Genetically engineered bacterium for efficiently synthesizing 5-aminolevulinic acid

Abstract

The invention relates to the field of genetic engineering, in particular to a genetically engineered bacterium for efficiently synthesizing 5-aminolevulinic acid. According to the invention, a synthetic biological technology and a genetic engineering means are utilized, escherichia coli E.coli BL21 (DE3) is taken as an original strain, a C5 way of the escherichia coli E.coli BL21 (DE3) is modified, and degradation of glutamic acid is reduced by knocking out a gadA gene and replacing a promoter of an argE gene; the synthesis of the 5-aminolevulinic acid is increased by enhancing the expression of gdhA, hemAS and hemL genes, replacing promoters and increasing the carbon flux flowing to the 5-aminolevulinic acid; in addition, by knocking out acetic acid synthesis genes ackA, poxB and pta, the loss of carbon flux flowing to TCA circulation is reduced, so that the purpose of increasing the yield of 5-aminolevulinic acid is achieved.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a genetic engineering bacterium for efficiently synthesizing 5-aminolevulinic acid. Background Art

[0002] Aminolevulinic acid (ALA) is a non-protein amino acid that is essential for the synthesis of heme, chlorophyll, cytochrome and vitamin B12. 12 The first identified intermediate in the common tetrapyrolytic pathway of glutamate. In fact, there are two known metabolic pathways in living organisms. One of the pathways is the C4 pathway, which condenses succinyl-CoA and glycine into ALA via ALA synthetase (ALAS). The C4 pathway is limited to mammals, fungi, and purple non-sulfur bacteria. The second pathway is the C5 pathway, which involves three enzymatic reactions leading to the biosynthesis of ALA from glutamate. The C5 pathway is active in most bacteria, all archaea, and plants.

[0003] Escherichia coli is the most widely used strain in the current microbial fermentation industry and has become an important choice. In Escherichia coli, the biosynthesis of ALA is carried out through the C5 pathway and is strictly regulated by the feedback inhibition of heme, the final product of the C5 pathway. In the current research on ALA-producing strains, the pathway strength of 5-aminolevulinic acid in Escherichia coli is mainly strengthened, while the carbon flow of the target product is ensured by knocking out the branch byproduct pathway. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a genetically engineered bacterium for efficiently synthesizing 5-aminolevulinic acid. Escherichia coli BL21 (DE3) is used as a starting strain, and its C5 pathway is modified to achieve the purpose of increasing the yield of 5-aminolevulinic acid.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a genetically engineered bacterium for efficiently synthesizing 5-aminolevulinic acid, wherein the genetically engineered bacterium is obtained by subjecting Escherichia coli to the following genetic modifications: knocking out the gadA gene, and replacing the promoter of the argE gene with any one of J23100, J23111, J23104, J23108, and J23103.

[0007] The present invention utilizes synthetic biology technology and genetic engineering to use Escherichia coli as the starting strain and to modify its own C5 pathway (such as Figure 1The present invention has found through a large number of experimental studies that by knocking out the gadA gene and replacing the promoter of argE, the degradation of glutamate can be reduced, thereby increasing the yield of 5-aminolevulinic acid.

[0008] More preferably, the genetically engineered bacteria are obtained by subjecting Escherichia coli to the following genetic modifications: knocking out the gadA gene, and replacing the promoter of the argE gene with J23108.

[0009] Preferably, the genetically engineered bacteria further include the following genetic modification: enhancing the gene expression or enzyme activity of at least one of gdhA, hemAS and heml.

[0010] The present invention has found through a large number of experimental studies that the synthesis of 5-aminolevulinic acid can be further increased by enhancing the expression or enzyme activity of gdhA, hemAS, and heml genes.

[0011] More preferably, the genetically engineered bacteria are obtained by genetically modifying Escherichia coli in the following manner: enhancing the gene expression or enzyme activity of hemAS and heml.

[0012] Preferably, the genetically engineered bacteria further comprises the following genetic modification: knocking out at least one of the ackA, poxB, and pta genes.

[0013] The present invention has found through a large number of experimental studies that the production of 5-aminolevulinic acid can be further increased by knocking out the acetate biosynthesis genes ackA, poxB, and pta in a superimposed manner to reduce the loss of carbon flux to the TCA cycle.

[0014] More preferably, the genetically engineered bacteria further comprises the following genetic modification: knocking out ackA, poxB and pta genes.

[0015] Preferably, the Escherichia coli is E. coli BL21 (DE3).

[0016] In a second aspect, the present invention provides the use of the above-mentioned genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid in the production of 5-aminolevulinic acid.

[0017] In a third aspect, the present invention provides a method for producing 5-aminolevulinic acid, wherein 5-aminolevulinic acid is obtained by inducing fermentation of the genetically engineered bacteria that efficiently synthesize 5-aminolevulinic acid.

[0018] The beneficial effects of the present invention are:

[0019] The invention utilizes synthetic biology technology and genetic engineering means, takes Escherichia coli BL21 (DE3) as a starting strain, transforms its C5 pathway, reduces the degradation of glutamate by knocking out the gadA gene and replacing the promoter of the argE gene; increases the synthesis of 5-aminolevulinic acid by increasing the carbon flux flowing to 5-aminolevulinic acid by enhancing the expression of gdhA, hemAS and heml genes and replacing the promoter; and reduces the loss of carbon flux flowing to the TCA cycle by knocking out the acetate synthesis genes ackA, poxB and pta, thereby achieving the purpose of increasing the yield of 5-aminolevulinic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the C5 pathway for ALA synthesis. DETAILED DESCRIPTION

[0021] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0022] The primers used in the present invention are all synthesized by biological companies, and the plasmids and strains used in the present invention can be purchased from the market.

[0023] The primers used in the embodiments of the present invention and the constructed homologous repair arm sequences are shown in Table 1.

[0024] Table 1

[0025]

[0026]

[0027]

[0028]

[0029] Embodiment 1:

[0030] An embodiment of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modification on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23100.

[0031] The construction method of the genetically engineered bacteria described in this embodiment is:

[0032] 1. Knockout of gadA gene:

[0033] The gadA gene of the genetically engineered bacterium Escherichia coli BL21 (DE3) was knocked out using the "CRISPR / Cas9" technology, including the following steps:

[0034] (a) The gadA gene sequence included in GenBank on NCBI was used as the target gene sequence, and the sgRNA of the gene was designed through online retrieval at http: / / chopchop.cbu.uib.no / . The sgRNA was connected to the plasmid pEcgRNA (synthesized by General Company), and the plasmid was transformed into DH5α and cultured overnight. The positive clones were selected for culture, and the plasmid was extracted using the Tiangen Biochemical Extraction Kit to obtain the plasmid pEcgRNA::gadA-N20.

[0035] (b) Preparation of homologous fragments: Primers required for gadA gene knockout were designed based on the GenBank genome information published in the NCBI database. Escherichia coli BL21 (DE3) genomic DNA was used as a template. The upstream homology arm primers gadA-up-F and gadA-up-R and the downstream homology arm primers gadA-down-F and gadA-down-R were used to amplify the homology arm sequences gadA-1 and gadA-2, respectively. After the products were purified and recovered, the fragments gadA-1 and gadA-2 were amplified and connected using the overlapping PCR technology with primers gadA-up-F and gadA-down-R to obtain the gadA gene homology repair arms.

[0036] (c) The pEcCas (synthesized by General Company) plasmid was transformed into Escherichia coli BL21(DE3) to obtain Escherichia coli BL21(DE3)::pEcCas, and positive clones were selected for electroporation competent preparation. The constructed plasmid pEcgRNA::gadA-N20 and the homologous repair arm of the gadA gene were co-electroporated into Escherichia coli BL21(DE3)::pEcCas and coated on 50 μg mL -1 Kanamycin and 50 μg mL -1 The cells were cultured on spectinomycin double-resistance plates at 37°C overnight, and the positive recombinants were identified by PCR using gadA-up-F and gadA-down-R.

[0037] (d) The strain with successful knockout was selected and cultured in LB liquid medium with 50 μg / mL kanamycin and 10 mmol / L rhamnose for 6 h to induce the elimination of plasmid pEcgRNA, subcultured at 37°C for 2 h, and streaked on a 10 g / L sucrose solid plate to eliminate plasmid pEcCas, thereby obtaining a strain with gadA gene knockout, which was named ALA-01.

[0038] 2. Replace the promoter of argE gene with J23100

[0039] (e) The primer pair argE-F1 and argE-R1 were designed to clone the argE gene from Escherichia coli, and the cloned argE fragment was ligated to the plasmid pETDuet-1 by homologous recombination technology to construct the plasmid pET-argE.

[0040] (f) Using “CRISPR / Cas9” technology to replace the promoter of the argE gene of the genetically engineered bacterium ALA-1:

[0041] The argE gene sequence included in GenBank on NCBI was used as the target gene sequence. The sgRNA of the gene was designed through online retrieval at http: / / chopchop.cbu.uib.no / . The sgRNA was connected to the plasmid pEcgRNA (synthesized by General Company), and the plasmid was transformed into DH5α and cultured overnight. The positive clones were selected for culture, and the plasmid was extracted using the Tiangen Biochemical Extraction Kit to obtain the plasmid pEcgRNA::argE-N20.

[0042] (g) Preparation of homologous fragments: promoter P J23100 The primer pairs argE-J23100-F and argE-J23100-R were designed and synthesized by General Company. The pET-argE plasmid was amplified by PCR using the primer pairs to obtain a linearized vector. The promoter sequences were connected to the plasmid pETDuet-1 by homologous recombination technology to obtain the plasmid pET-P J23100 -argE; then design primer pair argE-F2, argE-R2 for plasmid pET-P J23100 -argE was amplified by PCR to obtain fragment P J23100 -argE was used as the homologous repair arm of the argE gene. The pEcCas (synthesized by General Company) plasmid was transformed into the strain ALA-01 with the gadA gene knocked out obtained in step 1(d) to obtain ALA-01::pEcCas, and the positive clones were selected for electroporation competent preparation.

[0043] The constructed plasmid pEcgRNA::argE-N20 and the homology repair arm of argE gene were co-transfected into ALA-01::pEcCas and coated on 50 μg mL -1 Kanamycin 50 μg mL -1 The culture was cultured overnight at 37°C on a spectinomycin-resistant plate. The positive recombinants were identified by PCR using argE-J23100-F and argE-J23100-R. The knockout strains were selected and plated at 50 μg mL -1 Kanamycin, 10mmol·L -1The cells were cultured in rhamnose LB liquid medium for 6 h to induce elimination of plasmid pEcgRNA, subcultured at 37 °C for 2 h, and streaked at 10 g·L -1 Sucrose solid plate was used to eliminate the plasmid pEcCas to obtain the genetically engineered strain, which was named ALA-02.

[0044] Embodiment 2:

[0045] An embodiment of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modification on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23111.

[0046] The construction method of the genetically engineered bacteria described in this embodiment is:

[0047] The process of knocking out the gadA gene is the same as step (1) in Example 1.

[0048] In this example, the promoter of the argE gene was replaced with J23111. The step of replacing the promoter was referred to step (2). The designed primers included: argE-J23111-F and argE-J23111-R. The remaining steps were the same as in Example 1. The genetically engineered strain obtained in this example was named ALA-03.

[0049] Embodiment 3:

[0050] An embodiment of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modification on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23104.

[0051] The construction method of the genetically engineered bacteria described in this embodiment is:

[0052] The process of knocking out the gadA gene is the same as step (1) in Example 1.

[0053] In this example, the promoter of the argE gene was replaced with J23104. The step of replacing the promoter was referred to step (2). The designed primers included: argE-J23104-F and argE-J23104-R. The remaining steps were the same as in Example 1. The genetically engineered strain obtained in this example was named ALA-04.

[0054] Embodiment 4:

[0055] An embodiment of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modification on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23108.

[0056] The construction method of the genetically engineered bacteria described in this embodiment is:

[0057] The process of knocking out the gadA gene is the same as step (1) in Example 1.

[0058] In this example, the promoter of the argE gene was replaced with J23108. The step of replacing the promoter was referred to step (2). The designed primers included: argE-J23108-F and argE-J23108-R. The remaining steps were the same as in Example 1. The genetically engineered strain obtained in this example was named ALA-05.

[0059] Embodiment 5:

[0060] An embodiment of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modification on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23103.

[0061] The construction method of the genetically engineered bacteria described in this embodiment is:

[0062] The process of knocking out the gadA gene is the same as step (1) in Example 1.

[0063] In this example, the promoter of the argE gene was replaced with J23103. The step of replacing the promoter was referred to step (2). The designed primers included: argE-J23103-F and argE-J23103-R. The remaining steps were the same as in Example 1. The genetically engineered strain obtained in this example was named ALA-06.

[0064] Embodiment 6-8:

[0065] An embodiment of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to the present invention;

[0066] The genetically engineered bacteria of Example 6 are obtained by performing the following genetic modifications on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23108; and enhancing the gene expression or enzyme activity of gdhA.

[0067] The genetically engineered bacteria of Example 7 are obtained by performing the following genetic modifications on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23108; and enhancing the gene expression or enzyme activity of hemAS.

[0068] The genetically engineered bacteria of Example 8 are obtained by performing the following genetic modifications on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23108; and enhancing the gene expression or enzyme activity of heml.

[0069] The construction method of the genetically engineered bacteria is:

[0070] 1. Design primer pairs gdhA-F1, gdhA-R1, hemAS-F1, hemAS-R1, heml-F1, heml-R1 to clone the gdhA, hemAS, heml genes from Escherichia coli, respectively, and connect the cloned gdhA, hemAS, heml fragments to plasmid pETDuet-1 by homologous recombination technology to construct plasmids pET-gdhA, pET-hemAS, pET-hemL, respectively.

[0071] 2. Recombinant plasmid transformation: The plasmid pET-gdhA was mixed with ALA-05 competent cells and placed on ice for 30 min, then quickly heat-shocked (42°C, 90 s), and then quickly placed on ice and allowed to stand for 2 min. 1 mL of LB liquid culture medium was added under sterile conditions, and the culture was resumed at 37°C, 220 rpm for 1 h to obtain the genetically engineered strain, named ALA-07.

[0072] The same operation was performed as above, and the plasmid pET-hemAS was transformed into ALA-05 competent cells to obtain the genetically engineered strain, named ALA-08; the plasmid pET-hemL was transformed into ALA-05 competent cells to obtain the genetically engineered strain, named ALA-09.

[0073] Examples 6, 7, and 8 correspond to genetically engineered strains ALA-07, ALA-08, and ALA-09, respectively.

[0074] Embodiment 9:

[0075] An embodiment of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modification on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23108; and enhancing the gene expression or enzyme activity of gdhA and hemAS.

[0076] The construction method of the genetically engineered bacteria described in this embodiment is:

[0077] 1. The construction of plasmids pET-gdhA and pET-hemAS was the same as step (1) of Example 6-8.

[0078] 2. Design primer pairs PET-F1 and PET-R1, use the primer pairs to perform PCR amplification on the pET-hemAS plasmid to obtain a linearized vector, and connect the promoter sequence to the plasmid pET-gdhA through homologous recombination technology to construct the plasmid pET-gdhA-hemAS.

[0079] 3. Recombinant plasmid transformation: The same transformation method as step (2) in Example 6-8 was used to transform the plasmid pET-gdhA-hemAS into ALA-05 competent cells to obtain the genetically engineered strain, named ALA-10.

[0080] Embodiment 10:

[0081] An embodiment of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modification on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23108; and enhancing the gene expression or enzyme activity of gdhA and heml.

[0082] The construction method of the genetically engineered bacteria described in this embodiment is:

[0083] 1. The construction of plasmids pET-gdhA and pET-hemL was the same as step (1) of Example 6-8.

[0084] 2. Design primer pairs PET-F1 and PET-R1, use the primer pairs to perform PCR amplification on the pET-hemL plasmid to obtain a linearized vector, and connect the promoter sequence to the plasmid pET-gdhA through homologous recombination technology to construct the plasmid pET-gdhA-hemL.

[0085] 3. Recombinant plasmid transformation: The same transformation method as step (2) in Example 6-8 was used to transform the plasmid pET-gdhA-hemL into ALA-05 competent cells to obtain the genetically engineered strain, named ALA-11.

[0086] Embodiment 11:

[0087] An embodiment of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modifications on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23108; and enhancing the gene expression or enzyme activity of hemAS and heml.

[0088] The construction method of the genetically engineered bacteria described in this embodiment is:

[0089] 1. The construction of plasmids pET-hemAS and pET-hemL is the same as step (1) of Example 6-8.

[0090] 2. Design primer pairs PET-F1 and PET-R1, use the primer pairs to perform PCR amplification on the pET-hemAS plasmid to obtain linearized vectors, and connect the promoter sequences to the plasmid pET-hemL through homologous recombination technology to construct plasmid pET-hemAS-hemL.

[0091] 3. Recombinant plasmid transformation: The same transformation method as step (2) in Example 6-8 was used to transform the plasmid pET-hemAS-hemL into ALA-05 competent cells to obtain the genetically engineered strain, named ALA-12.

[0092] Embodiment 12:

[0093] An embodiment of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modification on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23108; and enhancing the gene expression or enzyme activity of gdhA, hemAS, and heml.

[0094] The construction method of the genetically engineered bacteria described in this embodiment is:

[0095] 1. The construction of plasmids pET-gdhA, pET-hemAS, and pET-hemL is the same as step (1) of Example 6-8.

[0096] 2. Design primer pairs PET-F1 and PET-R1, use the primer pairs to perform PCR amplification on the pET-hemAS plasmid, obtain linearized vectors, and connect the promoter sequences to the plasmid pET-gdhA through homologous recombination technology to construct the plasmid pET-gdhA-hemAS.

[0097] 3. Design primer pairs PET-F2 and PET-R2, use the primers to perform PCR amplification on the pET-hemL plasmid to obtain a linearized vector, and connect the promoter sequence to the plasmid pET-gdhA-hemAS through homologous recombination technology to construct the plasmid pET-gdhA-hemAS-hemL.

[0098] 4. Recombinant plasmid transformation: The same transformation method as step (2) in Example 6-8 was used to transform the plasmid pET-gdhA-hemAS-hemL into ALA-05 competent cells to obtain the genetically engineered strain, named ALA-13.

[0099] Embodiment 13:

[0100] An embodiment of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modifications on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23108; enhancing the gene expression or enzyme activity of hemAS and heml; and knocking out the ackA gene.

[0101] The construction method of the genetically engineered bacteria described in this embodiment is:

[0102] The ackA gene of the genetically engineered bacterium ALA-12 was knocked out using the "CRISPR / Cas9" technology, including the following steps:

[0103] 1. Use the ackA gene sequence included in GenBank on NCBI as the target gene sequence, search online at http: / / chopchop.cbu.uib.no / , design the sgRNA of the gene, connect the sgRNA with the plasmid pEcgRNA (synthesized by General Company), transform the plasmid into DH5α, and culture overnight. Select the positive clones for culture, extract the plasmid using the Tiangen Biochemical Extraction Kit, and obtain the plasmid pEcgRNA::ackA-N20.

[0104] 2. Preparation of homologous fragments: Primers required for ackA gene knockout were designed according to the GenBank genome information published in the NCBI database. Escherichia coli BL21 (DE3) genomic DNA was used as a template. The upstream homology arm primers ackA-up-F and ackA-up-R and the downstream homology arm primers ackA-down-F and ackA-down-R were used to amplify the homology arm sequences ackA-1 and ackA-2, respectively. After the products were purified and recovered, the fragments ackA-1 and ackA-2 were amplified and connected using overlapping PCR technology with primers ackA-up-F and ackA-down-R to obtain the homology repair arms of the ackA gene.

[0105] 3. Transform the pEcCas (synthesized by General Company) plasmid into genetically engineered bacteria ALA-12 to obtain ALA-12::pEcCas, and select positive clones for electroporation competent preparation. The constructed plasmid pEcgRNA::ackA-N20 and the homologous repair arm of the ackA gene were co-electroporated into ALA-12::pEcCas and coated on 50 μg mL -1 Kanamycin and 50 μg mL -1 The cells were cultured on spectinomycin double-resistance plates at 37°C overnight, and the positive recombinants were identified by PCR using ackA-up-F and ackA-down-R.

[0106] 4. The strain with successful knockout was selected and cultured in 50 μg / mL kanamycin and 10 mmol / L rhamnose LB liquid medium for 6 h to induce the elimination of plasmid pEcgRNA, subcultured at 37°C for 2 h, streaked on a 10 g / L sucrose solid plate, and the plasmid pEcCas was eliminated to obtain the genetically engineered strain, which was named ALA-14.

[0107] Embodiment 14:

[0108] An embodiment of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modifications on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23108; enhancing the gene expression or enzyme activity of hemAS and heml; and knocking out the poxB gene.

[0109] The difference between the construction method of the genetically engineered bacteria described in this example and that in Example 13 is that:

[0110] This example is to knock out the poxB gene. The steps of knocking out the gene refer to Example 13. The primers used in this example include: poxB-up-F, poxB-up-R, poxB-down-F, poxB-down-R, poxB-N20-F, poxB-N20-R, poxB-sgRNA. The genetically engineered strain obtained in this example is named ALA-15.

[0111] Embodiment 15:

[0112] An embodiment of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modifications on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23108; enhancing the gene expression or enzyme activity of hemAS and heml; and knocking out the pta gene.

[0113] The difference between the construction method of the genetically engineered bacteria described in this example and that in Example 13 is that:

[0114] This example is to knock out the pta gene. The steps of knocking out the gene refer to Example 13. The primers used in this example include: pta-up-F, pta-up-R, pta-down-F, pta-down-R, pta-N20-F, pta-N20-R, pta-sgRNA. The genetically engineered strain obtained in this example is named ALA-16.

[0115] Embodiment 16:

[0116] An embodiment of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modifications on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23108; enhancing the gene expression or enzyme activity of hemAS and heml; and knocking out the ackA and poxB genes.

[0117] The difference between the construction method of the genetically engineered bacteria described in this example and that in Example 13 is that:

[0118] This example is to knock out the ackA and poxB genes. The steps of knocking out the genes refer to Example 13. The primers used in this example include: ackA-up-F, ackA-up-R, ackA-down-F, ackA-down-R, ackA-N20-F, ackA-N20-R, ackA-sgRNA, poxB-up-F, poxB-up-R, poxB-down-F, poxB-down-R, poxB-N20-F, poxB-N20-R, poxB-sgRNA. The genetically engineered strain obtained in this example was named ALA-17.

[0119] Embodiment 17:

[0120] An embodiment of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modifications on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23108; enhancing the gene expression or enzyme activity of hemAS and heml; and knocking out the ackA and pta genes.

[0121] The difference between the construction method of the genetically engineered bacteria described in this example and that in Example 13 is that:

[0122] This example is to knock out the ackA and pta genes. The steps of knocking out the genes refer to Example 13. The primers used in this example include:

[0123] ackA-up-F, ackA-up-R, ackA-down-F, ackA-down-R, ackA-N20-F, ackA-N20-R, ackA-sgRNA, pta-up-F, pta-up-R, pta-down-F, pta-down-R, pta-N20-F, pta-N20-R, pta-sgRNA. The genetically engineered strain obtained in this example was named ALA-18.

[0124] Embodiment 18:

[0125] An embodiment of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modifications on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23108; enhancing the gene expression or enzyme activity of hemAS and heml; and knocking out the poxB and pta genes.

[0126] This example is to knock out the poxB and pta genes. The steps of knocking out the genes refer to Example 13. The primers used in this example include:

[0127] poxB-up-F, poxB-up-R, poxB-down-F, poxB-down-R, poxB-N20-F, poxB-N20-R, poxB-sgRNA, pta-up-F, pta-up-R, pta-down-F, pta-down-R, pta-N20-F, pta-N20-R, pta-sgRNA. The genetically engineered strain obtained in this example was named ALA-19.

[0128] Embodiment 19:

[0129] An embodiment of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modifications on Escherichia coli: knocking out the gadA gene; replacing the promoter of the argE gene with J23108; enhancing the gene expression or enzyme activity of hemAS and heml; and knocking out the ackA, poxB, and pta genes.

[0130] This example is to knock out ackA, poxB, and pta genes. The steps of knocking out genes refer to Example 13. The primers used in this example include:

[0131] ackA-up-F, ackA-up-R, ackA-down-F, ackA-down-R, ackA-N20-F, ackA-N20-R, ackA-sgRNA, poxB-up-F, poxB-up-R, poxB-down-F, poxB-down-R, poxB-N20-F, poxB-N20-R, poxB-sgRNA, pta-up-F, pta-up-R, pta-down-F, pta-down-R, pta-N20-F, pta-N20-R, pta-sgRNA. The genetically engineered strain obtained in this example was named ALA-20.

[0132] Test Example 1: Shake flask culture to detect the ability of strains to produce ALA

[0133] Test sample: genetically engineered bacteria ALA-2-ALA-20 constructed in Example 1-19.

[0134] Fermentation treatment of genetically engineered bacteria:

[0135] Prepare fermentation base in a 500 mL conical flask: 20 g / L glycerol, 0.020 g / L CaCl 2、 1.5g / L (NH4)2HPO4, 3g / L citric acid, 16g / L Na2HPO4·12H2O, 3g / L NH4Cl, 2g / L yeast extract and 10g / L tryptone.

[0136] Pick a single colony of genetically engineered bacteria and activate it in a test tube culture medium to obtain the OD 600 nm When the bacterial solution is close to 15, inoculate 1 mL of the bacterial solution into a conical flask containing the base material, and add 1.5 mL of glucose, 1.5 mL of MgSO4, and 2 mL of trace elements for shaking flask culture at 37°C.

[0137] After 5 hours of cultivation, the induction was carried out. During the induction, 150 mM IPTG and 4 g / L glutamic acid were added to the conical flask to induce fermentation. The temperature was 30° C., the rotation speed was maintained at 230 r / min, and the fermentation was carried out for 48 hours.

[0138] After fermentation, the ALA yield was determined by high performance liquid chromatography. The specific test method is as follows:

[0139] The samples to be tested were centrifuged at 8000 r / min for 10 min, and the supernatant was diluted to the concentration range of the standard curve.

[0140] Take 100 μL of sample dilution, add 100 μL of 0.5 M NaHCO3 solution (pH = 9.8) and 200 μL of 8 g / L DNS-Cl acetone solution, shake and mix, and place in a dark place at 30°C for derivatization for more than 1 hour. Use a 0.22 μM microporous filter membrane to filter the derivatized sample and inject it.

[0141] Chromatographic conditions: The chromatographic column was a ZORBAX SB-C18 column (46 mm × 150 mm, 5 um) from Agilent Technologies, USA; the mobile phase A was methanol, and the mobile phase B was tetrahydrofuran-methanol-50 mM sodium acetate (pH = 6.2), in a volume ratio of 5:75:428. The UV detection wavelength was 254 nm, the injection volume was 10 μL, the flow rate was 1 mL / min, and the column temperature was room temperature. The elution method was linear gradient elution, and the specific gradient elution program is shown in Table 2.

[0142] Table 2

[0143]

[0144]

[0145] Standard curve drawing: Prepare a 1.0 g / L 5-aminolevulinic acid standard mother solution, and then dilute it into 0.2 g / L, 0.4 g / L, 0.6 g / L, and 0.8 g / L standard solutions, and perform derivatization, filtration, and injection analysis. Draw a standard curve with the corresponding mass concentration as the horizontal axis and the chromatographic peak area of ​​5-ALA as the vertical axis.

[0146] The results of 5-ALA production are shown in Table 3.

[0147] Table 3

[0148]

[0149]

[0150] From the above table, we can see that the ALA yield of genetically engineered bacteria ALA-02-ALA-06 is compared, and the yield of ALA-05 is higher than that of other genetically engineered bacteria, indicating that the effect of replacing the promoter of argE with J23108 is better than that of other promoters J23100, J23111, J23104, and J23103.

[0151] Comparing the ALA yields of the genetically engineered bacteria ALA-07-ALA-13, the yield of ALA-12 is higher than that of other genetically engineered bacteria, indicating that the expression effect of the plasmid pET-hemAS-hemL is good. On the basis of ALA-05, enhancing the gene expression or enzyme activity of hemAS and heml is more conducive to increasing the yield of 5-aminolevulinic acid.

[0152] Comparison of ALA yields of genetically engineered bacteria ALA-14-ALA-20 shows that ALA-20 has a higher yield than other genetically engineered bacteria, indicating that the present invention further improves ALA yield by knocking out acetic acid synthesis genes ackA, poxB and pta.

[0153] Test Example 2: Testing the ALA production capacity of strains after 5L tank fermentation

[0154] Test sample: Genetically engineered bacteria ALA-20.

[0155] Fermentation of genetically engineered bacteria (in a 5L fermenter):

[0156] Prepare fermentation base material: 30g / L glycerol, 6g / L ammonium sulfate, 11g / L potassium dihydrogen phosphate trihydrate, 12g / L potassium dihydrogen phosphate, 0.6g / L citric acid, 10g / L peptone, 2g / L yeast powder, 5mL / 100L defoamer, add water to make up to 3.5L; then add mixed carbon source: 700g glucose, 640g glycerol, 6.3g magnesium sulfate heptahydrate, 9.4mL trace elements to make up to 2.5L; add 500mL25-28% ammonia water (0.22μm filtered for sterilization) and 60mL defoamer.

[0157] Pick a single colony of genetically engineered bacteria in a 50 mL test tube containing LB medium, culture at 37°C, 230 r / min for 8 hours to obtain a bacterial solution, inoculate 1.5 mL of the bacterial solution into 150 mL of medium for expansion culture for 6-7 hours. Then inoculate all of it into a conical flask containing the base material, and induce after 6 hours of culture. Induce fermentation: add 0.60mM IPTG, 5mg / L vitamin B1, 40mL trace elements (formula: 13.74g / L nitrilotriacetic acid sodium salt (add appropriate amount of alkali, prepared separately), 5.6g / L ammonium ferric citrate, 0.9g / L zinc sulfate heptahydrate, 0.2g / L CoCl2·6H2O, 1.0g / L manganese chloride tetrahydrate, 0.10g / L CuCl2·2H2O, 0.2g / L boric acid, 0.2g / L Na2MoO4·2H2O), 2g / L MgSO4·7H2O, 0.02g / L anhydrous CaCl2 into the fermenter; add 4g / L glutamic acid every 12h.

[0158] Feed formula: 640 g glucose, 960 g glycerol, 9 g trace elements, 6.7 mL magnesium sulfate heptahydrate, add water to make up to 2 L.

[0159] The fermentation parameters were controlled as follows:

[0160] (1) Liquid volume: 3.5L; Inoculation volume: 150mL; Fermentation time: about 120h;

[0161] (2) Control parameters: pH 7; temperature: 37°C, the temperature is maintained at 37°C after feeding;

[0162] (3) Ventilation volume: Initial ventilation volume 1:1 (2.5 L), intake pressure adjusted to 0.06 MPa;

[0163] (4) Speed: starting speed 200 rpm;

[0164] (5) Dissolved oxygen (DO) control: Before feeding, DO is linked to the speed: DO is set to 40% and the speed range is set to 200-1000 rpm; when the dissolved oxygen is lower than 30%, the ventilation is adjusted to 1:2 to control the dissolved oxygen at 30-40%.

[0165] (6) Dissolved oxygen 100% calibration: 200 rpm, 1:1 ventilation;

[0166] (7) Carbon source was fed at a constant rate of 6 g / L / h, and glycine was fed at a rate of 2.5 g / L / h, depending on the specific situation;

[0167] (8) For strain preparation, refer to the single strain entry position;

[0168] (9) The base material consumption time was set at 6-7 h. After 4 h of supplementation (640 g glucose + 960 g glycerol + 9 g trace elements + 6.7 mL magnesium sulfate heptahydrate, fixed to 2 L), IPTG was added alone for induction.

[0169] During the fermentation period, please note:

[0170] 1. Pay attention to accidents such as defoaming, peristaltic pump failure, power failure and tripping;

[0171] 2. Record every 2 hours and pay close attention to DO changes;

[0172] 3. Prevent the pH from increasing due to the active addition of ammonia water 6 hours before fermentation;

[0173] 4. Calibrate the pH electrode of the fermenter with a pH meter before inoculation.

[0174] After 120 h of fermentation, 2 mL of sample was taken to detect the ALA production.

[0175] The genetically engineered bacteria ALA-20 was induced to ferment in a fermenter, and its ALA production was measured to be 29.13 g / L after 120 hours of fermentation.

[0176] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A genetically engineered bacterium for efficiently synthesizing 5-aminolevulinic acid, characterized in that: The genetically engineered bacteria are obtained by performing the following gene modification on Escherichia coli: knocking out the gadA gene, and replacing the promoter of the argE gene with any one of J23100, J23111, J23104, J23108, and J23103.

2. The genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid as claimed in claim 1, characterized in that: The genetically engineered bacteria are obtained by performing the following gene modification on Escherichia coli: knocking out the gadA gene and replacing the promoter of the argE gene with J23108.

3. The genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid as claimed in claim 1, characterized in that: The genetically engineered bacteria also include the following genetic modification: enhancing the gene expression or enzyme activity of at least one of gdhA, hemAS and heml.

4. The genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid as claimed in claim 3, characterized in that: The genetically engineered bacteria also include the following genetic modification: enhancing the gene expression or enzyme activity of hemAS and heml.

5. The genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid as claimed in claim 3, characterized in that: The genetically engineered bacteria also include the following genetic modification: knocking out at least one of the ackA, poxB, and pta genes.

6. The genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid as claimed in claim 5, characterized in that: The genetically engineered bacteria also include the following gene modification: knocking out ackA, poxB and pta genes.

7. The genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid as claimed in claim 1, characterized in that: The Escherichia coli is E. coli BL21.

8. Use of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid according to any one of claims 1 to 7 in producing 5-aminolevulinic acid.

9. A method for producing 5-aminolevulinic acid, characterized in that: 5-Aminolevulinic acid is obtained by inducing fermentation of the genetically engineered bacteria for efficiently synthesizing 5-aminolevulinic acid as described in any one of claims 1 to 7.

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