Method for constructing 5-ALA high-yield engineering strain, engineering strain and method for producing 5-ALA through fermentation

Abstract

The invention relates to the field of engineering strain modification, in particular to a method for constructing a 5-ALA high-yield engineering strain, the engineering strain and a method for producing 5-ALA through fermentation. According to the invention, a C4 synthesis pathway of 5-ALA is introduced into a chassis strain, and the chassis strain is subjected to metabolic pathway modification. The engineering strain obtained by the method disclosed by the invention is subjected to fed-batch fermentation in a 5L fermentation tank, and the titer of the 5-aminolevulinic acid can reach 28g / L.

Description

Technical Field

[0001] The present invention relates to the field of engineering strain transformation, and in particular to a method for constructing an engineering strain with high 5-ALA production, an engineering strain and a method for producing 5-ALA by fermentation. Background Art

[0002] 5-aminolevulinic acid (5-ALA), also known as 5-amino-4-ketovaleric acid, is a functional non-protein amino acid that exists naturally in organisms. It is a common precursor for the biosynthesis of all porphyrin compounds (heme, chlorophyll, vitamin B12 and cytochrome). It is widely present in bacteria, fungi, animals and plants, and has an important impact on cell energy metabolism. ALA has the advantages of being biodegradable, non-toxic and residue-free, and is widely used in medicine, pesticides, chemicals and other fields.

[0003] In the agricultural field, 5-ALA promotes plant growth and can increase crop yield and quality; in the medical field, 5-ALA is suitable for the prevention, diagnosis and treatment of diabetes and cancer; in the cosmetic field, 5-ALA has a moisturizing function and helps to increase skin moisture and elasticity; in the feed field, 5-ALA can accelerate myosin synthesis, improve anemia, and thus enhance the immunity of poultry and livestock. Limited by production technology, cost and other factors, medicine and agriculture are currently the main application scenarios of 5-ALA, and other fields have not yet been effectively developed.

[0004] The production cost of synthesizing 5-ALA by microbial fermentation is high and still cannot meet the needs of the application market. There are two pathways for the biosynthesis of 5-ALA, C4 and C5. The C4 pathway: 5-ALA is generated by the condensation of succinyl CoA and glycine catalyzed by 5-aminolevulinic acid synthase (ALAS). The C5 pathway: 5-ALA is generated by three steps of glutamate as a substrate through glutamyl tRNA synthetase GluRS, NADPH-dependent glutamyl-tRNA-reductase HemA and glutamate-1-semialdehyde aminotransferase HemL. Among them, the C5 pathway uses tRNA-Glu as a synthetic intermediate and requires ATP and NADPH as cofactors. The limitation of these primary metabolites closely related to energy metabolism and protein synthesis limits the optimization of strain improvement. The substrate glycine and cofactor pyridoxal required for the C4 pathway can be added exogenously during the fermentation process. In the metabolic pathway, the 5-ALA yield can be increased by increasing the supply of succinyl CoA and the catalytic efficiency of ALAS through genetic modification, so the C4 pathway has greater potential.

[0005] Chinese patent application CN112980758A discloses a method for improving the production of 5-aminolevulinic acid synthesized by Corynebacterium glutamicum, by knocking out α-ketoglutarate inhibitor protein and succinate dehydrogenase and overexpressing 5-aminolevulinic acid synthase, recombining the construction of Corynebacterium glutamicum, and synthesizing 25.05g / L of 5-ALA in 48h. Chinese patent CN114369562B discloses a method for improving the expression of 5-aminolevulinic acid, which deletes the glutamate dehydrogenase gene gdhA in Corynebacterium glutamicum, overexpresses the ALA synthase gene hemA and the coding gene pykA of pyridoxal kinase, and the 5-ALA production is increased by 39.5%.

[0006] Although the prior art has disclosed a method for increasing the expression level of 5-aminolevulinic acid through biosynthesis, there is still a need to further improve the production efficiency of 5-aminolevulinic acid and further reduce the production cost. Summary of the invention

[0007] In order to solve the above technical problems, the present invention aims to provide a method for constructing a 5-ALA high-yield engineering strain.

[0008] Another object of the present invention is to provide an engineered strain with high 5-ALA production.

[0009] Another object of the present invention is to provide a method for producing 5-ALA by fermentation.

[0010] The method for constructing a 5-ALA high-yield engineered strain according to the present invention comprises the following steps:

[0011] Using Corynebacterium glutamicum as the starting strain, the DNA restriction modifier gene MRR was knocked out;

[0012] Knockout of succinyl-CoA synthetase gene SucC, glutamate dehydrogenase gene gdhA, α-ketoglutarate inhibitory protein gene odhI, isocitrate lyase gene aceA, and alanine synthetase gene alaT;

[0013] Integration of the pyridoxal kinase encoding gene pdK into the genome;

[0014] Weakened the expression of 5-aminolevulinic acid dehydratase gene hemB;

[0015] The 5-aminolevulinic acid synthase gene was introduced.

[0016] According to the method for constructing a 5-ALA high-yield engineered strain of the present invention, the method further comprises the following steps:

[0017] The NADPH-dependent FMN reductase gene frd was knocked out and the glycine transporter encoding gene cycA was introduced.

[0018] According to the method for constructing a 5-ALA high-yield engineered strain of the present invention, the method further comprises the following steps:

[0019] The transfected cells carry the 5-aminolevulinic acid synthase gene, the phosphoenolpyruvate carboxylase PPC gene, and the pantothenate kinase PTK gene.

[0020] According to the method for constructing a 5-ALA high-yield engineered strain of the present invention, the method further comprises the following steps:

[0021] The 5-ALA efflux protein encoding gene eamA and the DNA oxidative damage repair protein encoding gene were introduced.

[0022] According to the method for constructing a 5-ALA high-yield engineering strain of the present invention, the expression of the 5-aminolevulinic acid dehydratase gene hemB is weakened by introducing a siRNA interference gene of the HemB gene and an RNA interference auxiliary protein encoding gene Hfq.

[0023] The method for constructing a 5-ALA high-yield engineered strain according to the present invention comprises the following steps:

[0024] Using Corynebacterium glutamicum as the starting strain, the DNA restriction modification gene MRR, the succinyl-CoA synthetase gene SucC (SEQ ID No: 12), the glutamate dehydrogenase gene gdhA (SEQ ID No: 13), the α-ketoglutarate inhibitory protein gene odhI (SEQ ID No: 14), the isocitrate lyase gene aceA (SEQ ID No: 15) and the alanine synthetase gene alaT (SEQ ID No: 16) were knocked out, and the pyridoxal kinase encoding gene pdK (SEQ ID No: 8) was integrated into the genome to construct the chassis strain D2;

[0025] Based on the chassis strain D2, the expression of 5-aminolevulinic acid dehydratase gene (hemB) was weakened to construct the chassis strain D3;

[0026] On the basis of the chassis strain D3, the 5-aminolevulinic acid synthase gene (SEQ ID No: 2) was introduced to construct the expression strain V3.

[0027] The method for constructing a 5-ALA high-yield engineered strain according to the present invention further comprises the following steps:

[0028] Based on the chassis strain D3, the NADPH-dependent FMN reductase gene frd (SEQ ID No: 11) was knocked out, and the glycine transporter encoding gene cycA (SEQ ID No: 7) was introduced to construct the chassis strain D4.

[0029] The method for constructing a 5-ALA high-yield engineering strain according to the present invention further comprises the following steps: on the basis of the chassis strain D4, after introducing a plasmid carrying a 5-aminolevulinic acid synthase gene (SEQ ID No: 2), a phosphoenolpyruvate carboxylase gene (SEQ ID No: 3) and a pantothenate kinase gene (SEQ ID No: 5), an expression strain V5 is constructed.

[0030] The method for constructing a 5-ALA high-yield engineered strain according to the present invention further comprises the following steps:

[0031] On the basis of the expression strain V5, a plasmid carrying a 5-ALA efflux protein encoding gene (SEQ ID No: 4) and a DNA oxidative damage repair protein encoding gene (SEQ ID No: 6) was introduced to construct the expression strain V6.

[0032] According to the method for constructing a 5-ALA high-yield engineering strain of the present invention, the expression of the 5-aminolevulinic acid dehydratase gene (hemB) is weakened by introducing a siRNA interference gene (SEQ ID No: 10) of the HemB gene and an RNA interference auxiliary protein encoding gene Hfq (SEQ ID No: 9).

[0033] The present invention provides an engineered strain constructed by the above method.

[0034] Beneficial effects of the present invention:

[0035] 1. The engineered strain obtained by the method of the present invention can produce 5-aminolevulinic acid at a titer of up to 28 g / L in a 5L fermenter after fed-batch fermentation.

[0036] 2. The method of the present invention changes the metabolic pathway of the host cell so that 5-ALA can be accumulated, and obtains a favorable 5-ALA product by enhancing the expression of 5-ALA efflux protein.

[0037] 3. The prior art also has methods for increasing 5-ALA production through genetic engineering techniques, but these methods are different from the methods used in this application to solve the problem:

[0038] The present invention not only further improves the supply of succinate-CoA by genome modification (further knocking out the alanine synthetase gene alaT);

[0039] The technical solution of the present invention increases the in vivo transport of glycine and improves the supply of glycine. Glycine can destroy cell walls. Increasing glycine can increase the permeability of cell walls and increase the yield of product 5-ALA through high-concentration substrates. High-concentration glycine affects the subsequent extraction and purification efficiency. In addition, high-concentration glycine affects cell growth.

[0040] The final yield of 5-ALA is mainly affected by the strain's tolerance to 5-ALA. The peroxides produced by high concentrations of 5-ALA are toxic to cells and cause cell death. By knocking out the NADPH-dependent peroxide synthesis gene, the production of peroxides during fermentation is reduced and the strain's tolerance to high concentrations of 5-ALA is enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The fermentation titer of 5-ALA expressing strain in 5L fermenter is shown;

[0042] Figure 2 This is a schematic diagram of the technical solution of this application. DETAILED DESCRIPTION

[0043] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0044] Unless otherwise specified, the biological materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or by existing known methods. The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the specific methods listed in the book Molecular Cloning Laboratory Manual (3rd Edition) by J. Sambrook, or according to the kits and product instructions.

[0045] The invention introduces a C4 synthesis pathway of 5-ALA into a chassis strain, and performs metabolic pathway modification on the chassis strain: knocking out a succinyl-CoA synthetase gene (sucC), a glutamate dehydrogenase gene (gdhA), an alpha-ketoglutarate inhibitory protein gene (odhI), an isocitrate lyase gene (aceA), an alanine synthetase gene (alaT), an NADPH-dependent FMN reductase gene (frd), and a DNA restriction modifier gene MRR; and enhancing the expression of a 5-ALA efflux protein (eamA), a pyridoxal kinase (pdK), a glycine transporter (cycA), a phosphoenolpyruvate carboxylase (PPC), a pantothenate kinase (PTK), and a DNA oxidative damage repair protein (grxA) by increasing the gene copy number and enhancing gene transcription and translation.

[0046] SEQ ID No: 1 is the amino acid sequence of 5-aminolevulinic acid synthetase. 5-aminolevulinic acid synthetase uses glycine and succinate coenzyme A as substrates and pyridoxal phosphate as a cofactor to catalyze the synthesis of 5-ALA and promote the synthesis of 5-ALA.

[0047] SEQ ID No: 2 is 5-aminolevulinic acid synthase gene

[0048] SEQ ID No: 3 is the gene sequence of phosphoenolpyruvate carboxylase. The carboxylase catalyzes the reaction of phosphoenolpyruvate and carbon dioxide to generate oxaloacetate, supplies oxaloacetate for the tricarboxylic acid cycle, and improves the substrate supply for 5-ALA production.

[0049] SEQ ID No: 4 is the gene sequence encoding 5-ALA efflux protein, which promotes 5-ALA efflux and improves the strain's tolerance to 5-ALA.

[0050] SEQ ID No: 5 is the gene sequence encoding pantothenate kinase, which is a key enzyme in the synthesis of coenzyme A and promotes the synthesis of 5-ALA.

[0051] SEQ ID No: 6 is a gene sequence encoding a DNA oxidative damage repair protein;

[0052] The above-mentioned 5-aminolevulinic acid synthase gene, phosphoenolpyruvate carboxylase gene, and pantothenate kinase encoding gene are carried by the high-copy plasmid pXMJ19; the above-mentioned 5-ALA efflux protein encoding gene and DNA oxidative damage repair protein encoding gene are carried by the high-copy plasmid pEC-XK99E.

[0053] SEQ ID No: 7 is a gene encoding a glycine transporter. As the name implies, a glycine transporter is used for intracellular transport of glycine. The present invention enhances the expression of the glycine transporter, helps to improve the utilization rate of glycine, and improves the substrate utilization rate of 5-ALA production.

[0054] SEQ ID No: 8 is a gene encoding pyridoxal kinase. Pyridoxal kinase converts exogenously added pyridoxal into active pyridoxal phosphate, promoting the synthesis of 5-ALA.

[0055] SEQ ID No: 9 is the RNA molecular chaperone Hfq gene in the siRNA interference system.

[0056] SEQ ID No: 10 is the siRNA interference gene of 5-aminolevulinic acid dehydratase gene.

[0057] SEQ ID No: 11 is a NADPH-dependent FMN reductase gene, which helps to reduce superoxide production and improve the strain's tolerance to 5-ALA.

[0058] SEQ ID No: 12 is a succinyl-CoA synthetase gene. Knocking out or weakening succinyl-CoA synthetase is beneficial to increasing the supply of substrate succinate-CoA and promoting the synthesis of 5-ALA.

[0059] SEQ ID No: 13 is a glutamate dehydrogenase gene. Knocking out or weakening glutamate dehydrogenase is beneficial to increasing the supply of substrate succinate coenzyme A and promoting the synthesis of 5-ALA.

[0060] SEQ ID No: 14 is the α-ketoglutarate inhibitory protein gene. Knocking out or weakening the α-ketoglutarate inhibitory protein is beneficial to increasing the supply of substrate succinate coenzyme A and promoting the synthesis of 5-ALA.

[0061] SEQ ID No: 15 is the isocitrate lyase gene. Knocking out or weakening isocitrate lyase is beneficial to increasing the supply of substrate succinate coenzyme A and promoting the synthesis of 5-ALA.

[0062] SEQ ID No: 16 is the alanine synthetase gene. Knocking out or weakening alanine synthetase is beneficial to increasing the supply of substrate succinate coenzyme A and promoting the synthesis of 5-ALA.

[0063] SEQ ID No: 17 is a DNA restriction modification gene MRR, which improves the stability of foreign genes.

[0064] SEQ ID No: 18 is the 5-aminolevulinic acid dehydratase gene (hemB).

[0065] Experimental methods:

[0066] 1. Preparation of competent cells of Corynebacterium glutamicum

[0067] Seeds of Corynebacterium glutamicum were inoculated into seed culture medium and cultured overnight at 200rpm and 30℃. Transferred to 100mL culture medium at a ratio of 10%, so that the initial cell OD reached 0.3, and cultured at 200rpm and 30℃ for 3-5h until the OD reached 0.6-0.9. All bacterial liquids were placed in a 50ml centrifuge tube and ice-bathed for 15min, and centrifuged at 4000rpm and 4℃ for 10min. Take about 30ml of pre-cooled 10% glycerol, fully suspend the bacteria, and centrifuge at 4000rpm and 4℃ for 10min. Take pre-cooled 10% glycerol again and repeat the wash twice. Resuspend the cells with 400uL of pre-cooled 10% glycerol, and dispense into 1.5ml centrifuge tubes, 80uL per tube, and store at -70℃ or click transformation.

[0068] 2. Electrotransformation of Corynebacterium glutamicum

[0069] Place freshly prepared (or taken out of the refrigerator) competent cells and ligation products on ice, and flick the tube wall to mix. Pipette 5ul of pre-cooled plasmid on ice and add it to the competent cells, flick the tube wall to mix, and then place on ice for 5-10min. Add to the pre-cooled 0.1cm electroporation cup, 1.8kv, 5ms electroporation. Add recovery medium LBHISml, mix and place in a 46℃ water bath for 6min. Incubate at 30℃, 100rpm for 1h. Spread on a plate containing antibiotics and incubate at 30℃ overnight for observation.

[0070] 3. Gene knockout of Corynebacterium glutamicum

[0071] PCR amplified 500bp of the upstream and downstream sequences of the gene to be knocked out, connected to the HindIII-EcoRI site of the pK18mobSacB plasmid to construct a gene knockout plasmid, and the gene knockout plasmid was electrotransformed into Corynebacterium glutamicum to screen for transformants with kanamycin resistance. Pick the transformants to non-antibiotic liquid culture medium, culture overnight at 30°C, dilute the cultured bacterial solution by an appropriate multiple and spread it on the LB-10% sucrose solid plate, and culture it upside down at 30°C overnight until a single clone grows. The grown single colony is plated to a non-antibiotic LB plate and cultured upside down in a 30°C incubator until a colony grows. Mark the colonies and plate them one by one to the LB-resistant plate, and culture them upside down in a 30°C incubator overnight. Screen out the colonies that do not grow bacteria in the LB-resistant plate, and find the corresponding numbered colonies in the non-antibiotic LB plate for PCR verification.

[0072] 4. 5-ALA Fermentation

[0073] Fermentation broth: peptone 10g / L, yeast extract 15g / L, sodium chloride 2.5g / L, glycine 7.5g / L, glucose (sterilized separately) 50g / L.

[0074] Fermentation process: The initial fermentation temperature is 30°C, the ventilation volume is 30L / min, the initial speed is 400rpm, the pH is 7.0, and the speed-linked dissolved oxygen value is not less than 30% when the bacteria grow. When the wet weight of the bacteria reaches about 100g / L (the fermentation time is generally 10h), add IPTG induction (0.5mM / L), pH 6.5, flow glycine (100g / L) and succinic acid (100g / L) (cycle 600s, opening 1s), after 2h of induction, add calcium pantothenate 0.2g / L, pyridoxal hydrochloride 0.2g / L, ascorbic acid (VC) 10mg / L, FeSO4 0.01g / L, MnSO4 0.01g / L, MgSO4 0.2g / L. The speed-linked dissolved oxygen value is set at about 10%, and the fermentation time is roughly within 48h.

[0075] Example 1 Verification of the effect of knocking out the DNA restriction modification gene cluster MRR on 5-ALA production

[0076] The starting strain ATCC13032 was introduced with the pXMJ19 plasmid carrying the 5-aminolevulinic acid synthase gene (SEQ ID No: 2) to construct the expression strain V0. The expression strain V1 was subjected to fed-batch fermentation in a 5L tank to produce 5-ALA. After 48h of fermentation according to the fermentation process, the 5-ALA titer was 1.4g / L.

[0077] Using Corynebacterium glutamicum ATCC13032 (ATCC) as the starting strain, the DNA restriction modification gene cluster MRR was knocked out to construct the chassis strain D1.

[0078] After the pXMJ19 plasmid carrying the 5-aminolevulinic acid synthase gene (SEQ ID No: 2) was introduced into the chassis strain D1, the expression strain V1 was constructed.

[0079] The expression strain V1 was subjected to fed-batch fermentation in a 5 L tank to produce 5-ALA. After 48 h of fermentation according to the fermentation process, the 5-ALA titer was 1.6 g / L.

[0080] Example 2: Increasing the supply of substrate succinyl-CoA by knocking out the competitive pathway that affects the production of succinyl-CoA. And the effect of increasing the supply of cofactor pyridoxal phosphate on 5-ALA production.

[0081] Based on the chassis strain D1, the succinyl-CoA synthetase gene SucC (SEQ ID No: 12), the glutamate dehydrogenase gene gdhA (SEQ ID No: 13), the α-ketoglutarate inhibitory protein gene odhI (SEQ ID No: 14), the isocitrate lyase gene aceA (SEQ ID No: 15) and the alanine synthetase gene alaT (SEQ ID No: 16) were knocked out in sequence, and the pyridoxal kinase encoding gene pdK (SEQ ID No: 8) was integrated into the genome to construct the chassis strain D2.

[0082] The expression strain V2 was constructed by introducing the pXMJ19 plasmid carrying the 5-aminolevulinic acid synthase gene (SEQ ID No: 2) into the chassis strain D2.

[0083] The expression strain V2 was subjected to fed-batch fermentation in a 5 L tank to produce 5-ALA. After 48 h of fermentation according to the fermentation process, the 5-ALA titer was 5.1 g / L.

[0084] Example 3 Verification of the effect of the weakening method of the key gene HemB on 5-ALA production

[0085] On the basis of chassis strain D2, the siRNA interference gene of HemB gene (SEQ ID No: 10) and the RNA interference auxiliary protein encoding gene Hfq (SEQ ID No: 9) were introduced to construct chassis strain D3.

[0086] The expression strain V3 was constructed by introducing the pXMJ19 plasmid carrying the 5-aminolevulinic acid synthase gene (SEQ ID No: 2) into the chassis strain D3.

[0087] The expression strain V3 was subjected to fed-batch fermentation in a 5 L tank to produce 5-ALA. After 48 h of fermentation according to the fermentation process, the 5-ALA titer was 8 g / L.

[0088] Example 4 Effect of reducing self-peroxide production and increasing glycine utilization on 5-ALA production

[0089] Based on the chassis strain D3, the NADPH-dependent FMN reductase gene frd (SEQ ID No: 11) was knocked out, and the glycine transporter encoding gene cycA (SEQ ID No: 7) was introduced to construct the chassis strain D4.

[0090] The expression strain V4 was constructed by introducing the pXMJ19 plasmid carrying the 5-aminolevulinic acid synthase gene (SEQ ID No: 2) into the chassis strain D4.

[0091] The expression strain V4 was subjected to fed-batch fermentation in a 5 L tank to produce 5-ALA. After 48 h of fermentation according to the fermentation process, the 5-ALA titer was 13 g / L and the fermentation liquid was light yellow.

[0092] Example 5: Improving the expression of Supply of succinyl-CoA.

[0093] The substrate of A5-ALA comes from the TCA cycle. An important method to strengthen the TCA cycle is to overexpress the phosphoenolpyruvate carboxylase gene, which can catalyze phosphoenolpyruvate and carbon dioxide to produce oxaloacetate, thereby enhancing the TCA cycle. Pantothenate kinase is a key gene for the synthesis of coenzyme A, which is an important precursor for the synthesis of succinyl-CoA.

[0094] Based on the chassis strain D4, the expression strain V5 was constructed by introducing the pXMJ19 plasmid carrying the 5-aminolevulinic acid synthase gene (SEQ ID No: 2), the phosphoenolpyruvate carboxylase gene (SEQ ID No: 3) and the pantothenate kinase gene (SEQ ID No: 5).

[0095] The expression strain V5 was subjected to fed-batch fermentation in a 5 L tank to produce 5-ALA. After 48 h of fermentation according to the fermentation process, the 5-ALA titer was 16 g / L.

[0096] Example 6 Effects of enhancing 5-ALA efflux and DNA damage repair on 5-ALA production

[0097] Based on the expression strain V5, the pEC-XK99E plasmid carrying the 5-ALA efflux protein encoding gene (SEQ ID No: 4) and the DNA oxidative damage repair protein encoding gene (SEQ ID No: 6) was introduced to construct the expression strain V6. The V6 strain was fermented in a 5L tank with fed-batch fermentation. After 48 hours of fermentation according to the fermentation process, the 5-ALA titer was 28 g / L.

[0098] The above embodiments are only used to understand the technical solution of the present application and do not limit the protection scope of the present application.

Claims

1. A method for constructing a 5-ALA high-yield engineering strain, characterized in that: The method comprises the following steps: Using Corynebacterium glutamicum as the starting strain, the DNA restriction modifier gene MRR was knocked out; Knockout of succinyl-CoA synthetase gene SucC, glutamate dehydrogenase gene gdhA, α-ketoglutarate inhibitory protein gene odhI, isocitrate lyase gene aceA, and alanine synthetase gene alaT; Integration of the pyridoxal kinase encoding gene pdK into the genome; Weakened the expression of 5-aminolevulinic acid dehydratase gene hemB; The 5-aminolevulinic acid synthase gene was introduced.

2. The method for constructing a 5-ALA high-yield engineering strain according to claim 1, characterized in that: The method further comprises the following steps: The NADPH-dependent FMN reductase gene frd was knocked out and the glycine transporter encoding gene cycA was introduced.

3. The method for constructing a 5-ALA high-yield engineering strain according to claim 2, characterized in that: The method further comprises the following steps: The transfected cells carry the 5-aminolevulinic acid synthase gene, the phosphoenolpyruvate carboxylase PPC gene, and the pantothenate kinase PTK gene.

4. The method for constructing a 5-ALA high-yield engineering strain according to claim 3, characterized in that: The method further comprises the following steps: The 5-ALA efflux protein encoding gene eamA and the DNA oxidative damage repair protein encoding gene were introduced.

5. The method for constructing a 5-ALA high-yield engineering strain according to claim 1, characterized in that: The expression of the 5-aminolevulinic acid dehydratase gene hemB is weakened by introducing the siRNA interference gene of the HemB gene and the RNA interference auxiliary protein encoding gene Hfq.

6. An engineered strain constructed by the method for constructing a 5-ALA high-yielding engineered strain according to any one of claims 1 to 5.

7. A method for producing 5-ALA by fermentation, characterized in that: The method comprises the step of fermenting using the engineered strain according to claim 6.

8. The method for producing 5-ALA by fermentation according to claim 7, characterized in that: The formula of the fermentation broth used in the fermentation process is: peptone 10g / L, yeast extract 15g / L, sodium chloride 2.5g / L, glycine 7.5g / L, glucose 50g / L.

9. The method for producing 5-ALA by fermentation according to claim 7, characterized in that: The initial fermentation temperature was 30°C, the ventilation volume was 30 L / min, the initial speed was 400 rpm, the pH was 7.0, and the dissolved oxygen value linked to the speed was not less than 30% during bacterial growth. When the wet weight of the bacterial cells reached 100 g / L, IPTG was added for induction. The pH was 6.5, and 100 g / L of glycine and 100 g / L of succinic acid were added. The cycle was 600 s and the opening was 1 s. After 2 hours of induction, 0.2 g / L of calcium pantothenate was added. Pyridoxal hydrochloride 0.2g / L, ascorbic acid 10mg / L, FeSO4 0.01g / L, MnSO4 0.01g / L, MgSO4 0.2g / L, The fermentation time is within 48 hours.

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