Application of transcription factor ArnR in promotion of biosynthesis of 5-aminolevulinic acid

Abstract

The invention belongs to the technical field of bioengineering and application, and particularly relates to application of a transcription factor ArnR in promoting biosynthesis of 5-aminolevulinic acid. The amino acid sequence of the transcription factor ArnR is as shown in SEQ ID NO. 1. The biological synthesis efficiency of the 5-ALA is improved by up-regulating the expression level of the transcription factor ArnR, so that the large-scale application of synthesizing the 5-ALA by a biological method is promoted.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering technology and application technology, and particularly relates to the application of transcription factor ArnR in promoting the biosynthesis of 5-aminolevulinic acid. Background Art

[0002] 5-aminolevulinic acid (5-ALA), also known as 5-aminolevulinic acid, is an essential nutrient in the synthesis of chlorophyll, hemoglobin and vitamin B1 12 5-ALA is a common precursor of pyrrole compounds such as 5-ALA, and is widely present in microbial, animal, and plant cells. 5-ALA is non-toxic to humans and animals, easily degrades in the environment, and leaves no residue. It is widely used in medicine and agriculture as a photodynamic drug, biodegradable herbicide, plant growth regulator, and feed additive. Currently, large-scale production of 5-ALA is primarily based on chemical methods. However, chemical synthesis methods are costly, involve significant pollution during the synthesis process, have low yields, and employ harsh reaction conditions, severely hindering the industrial production and widespread application of 5-ALA and its related derivatives. Consequently, microbial synthesis methods have emerged, and with the rise of synthetic biology, the biological synthesis of 5-ALA has further developed.

[0003] Currently, 5-ALA production in Corynebacterium glutamicum typically involves identifying key targets within pathway-related genes. Efficient 5-ALA synthesis is achieved through directed evolution of key enzymes, improved precursor supply, and cofactor metabolic engineering. However, due to the complexity and diversity of metabolic pathways, modifying a single gene can ultimately disrupt cellular homeostasis, resulting in low product synthesis efficiency and hindering large-scale application. Summary of the Invention

[0004] To address the above issues, the present invention provides the use of the transcription factor ArnR in promoting the biosynthesis of 5-aminolevulinic acid. Through research, the present invention first discovered that the expression level of the transcription factor ArnR can regulate the production efficiency of 5-ALA. Upregulating the expression level of the transcription factor ArnR can significantly increase the production of 5-aminolevulinic acid.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides the use of the transcription factor ArnR in promoting the biosynthesis of 5-aminolevulinic acid. The amino acid sequence of the transcription factor ArnR is shown in SEQ ID NO.1.

[0006] In existing research reports, the transcription factor ArnR mainly regulates the nitrate reductase operon narKGHJI The expression of the transcription factor ArnR controls nitrate respiration. arnR As a global transcriptional regulator of microbial nitrogen metabolism, ArnR has been successfully used to improve nitrogen transport and utilization. However, there are no reports on increasing 5-ALA synthesis by regulating the expression of the transcription factor ArnR.

[0007] The present invention can significantly improve the efficiency of synthesizing 5-ALA by upregulating the expression level of the transcription factor ArnR of the strain, thereby helping to promote the large-scale application of 5-ALA synthesis by biological methods.

[0008] Preferably, the application is to promote the biosynthesis of 5-aminolevulinic acid by upregulating the expression level of the transcription factor ArnR.

[0009] Preferably, the application is to promote the biosynthesis of 5-aminolevulinic acid by upregulating the expression level of the transcription factor ArnR of Corynebacterium glutamicum.

[0010] Preferably, the method for upregulating the expression level of the transcription factor ArnR is: transferring a plasmid containing the gene encoding the transcription factor ArnR into Corynebacterium glutamicum, wherein the nucleotide sequence of the gene encoding the transcription factor ArnR is shown in SEQ ID NO. 2.

[0011] Further preferably, the plasmid is a pX-ArnR plasmid, and the construction method of the pX-ArnR plasmid is: Using pXMJ19 plasmid as template and pX-ArnR-F and pX-ArnR-R as upstream and downstream primers, PCR amplification was performed to obtain linearized vector fragment 1; the pX-ArnR - The nucleotide sequence of F is shown in SEQ ID NO. 3, and the nucleotide sequence of pX-ArnR-R is shown in SEQ ID NO. 4; Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification was performed using ArnR-F and ArnR-R as upstream and downstream primers to obtain linearized gene fragment 2; the nucleotide sequence of the ArnR-F is shown in SEQ ID NO. 5, and the nucleotide sequence of the ArnR-R is shown in SEQ ID NO. 6; The fragment 1 and the fragment 2 were connected together and verified by colony PCR to obtain the pX-ArnR plasmid.

[0012] The second aspect of the present invention provides a pX-ArnR plasmid, the construction method of which is as follows: Using pXMJ19 plasmid as template and pX-ArnR-F and pX-ArnR-R as upstream and downstream primers, PCR amplification was performed to obtain linearized vector fragment 1; the pX-ArnR -The nucleotide sequence of F is shown in SEQ ID NO. 3, and the nucleotide sequence of pX-ArnR-R is shown in SEQ ID NO. 4; Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification was performed using ArnR-F and ArnR-R as upstream and downstream primers to obtain linearized gene fragment 2; the nucleotide sequence of the ArnR-F is shown in SEQ ID NO. 5, and the nucleotide sequence of the ArnR-R is shown in SEQ ID NO. 6; The fragment 1 and the fragment 2 were connected together and verified by colony PCR to obtain the pX-ArnR plasmid.

[0013] Preferably, the reaction system for PCR amplification is: 2 μL of template, 2 μL of upstream and downstream primers, 1 μL of dNTP, 25 μL of phanta buffer, 17 μL of sterilized double-distilled water, 1 μL of DNA polymerase, and a total volume of 50 μL; the reaction conditions are: 95°C for 3 min; 95°C for 20 s, 58°C for 20 s, and 72°C for 1 min, 30 cycles; 72°C for 10 min, and 4°C for 10 min.

[0014] The third aspect of the present invention provides a recombinant bacterium comprising the above-mentioned pX-ArnR plasmid.

[0015] Preferably, the recombinant bacteria is derived from Corynebacterium glutamicum.

[0016] Preferably, the recombinant bacteria is integrated with hemA C132A The recombinant strain of Corynebacterium glutamicum containing the gene is the starting strain.

[0017] A fourth aspect of the present invention provides a recombinant strain of Corynebacterium glutamicum Cgh3-pX-ArnR, which is constructed by transforming the above-mentioned pX-ArnR plasmid into the recombinant strain of Corynebacterium glutamicum Cgh3. The construction method of the recombinant strain of Corynebacterium glutamicum Cgh3 is as follows: knocking out the recombinant strain of Corynebacterium glutamicum CGL11-RM-BEI (the strain and its construction method have been disclosed in the patent application number CN202010333584.X) sucCD gene, and obtain the recombinant strain Cgh of Corynebacterium glutamicum; on the chromosome of the recombinant strain Cgh of Corynebacterium glutamicum aceA 、 catA 、 pqo Site integration 3 copies hemA C132A gene, and obtained strain Cgh3.

[0018] Preferably, the construction method of the recombinant strain of Corynebacterium glutamicum Cgh3-pX-ArnR is: integrating the pX-ArnR plasmid into the recombinant strain of Corynebacterium glutamicum Cgh3 by electroporation, and verifying it by colony PCR to obtain the recombinant strain of Corynebacterium glutamicum Cgh3-pX-ArnR.

[0019] A fifth aspect of the present invention provides the use of the recombinant bacteria containing the pX-ArnR plasmid or the recombinant strain Cgh3-pX-ArnR of Corynebacterium glutamicum in the biosynthesis of 5-aminolevulinic acid.

[0020] The sixth aspect of the present invention provides a method for biosynthesizing 5-aminolevulinic acid: after the recombinant bacteria containing the pX-ArnR plasmid or the recombinant strain Cgh3-pX-ArnR of Corynebacterium glutamicum are expanded, inoculated into CGIII medium containing glucose, fermented and cultured at 25~31°C, and glycine is added during the fermentation culture.

[0021] Preferably, the concentration of glucose in the CGIII medium is 30 g / L.

[0022] Preferably, the composition of the CGIII medium is: yeast extract 10 g / L, tryptone 10 g / L, MOPS 21 g / L, NaCl 2.5 g / L, pH=7.

[0023] Alternatively, the expansion method is as follows: streak the recombinant bacteria on BHIS solid medium and incubate in a 30°C incubator for approximately 18 hours. Pick a single colony from the plate and inoculate it into 5 mL of BHIS liquid medium, incubate at 30°C, 220 rpm for 12 to 16 hours, and then transfer 1 mL to CGIII medium and continue incubating for 12 hours.

[0024] The present invention has the beneficial effect of discovering for the first time that the expression level of the transcription factor ArnR can regulate the production efficiency of 5-ALA. Experiments have shown that upregulating the expression level of the transcription factor ArnR in Corynebacterium glutamicum can effectively promote the biosynthesis of 5-ALA. The recombinant Corynebacterium glutamicum strain Cgh3-pX-ArnR, which upregulates the expression level of the transcription factor ArnR, produced 3.73 g / L of 5-ALA under shake flask fermentation conditions, an increase of approximately 2.36 times compared to the control strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the plasmid map of pX-ArnR in Example 1; Figure 2 is the plasmid map of pD-sucCD in Example 2; Figure 3This is a graph showing the production of 5-ALA by the recombinant strain Cgh3-pX-ArnR of Corynebacterium glutamicum under shake flask conditions in Example 3. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] Directed evolution of key enzymes in Corynebacterium glutamicum, improving the supply of precursors, and cofactor metabolic engineering are the main methods currently used to improve the efficiency of 5-ALA synthesis. However, the modification of a single gene may ultimately disrupt cell homeostasis, resulting in lower product synthesis efficiency. To address this problem, an embodiment of the present invention provides the use of the transcription factor ArnR in promoting the biosynthesis of 5-aminolevulinic acid, and the amino acid sequence of the transcription factor ArnR is shown in SEQ ID NO.1. There are currently no reports on improving 5-ALA synthesis by regulating the expression of the transcription factor ArnR. The present invention proposes for the first time to improve the production efficiency of 5-ALA by regulating the expression level of the transcription factor ArnR, providing a new method for high-yield 5-ALA that is feasible and can be used for large-scale production.

[0028] The embodiment of the present invention also provides a pX-ArnR plasmid containing the above gene.

[0029] The present invention also provides a recombinant strain comprising the pX-ArnR plasmid. As a specific embodiment, the present invention provides a recombinant strain of Corynebacterium glutamicum Cgh3-pX-ArnR comprising the pX-ArnR plasmid.

[0030] The present invention also provides the use of the recombinant bacteria in the biosynthesis of 5-aminolevulinic acid.

[0031] The solutions of the present invention are described below through specific embodiments.

[0032] The original strain Corynebacterium glutamicum used in the following examples ( Corynebacterium glutamicum ATCC 13032 was purchased from ATCC (The Global Bioresource Center, http: / / www.atcc.org / ) in October 2012.

[0033] The recombinant strain CGL11-RM-BEI of Corynebacterium glutamicum used in the following examples and its construction method are described in the patent "Corynebacterium glutamicum transcriptional regulatory factor IpsA mutant and its application" (application number: CN202010333584.X). The genotype of the recombinant strain CGL11-RM-BEI of Corynebacterium glutamicum is C. glutamicum ATCC13032 ΔldhA ΔackA-ptaΔpqo Δcat P pyc ::P sod P ppc ::P sod P tal ::P sod P tkt ::P sod P araE ::P tuf (del 21 bp) P ackA-pta ::P sod ldhA::araE ackA-pta::xylAB:C131T NCgl2538:C331T.

[0034] The integration vector pD-aceA::hemA involved in the following examples C132A 、pD-catA::hemA C132A 、pD-pqo::hemA C132A For the specific operation method and primers used for the construction, please refer to the patent "5-aminolevulinic acid-producing Corynebacterium glutamicum engineered bacteria and construction method" (application number: CN202410677943.1).

[0035] The strong promoter P involved in the construction of Cgh3 in the following examples sod-sx The construction method of the patent is "A high-strength promoter P of Corynebacterium glutamicum" sod-sx and Applications” (Application Number: CN202010351748.1).

[0036] The hemA involved in the construction of Cgh3 in the following examples C132A Genes and can be used to amplify genes and P sod* promoter-pEC-ΔlacIq-Δtrc-P sod* -hemA-C132A plasmid, please refer to the 5-ALA synthase mutant and pEC-ΔlacIq-Δtrc-P in the patent "5-aminolevulinic acid synthase mutant of Rhodopseudomonas palustris and its application" (application number: CN202210142094.0). sod* -hemA-C132A plasmid construction method.

[0037] In the following examples, the genotype of Cgh3 is C. glutamicum ATCC13032∆ldhA∆pqo:: P sod- sx hemA C132A ∆cat:: P sod-sx hemA C132A ∆pta∆ackA P sod pyc P sod ppc ∆aceA:: P sod-sx hemA C132A ,P sod tal P sod tkt, P tuf araE (del 21 bp), ackA-pta::P sod xylAB:C131T, NCgl2538:C311T.

[0038] The specific operation methods of the scarless knockout operation technology of Corynebacterium glutamicum and the construction of the tool vector pD-sacB involved in the following examples refer to the patent "High-yield chiral D-(-)-acetoin Corynebacterium glutamicum strain and construction and application" (application number: CN201710215459.7).

[0039] Molecular biological reagents such as DNA ligase used in the following examples were purchased from Thermo Fisher Scientific (http: / / www.thermoscientificbio.com / fermentas), and other biochemical reagents were purchased from Sangon Biotech (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0040] The Escherichia coli used in the following examples ( E. coli ) DH5α competent cells were prepared by the conventional CaCl2 method.

[0041] The culture medium used in the following examples is as follows: CGIII medium: yeast extract 10 g / L, tryptone 10 g / L, MOPS 21 g / L, NaCl 2.5 g / L, pH adjusted to 7 with 5 M NaOH aqueous solution.

[0042] BHIS liquid medium: Brain Heart Infusion Broth powder 74 g / L. BHIS solid medium: Add 2% agar powder to BHIS liquid medium.

[0043] The 5-ALA detection method described in the following examples is as follows: 250 μL of 5-ALA standard solution (final concentrations of 1, 2, 4, 6, and 8 mg / L) or diluted fermentation broth is added to 125 μL of sodium acetate buffer (pH 4.6), followed by 62.5 μL of acetylacetone. The mixture is incubated in a 100°C metal bath for 15 minutes. After cooling to room temperature, 440 μL of freshly prepared Modified Ehrlich's reagent (0.2 g p-dimethylaminobenzaldehyde, 1 mL glacial acetic acid, 1 mL perchloric acid, and the volume is adjusted to 10 mL with glacial acetic acid) is added and mixed thoroughly. The reaction is allowed to react at room temperature for 20 minutes, and the absorbance of the reaction solution is measured at 554 nm. The 5-ALA content in the fermentation broth is calculated using a standard curve generated using the 5-ALA standard. 5-ALA standards are purchased from Sigma (http: / / www.sigmaaldrich.com / sigmaaldrich).

[0044] Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the pXMJ19 plasmid and other materials and reagents used in the following examples can be obtained from commercial channels.

[0045] Example 1 This example provides the pX-ArnR plasmid and its construction process.

[0046] Using the pXMJ19 plasmid as a template and pX-ArnR-F (SEQ ID NO. 3) and pX-ArnR-R (SEQ ID NO. 4) as upstream and downstream primers, PCR amplification was performed to obtain linearized vector fragment 1; Using the genome of Corynebacterium glutamicum ATCC13032 as a template and ArnR-F (SEQ ID NO. 5) and ArnR-R (SEQ ID NO. 6) as upstream and downstream primers, PCR amplification was performed to obtain linearized gene fragment 2; Fragment 1 and fragment 2 were connected using seamless cloning technology, and the obtained connection product was verified by colony PCR using the primer pair test-px-f (SEQ ID NO. 7) / test-px-r (SEQ ID NO. 8). The resulting pX-ArnR plasmid has a map as shown below. Figure 1 shown.

[0047] PCR amplification system: 2 μL template, 2 μL upstream and downstream primers, 1 μL dNTP, 25 μL phanta buffer, 17 μL sterile double-distilled water, 1 μL DNA polymerase, total volume 50 μL.

[0048] PCR reaction conditions were: 95°C for 3 min, 30 cycles (95°C for 20 s, 58°C for 20 s, 72°C for 1 min), 72°C for 10 min, and 4°C for 10 min. PCR products were recovered and purified using a gel recovery kit.

[0049] Example 2 This example provides a recombinant strain of Corynebacterium glutamicum Cgh3-pX-ArnR and its construction process.

[0050] 1. Construction of the recombinant strain Cgh3 of Corynebacterium glutamicum (1) Construction of sucCD knockout plasmid pD-sucCD PCR amplification was performed using pD-sacB as a template and up-sucCD-F (SEQ ID NO. 9) and up-sucCD-R (SEQ ID NO. 10) as upstream and downstream primers to obtain the upstream fragment; Using pD-sacB as a template and down-sucCD-F (SEQ ID NO. 11) and down-sucCD-R (SEQ ID NO. 12) as upstream and downstream primers, PCR amplification was performed to obtain the downstream fragment; PCR amplification was performed using pD-sacB as a template and pD-F (SEQ ID NO. 13) and pD-R (SEQ ID NO. 14) as upstream and downstream primers to obtain the pD vector fragment; After the upstream fragment, downstream fragment and pD vector fragment were recovered and purified, the upstream fragment, downstream fragment and pD vector fragment were connected by molecular cloning using 2X MultiF Seamless Assembly Mix reagent and transformed into Escherichia coli DH5α competent cells. The obtained transformants were verified by colony PCR using the primer pair test-pD-F (SEQ ID NO. 15) / test-pD-R (SEQ ID NO. 16). After sequencing verification, the pD-sucCD plasmid was obtained. The pD-sucCD plasmid map is shown in Figure 1. Figure 2 shown.

[0051] (2) Construction of the recombinant strain Cgh of Corynebacterium glutamicum The pD-sucCD plasmid was introduced into the recombinant strain CGL11-RM-BEI of Corynebacterium glutamicum by electroporation, and the CGL11-RM-BEI genome was knocked out according to the scarless knockout method of Corynebacterium glutamicum in patent CN201710215459.7. sucCD gene, and obtained the recombinant strain Cgh of Corynebacterium glutamicum.

[0052] (3) Construction of the recombinant strain Cgh3 of Corynebacterium glutamicum Integration vector pD-aceA::hemA C132A 、pD-catA::hemA C132A 、pD-pqo::hemA C132A The recombinant strain Cgh of Corynebacterium glutamicum was introduced into the recombinant strain Cgh of Corynebacterium glutamicum by electroporation. According to the scarless knockout method of Corynebacterium glutamicum in patent CN201710215459.7, the recombinant strain Cgh of Corynebacterium glutamicum was cloned into the chromosome of the recombinant strain Cgh of Corynebacterium glutamicum. aceA 、 catA 、 pqo Site integration 3 copies hemA C132A gene, and obtained the recombinant strain Cgh3 of Corynebacterium glutamicum.

[0053] 2. Construction of the recombinant strain Cgh3-pX-ArnR of Corynebacterium glutamicum The pX-ArnR plasmid constructed in Example 1, which showed a correct sequencing result, was integrated into the C. glutamicum recombinant strain Cgh3 via electroporation and evenly plated onto chloramphenicol-resistant BHIS plates (final concentration 10 μg / mL). Single colonies were selected for PCR verification using the following primers: test-px-f (SEQ ID NO. 7) / test-px-r (SEQ ID NO. 8). Those that showed a correct sequencing result were confirmed as recombinant C. glutamicum strains Cgh3-pX-ArnR that had successfully inserted the plasmid and upregulated the transcription factor ArnR in C. glutamicum.

[0054] Example 3 This example provides a method for producing 5-ALA using the recombinant Corynebacterium glutamicum Cgh3-pX-ArnR constructed in Example 2.

[0055] The recombinant strain of Corynebacterium glutamicum Cgh3-pX-ArnR was streaked onto BHIS solid medium and cultured in a 30°C incubator for approximately 18 h. Single colonies were picked from the plate for colony PCR. The verified strains were inoculated into 5 mL of BHIS liquid medium and cultured at 30°C and 220 rpm for 12-16 h. 1 mL was then transferred to CGIII medium and cultured for another 12 h. The initial OD was 0. 600 The cells were cultured in a CGIII medium with a final glucose concentration of 30 g / L at 0.5% OD and a constant temperature shaker at 30°C and 220 rpm for 4 h until the OD 600 When the pH was 5.0, 7.5 g / L of the precursor substance glycine was added, and the 5-ALA production was measured during the culture period.

[0056] The same method was used to investigate the production of 5-ALA by the control strain Cgh3-pX (a strain obtained by integrating the pXMJ19 plasmid into the recombinant strain Cgh3 of Corynebacterium glutamicum by electroporation) under shake flask conditions.

[0057] The results are as follows Figure 3 As shown, after 72 h of culture, the yield of 5-ALA produced by the recombinant strain Cgh3-pX-ArnR of Corynebacterium glutamicum, which upregulated the expression of the transcription factor ArnR in Corynebacterium glutamicum, in shake flask fermentation was 3.73 g / L, which was about 1.36 times higher than that of the control strain Cgh3-pX.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. The application of the transcription factor ArnR in promoting the biosynthesis of 5-aminolevulinic acid, characterized in that: The amino acid sequence of the transcription factor ArnR is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The application is to promote the biosynthesis of 5-aminolevulinic acid by upregulating the expression level of the transcription factor ArnR; or The application is to promote the biosynthesis of 5-aminolevulinic acid by up-regulating the expression level of the transcription factor ArnR of Corynebacterium glutamicum; or The method for increasing the expression level of the transcription factor ArnR is as follows: a plasmid containing the gene encoding the transcription factor ArnR is transferred into Corynebacterium glutamicum, wherein the nucleotide sequence of the gene encoding the transcription factor ArnR is shown in SEQ ID NO.

2.

3. The use according to claim 2, characterized in that The plasmid is pX-ArnR plasmid, and the construction method of the pX-ArnR plasmid is: Using pXMJ19 plasmid as template and pX-ArnR-F and pX-ArnR-R as upstream and downstream primers, PCR amplification was performed to obtain linearized vector fragment 1; the pX-ArnR - The nucleotide sequence of F is shown in SEQ ID NO. 3, and the nucleotide sequence of pX-ArnR-R is shown in SEQ ID NO. 4; Using the genome of Corynebacterium glutamicum ATCC 13032 as a template and ArnR-F and ArnR-R as upstream and downstream primers, PCR amplification was performed to obtain linearized gene fragment 2; the nucleotide sequence of the ArnR-F is shown in SEQ ID NO. 5, and the nucleotide sequence of the ArnR-R is shown in SEQ ID NO. 6; The fragment 1 and the fragment 2 were connected together and verified by colony PCR to obtain the pX-ArnR plasmid.

4. A pX-ArnR plasmid, characterized in that Its construction method is: Using pXMJ19 plasmid as template and pX-ArnR-F and pX-ArnR-R as upstream and downstream primers, PCR amplification was performed to obtain linearized vector fragment 1; the pX-ArnR - The nucleotide sequence of F is shown in SEQ ID NO. 3, and the nucleotide sequence of pX-ArnR-R is shown in SEQ ID NO. 4; Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification was performed using ArnR-F and ArnR-R as upstream and downstream primers to obtain linearized gene fragment 2; the nucleotide sequence of the ArnR-F is shown in SEQ ID NO. 5, and the nucleotide sequence of the ArnR-R is shown in SEQ ID NO. 6; The fragment 1 and the fragment 2 were connected together and verified by colony PCR to obtain the pX-ArnR plasmid. A recombinant bacterium comprising the pX-ArnR plasmid according to claim 4 .

6. The recombinant bacterium according to claim 5, characterized in that The recombinant bacteria uses Corynebacterium glutamicum as the starting strain.

7. The recombinant bacterium according to claim 5, characterized in that The recombinant bacteria are integrated hemA C132A The recombinant strain of Corynebacterium glutamicum containing the gene is the starting strain.

8. A recombinant strain of Corynebacterium glutamicum Cgh3-pX-ArnR, characterized in that The recombinant strain Cgh3-pX-ArnR of Corynebacterium glutamicum is constructed by transforming the pX-ArnR plasmid of claim 4 into the recombinant strain Cgh3 of Corynebacterium glutamicum. The construction method of the recombinant strain Cgh3 of Corynebacterium glutamicum is as follows: knocking out the pX-ArnR plasmid of Corynebacterium glutamicum recombinant strain CGL11-RM-BEI. sucCD gene, and obtained the recombinant strain Cgh of Corynebacterium glutamicum; on the chromosome of the recombinant strain Cgh of Corynebacterium glutamicum aceA 、 catA 、 pqo 3 copies integrated into the site hemA C132A gene, and obtained strain Cgh3.

9. Use of the recombinant bacterium according to any one of claims 5 to 7 or the recombinant strain Cgh3-pX-ArnR of Corynebacterium glutamicum according to claim 8 in the biosynthesis of 5-aminolevulinic acid.

10. A method for biosynthesizing 5-aminolevulinic acid, characterized in that: The recombinant bacteria according to any one of claims 5 to 7 or the recombinant strain Cgh3-pX-ArnR of Corynebacterium glutamicum according to claim 8 is expanded and inoculated into a CGIII medium containing glucose, and fermented at 25 to 31° C., and glycine is added during the fermentation.

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