Genetically engineered bacteria for synthesizing 5-amino levulinic acid

Abstract

The present application relates to the field of genetic engineering, and more particularly to a genetically engineered bacterium for synthesizing 5-amino levulinic acid. The present application uses synthetic biology technology and genetic engineering means, takes E. coli BL21 (DE3) as a starting strain, modifies the C5 pathway, reduces the degradation of glutamic acid by knocking out the gadA gene and replacing the promoter of the argE gene, enhances the expression of the gdhA, hemAS and hemL genes, replaces the promoter, increases the carbon flux to 5-amino levulinic acid, and increases the synthesis of 5-amino levulinic acid. In addition, the loss of carbon flux to the TCA cycle is reduced by knocking out the acetic acid synthesis genes ackA, poxB and pta, so as to achieve the purpose of increasing the yield of 5-amino levulinic acid.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and more particularly to a genetically engineered bacterium that synthesizes 5-aminolevulinic acid. Background Technology

[0002] Aminolevulinic acid (ALA) is a non-protein amino acid that is essential for the synthesis of heme, chlorophyll, cytochromes, and vitamin B. 12 The first identified intermediate in the common tetrapyrrolidone pathway. In fact, two known metabolic pathways exist in organisms. One is the C4 pathway, which involves the condensation of succinyl-CoA and glycine into ALA via ALA synthase (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, as the most widely used strain in the microbial fermentation industry, has become an important choice. In E. coli, ALA biosynthesis occurs through the C5 pathway and is strictly regulated by feedback inhibition from the C5 pathway's final product, heme. Current research on ALA-producing strains mainly focuses on strengthening the 5-aminolevulinic acid pathway in E. coli while simultaneously knocking out byproduct pathways to ensure the carbon flow of the target product. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a genetically engineered bacterium for synthesizing 5-aminolevulinic acid, using *Escherichia coli*. E.coli BL21(DE3) was used as the starting strain, and its C5 pathway was modified to increase the production of 5-aminolevulinic acid.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a genetically engineered bacterium for synthesizing 5-aminolevulinic acid, wherein the genetically engineered bacterium is obtained by modifying Escherichia coli by the following gene modification: knocking out the gadA gene and replacing the promoter of the argE gene with any one of J23100, J23111, J23104, J23108, and J23103.

[0006] This invention utilizes synthetic biology techniques and genetic engineering methods, using *Escherichia coli* as the starting strain, to target its C5 pathway (e.g., Figure 1The invention modifies the gadA gene (as shown) to increase the production of 5-aminolevulinic acid. Extensive experimental research has revealed that knocking out the gadA gene and replacing the argE promoter reduces glutamate degradation, thereby increasing the production of 5-aminolevulinic acid.

[0007] More preferably, the genetically engineered bacteria are obtained by genetically modifying Escherichia coli as follows: knocking out the gadA gene and replacing the promoter of the argE gene with J23108.

[0008] Preferably, the genetically engineered bacteria further includes the following gene modification: enhancing the gene expression or enzyme activity of at least one of gdhA, hemAS, and hemL.

[0009] Through extensive experimental research, this invention has discovered that enhancing the expression of gdhA, hemAS, and hemL genes or enzyme activity can further increase the synthesis of 5-aminolevulinic acid.

[0010] More preferably, the genetically engineered bacteria are obtained by genetically modifying Escherichia coli in the following way: enhancing the gene expression or enzyme activity of hemAS and hemL.

[0011] Preferably, the genetically engineered bacteria further includes the following gene modification: knocking out at least one of the ackA, poxB, and pta genes.

[0012] Through extensive experimental research, this invention has found that by superimposing the knockout of acetic acid synthesis genes ackA, poxB, and pta to reduce the loss of carbon flux flowing to the TCA cycle, the production of 5-aminolevulinic acid can be further increased.

[0013] More preferably, the genetically engineered bacteria further includes the following gene modification: knockout of the ackA, poxB and pta genes.

[0014] Preferably, the *Escherichia coli* is... E.coli BL21 (DE3).

[0015] Secondly, the present invention provides the application of the above-mentioned genetically engineered bacteria for synthesizing 5-aminolevulinic acid in the production of 5-aminolevulinic acid.

[0016] Thirdly, the present invention provides a method for producing 5-aminolevulinic acid, which is obtained by inducing fermentation of the above-mentioned genetically engineered bacteria for synthesizing 5-aminolevulinic acid.

[0017] The beneficial effects of this invention are as follows: This invention utilizes synthetic biology techniques and genetic engineering methods, using Escherichia coli... E.coliBL21(DE3) was used as the starting strain. Its C5 pathway was modified by knocking out the gadA gene and replacing the promoter of the argE gene to reduce glutamate degradation; by enhancing the expression of the gdhA, hemAS and hemL genes and replacing the promoter, the carbon flux to 5-aminolevulinic acid was increased, thereby increasing the synthesis of 5-aminolevulinic acid; in addition, the loss of carbon flux to the TCA cycle was reduced by knocking out the acetic acid synthesis genes ackA, poxB and pta, thereby increasing the yield of 5-aminolevulinic acid. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the C5 pathway for ALA synthesis. Detailed Implementation

[0019] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0020] The primers used in this invention were all synthesized by a biotechnology company, and the plasmids and strains used in this invention can be purchased from the market.

[0021] The primers used in the embodiments of this invention and the constructed homologous repair arm sequences are shown in Table 1.

[0022] Table 1 Example 1: An embodiment of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria are obtained by genetic modification of Escherichia coli as follows: knocking out the gadA gene; replacing the promoter of the argE gene with J23100.

[0023] The method for constructing the genetically engineered bacteria described in this embodiment is as follows: 1. Knock out the gadA gene: Using CRISPR / Cas9 technology to knock out genetically engineered bacteria Escherichia coli The gadA gene of BL21(DE3) includes the following steps: (a) Using the gadA gene sequence indexed in GenBank on NCBI as the target gene sequence, the sgRNA of the gene was designed through an online search at http: / / chopchop.cbu.uib.no / . The sgRNA was ligated with the plasmid pEcgRNA (synthesized by GE), and the plasmid was transformed into DH5α and cultured overnight. Positive clones were selected and cultured, and the plasmid was extracted using the Tiangen Biotech Mini-Prep Kit to obtain the plasmid pEcgRNA::gadA-N20.

[0024] (b) Homologous fragment preparation: Primers required for gadA gene knockout were designed based on the GenBank genome information published in the NCBI database, and selected... Escherichia coli Using BL21(DE3) genomic DNA as a template, homologous arm sequences gadA-1 and gadA-2 were amplified using upstream homologous arm primers gadA-up-F and gadA-up-R, and downstream homologous arm primers gadA-down-F and gadA-down-R, respectively. After purification and recovery of the products, overlapping PCR was used to amplify and ligate fragments gadA-1 and gadA-2 together with primers gadA-up-F and gadA-down-R to obtain the homologous repair arm of the gadA gene.

[0025] (c) Transform the pEcCas (synthesized by General Electric) plasmid Escherichia coli BL21 (DE3), obtained Escherichia coli BL21(DE3)::pEcCas was used to select positive clones for electroporation competence preparation. The constructed plasmid pEcgRNA::gadA-N20 and the homologous repair arm of the gadA gene were co-electroporated. Escherichia coli BL21(DE3)::pEcCas, coated on 50 μg·mL -1 Kanamycin and 50 μg mL -1 The spectinomycin-containing double antibody plates were incubated overnight at 37°C, and the positive recombinants were identified by PCR using gadA-up-F and gadA-down-R.

[0026] (d) Select the successfully knocked-out strains and culture them in LB liquid medium of 50 μg / mL kanamycin and 10 mmol / L rhamnose for 6 h to induce the elimination of plasmid pEcgRNA. After subculturing at 37℃ for 2 h, the strains were streaked onto 10 g / L sucrose solid plates to eliminate plasmid pEcCas, and the strain with the gadA gene knocked out was obtained and named ALA-01.

[0027] 2. Replace the promoter of the argE gene with J23100. (e) Design primer pairs argE-F1 and argE-R1 to clone the argE gene from Escherichia coli, and use homologous recombination technology to link the cloned argE fragment to plasmid pETDuet-1 to construct plasmid pET-argE.

[0028] (f) The promoter of the argE gene in the genetically engineered bacterium ALA-1 was replaced using CRISPR / Cas9 technology: Using the argE gene sequence indexed in GenBank on NCBI as the target gene sequence, an sgRNA for this gene was designed via an online search at http: / / chopchop.cbu.uib.no / . The sgRNA was ligated with the plasmid pEcgRNA (synthesized by GE), and the plasmid was transformed into DH5α and cultured overnight. Positive clones were selected and cultured, and the plasmid was extracted using the Tiangen Biotech Mini-Prep Kit to obtain the plasmid pEcgRNA::argE-N20.

[0029] (g) Preparation of homologous fragments: promoter P J23100 Synthesized by General Electric, primer pairs argE-J23100-F and argE-J23100-R were designed. PCR amplification of the pET-argE plasmid was performed using these primer pairs to obtain a linearized vector. The promoter sequence was then ligated to the plasmid pETDuet-1 via homologous recombination technology to obtain the plasmid pET-P. J23100 -argE; then design primer pairs argE-F2 and argE-R2 for plasmid pET-P J23100 -argE is used for PCR amplification to obtain fragment P J23100 -argE serves as the homologous repair arm for the argE gene. The pEcCas plasmid (synthesized by General Electric) was transformed into the ALA-01 strain with the gadA gene knocked out in step 1 (d) to obtain ALA-01::pEcCas. Positive clones were selected for electroporation to prepare competent cells.

[0030] The constructed plasmid pEcgRNA::argE-N20 and the homologous repair arm of the argE gene were co-electroporated to ALA-01::pEcCas and plated on 50 μg·mL⁻¹. -1 Kanamycin 50 μg / mL -1 The spectinomycin-containing double antibody plates were incubated overnight at 37°C. Positive recombinants were identified by PCR using argE-J23100-F and argE-J23100-R. Successfully knocked-out strains were then cultured in 50 μg / mL... -1 Kanamycin, 10 mmol·L -1 In rhamnose LB broth, cultured for 6 h to induce elimination of plasmid pEcgRNA, subcultured at 37 ℃ for 2 h, and streaked onto 10 g·L⁻¹ medium.-1 The genetically engineered strain was obtained by eliminating plasmid pEcCas on a sucrose solid plate and named ALA-02.

[0031] Example 2: An embodiment of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria are obtained by genetic modification of Escherichia coli as follows: knocking out the gadA gene; replacing the promoter of the argE gene with J23111.

[0032] The method for constructing the genetically engineered bacteria described in this embodiment is as follows: The process of knocking out the gadA gene is the same as step (1) in Example 1.

[0033] In this embodiment, the promoter of the argE gene is replaced with J23111. The steps for replacing the promoter are the same as in step (2). The designed primers include argE-J23111-F and argE-J23111-R. The remaining steps are the same as in Example 1. The genetically engineered strain obtained in this embodiment is named ALA-03.

[0034] Example 3: An embodiment of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria are obtained by genetic modification of Escherichia coli as follows: knocking out the gadA gene; replacing the promoter of the argE gene with J23104.

[0035] The method for constructing the genetically engineered bacteria described in this embodiment is as follows: The process of knocking out the gadA gene is the same as step (1) in Example 1.

[0036] In this embodiment, the promoter of the argE gene is replaced with J23104. The steps for replacing the promoter are the same as in step (2). The designed primers include argE-J23104-F and argE-J23104-R. The remaining steps are the same as in Example 1. The genetically engineered strain obtained in this embodiment is named ALA-04.

[0037] Example 4: An embodiment of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria are obtained by genetic modification of Escherichia coli as follows: knocking out the gadA gene; replacing the promoter of the argE gene with J23108.

[0038] The method for constructing the genetically engineered bacteria described in this embodiment is as follows: The process of knocking out the gadA gene is the same as step (1) in Example 1.

[0039] In this embodiment, the promoter of the argE gene is replaced with J23108. The steps for replacing the promoter are the same as in step (2). The designed primers include argE-J23108-F and argE-J23108-R. The remaining steps are the same as in Example 1. The genetically engineered strain obtained in this embodiment is named ALA-05.

[0040] Example 5: An embodiment of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria are obtained by genetic modification of Escherichia coli as follows: knocking out the gadA gene; replacing the promoter of the argE gene with J23103.

[0041] The method for constructing the genetically engineered bacteria described in this embodiment is as follows: The process of knocking out the gadA gene is the same as step (1) in Example 1.

[0042] In this embodiment, the promoter of the argE gene is replaced with J23103. The steps for replacing the promoter are the same as in step (2). The designed primers include argE-J23103-F and argE-J23103-R. The remaining steps are the same as in Example 1. The genetically engineered strain obtained in this embodiment is named ALA-06.

[0043] Examples 6-8: Examples of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid as described in this invention; The genetically engineered bacteria in Example 6 were obtained by modifying Escherichia coli as follows: knocking out the gadA gene; replacing the promoter of the argE gene with J23108; and enhancing the gene expression or enzyme activity of gdhA.

[0044] The genetically engineered bacteria in Example 7 were obtained by modifying Escherichia coli as follows: knocking out the gadA gene; replacing the promoter of the argE gene with J23108; and enhancing the gene expression or enzyme activity of hemAS.

[0045] The genetically engineered bacteria in Example 8 were obtained by modifying Escherichia coli as follows: knocking out the gadA gene; replacing the promoter of the argE gene with J23108; and enhancing the gene expression or enzyme activity of hemL.

[0046] The method for constructing the genetically engineered bacteria is as follows: 1. Primer pairs gdhA-F1, gdhA-R1, hemAS-F1, hemAS-R1, hemL-F1, and hemL-R1 were designed to clone the gdhA, hemAS, and hemL genes from Escherichia coli, respectively. The cloned gdhA, hemAS, and hemL fragments were then ligated into plasmid pETDuet-1 using homologous recombination technology to construct plasmids pET-gdhA, pET-hemAS, and pET-hemL, respectively.

[0047] 2. Recombinant plasmid transformation: After mixing plasmid pET-gdhA with ALA-05 competent cells, place them on ice for 30 min, then quickly heat shock (42℃, 90 s), and then quickly place them on ice for 2 min. Under aseptic conditions, add 1 mL of LB liquid medium and restore culture at 37℃ and 220 rpm for 1 h to obtain the genetically engineered strain, named ALA-07.

[0048] Following the same procedure, plasmid pET-hemAS was transformed into ALA-05 competent cells to obtain the genetically engineered strain, named ALA-08; plasmid pET-hemL was transformed into ALA-05 competent cells to obtain the genetically engineered strain, named ALA-09.

[0049] Examples 6, 7, and 8 correspond to genetically engineered strains ALA-07, ALA-08, and ALA-09, respectively.

[0050] Example 9: An embodiment of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria are obtained by genetic modification of Escherichia coli as follows: 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.

[0051] The method for constructing the genetically engineered bacteria described in this embodiment is as follows: 1. The construction of plasmids pET-gdhA and pET-hemAS is the same as step (1) in Examples 6-8.

[0052] 2. Design primer pairs PET-F1 and PET-R1, and use the primer pairs to perform PCR amplification on the pET-hemAS plasmid to obtain a linearized vector. Then, use homologous recombination technology to ligate the promoter sequence to the plasmid pET-gdhA to construct the plasmid pET-gdhA-hemAS.

[0053] 3. Transformation of recombinant plasmid: Using the same transformation method as step (2) in Examples 6-8, the plasmid pET-gdhA-hemAS was transformed into ALA-05 competent cells to obtain the genetically engineered strain, which was named ALA-10.

[0054] Example 10: An embodiment of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria are obtained by genetic modification of Escherichia coli as follows: 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.

[0055] The method for constructing the genetically engineered bacteria described in this embodiment is as follows: 1. The construction of plasmids pET-gdhA and pET-hemL is the same as step (1) in Examples 6-8.

[0056] 2. Design primer pairs PET-F1 and PET-R1, and use the primer pairs to perform PCR amplification on the pET-hemL plasmid to obtain a linearized vector. Then, use homologous recombination technology to ligate the promoter sequence to the plasmid pET-gdhA to construct the plasmid pET-gdhA-hemL.

[0057] 3. Transformation of recombinant plasmid: Using the same transformation method as step (2) in Examples 6-8, transform the plasmid pET-gdhA-hemL into ALA-05 competent cells to obtain the genetically engineered strain, named ALA-11.

[0058] Example 11: An embodiment of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria are obtained by genetic modification of Escherichia coli as follows: 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.

[0059] The method for constructing the genetically engineered bacteria described in this embodiment is as follows: 1. The construction of plasmids pET-hemAS and pET-hemL is the same as step (1) in Examples 6-8.

[0060] 2. Design primer pairs PET-F1 and PET-R1, and use the primer pairs to perform PCR amplification on the pET-hemAS plasmid to obtain a linearized vector. Then, use homologous recombination technology to connect the promoter sequence to the plasmid pET-hemL to construct the plasmid pET-hemAS-hemL.

[0061] 3. Recombinant plasmid transformation: Using the same transformation method as step (2) in Examples 6-8, the plasmid pET-hemAS-hemL was transformed into ALA-05 competent cells to obtain the genetically engineered strain, which was named ALA-12.

[0062] Example 12: An embodiment of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria are obtained by genetic modification of Escherichia coli as follows: 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.

[0063] The method for constructing the genetically engineered bacteria described in this embodiment is as follows: 1. The construction of plasmids pET-gdhA, pET-hemAS, and pET-hemL is the same as step (1) in Examples 6-8.

[0064] 2. Design primer pairs PET-F1 and PET-R1, and use the primer pairs to perform PCR amplification on the pET-hemAS plasmid to obtain a linearized vector. Then, use homologous recombination technology to ligate the promoter sequence to the plasmid pET-gdhA to construct the plasmid pET-gdhA-hemAS.

[0065] 3. Design primer pairs PET-F2 and PET-R2, and use primer pairs to amplify the pET-hemL plasmid by PCR to obtain a linearized vector. Then, use homologous recombination technology to connect the promoter sequence to the plasmid pET-gdhA-hemAS to construct the plasmid pET-gdhA-hemAS-hemL.

[0066] 4. Transformation of recombinant plasmid: Using the same transformation method as step (2) in Examples 6-8, transform the plasmid pET-gdhA-hemAS-hemL into ALA-05 competent cells to obtain the genetically engineered strain, named ALA-13.

[0067] Example 13: An embodiment of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria are obtained by genetic modification of Escherichia coli as follows: 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; knocking out the ackA gene.

[0068] The method for constructing the genetically engineered bacteria described in this embodiment is as follows: The ackA gene of the genetically engineered bacterium ALA-12 was knocked out using CRISPR / Cas9 technology, including the following steps: 1. Using the ackA gene sequence indexed in GenBank on NCBI as the target gene sequence, the sgRNA of the gene was designed through an online search at http: / / chopchop.cbu.uib.no / . The sgRNA was ligated with the plasmid pEcgRNA (synthesized by GE), and the plasmid was transformed into DH5α and cultured overnight. Positive clones were selected and cultured, and the plasmid was extracted using the Tiangen Biotech Mini-Prep Kit to obtain the plasmid pEcgRNA::ackA-N20.

[0069] 2. Homologous fragment preparation: Primers for ackA gene knockout were designed based on the GenBank genome information published in the NCBI database. Escherichia coli Using BL21(DE3) genomic DNA as a template, homologous arm sequences ackA-1 and ackA-2 were amplified using upstream homologous arm primers ackA-up-F and ackA-up-R and downstream homologous arm primers ackA-down-F and ackA-down-R, respectively. After purification and recovery of the products, overlapping PCR was used to amplify and ligate fragments ackA-1 and ackA-2 together with primers ackA-up-F and ackA-down-R to obtain the homologous repair arm of the ackA gene.

[0070] 3. Transform the pEcCas plasmid (synthesized by GE) into the engineered bacterium ALA-12 to obtain ALA-12::pEcCas. Select positive clones for electroporation to prepare competent cells. Co-electroporate the constructed plasmid pEcgRNA::ackA-N20 and the homologous repair arm of the ackA gene into ALA-12::pEcCas, and plate the plasmid onto a 50 μg·mL⁻¹ plate. -1 Kanamycin and 50 μg mL -1 The spectinomycin-containing double antibody plates were incubated overnight at 37°C, and the positive recombinants were identified by PCR using ackA-up-F and ackA-down-R.

[0071] 4. Select the successfully knocked-out strains and culture them in LB liquid medium containing 50 μg / mL kanamycin and 10 mmol / L rhamnose for 6 h to induce the elimination of plasmid pEcgRNA. After subculturing at 37℃ for 2 h, the culture was streaked onto 10 g / L sucrose solid plates to eliminate plasmid pEcCas, thus obtaining the genetically engineered strain, named ALA-14.

[0072] Example 14: An embodiment of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria are obtained by genetic modification of Escherichia coli as follows: 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; knocking out the poxB gene.

[0073] The method for constructing the genetically engineered bacteria described in this embodiment differs from that in Example 13 in that: This embodiment describes the knockout of the poxB gene. The knockout procedure is the same as in Example 13. The primers used in this embodiment include: poxB-up-F, poxB-up-R, poxB-down-F, poxB-down-R, poxB-N20-F, poxB-N20-R, and poxB-sgRNA. The genetically engineered strain obtained in this embodiment is named ALA-15.

[0074] Example 15: An embodiment of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria are obtained by genetic modification of Escherichia coli as follows: 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; knocking out the pta gene.

[0075] The method for constructing the genetically engineered bacteria described in this embodiment differs from that in Example 13 in that: This embodiment describes the knockout of the pta gene. The knockout procedure is the same as in Example 13. The primers used in this embodiment include: pta-up-F, pta-up-R, pta-down-F, pta-down-R, pta-N20-F, pta-N20-R, and pta-sgRNA. The genetically engineered strain obtained in this embodiment is named ALA-16.

[0076] Example 16: An embodiment of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria are obtained by genetic modification of Escherichia coli as follows: 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; knocking out the ackA and poxB genes.

[0077] The method for constructing the genetically engineered bacteria described in this embodiment differs from that in Example 13 in that: This embodiment describes the knockout of the ackA and poxB genes. The gene knockout steps are the same as in Example 13. The primers used in this embodiment 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, and poxB-sgRNA. The genetically engineered strain obtained in this embodiment is named ALA-17.

[0078] Example 17: An embodiment of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria are obtained by genetic modification of Escherichia coli as follows: 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; knocking out the ackA and pta genes.

[0079] The method for constructing the genetically engineered bacteria described in this embodiment differs from that in Example 13 in that: This embodiment involves knocking out the ackA and pta genes. The gene knockout steps are the same as in Example 13. The primers used in this embodiment include: 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 is named ALA-18.

[0080] Example 18: An embodiment of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria are obtained by genetic modification of Escherichia coli as follows: 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; knocking out the poxB and pta genes.

[0081] This embodiment involves knocking out the poxB and pta genes. The gene knockout steps are the same as in Example 13. The primers used in this embodiment include: 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 is named ALA-19.

[0082] Example 19: An embodiment of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria are obtained by genetic modification of Escherichia coli as follows: 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; knocking out the ackA, poxB, and pta genes.

[0083] This embodiment involves knocking out the ackA, poxB, and pta genes. The gene knockout steps are the same as in Example 13. The primers used in this embodiment 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, 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-20.

[0084] Test Example 1: Testing the ALA Production Capacity of Strains in Shake Flask Culture Test sample: ALA-2-ALA-20, a genetically engineered bacterium constructed in Examples 1-19.

[0085] Fermentation treatment of genetically engineered bacteria: Prepare the fermentation substrate in a 500 mL Erlenmeyer flask: 20 g / L glycerol, 0.020 g / L CaCl₂ 2、 1.5 g / L (NH4)2HPO4, 3 g / L citric acid, 16 g / L Na2HPO4·12H2O, 3 g / L NH4Cl, 2 g / L yeast extract and 10 g / L tryptone.

[0086] Single colonies of genetically engineered bacteria were selected and activated in test tube culture medium to obtain OD. 600 nm With a bacterial culture volume close to 15, inoculate 1 mL of the bacterial culture into an Erlenmeyer flask containing the substrate, and add 1.5 mL of glucose, 1.5 mL of MgSO4, and 2 mL of trace elements. Shake the flask for incubation at 37℃.

[0087] After culturing for 5 h, induction was performed by adding 150 mM IPTG and 4 g / L glutamic acid to the conical flask to induce fermentation at 30℃ and 230 r / min for 48 h.

[0088] After fermentation, the yield of ALA was determined by high performance liquid chromatography. The specific test method is as follows: Centrifuge the sample to be tested at 8000 r / min for 10 min, and take the supernatant to dilute to the concentration range of the standard curve.

[0089] 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 to mix, and derivatize at 30℃ in the dark for at least 1 h. Filter the derivatized sample using a 0.22 μM microporous membrane and inject the sample.

[0090] Chromatographic conditions: The column was a ZORBAX SB-C18 column (46 mm × 150 mm, 5 μm), manufactured by Agilent Technologies, USA. Mobile phase A was methanol, and mobile phase B was tetrahydrofuran-methanol-50 mM sodium acetate (pH=6.2) in a volume ratio of 5:75:428. UV detection wavelength was 254 nm, injection volume was 10 μL, flow rate was 1 mL / min, and column temperature was room temperature. Linear gradient elution was used; the specific gradient elution program is shown in Table 2.

[0091] Table 2 Standard curve construction: Prepare a 1.0 g / L stock solution of 5-aminolevulinic acid (ALA), and then dilute it to concentrations of 0.2 g / L, 0.4 g / L, 0.6 g / L, and 0.8 g / L, respectively. Perform derivatization, filtration, and injection analysis on each solution. Plot a standard curve with the corresponding mass concentration on the x-axis and the peak area of ​​5-ALA on the y-axis.

[0092] The results of 5-ALA yield determination are shown in Table 3.

[0093] Table 3 As shown in the table above, when comparing the ALA production of genetically engineered bacteria ALA-02-ALA-06, ALA-05 produced a higher ALA yield than other genetically engineered bacteria. This indicates that replacing the argE promoter with J23108 is more effective than other promoters J23100, J23111, J23104, and J23103.

[0094] A comparison of ALA production among genetically engineered bacteria ALA-07-ALA-13 showed that ALA-12 produced higher ALA than other genetically engineered bacteria. This indicates that the expression of plasmid pET-hemAS-hemL is effective. Based on ALA-05, enhancing the gene expression or enzyme activity of hemAS and hemL is more conducive to increasing the production of 5-aminolevulinic acid.

[0095] A comparison of ALA production among genetically engineered bacteria ALA-14 and ALA-20 showed that ALA-20 produced higher ALA than other genetically engineered bacteria, indicating that the present invention achieved a further increase in ALA production by knocking out the acetic acid synthesis genes ackA, poxB, and pta.

[0096] Test Example 2: Testing the ALA production capacity of the strain after fermentation in a 5 L tank. Test sample: Genetically engineered bacteria ALA-20.

[0097] Fermentation of genetically engineered bacteria (carried out in a 5 L fermenter): Prepare the fermentation base: 30 g / L glycerol, 6 g / L ammonium sulfate, 11 g / L dipotassium hydrogen phosphate trihydrate, 12 g / L potassium dihydrogen phosphate, 0.6 g / L citric acid, 10 g / L peptone, 2 g / L yeast powder, and 5 mL / 100 L defoamer. Add water to a final volume of 3.5 L. Then add the mixed carbon source: 700 g glucose, 640 g glycerol, 6.3 g magnesium sulfate heptahydrate, and 9.4 mL trace elements to a final volume of 2.5 L. Add 500 mL of 25-28% ammonia water (sterilized by 0.22 μm filtration) and 60 mL of defoamer.

[0098] Single colonies of the genetically engineered bacteria were picked and cultured in 50 mL test tubes containing LB medium at 37°C and 230 r / min for 8 h to obtain a bacterial suspension. 1.5 mL of the bacterial suspension was then inoculated into 150 mL of medium for expansion culture for 6-7 h. The entire suspension was then inoculated into Erlenmeyer flasks containing substrate and cultured for 6 h before induction. Induced fermentation: Add 0.60 mM IPTG, 5 mg / L vitamin B1, 40 mL trace elements (formulation: 13.74 g / L sodium hypotriacetate (prepared separately with appropriate alkali), 5.6 g / L ferric ammonium citrate, 0.9 g / L zinc sulfate heptahydrate, 0.2 g / L CoCl2•6H2O, 1.0 g / L manganese chloride tetrahydrate, 0.10 g / L CuCl2•2H2O, 0.2 g / L boric acid, 0.2 g / L Na2MoO4•2H2O), 2 g / L MgSO4•7H2O, and 0.02 g / L anhydrous CaCl2 to the fermenter; add 4 g / L glutamic acid every 12 h.

[0099] Feeding 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.

[0100] The fermentation parameters are controlled as follows: (1) Liquid volume: 3.5 L; Inoculum volume: 150 mL; Fermentation time: approximately 120 h; (2) Control parameters: pH 7; temperature: 37℃, and the temperature is maintained at 37℃ after feeding; (3) Ventilation rate: Initial ventilation rate 1:1 (2.5 L), inlet pressure adjusted to 0.06 MPa; (4) Rotational speed: Starting speed 200 rpm; (5) Dissolved oxygen (DO) control: Before feeding, DO is linked to the rotation speed: DO is set to 40%, and the rotation speed range is set to 200-1000 rpm; when the dissolved oxygen is below 30%, the ventilation ratio is adjusted to 1:2 to control the dissolved oxygen at 30-40%.

[0101] (6) Dissolved oxygen 100% calibration: 200 rpm, 1:1 ventilation rate; (7) The carbon source is fed at a constant rate of 6 g / L / h, and the glycine feeding rate is 2.5 g / L / h, depending on the specific situation; (8) For strain preparation, see the single strain entry station; (9) The base material is consumed in 6-7 hours. After replenishing the material (640 g glucose + 960 g glycerol + 9 g trace elements + 6.7 mL magnesium sulfate heptahydrate, and bringing the volume to 2 L) for 4 hours, IPTG is added separately for induction.

[0102] During fermentation, please note the following: 1. Be aware of potential accidents such as defoaming, peristaltic pump malfunctions, and power outages / tripping. 2. Record every 2 hours, paying close attention to changes in DO; 3. Prevent the active addition of ammonia water 6 hours before fermentation, which would cause the pH to increase; 4. Calibrate the pH electrode of the fermenter with a pH meter before inoculation.

[0103] After 120 h of fermentation, 2 mL of sample was taken to detect the yield of ALA.

[0104] The genetically engineered bacterium ALA-20 was induced to ferment in a fermenter, and its ALA yield was measured to be 29.13 g / L after 120 h of fermentation.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A genetically engineered bacterium for synthesizing 5-aminolevulinic acid, characterized in that, The genetically engineered bacteria were obtained by modifying Escherichia coli as follows: the gadA gene was knocked out, and the promoter of the argE gene was replaced with any one of J23100, J23111, J23104, J23108, and J23103.

2. The genetically engineered bacterium for synthesizing 5-aminolevulinic acid as described in claim 1, characterized in that, The genetically engineered bacteria were obtained by modifying Escherichia coli as follows: the gadA gene was knocked out, and the promoter of the argE gene was replaced with J23108.

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

4. The genetically engineered bacterium for synthesizing 5-aminolevulinic acid as described in claim 3, characterized in that, The genetically engineered bacteria also include the following gene modifications: enhancing the gene expression or enzyme activity of hemAS and hemL.

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

6. The genetically engineered bacterium for synthesizing 5-aminolevulinic acid as described in claim 5, characterized in that, The genetically engineered bacteria also include the following gene modifications: knockout of ackA, poxB and pta genes.

7. The genetically engineered bacterium for synthesizing 5-aminolevulinic acid as described in claim 1, characterized in that, The Escherichia coli is E. coli BL21.

8. The use of the genetically engineered bacteria for synthesizing 5-aminolevulinic acid as described in any one of claims 1-7 in the production of 5-aminolevulinic acid.

9. A method for producing 5-aminolevulinic acid, characterized in that, 5-Aminolevulinic acid is produced by induced fermentation of the genetically engineered bacteria that synthesize 5-aminolevulinic acid according to any one of claims 1-7.

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