Genetically engineered bacterium for high yield of 5-aminolevulinic acid

Abstract

The invention relates to the field of genetic engineering, in particular to a genetically engineered bacterium for high yield of 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 pathway of escherichia coli is modified, ROS repair capability of escherichia coli is enhanced in the C5 pathway, a synthesis pathway of 5-ALA is enhanced, resistance of escherichia coli to 5-ALA is enhanced, carbon flux flowing to 5-ALA is increased, degradation of heme is accelerated, and the escherichia coli E.coli BL21 (DE3) can be used for preparing the recombinant escherichia coli. The synthesis pressure of the 5-ALA is reduced, and the genetically engineered bacterium for high yield of the 5-aminolevulinic acid is obtained, so that the purpose of increasing the yield of the 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 with high production of 5-aminolevulinic acid. Background Art

[0002] There are two main methods for the synthesis of 5-aminolevulinic acid (5-ALA), namely chemical synthesis and biosynthesis. At present, 5-ALA is mainly produced by chemical synthesis, but the high complexity and high pollution of the chemical synthesis process limit the scale of its industrial production; multi-step catalytic reactions and low product yields also further increase the production cost of the product, thereby limiting its large-scale application and promotion in various fields. The biosynthesis method mainly uses enzyme catalysis and fermentation. The fermentation method requires the construction of a biological metabolic pathway of 5-aminolevulinic acid in the strain. In nature, the biosynthesis of 5-ALA is mainly carried out through the C4 and C5 pathways. Among them, in the C4 pathway, ALA synthase directly converts glycine and succinyl coenzyme A into 5-aminolevulinic acid; while in the C5 pathway, first, glutamyl-tRNA synthase catalyzes glutamate to form glutamyl-tRNA. Then, glutamate-1-semialdehyde is formed under the action of glutamyl-tRNA reductase. Finally, glutamate-1-semialdehyde is synthesized into 5-ALA under the catalysis of glutamate-1-semialdehyde aminotransferase.

[0003] In existing studies, the metabolic pathway of 5-aminolevulinic acid in Escherichia coli is usually constructed by strengthening the strength of the target product pathway, while knocking out the branch byproduct pathway to ensure the carbon flow of the target product. However, in this process, 5-ALA causes serious cell damage and morphological changes in Escherichia coli by producing reactive oxygen species (ROS). Excessive ROS can cause oxidative damage to proteins, DNA and lipids, thereby inhibiting cell metabolism. Since 5-ALA is an essential precursor for heme biosynthesis, its biosynthesis is also strictly regulated by heme. Therefore, it is urgent to find the most suitable 5-aminolevulinic acid transporter, and to strengthen the ability of the strain to resist 5-aminolevulinic acid and accelerate the removal of ROS to obtain Escherichia coli with high yield of 5-aminolevulinic acid. 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 that produces high 5-aminolevulinic acid, wherein the genetically engineered bacterium is obtained by subjecting Escherichia coli to the following genetic modifications: replacing RBS and enhancing the gene expression of cyoE or the corresponding enzyme activity.

[0007] Escherichia coli is the most widely used strain in the current microbial fermentation industry. The microbial fermentation method using Escherichia coli as the host can use glucose as a substrate to achieve the de novo synthesis of 5-aminolevulinic acid. Through reasonable metabolic regulation and precise fermentation condition control, high yield of 5-aminolevulinic acid can be achieved. The present invention uses synthetic biology technology and genetic engineering to use Escherichia coli as the starting strain, and its own C5 pathway (such as Figure 1 The present invention has been modified through a large number of experimental studies. It has been found that the heme O synthase gene cyoE catalyzes heme to produce heme O. By enhancing the expression of different RBS, the expression of cyoE can be increased, the degradation of heme can be accelerated, and the inhibition of heme on the synthesis of 5-aminolevulinic acid can be reduced, thereby obtaining a genetically engineered bacterium with a high yield of 5-aminolevulinic acid.

[0008] Preferably, the replacing of the RBS includes replacing the RBS with any one of RBS2, RBS3, RBS5 and RBS8.

[0009] More preferably, the RBS is replaced with RBS2.

[0010] Preferably, the genetically engineered bacteria further include the following genetic modification: enhancing the gene expression or corresponding enzyme activity of at least one of hemAS and heml.

[0011] The invention increases the synthesis of 5-aminolevulinic acid by enhancing the expression of glutamyl-tRNA reductase (hemAS) and glutamate-1-semialdehyde aminotransferase (hemL) (hemAS catalyzes glutamate tRNA to generate glutamate-1-semialdehyde, and heml catalyzes glutamate-1-semialdehyde to generate ALA), thereby increasing the yield of 5-aminolevulinic acid.

[0012] More preferably, the genetically engineered bacteria further include the following genetic modification: enhancing the gene expression of hemAS and heml or the corresponding enzyme activity.

[0013] Preferably, the genetically engineered bacteria further include the following genetic modification: enhancing the gene expression or corresponding enzyme activity of at least one of ppC and pyC.

[0014] The present invention further increases the yield of 5-aminolevulinic acid by increasing the expression of ppC (5-aminolevulinic acid phosphoenolpyruvate carboxylase gene derived from Escherichia coli) and pyC (pyruvate carboxylase gene derived from Corynebacterium glutamicum).

[0015] More preferably, the genetically engineered bacteria further include the following genetic modification: enhancing the gene expression of ppC and pyC or the corresponding enzyme activity.

[0016] Preferably, the genetically engineered bacteria further comprises any one of the following genetic modifications (i)-(ii):

[0017] (i) enhancing the gene expression or corresponding enzyme activity of either rhtA or eamA;

[0018] (ii) enhancing the gene expression or corresponding enzyme activity of any one of rhtA and eamA, and enhancing the gene expression or corresponding enzyme activity of any one of kate, sodA, yfcF, htpG, sodB, and katG.

[0019] The present invention increases the efflux of 5-aminolevulinic acid by enhancing the expression of 5-aminolevulinic acid transporter rhtA or eamA, thereby further increasing the yield of 5-aminolevulinic acid. The present invention has found through a large number of experimental studies that the ability of the strain to produce 5-aminolevulinic acid can be improved by enhancing the expression of genes involved in oxidative damage repair and ROS scavenging: kate, sodA, yfcF, htpG, sodB or katG to enhance the tolerance of the strain to ROS.

[0020] More preferably, the genetically engineered bacteria further comprises the following genetic modification: enhancing the gene expression of rhtA or the corresponding enzyme activity.

[0021] More preferably, the genetically engineered bacteria further include the following genetic modification: enhancing the gene expression of rhtA and kate or the corresponding enzyme activity.

[0022] More preferably, the genetically engineered bacteria further include the following genetic modification: enhancing the gene expression of hemAS, heml, ppC, pyC, rhtA, kate or the corresponding enzyme activity.

[0023] Preferably, the Escherichia coli is E. coli BL21 (DE3).

[0024] In a second aspect, the present invention provides the use of the above-mentioned genetically engineered bacteria with high 5-aminolevulinic acid production in the production of 5-aminolevulinic acid.

[0025] Preferably, the application is to inoculate the genetically engineered bacteria with high 5-aminolevulinic acid production into a fermentation medium to ferment and produce 5-aminolevulinic acid.

[0026] 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 above-mentioned genetically engineered bacteria with high 5-aminolevulinic acid production.

[0027] The beneficial effects of the present invention are:

[0028] The invention utilizes synthetic biology technology and genetic engineering means, takes Escherichia coli BL21 (DE3) as a starting strain, and transforms its own C5 pathway, thereby reducing damage to cells by enhancing the efflux of 5-aminolevulinic acid in the C5 pathway; enhancing the expression of ROS repair genes, strengthening the ROS repair ability of Escherichia coli, and enhancing the strain's tolerance to ROS; simultaneously enhancing the synthesis pathway of 5-aminolevulinic acid, enhancing the resistance of Escherichia coli to 5-ALA, and increasing the carbon flux flowing to 5-aminolevulinic acid; accelerating the degradation of heme, and reducing the inhibition of heme on the synthesis of 5-aminolevulinic acid, thereby obtaining a genetically engineered bacterium with high yield of 5-aminolevulinic acid, thereby achieving the purpose of increasing the yield of 5-aminolevulinic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the ALA synthesis pathway. DETAILED DESCRIPTION

[0030] 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.

[0031] The examples used in the present invention and the primers used in the examples are shown in Table 1.

[0032] Table 1

[0033]

[0034]

[0035] Embodiment 1:

[0036] An embodiment of the genetically engineered bacteria with high 5-aminolevulinic acid production according to the present invention; the genetically engineered bacteria is obtained by subjecting Escherichia coli to the following genetic modifications: replacing RBS with RBS2 and enhancing cyoE gene expression or corresponding enzyme activity.

[0037] The construction method of the genetically engineered bacteria described in this embodiment is:

[0038] Primer pairs cyoE-F and cyoE-R were designed to clone the cyoE gene from Escherichia coli, and the cloned cyoE fragment was connected to the plasmid pETDuet-1 by homologous recombination technology to construct the plasmid pET-cyoE;

[0039] Primer pairs RBS2-F and RBS2-R were designed to perform PCR amplification on pET-cyoE, and the cloned fragment was connected to plasmid pETDuet-1 by homologous recombination technology to construct plasmid pET-PT7-RBS2-cyoE.

[0040] Using E. coli BL21 (DE3) as the competent cell, the obtained plasmid pET-PT7-RBS2-cyoE was mixed with the competent cells and placed on ice for 30 minutes, then quickly heat-shocked (42°C, 90s), and then quickly placed on ice for 2 minutes. 1 mL of LB liquid culture medium was added under sterile conditions, and the culture was resumed at 37°C and 220rpm for 1 hour to obtain a genetically engineered bacterium named ALA-01.

[0041] Embodiment 2:

[0042] An embodiment of the genetically engineered bacteria with high 5-aminolevulinic acid production according to the present invention; the genetically engineered bacteria is obtained by subjecting Escherichia coli to the following genetic modifications: replacing RBS with RBS3 and enhancing cyoE gene expression or corresponding enzyme activity.

[0043] The construction method of the genetically engineered bacteria described in this embodiment is:

[0044] Primer pairs cyoE-F and cyoE-R were designed to clone the cyoE gene from Escherichia coli, and the cloned cyoE fragment was connected to the plasmid pETDuet-1 by homologous recombination technology to construct the plasmid pET-cyoE;

[0045] Primer pairs RBS3-F and RBS3-R were designed to perform PCR amplification on pET-cyoE, and the cloned fragment was connected to plasmid pETDuet-1 by homologous recombination technology to construct plasmid pET-PT7-RBS3-cyoE.

[0046] Using E. coli BL21 (DE3) as the competent cell, the obtained plasmid pET-PT7-RBS3-cyoE was mixed with the competent cells and placed on ice for 30 minutes, then quickly heat-shocked (42°C, 90s), and then quickly placed on ice for 2 minutes. 1 mL of LB liquid culture medium was added under sterile conditions, and the culture was resumed at 37°C and 220rpm for 1 hour to obtain a genetically engineered bacterium named ALA-02.

[0047] Embodiment 3:

[0048] An embodiment of the genetically engineered bacteria with high 5-aminolevulinic acid production according to the present invention; the genetically engineered bacteria is obtained by subjecting Escherichia coli to the following genetic modifications: replacing RBS with RBS5 and enhancing cyoE gene expression or corresponding enzyme activity.

[0049] The construction method of the genetically engineered bacteria described in this embodiment is:

[0050] Primer pairs cyoE-F and cyoE-R were designed to clone the cyoE gene from Escherichia coli, and the cloned cyoE fragment was connected to the plasmid pETDuet-1 by homologous recombination technology to construct the plasmid pET-cyoE;

[0051] Primer pair RBS5-F and RBS5-R were designed to perform PCR amplification on pET-cyoE, and the cloned fragment was connected to plasmid pETDuet-1 by homologous recombination technology to construct plasmid pET-PT7-RBS5-cyoE.

[0052] Using E. coli BL21 (DE3) as the competent cell, the obtained plasmid pET-PT7-RBS5-cyoE was mixed with the competent cells and placed on ice for 30 minutes, then quickly heat-shocked (42°C, 90s), and then quickly placed on ice for 2 minutes. 1 mL of LB liquid culture medium was added under sterile conditions, and the culture was resumed at 37°C and 220rpm for 1 hour to obtain a genetically engineered bacterium named ALA-03.

[0053] Embodiment 4:

[0054] An embodiment of the genetically engineered bacteria with high 5-aminolevulinic acid production according to the present invention; the genetically engineered bacteria is obtained by subjecting Escherichia coli to the following genetic modifications: replacing RBS with RBS8 and the gene expression or corresponding enzyme activity of cyoE.

[0055] The construction method of the genetically engineered bacteria described in this embodiment is:

[0056] Primer pairs cyoE-F and cyoE-R were designed to clone the cyoE gene from Escherichia coli, and the cloned cyoE fragment was connected to the plasmid pETDuet-1 by homologous recombination technology to construct the plasmid pET-cyoE;

[0057] Primer pairs RBS8-F and RBS8-R were designed to perform PCR amplification on pET-cyoE, and the cloned fragment was connected to plasmid pETDuet-1 by homologous recombination technology to construct plasmid pET-PT7-RBS8-cyoE.

[0058] Using E. coli BL21 (DE3) as the competent cell, the obtained plasmid pET-PT7-RBS8-cyoE was mixed with the competent cells and placed on ice for 30 minutes, then quickly heat-shocked (42°C, 90s), and then quickly placed on ice for 2 minutes. 1 mL of LB liquid culture medium was added under sterile conditions, and the culture was resumed at 37°C and 220rpm for 1 hour to obtain a genetically engineered bacterium named ALA-04.

[0059] Embodiment 5:

[0060] An embodiment of the genetically engineered bacteria with high 5-aminolevulinic acid production according to the present invention; the genetically engineered bacteria is obtained by subjecting Escherichia coli to the following genetic modification: replacing RBS with RBS2, and enhancing the gene expression of cyoE and hemAS or the corresponding enzyme activity.

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

[0062] Primer pairs hemAS-F and hemAS-R were designed to clone the glutamyl-tRNA reductase gene hemAS from Escherichia coli, and the cloned hemAS fragment was connected to the plasmid pET-PT7-RBS2-cyoE (plasmid construction see Example 1, synthesized by General Motors, the same below) by homologous recombination technology to construct the plasmid pET-PT7-RBS2-cyoE-PT7-hemAS.

[0063] The obtained plasmid pET-PT7-RBS2-cyoE-PT7-hemAS was mixed with ALA-01 competent cells and placed on ice for 30 minutes, followed by rapid heat shock (42°C, 90s), and then quickly placed on ice for 2 minutes. 1 mL of LB liquid culture medium was added under sterile conditions, and the culture was resumed at 37°C and 220rpm for 1 hour to obtain the genetically engineered bacteria, named ALA-05.

[0064] Embodiment 6:

[0065] An embodiment of the genetically engineered bacteria with high 5-aminolevulinic acid production according to the present invention; the genetically engineered bacteria is obtained by subjecting Escherichia coli to the following genetic modification: replacing RBS with RBS2, and enhancing the gene expression of cyoE and heml or the corresponding enzyme activity.

[0066] The construction method of the genetically engineered bacteria described in this embodiment is:

[0067] Primer pairs heml-F and heml-R were designed to clone the glutamate-1-semialdehyde aminotransferase gene heml from Escherichia coli, and the cloned heml fragment was ligated to the plasmid pET-PT7-RBS2-cyoE by homologous recombination technology to construct the plasmid pET-PT7-RBS2-cyoE-PT7-hemL.

[0068] The obtained plasmid pET-PT7-RBS2-cyoE-PT7-hemL was mixed with ALA-01 competent cells and placed on ice for 30 minutes, then quickly heat-shocked (42°C, 90s), and then quickly placed on ice for 2 minutes. 1 mL of LB liquid culture medium was added under sterile conditions, and the culture was resumed at 37°C and 220rpm for 1 hour to obtain a genetically engineered bacterium named ALA-06.

[0069] Embodiment 7:

[0070] An embodiment of the genetically engineered bacteria with high 5-aminolevulinic acid production according to the present invention; the genetically engineered bacteria is obtained by genetically modifying Escherichia coli as follows: replacing RBS with RBS2, and enhancing the gene expression of cyoE, hemAS, and heml or the corresponding enzyme activity.

[0071] The construction method of the genetically engineered bacteria described in this embodiment is:

[0072] Primer pairs heml-F and heml-R were designed to clone the glutamate-1-semialdehyde aminotransferase gene heml from Escherichia coli, and the cloned heml fragment was ligated to the plasmid pET-PT7-RBS2-cyoE-PT7-hemAS (plasmid construction see Example 5, synthesized by General Motors) by homologous recombination technology to construct the plasmid pET-PT7-RBS2-cyoE-PT7-hemAS-PT7-hemL.

[0073] The obtained plasmid pET-PT7-RBS2-cyoE-PT7-hemAS-PT7-hemL was mixed with ALA-01 competent cells and placed on ice for 30 minutes, followed by rapid heat shock (42°C, 90s), and then quickly placed on ice for 2 minutes. 1 mL of LB liquid culture medium was added under sterile conditions, and the culture was resumed at 37°C and 220rpm for 1 hour to obtain a genetically engineered bacterium named ALA-07.

[0074] Embodiment 8:

[0075] An embodiment of the genetically engineered bacteria with high production of 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by genetically modifying Escherichia coli as follows: replacing RBS with RBS2, and enhancing the gene expression of cyoE, hemAS, heml, pyC or the corresponding enzyme activity.

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

[0077] The pyruvate carboxylase gene pyC from Corynebacterium glutamicum with T7 promoter was synthesized by General Motors and connected to plasmid pCDFDuet-1 to obtain plasmid pCD-PT7-pyC.

[0078] ALA-07 was used as the competent cell. The obtained plasmid pCD-PT7-pyC was mixed with the competent cells and placed on ice for 30 min. Then, the mixture was rapidly heat-shocked (42°C, 90 s). The mixture was then quickly placed on ice for 2 min. 1 mL of LB liquid culture medium was added under sterile conditions. The culture was resumed at 37°C and 220 rpm for 1 h to obtain the genetically engineered bacteria, which was named ALA-08.

[0079] Embodiment 9:

[0080] An embodiment of the genetically engineered bacteria with high production of 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by genetically modifying Escherichia coli as follows: replacing RBS with RBS2, and enhancing the gene expression of cyoE, hemAS, heml, ppC or the corresponding enzyme activity.

[0081] The construction method of the genetically engineered bacteria described in this embodiment is:

[0082] Primer pairs ppC-F and ppC-R were designed to clone the 5-aminolevulinic acid phosphoenolpyruvate carboxylase gene ppC from Escherichia coli, and the cloned ppC fragment was ligated to plasmid pCDFDuet-1 by homologous recombination technology to construct plasmid pCD-T7-ppC.

[0083] ALA-07 was used as the competent cell. The obtained plasmid pCD-T7-ppC was mixed with the competent cells and placed on ice for 30 min. Then, the mixture was rapidly heat-shocked (42°C, 90 s) and then quickly placed on ice for 2 min. 1 mL of LB liquid culture medium was added under sterile conditions. The culture was resumed at 37°C and 220 rpm for 1 h to obtain the genetically engineered bacteria, which was named ALA-09.

[0084] Embodiment 10:

[0085] An embodiment of the genetically engineered bacteria with high production of 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by subjecting Escherichia coli to the following genetic modification: replacing RBS with RBS2, enhancing the gene expression of cyoE, hemAS, heml, ppC, pyC or the corresponding enzyme activity.

[0086] The construction method of the genetically engineered bacteria described in this embodiment is:

[0087] Design primer pairs pyC-F and pyC-R, clone the pyruvate carboxylase gene pyC of pCD-PT7-pyC, and ligate to the plasmid pCD-PT7-ppC (see Example 9 for plasmid construction) to obtain plasmid pCD-PT7-ppC-PT7-pyC.

[0088] ALA-07 was used as the competent cell, and the obtained plasmid pCD-PT7-ppC-PT7-pyC was mixed with the competent cells and placed on ice for 30 minutes, followed by rapid heat shock (42°C, 90s), and then quickly placed on ice for 2 minutes. 1 mL of LB liquid culture medium was added under sterile conditions, and the culture was resumed at 37°C and 220rpm for 1 hour to obtain the genetically engineered bacteria, named ALA-10.

[0089] Embodiment 11:

[0090] An embodiment of the genetically engineered bacteria with high production of 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modification on Escherichia coli: replacing RBS with RBS2, enhancing the gene expression of cyoE, hemAS, heml, ppC, pyC, and rhtA or the corresponding enzyme activity.

[0091] The construction method of the genetically engineered bacteria described in this embodiment is:

[0092] Primer pair rhtA-F / rhtA-R was designed to clone the 5-aminolevulinic acid transporter rhtA from Escherichia coli, and the cloned rhtA fragment was ligated into plasmid pRSFDuet-1 by homologous recombination technology to construct plasmid pRSF-T7-rhtA.

[0093] ALA-10 was used as the competent cell, and the obtained plasmid pRSF-T7-rhtA was mixed with the competent cells and placed on ice for 30 minutes, followed by rapid heat shock (42°C, 90s), and then quickly placed on ice for 2 minutes. 1 mL of LB liquid culture medium was added under sterile conditions, and the culture was resumed at 37°C and 220rpm for 1 hour to obtain the genetically engineered bacteria, named ALA-11.

[0094] Embodiment 12:

[0095] An embodiment of the genetically engineered bacteria with high production of 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modification on Escherichia coli: replacing RBS with RBS2, enhancing the gene expression of cyoE, hemAS, heml, ppC, pyC, eamA or the corresponding enzyme activity.

[0096] The construction method of the genetically engineered bacteria described in this embodiment is:

[0097] Primer pair eamA-F / eamA-R was designed to clone the 5-aminolevulinic acid transporter eamA from Escherichia coli, and the cloned eamA fragment was ligated into plasmid pRSFDuet-1 by homologous recombination technology to construct plasmid pRSF-T7-eamA.

[0098] ALA-10 was used as the competent cell, and the obtained plasmid pRSF-T7-eamA was mixed with the competent cells and placed on ice for 30 minutes, followed by rapid heat shock (42°C, 90s), and then quickly placed on ice for 2 minutes. 1 mL of LB liquid culture medium was added under sterile conditions, and the culture was resumed at 37°C and 220rpm for 1 hour to obtain the genetically engineered bacteria, named ALA-12.

[0099] Embodiment 13:

[0100] An embodiment of the genetically engineered bacteria with high production of 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modification on Escherichia coli: replacing RBS with RBS2, enhancing the gene expression of cyoE, hemAS, heml, ppC, pyC, rhtA, and kate or the corresponding enzyme activity.

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

[0102] Primer pairs kate-F and kate-R were designed to clone kate from E. coli, and the cloned kate fragment was ligated to plasmid pRSF-T7-rhtA (see Example 11 for plasmid construction, the same below) by homologous recombination technology to construct plasmid pRSF-T7-rhtA-T7-kate.

[0103] ALA-11 was used as the competent cell, and the obtained plasmid pRSF-T7-rhtA-T7-kate was mixed with the competent cells and placed on ice for 30 minutes, followed by rapid heat shock (42°C, 90s), and then quickly placed on ice for 2 minutes. 1 mL of LB liquid culture medium was added under sterile conditions, and the culture was resumed at 37°C and 220rpm for 1 hour to obtain the genetically engineered bacteria, named ALA-13.

[0104] Embodiment 14:

[0105] An embodiment of the genetically engineered bacteria with high production of 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modification on Escherichia coli: replacing RBS with RBS2, enhancing the gene expression of cyoE, hemAS, heml, ppC, pyC, rhtA, sodA or the corresponding enzyme activity.

[0106] The construction method of the genetically engineered bacteria described in this embodiment is:

[0107] Primer pairs sodA-F and sodA-R were designed to clone sodA from Escherichia coli, and the cloned sodA fragment was ligated to plasmid pRSF-T7-rhtA by homologous recombination technology to construct plasmid pRSF-T7-rhtA-T7-sodA.

[0108] ALA-11 was used as the competent cell, and the obtained plasmid pRSF-T7-rhtA-T7-sodA was mixed with the competent cells and placed on ice for 30 minutes, followed by rapid heat shock (42°C, 90s), and then quickly placed on ice for 2 minutes. 1 mL of LB liquid culture medium was added under sterile conditions, and the culture was resumed at 37°C and 220rpm for 1 hour to obtain the genetically engineered bacteria, named ALA-14.

[0109] Embodiment 15:

[0110] An embodiment of the genetically engineered bacteria with high production of 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modification on Escherichia coli: replacing RBS with RBS2, enhancing the gene expression of cyoE, hemAS, heml, ppC, pyC, rhtA, yfcF or the corresponding enzyme activity.

[0111] The construction method of the genetically engineered bacteria described in this embodiment is:

[0112] Primer pairs yfcF-F and yfcF-R were designed to clone yfcF from Escherichia coli, and the cloned yfcF fragment was ligated to plasmid pRSF-T7-rhtA by homologous recombination technology to construct plasmid pRSF-T7-rhtA-T7-yfcF.

[0113] ALA-11 was used as the competent cell, and the obtained plasmid pRSF-T7-rhtA-T7-yfcF was mixed with the competent cells and placed on ice for 30 minutes, followed by rapid heat shock (42°C, 90s), and then quickly placed on ice for 2 minutes. 1 mL of LB liquid culture medium was added under sterile conditions, and the culture was resumed at 37°C and 220rpm for 1 hour to obtain the genetically engineered bacteria, named ALA-15.

[0114] Embodiment 16:

[0115] An embodiment of the genetically engineered bacteria with high production of 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modification on Escherichia coli: replacing RBS with RBS2, enhancing the gene expression of cyoE, hemAS, heml, ppC, pyC, rhtA, and htpG or the corresponding enzyme activity.

[0116] The construction method of the genetically engineered bacteria described in this embodiment is:

[0117] Primer pairs htpG-F and htpG-R were designed to clone htpG from Escherichia coli, and the cloned htpG fragment was ligated to plasmid pRSF-T7-rhtA by homologous recombination technology to construct plasmid pRSF-T7-rhtA-T7-htpG.

[0118] ALA-11 was used as the competent cell, and the obtained plasmid pRSF-T7-rhtA-T7-htpG was mixed with the competent cells and placed on ice for 30 minutes, followed by rapid heat shock (42°C, 90s), and then quickly placed on ice for 2 minutes. 1 mL of LB liquid culture medium was added under sterile conditions, and the culture was resumed at 37°C and 220rpm for 1 hour to obtain the genetically engineered bacteria, named ALA-16.

[0119] Embodiment 17:

[0120] An embodiment of the genetically engineered bacteria with high production of 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modification on Escherichia coli: replacing RBS with RBS2, enhancing the gene expression of cyoE, hemAS, heml, ppC, pyC, rhtA, sodB or the corresponding enzyme activity.

[0121] The construction method of the genetically engineered bacteria described in this embodiment is:

[0122] Primer pairs sodB-F and sodB-R were designed to clone sodB from Escherichia coli, and the cloned sodB fragment was ligated to plasmid pRSF-T7-rhtA by homologous recombination technology to construct plasmid pRSF-T7-rhtA-T7-sodB.

[0123] ALA-11 was used as the competent cell, and the obtained plasmid pRSF-T7-rhtA-T7-sodB was mixed with the competent cells and placed on ice for 30 minutes, followed by rapid heat shock (42°C, 90s), and then quickly placed on ice for 2 minutes. 1 mL of LB liquid culture medium was added under sterile conditions, and the culture was resumed at 37°C and 220rpm for 1 hour to obtain the genetically engineered bacteria, named ALA-17.

[0124] Embodiment 18:

[0125] An embodiment of the genetically engineered bacteria with high production of 5-aminolevulinic acid according to the present invention; the genetically engineered bacteria is obtained by performing the following genetic modification on Escherichia coli: replacing RBS with RBS2, enhancing the gene expression of cyoE, hemAS, heml, ppC, pyC, rhtA, katG or the corresponding enzyme activity.

[0126] The construction method of the genetically engineered bacteria described in this embodiment is:

[0127] Primer pairs katG-F and katG-R were designed to clone katG from Escherichia coli, and the cloned katG fragment was ligated to plasmid pRSF-T7-rhtA by homologous recombination technology to construct plasmid pRSF-T7-rhtA-T7-katG.

[0128] ALA-11 was used as the competent cell, and the obtained plasmid pRSF-T7-rhtA-T7-katG was mixed with the competent cells and placed on ice for 30 minutes, followed by rapid heat shock (42°C, 90s), and then quickly placed on ice for 2 minutes. 1 mL of LB liquid culture medium was added under sterile conditions, and the culture was resumed at 37°C and 220rpm for 1 hour to obtain the genetically engineered bacteria, named ALA-18.

[0129] Test Example 1: Shake flask test of strain ALA production capacity

[0130] Test sample: the genetically engineered bacteria constructed in Example 1-18.

[0131] Fermentation treatment of genetically engineered bacteria:

[0132] 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.

[0133] 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 5, 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.

[0134] 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.

[0135] After fermentation, the ALA yield was determined by high performance liquid chromatography. The specific test method is as follows:

[0136] 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.

[0137] 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.

[0138] 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.

[0139] Table 2

[0140]

[0141] 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.

[0142] The results of 5-ALA yield determination are shown in Table 3-5.

[0143] Table 3

[0144]

[0145]

[0146] From the above table, we can see that the ALA yield of the genetically engineered bacteria ALA-01, ALA-02, ALA-03, and ALA-04 is compared, and the yield of ALA-01 is higher than that of ALA-02, ALA-03, and ALA-04; this indicates that the expression effect of plasmid pET-PT7-RBS2-cyoE is better than that of plasmid pET-PT7-RBS3-cyoE, pET-PT7-RBS5-cyoE, and pET-PT7-RBS8-cyoE, and the yield of 5-aminolevulinic acid is significantly improved.

[0147] Table 4

[0148] Genetically engineered bacteria ALA yield (g / L) ALA-05 1.21 ALA-06 1.37 ALA-07 1.45 ALA-08 2.13 ALA-09 2.26 ALA-10 2.37

[0149] Comparing the ALA yields of genetically engineered bacteria ALA-05, ALA-06, and ALA-07, the yield of ALA-07 was higher than that of other genetically engineered bacteria, indicating that the expression effect of plasmid pET-PT7-RBS2-cyoE-PT7-hemAS-PT7-hemL was better than that of plasmid pET-PT7-RBS2-cyoE-PT7-hemAS and pET-PT7-RBS2-cyoE-PT7-hemL, and the yield of 5-aminolevulinic acid was significantly improved.

[0150] Comparing the ALA yields of the genetically engineered bacteria ALA-08, ALA-09, and ALA-10, the yield of ALA-10 was higher than that of ALA-08 and ALA-09, indicating that the expression effect of the plasmid pCD-PT7-ppC-PT7-pyC was better than that of the plasmid pCD-PT7-pyC and pCD-T7-ppC, and the yield of 5-aminolevulinic acid was significantly improved.

[0151] Table 5

[0152]

[0153]

[0154] Comparing the ALA production of genetically engineered bacteria ALA-11 and ALA-12, the production of ALA-11 was higher than that of ALA-12, indicating that the expression effect of plasmid pRSF-T7-rhtA was better than that of plasmid pRSF-T7-eamA, and the production of 5-aminolevulinic acid was significantly improved.

[0155] Comparing the ALA yields of genetically engineered bacteria ALA-13, ALA-14, ALA-15, ALA-16, ALA-17, and ALA-18, the yield of ALA-13 was higher than that of other genetically engineered bacteria, indicating that the expression effect of plasmid pRSF-T7-rhtA-T7-kate was better than that of plasmids pRSF-T7-rhtA-T7-sodA, pRSF-T7-rhtA-T7-yfcF, pRSF-T7-rhtA-T7-htpG, pRSF-T7-rhtA-T7-sodB, and pRSF-T7-rhtA-T7-katG, and the yield of 5-aminolevulinic acid was significantly improved.

[0156] Test Example 2: Testing the ALA production capacity of strains in 5L tanks

[0157] Test sample: the genetically engineered bacteria constructed in Example 18.

[0158] Fermentation of genetically engineered bacteria (in a 5L fermenter):

[0159] Prepare 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; then add mixed carbon source: 700g glucose, 640g glycerol, 6.3g magnesium sulfate heptahydrate, 9.4mL trace elements to make up to 3.5L; add 500mL 25-28% ammonia water (0.22μm filtered for sterilization) and 60mL defoamer.

[0160] 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 / LCoCl2·6H2O, 1.0g / L manganese chloride tetrahydrate, 0.10g / L CuCl2·2H2O, 0.2g / L boric acid, 0.2g / LNa2MoO4·2H2O), 2g / L MgSO4·7H2O, 0.02g / L anhydrous CaCl2; add 4g / L glutamic acid every 12h.

[0161] 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.

[0162] The fermentation parameters were controlled as follows:

[0163] (1) Liquid volume: 3.5L; Inoculation volume: 150mL; Fermentation time: about 120h;

[0164] (2) Control parameters: pH 7; temperature: 37°C, the temperature is maintained at 37°C after feeding;

[0165] (3) Ventilation volume: Initial ventilation volume 1:1 (2.5 L), intake pressure adjusted to 0.06 MPa;

[0166] (4) Speed: starting speed 200 rpm;

[0167] (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%.

[0168] (6) Dissolved oxygen 100% calibration: 200 rpm, 1:1 ventilation;

[0169] (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;

[0170] (8) For strain preparation, refer to the single strain entry position;

[0171] (9) The base feed consumption time is set at 6-7 hours. After 4 hours of supplementation, IPTG is added for induction.

[0172] During the fermentation period, please note:

[0173] 1. Pay attention to accidents such as defoaming, peristaltic pump failure, power failure and tripping;

[0174] 2. Record every 2 hours and pay close attention to DO changes;

[0175] 3. Prevent the pH from increasing due to the active addition of ammonia water 6 hours before fermentation;

[0176] 4. Calibrate the pH electrode of the fermenter with a pH meter before inoculation.

[0177] Fermentation detection indicators: After 120 hours of fermentation, take 2 mL of sample and detect the ALA production.

[0178] The genetically engineered bacteria ALA-18 was induced to ferment in a fermenter, and its ALA production was measured to be 30.14 g / L after 120 hours of fermentation.

[0179] 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 that produces high yield of 5-aminolevulinic acid, characterized in that: The genetically engineered bacteria are obtained by performing the following gene modification on Escherichia coli: replacing RBS and enhancing the gene expression of cyoE or the corresponding enzyme activity.

2. The genetically engineered bacterium for high production of 5-aminolevulinic acid as claimed in claim 1, characterized in that: The replacing of the RBS includes replacing the RBS with any one of RBS2, RBS3, RBS5, and RBS8.

3. The genetically engineered bacterium for high production of 5-aminolevulinic acid as claimed in claim 2, characterized in that: The RBS is replaced with RBS2.

4. The genetically engineered bacterium for high production of 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 corresponding enzyme activity of at least one of hemAS and heml.

5. The genetically engineered bacterium for high production of 5-aminolevulinic acid as claimed in claim 4, characterized in that: The genetically engineered bacteria also include the following genetic modification: enhancing the gene expression or corresponding enzyme activity of at least one of ppC and pyC.

6. The genetically engineered bacterium for high production of 5-aminolevulinic acid according to claim 5, characterized in that: The genetically engineered bacteria also include any one of the following genetic modifications (i)-(ii): (i) enhancing the gene expression or corresponding enzyme activity of either rhtA or eamA; (ii) enhancing the gene expression or corresponding enzyme activity of any one of rhtA and eamA, and enhancing the gene expression or corresponding enzyme activity of any one of kate, sodA, yfcF, htpG, sodB, and katG.

7. The genetically engineered bacterium for high production of 5-aminolevulinic acid according to claim 6, characterized in that: The genetically engineered bacteria also include the following genetic modification: enhancing the gene expression of rhtA or the corresponding enzyme activity.

8. The genetically engineered bacterium for high production of 5-aminolevulinic acid according to claim 6, characterized in that: The genetically engineered bacteria also include the following genetic modification: enhancing the gene expression of rhtA and kate or the corresponding enzyme activity.

9. Use of the genetically engineered bacteria with high 5-aminolevulinic acid production according to any one of claims 1 to 8 in producing 5-aminolevulinic acid.

10. A method for producing 5-aminolevulinic acid, characterized in that: 5-Aminolevulinic acid is obtained by inducing fermentation of the genetically engineered bacteria with high 5-aminolevulinic acid production according to any one of claims 1 to 8.

Images