A method for screening an engineered strain with high production of 5-aminolevulinic acid, an engineered strain and its application

Abstract

The present invention relates to a method for screening an engineering strain with high yield of 5-aminolevulinic acid, an engineering bacterium and an application thereof, and belongs to the field of engineering microorganisms. The present invention uses a biosensor based on a change in intracellular cAMP level in response to ALA, and screens an Escherichia coli genome mutant library by fluorescence activated cell sorting (FACS), thereby obtaining a mutant Escherichia coli with high ALA yield. The results show that the mutant Escherichia coli screened by the present invention has a higher ALA yield than wild-type Escherichia coli.

Description

Technical Field

[0001] The present invention belongs to the technical field of engineering microorganisms, and specifically relates to a method for screening an engineering strain with high yield of 5-aminolevulinic acid, an engineering bacterium with high yield of 5-aminolevulinic acid and a 5-aminolevulinic acid biosynthesis method based on the strain. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] 5-Aminolevulinic acid (ALA) is a non-protein amino acid and an essential precursor for the synthesis of tetrapyrrole compounds, including heme, porphyrin, chlorophyll, and vitamin B12, which play a key role in maintaining normal physiological functions of the body. Due to its safety, environmental compatibility, and biodegradability, ALA has been widely used in agriculture, animal husbandry, and medicine. Nowadays, the production of ALA by microbial fermentation is an environmentally friendly, simple, inexpensive, and sustainable method that avoids the disadvantages of complex reaction steps, high cost, and environmental pollution in chemical synthesis.

[0004] The biosynthetic pathway of ALA is widely present in plants, animals and microorganisms, and its biosynthetic pathway is divided into the C4 (Shemin) pathway and the C5 pathway. In the C4 pathway, ALA is produced by one-step condensation of succinyl-CoA and glycine through ALA synthetase (hemA). In the C5 pathway, glutamate undergoes three sequential enzymatic reactions of glutamyl tRNA synthetase, glutamyl tRNA reductase (hemA) and glutamate-1-semialdehyde transaminase (hemL) to produce ALA, which is more complicated than the C4 pathway. So far, enzyme screening, pathway engineering, and fermentation process optimization have been studied, and the microbial production of ALA has also been significantly improved.

[0005] As an amino ketone compound, ALA is highly reactive and unstable and can generate reactive oxygen species (ROS). The reactive oxygen species generation pathways include: (1) ALA undergoes enolization and aerobic oxidation to generate ROS; (2) ALA spontaneously dimerizes and irreversibly decomposes into 2,5-(β-carboxyethyl)pyrazine, generating ROS at the same time; (3) ALA's downstream product, protoporphyrin IX, generates ROS after irradiation. A large amount of ROS induces oxidative stress and affects the physiological state of cells. In the microbial production of ALA, the production of ALA will affect the physiological state of the production host and inhibit the growth and physiological activity of the host, thereby limiting the ALA production efficiency. Studies have shown that when the tolerance of E. coli to ALA is increased, the growth state of E. coli during the fermentation process can be significantly improved and the production of ALA can be significantly promoted. Similar regulatory methods can be applied to other microorganisms, which can also significantly increase the tolerance of microorganisms to products and promote microbial production. At the same time, the regulation of metabolic flux in the production chassis cells is also a common means to improve related products, and there have been a large number of reports on introducing metabolic flux into the ALA production pathway, such as: introducing metabolic flux into the TCA cycle, inhibiting the metabolic flux of the downstream pathway of ALA, etc. The relevant research is relatively complete.

[0006] The above are all rational design strategies for ALA production chassis cells, but there are few reports on irrational design strategies for ALA production chassis cells. Compared with rational design, the advantages of irrational design include: 1. A wider search space: rational design can only adjust specific genes or gene fragments, while random mutations in irrational design can involve the entire genome, thereby exploring a wider range of possibilities. 2. A higher probability of discovering unknown functions: when researchers do not fully understand a gene, random mutation methods can discover other (unknown) functions of the gene. 3. Higher efficiency: random mutation methods do not require prior understanding of complex regulatory mechanisms and gene functions, reducing research costs and time. At the same time, based on high-throughput screening methods, screened strains can be quickly identified.

[0007] Therefore, in the microbial production process of ALA, the tolerance of the bottom plate bacteria to ALA plays a relatively important role in the microbial fermentation production efficiency. Obtaining bottom plate bacteria that are more suitable for ALA microbial fermentation is of great significance to improving the ALA fermentation efficiency. Summary of the invention

[0008] In order to obtain chassis cells that are more suitable for ALA microbial fermentation production, the present invention constructed and screened a genomic mutant library of Escherichia coli based on a irrational design strategy, and finally obtained an Escherichia coli DM16 with high ALA production.

[0009] Based on the above research results, the present invention provides the following technical solutions:

[0010] The present invention firstly provides a method for screening high-yield 5-aminolevulinic acid engineering bacteria, comprising the following steps: using Escherichia coli as a starting strain, overexpressing ALA synthase (hemA) in the starting strain, introducing a mutant dnaQ* of DNA polymerase III and screening plasmids to induce Escherichia coli genome mutation to obtain a mutant library, and screening ALA high-yield strains from the mutant library.

[0011] Wild-type Escherichia coli can only synthesize 5-ALA through the C5 pathway, with glutamate as a prerequisite, and finally generate 5-ALA under the catalysis of glutamyl tRNA synthetase, glutamyl tRNA reductase, and glutamate-1-semialdehyde transaminase series enzymes. In the above three-step reaction, the rate-limiting step is the reaction of glutamyl tRNA reduction to GSA catalyzed by glutamyl tRNA reductase. In order to obtain an engineered bacterium suitable for ALA fermentation, the present invention introduces the C4 synthesis pathway into Escherichia coli, using glycine and succinyl coenzyme A as precursors, PLP as a cofactor, and one-step reaction to generate ALA. And by constructing a mutant library, screen the strains that can efficiently synthesize ALA.

[0012] DNA polymerase III (dnaQ) has both 5'→3' polymerase activity and 3'→5' exonuclease activity. The activity of this enzyme is relatively strong, 15 times that of DNA polymerase I and 300 times that of DNA polymerase II. It can extend the new DNA chain at a speed of about 50,000 nucleotides per minute on the 3'-OH of the primer. It is the leading polymerase in the chain extension reaction in Escherichia coli DNA replication. The present invention first constructs a mutant dnaQ* of dnaQ, which has lower proofreading activity than dnaQ. Therefore, when dnaQ* is used for genome replication of Escherichia coli, some mutation sites will be retained on the genome, and a genomic mutant library of Escherichia coli is obtained in this way. First, the mutants are sorted based on fluorescence intensity and resistance. After sorting, the mutant strains are fermented by shaking flasks to screen mutant strains with high ALA production.

[0013] The screening plasmid is a biosensor that responds to ALA based on changes in intracellular cAMP levels. By introducing the plasmid, technicians can conveniently screen strains with high ALA expression based on fluorescence intensity.

[0014] The present invention screened and obtained the strain with the highest yield by the above method, and preserved it after losing the recombinant plasmid, and named it DM16.

[0015] Therefore, the present invention also provides an engineered bacterium with high production of 5-aminolevulinic acid, the strain is named Escherichia coli DM16, and the strain has been deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, on April 26, 2023, with an address of: Wuhan University, Wuhan, China, and its biological preservation number is: CCTCC NO: M2023631.

[0016] The above strain can be used for the biosynthesis of 5-aminolevulinic acid. DM16 is used as a production strain for ALA C4 pathway production. The hemA gene is overexpressed in DM16 so that the DM16 strain can produce ALA through the C4 pathway. The DM16 strain containing the hemA gene supplements glycine for the synthesis of ALA during the fermentation process of ALA. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0018] Figure 1 Comparison of results after FACS screening;

[0019] Light-colored scattered dots represent the single-cell fluorescence level of the initial library strains, and dark-colored scattered dots represent the single-cell fluorescence level of the library strains after sorting;

[0020] Figure 2 The ALA production of single colonies in the well plate after FACS sorting;

[0021] Dark colors represent the ALA production of the wild-type strain, and light colors represent the ALA production of the picked mutant single colonies;

[0022] Figure 3 The results of shake flask fermentation of mutant strains;

[0023] Dark color represents ALA production, and light color represents biomass;

[0024] Figure 4 To verify the ALA production capacity of the plasmid-lost strain DM16;

[0025] Dark color represents ALA production, and light color represents biomass. DETAILED DESCRIPTION

[0026] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0027] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0028] As introduced in the background technology, in the microbial production process of ALA, the strain's tolerance to ALA plays an important role in the microbial fermentation production of ALA by Escherichia coli. In order to screen and obtain chassis cells that are more suitable for the microbial fermentation production of ALA, the present invention uses a biosensor based on changes in intracellular cAMP levels to respond to ALA to screen the Escherichia coli genome mutant library through fluorescence activated cell sorting (FACS), and obtains a mutant Escherichia coli with high ALA production.

[0029] The specific plan is as follows:

[0030] In a first aspect, a method for screening an engineered strain with high 5-aminolevulinic acid production is provided, comprising the following steps: using Escherichia coli as a starting strain, overexpressing ALA synthetase (hemA) in the starting strain, introducing a mutant dnaQ* of DNA polymerase III, and screening plasmids to induce mutations in the Escherichia coli genome to obtain a mutant library, and screening an ALA high-producing strain from the mutant library.

[0031] The above screening method also has the following preferred embodiments:

[0032] Starting strain. The above screening method uses Escherichia coli as the starting strain. Since Escherichia coli may have similar metabolic flows, any Escherichia coli can be selected as the starting strain in theory. Considering cost economy, conventional model bacteria can be used. In one embodiment verified by the present invention, it is Escherichia coli DH5α.

[0033] ALA synthase (hemA), derived from Rhodopseudomonas palustris KUGB306, has a sequence as shown in SEQ ID NO: 1, and is transferred into the starting strain by constructing a recombinant plasmid.

[0034] The mutant of DNA polymerase III, the sequence of dnaQ* is shown in SEQ ID NO: 2, and is also transformed into the starting strain by constructing a recombinant plasmid.

[0035] The screening plasmid has the expression cassette shown in SEQ ID NO.5.

[0036] The steps for screening ALA high-producing strains from the mutant library are as follows:

[0037] (1) The strains in the mutant library were inoculated into LB medium for cultivation. During the cultivation process, dehydrated tetracycline was added to induce the expression of dnaQ*. The bacteria were collected and strains with lower fluorescence intensity were sorted by flow cytometry. The sorted strains were inoculated into resistance medium for cultivation and the above screening process was repeated until a strain with significantly reduced fluorescence level was obtained;

[0038] (2) The strains screened in step (1) were screened again based on their ALA fermentation capacity, and strains with higher ALA production were selected for shake flask fermentation verification.

[0039] The present invention removes plasmids from the strain with the highest yield (containing the three plasmids mentioned above) obtained by shake flask fermentation, wherein the strain with the highest yield is DM16, which is a non-resistant and plasmid-free strain. Subsequently, the present invention verifies the performance of ALA production of DM16 strain (randomly picking four DM16 single colonies that lost plasmids), re-transforms the hemA gene for ALA fermentation, and proves that the strain has good tolerance to ALA, can stably and highly produce ALA, and can be used as an engineered bacterium for 5-aminolevulinic acid in industrial fermentation.

[0040] In the second aspect, an engineered bacterium with high production of 5-aminolevulinic acid is provided. The strain is named Escherichia coli DM16, which was deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, on April 26, 2023. The address is: Wuhan University, Wuhan, China, and its biological preservation number is: CCTCCNO: M2023631.

[0041] In a third aspect, a bacterial agent is provided, which includes the engineered bacteria described in the second aspect, or a fermentation culture of the bacteria.

[0042] The fermentation culture of the above bacteria, that is, the fermentation product of the engineered bacteria described in the second aspect, contains a considerable amount of ALA.

[0043] In addition, the above-mentioned bacterial agent can be a solid or liquid preparation, and also includes a pharmaceutically necessary carrier, such as a solid preparation such as bacterial powder and granules, and a liquid preparation such as a water suspension and a dispersible oil suspension; the pharmaceutically acceptable carrier is selected from one or more of a dispersant, a wetting agent, a disintegrant, a binder, a defoaming agent, an antifreeze agent, a thickener, a filler and a solvent. The present invention has no special restrictions on the source of the pesticide-acceptable excipients, and generally commercial products can be used.

[0044] In a fourth aspect, a method for the biosynthesis of 5-aminolevulinic acid is provided, wherein ALA synthase (hemA) in Escherichia coli DM16 is overexpressed to prepare a recombinant bacterium, and glycine is supplemented during the fermentation process of the recombinant bacterium to synthesize 5-aminolevulinic acid.

[0045] The specific steps are as follows:

[0046] Exogenous hemA was introduced into Escherichia coli DM16 by plasmid transfection, and the modified strain was transferred into LB medium for seed culture, and then transferred into fermentation medium for fermentation culture; the composition of the fermentation medium is as follows: 18-22 g / L glucose, 1-3 g / L yeast extract, 8-12 g / L succinic acid, 3-5 g / L glycine, 16-17 g / L (NH 4 ) 2 SO 4 ,1~4g / L KH 2 PO 4 , 15~18g / LNa 2 HPO 4 12H 2 O, 0.8~1.2g / L MgSO 4 7H 2 O and 0.01 g / L MnSO 4 7H 2 O fermentation medium, supplemented with 3-5 g / L glycine every 12 h.

[0047] Preferably, the fermentation is carried out in a shake flask, the fermentation temperature is 35-38° C., and the rotation speed is 200-250 rpm.

[0048] Preferably, during the above fermentation process, IPTG and / or dehydrotetracycline may be added to the culture medium;

[0049] Furthermore, during the fermentation process, when OD 600 When the concentration of 0.1 mM IPTG was added at 0.6, the cells were induced.

[0050] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0051] Example 1

[0052] 1. Experimental Methods

[0053] 1.1 Strains, culture media and culture conditions

[0054] E. coli DH5α cells were selected for plasmid construction and E. coli genome mutant library construction. The following culture medium was used for E. coli DH5α and the strains in the mutant library:

[0055] LB medium: 10 g / L peptone, 5 g / L yeast extract and 5 g / L NaCl, the balance is water.

[0056] 1.2 Plasmid construction

[0057] 1.2.1 hemA plasmid construction

[0058] According to the protein sequence of ALA synthase hemA in Rhodopseudomonas palustris KUGB306, as shown in SEQ ID NO: 1. Escherichia coli was used as the expression host for codon optimization and gene synthesis, and it was amplified by using PhantaMax Super-Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China) and corresponding primers, and the amplified gene fragment was inserted into the pTrc99a plasmid using ClonExpress II One Step Cloning Kit (VazymeBiotech, Nanjing, China) to obtain the overexpression plasmid of hemA. The primer sequences involved in the above construction process are shown in Table 1 below:

[0059] Table 1

[0060]

[0061] 1.2.2 Construction of plasmid for random genome mutation

[0062] Escherichia coli MG1655 was purchased from Shanghai Weidi Biotechnology. The target fragment was amplified from the genomic DNA of Escherichia coli MG1655 using Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China) and corresponding primers. The dnaQ fragment was purified and recovered and inserted into the pACYC184 plasmid for expression induced by dehydrated tetracycline using ClonExpress II One Step Cloning Kit (Vazyme Biotech, Nanjing, China). Subsequently, the point mutation D12A was introduced into dnaQ by the inverse PCR method to obtain the mutant expression plasmid of dnaQ*. Restriction endonucleases were purchased from Thermo Fisher Scientific (Waltham, MA). Primer synthesis and Sanger sequencing were performed by Tsingke (Beijing, China).

[0063] The wild-type dnaQ sequence in the starting strain Escherichia coli MG1655 is as follows:

[0064] mstaitrqiv ldtettgmnq igahyeghki ieigavevvn rrltgnnfhv ylkpdrlvdpeafgvhgiad

[0065] eflldkptfa evadefmdyi rgaelvihna ​​afdigfmdye fsllkrdipk tntfckvtdslavarkmfpg

[0066] krnsldalca ryeidnskrt lhgalldaqi laevylamtg gqtsmafame getqqqqgeatiqrivrqas

[0067] klrvvfatde eiaahearld lvqkkggscl wra(SEQ ID NO.2);

[0068] The sequence of the mutant dnaQ* constructed in this example is as follows:

[0069] mstaitrqiv latettgmnq igahyeghki ieigavevvn rrltgnnfhv ylkpdrlvdpeafgvhgiad

[0070] eflldkptfa evadefmdyi rgaelvihna ​​afdigfmdye fsllkrdipk tntfckvtdslavarkmfpg

[0071] krnsldalca ryeidnskrt lhgalldaqi laevylamtg gqtsmafame getqqqqgeatiqrivrqas

[0072] klrvvfatde eiaahearld lvqkkggscl wra (SEQ ID NO. 3).

[0073]

[0074] 1.2.3 Construction of screening plasmid

[0075] The inventor's previous studies have shown that ROS caused by ALA can reduce the intracellular cAMP level of Escherichia coli and is related to the ALA concentration. The promoter specifically regulated by CRP can characterize the relationship between ALA and intracellular cAMP, thereby reflecting the intracellular ALA level of bacteria. The present invention adds a strong CRP binding site at three positions upstream of the promoter -83.5, which has good responsiveness to cAMP dose and ALA dose. The relevant research is recorded in the patent document with application number (2022117303171). Based on the conclusions of this study, a screening plasmid was constructed:

[0076] The target promoter was amplified from Escherichia coli MG1655 genomic DNA using Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China) and corresponding primers. The promoter and GFP sequence were connected together by fusion PCR, and the target promoter was used to regulate the expression of GFP. Subsequently, ClonExpress II One Step Cloning Kit (Vazyme Biotech, Nanjing, China) was used to insert it into the pCDF-duet-1 plasmid to obtain the screening plasmid, wherein the expression cassette of the screening plasmid includes: promoter-ribosome binding site (RBS)-GFP, wherein the promoter sequence is shown in SEQ ID NO.4, and the expression cassette sequence is shown in SEQ ID NO.5.

[0077] 1.3 Analysis method of ALA

[0078] 5-ALA fermentation medium: 20 g / L glucose, 2 g / L yeast extract, 10 g / L succinic acid, 4 g / L glycine, 16 g / L (NH 4 ) 2 SO 4 ,3g / L KH 2 PO 4 , 16g / L Na 2 HPO 4 12H 2 O,1g / LMgSO 4 7H 2 O and 0.01 g / L MnSO 4 7H 2 O fermentation medium, supplemented with 4 g / L glycine every 12 h.

[0079] Fermentation conditions: Cultivation and fermentation were carried out at 37°C and 220 rpm. Appropriate concentrations of antibiotics were added to the culture medium when necessary, including chloramphenicol (34 μg / mL) and kanamycin (50 μg / mL). 0.1 mM isopropyl-β-d-thiobeta-lactoside (IPTG) was added to induce the expression of hemA or 200 μg / L of anhydrotetracycline was used to induce the expression of dnaQ* genes.

[0080] Transfer the fermentation supernatant into a new centrifuge tube. Dilute according to a certain ratio. Take 400 μL of the dilution, add 200 μL of sodium acetate buffer and 100 μL of acetylacetone, boil and react for 15 minutes. Cool to room temperature, add Modified Ehrlich's reagent and react for 20 minutes, then use a 1 cM cuvette to detect the OD value with a spectrophotometer at a wavelength of 554 nm. According to the ALA / OD 554 The concentration of ALA was calculated using the standard curve.

[0081] 2. Experimental Results

[0082] The DH5α strain with ALA production plasmid (hemA), genome random mutation plasmid (dnaQ*) and screening plasmid (for subsequent ALA high-throughput screening) was inoculated into a shake flask containing LB medium for culture. During the culture period, 200 μg / L dehydrated tetracycline was added to induce the expression of dnaQ*, and the cells were transferred every 12 hours. After 3-4 transfers, the cells were collected, washed 3 times with PBS, and resuspended with PBS. Then a certain amount of bacterial liquid was taken and diluted to an appropriate concentration for subsequent flow cytometric sorting. During the sorting process, the sorting area was selected according to the fluorescence intensity of the bacteria, and a sorting gate was circled to screen the strains with low green fluorescence falling in the gate. The sorted bacterial liquid was inoculated into LB medium containing the corresponding resistance for culture for the next continued sorting. After sorting, the mutant strain library was analyzed by flow cytometry, and it can be clearly seen that its overall fluorescence level has a significant decrease compared with the fluorescence level of the initial library ( Figure 1 ).

[0083] The final library was streaked on an agar plate and single colonies were inoculated into a well plate for primary screening. According to the results of the measured ALA production, it can be seen that the production of some mutant strains is significantly higher than that of the wild-type strain ( Figure 2 ). The present invention selected the top 15 strains with the highest yield and inoculated them into shaking flasks for rescreening, among which the yields of the three strains DM14, DM15 and DM16 were higher than those of the wild-type strain ( Figure 3 ).

[0084] Then, in this example, the best performing DM16 strain was subjected to plasmid loss, and four DM16 strains that had lost plasmids were selected for transformation. 4 The plasmid of the production pathway was transformed into the DM16 strain for fermentation verification. The results showed that the ALA yield of the DM16 strain finally obtained in this example (DM16-1: 4.14±0.22g / L DM16-2: 4.14±0.29g / L DM16-3: 4.26±0.30g / L DM16-4: 3.99±0.24g / L) was significantly higher than that of the control strain (2.91±0.23g / L). Finally, this example obtained a high-yield ALA strain DM16 obtained by DH5α mutation, which has been deposited with CCTCC, with the deposit number: CCTCC M 2023631.

[0085] Example 2

[0086] In this embodiment, a method for synthesizing ALA based on the above-mentioned preserved strain is provided, comprising the following steps:

[0087] The hemA gene was transformed into the DM16 strain, and a single colony was picked and inoculated into LB medium for overnight culture at 37°C and 220rpm. The bacterial liquid was inoculated into a shake flask containing 50ml LB liquid at a 2% inoculum as a seed for culture. After culturing for 12-16 hours, it was inoculated into ALA fermentation medium at a 2% inoculum for ALA fermentation. The fermentation medium components are as follows: 20g / L glucose, 2g / l yeast extract, 10g / L succinic acid, 4g / L glycine, 16g / L (NH 4 ) 2 SO 4 ,3g / L KH 2 PO 4 , 16g / LNa 2 HPO 4 12H 2 O,1g / L MgSO 4 7H 2 O and 0.01 g / L MnSO 4 7H 2 O fermentation medium, supplemented with 4 g / L glycine every 12 h. Fermentation conditions: culture and fermentation were carried out at 37°C and 220 rpm.

[0088] The maximum ALA production of the strain was 4.26±0.30 g / L.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for screening a high-yield 5-aminolevulinic acid engineering strain, characterized in that: The method comprises the following steps: using Escherichia coli as a starting strain, overexpressing ALA synthase in the starting strain, introducing a mutant dnaQ* of DNA polymerase III, and screening plasmids to induce mutations in the Escherichia coli genome to obtain a mutant library, and screening an ALA high-yielding strain from the mutant library; The ALA synthase is derived from Rhodopseudomonas palustris ( Rhodopseudomonas palustris ) KUGB306, the amino acid sequence of which is shown in SEQ ID NO: 1; The amino acid sequence of the dnaQ* is shown in SEQ ID NO: 2; The screening plasmid has an expression cassette with a nucleotide sequence as shown in SEQ ID NO.5; The steps for screening ALA high-producing strains from the mutant library are as follows: (1) The strains in the mutant library were inoculated into LB medium for cultivation. During the cultivation process, dehydrated tetracycline was added to induce the expression of dnaQ*. The bacteria were collected and strains with lower green fluorescence intensity were sorted by flow cytometry. The sorted strains were inoculated into resistance medium for cultivation and the above screening process was repeated until a strain with significantly reduced fluorescence level was obtained; (2) The strains screened in step (1) were screened again based on their ALA fermentation capacity, and strains with higher ALA production were selected for shake flask fermentation verification.

2. An engineered bacterium that produces high yield of 5-aminolevulinic acid, characterized in that: The strain was named Escherichia coli ( Escherichia coli )DM16, the engineered bacteria has been deposited in the China Center for Type Culture Collection, referred to as CCTCC, on April 26, 2023, at Wuhan University, Wuhan, China, and its biological deposit number is: CCTCC NO:M2023631.

3. A bacterial agent, characterized in that: The bacterial agent includes the engineered bacteria according to claim 2.

4. The bacterial agent according to claim 3, characterized in that The bacterial agent is a solid or liquid preparation, and also includes a pharmaceutically necessary carrier. The solid preparation is selected from bacterial powder and granules; the liquid preparation is selected from water suspension and dispersible oil suspension; the pharmaceutically acceptable carrier is selected from one or more of a dispersant, a wetting agent, a disintegrant, a binder, a defoaming agent, an antifreeze agent, a thickener, a filler and a solvent.

5. A method for biosynthesis of 5-aminolevulinic acid, characterized in that: Overexpressing ALA synthase in the Escherichia coli DM16 as claimed in claim 2 to prepare a recombinant bacterium, and then supplementing glycine during the fermentation of the recombinant bacterium to synthesize 5-aminolevulinic acid; The amino acid sequence of the ALA synthase is shown in SEQ ID NO: 1; The specific steps are as follows: By plasmid transfection into the Escherichia coli as claimed in claim 2 ( Escherichia coli ) ALA synthase with an amino acid sequence as shown in SEQ ID NO: 1 was introduced into DM16, the modified strain was transferred into LB medium for seed culture, and then transferred into fermentation medium for fermentation culture; the fermentation medium comprises the following components: 18-22 g / L glucose, 1-3 g / L yeast extract, 8-12 g / L succinic acid, 3-5 g / L glycine, 16-17 g / L (NH4)2SO4, 1-4 g / L KH2PO4, 15-18 g / L Na2HPO4•12H2O, 0.8-1.2 g / L MgSO4•7H2O and 0.01 g / L MnSO4•7H2O, and 3-5 g / L glycine is supplemented every 12 hours.

6. The method for biosynthesis of 5-aminolevulinic acid according to claim 5, characterized in that: The fermentation process was carried out by shaking flask fermentation, the fermentation temperature was 35-38℃, and the rotation speed was 200-250rpm. 600 When the concentration of the cells was ~0.6, 0.1 mM IPTG was added for induction.

Images