycin was replaced by 20 mg / L glyphosate) and cultured under light at 26°C. After the seedlings took root, they were hardened and transplanted to obtain transgenic rice transformants. The p1300-g10-transfected strain and the rice variety Xiushui 134 were used as controls. During the differentiation to rooting stage, some callus lines could not differentiate into seedlings, and some callus lines took longer to differentiate into seedlings than the control group, and the growth of the seedlings was relatively slow. This may be because the overexpression of hemoglobin has a certain effect on plant growth.
[0054] After the transgenic rice matured, the rice seeds were harvested and observed after shelling. The results showed that no red color was observed in the seeds of all transgenic lines and non-transgenic controls. The heme content in the transgenic rice seeds was measured by the method described in Example 5. The results showed that the heme content in the transgenic rice seeds was no different from that in the non-transgenic control rice seeds.
[0055] The above results indicate that using rice callus to express heme is a good choice. These callus can highly express heme without the need to differentiate into rice seedlings, thus avoiding the effects of excessive heme on the plants.
Claims
1. A method for producing heme using transgenic plant callus, characterized in that: The method comprises the following steps: transferring an expression vector containing expression frames of 9 enzymes in a heme synthesis pathway into rice callus tissue and expressing the expression vector to obtain callus tissue rich in heme, separating and extracting the heme, wherein the 9 enzymes are: glutamyl t-RNA reductase GluTR, glutamate-1-semialdehyde aminotransferase GSAM, porphobilinogen synthase PBGS, porphobilinogen deaminase PBGD, uroporphyrinogen III synthase UROS, uroporphyrinogen decarboxylase UROD, coproporphyrinogen III oxidase CPO, protoporphyrin oxidase PPO and ferrochelatase FECH.
2. The method according to claim 1, characterized in that The enzyme expression frame includes a promoter, an enzyme gene, and a terminator.
3. The method according to claim 2, characterized in that The promoters include maize ubiquitin promoter pZmUbi, rice actin promoter pActin, and cauliflower mosaic virus 35S promoter pCaMV35S.
4. The method according to claim 2, characterized in that The terminators include NOS terminator T-NOS, cauliflower mosaic virus 35S terminator, and potato proteinase inhibitor II terminator T-pinII.
5. The method according to claim 2, characterized in that The amino acid sequences of the enzyme genes are shown in SEQ ID NO: 13 to SEQ ID NO: 21 respectively.
6. The method according to claim 1, characterized in that The enzyme expression frame also includes a connecting peptide; the connecting peptide includes the porcine teschovirus 2A peptide segment P2A or the hand, foot and mouth disease virus 2A peptide segment F2A, and the amino acid sequence is shown in SEQ ID NO:22 or SEQ ID NO:
23.
7. The method according to claim 1, characterized in that The expression vector also includes a screening marker gene expression frame; the screening marker gene expression frame consists of a promoter, a herbicide-resistant gene, and a terminator; the herbicide-resistant gene includes a glyphosate-resistant gene, a glufosinate-resistant gene, and a flazasulfuron-resistant gene.
8. A transgenic rice callus and cell constructed with the expression vector according to claim 1.