Strain with high yield of 5-amino levulinic acid, screening method and application thereof

Abstract

The present application belongs to the technical field of synthetic biology and microbial metabolic engineering, and particularly relates to a high-yield 5-amino levulinic acid (ALA) strain, a screening method and application thereof. The present application discloses that 5-amino levulinic acid (ALA) causes higher levels of oxidative stress in a dose-dependent manner, and the oxidative stress caused by ALA reduces the intracellular cAMP level of Escherichia coli. Based on this, the present application constructs cAMP response promoters with different kinetic ranges, and establishes a high-throughput screening system of ALA high-yield strains on the basis. The present application uses the system to screen enzyme high-yield mutants in two ALA production pathways (C4 pathway and C5 pathway) through fluorescence-activated cell sorting (FACS), and successfully screens excellent mutant strains of ALA production pathway enzymes, and therefore has good practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of synthetic biology and microbial metabolic engineering, and particularly relates to a 5-amino levulinic acid (ALA) high-yield strain, a screening method thereof and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art already known to a person of ordinary skill in the art.

[0003] 5-amino levulinic acid (ALA) is a non-protein amino acid, which is a necessary precursor for the synthesis of tetrapyrrole compounds (including hemoglobin, 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 a wide range of applications in agriculture, animal husbandry and medicine. Today, microbial fermentation for the production of ALA is an environmentally friendly, simple, inexpensive and sustainable method, which 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 C4 (Shemin) pathway and C5 pathway. In the C4 pathway, ALA is produced by one-step condensation of succinyl coenzyme A and glycine by ALA synthase. In the C5 pathway, glutamate undergoes three sequential enzymatic reactions of glutamyl tRNA synthetase, glutamyl tRNA reductase and glutamate-1-semialdehyde aminotransferase to produce ALA, which is more complex than the C4 pathway. So far, enzyme screening, pathway engineering, fermentation process optimization and tolerance engineering have been studied, and the microbial production of ALA has been significantly improved. In the aspect of protein engineering, Kang et al. significantly improved the ALA production of E. coli C5 pathway by modifying the N-terminal amino acid sequence of hemA of Salmonella enterica. Tan et al. reduced the hemoglobin inhibition of ALA synthase of Rhodopseudomonas palustris by computer-aided protein rational design, and the yield of ALA in microbial fermentation was higher than that of wild type. This shows that protein engineering of ALA production pathway enzymes is an effective strategy to improve the microbial production of ALA. However, the inventors found that there are still few reports on high-throughput screening methods for ALA production pathway enzymes. Therefore, the lack of a method for high-throughput screening of excellent mutants of ALA production pathway enzymes is a limiting factor for high ALA production in microorganisms. SUMMARY

[0005] In view of the problems in the prior art, the present application provides a 5-amino levulinic acid (5-ALA) high-yield strain and a screening method and application thereof. The present application successfully constructs cAMP response promoters with different kinetic ranges, and on this basis, establishes a high-throughput screening system for ALA high-yield strains. Fluorescence-activated cell sorting (FACS) is used to screen enzyme high-yield mutants in two ALA production pathways (C4 pathway and C5 pathway), and excellent mutant strains of ALA production pathway enzymes are screened out. Based on the above research results, the present application is completed.

[0006] Specifically, the present application relates to the following technical solutions:

[0007] In a first aspect of the present application, a high-throughput screening system for 5-amino levulinic acid (5-ALA) high-yield strains is provided, which at least comprises cAMP, cAMP receptor protein (CRP), and a promoter and a reporter gene regulated by CRP in response to 5-amino levulinic acid, wherein the reporter gene is located downstream of the promoter and is regulated by the promoter.

[0008] The promoter includes but is not limited to P cspE , P epd , P glpD , P gntK , P cspP , P cdd , P mglB , P gapA , P galS , and P galP . It has been proved by experiments that the above-mentioned promoters have good dose-response relationship with cAMP and ALA, etc.

[0009] In a second aspect of the present application, a recombinant expression vector is provided, which carries the above-mentioned high-throughput screening system.

[0010] In a third aspect of the present application, a cell is provided, which comprises the above-mentioned recombinant expression vector or carries the above-mentioned high-throughput screening system.

[0011] The cell can be a prokaryotic cell or a eukaryotic cell.

[0012] In a fourth aspect of the present application, the above-mentioned high-throughput screening system, recombinant expression vector or cell is applied to high-throughput screening of a strain for metabolically producing 5-amino levulinic acid or its upstream and downstream products.

[0013] In a fifth aspect of the present application, a method for high-throughput screening of a 5-amino levulinic acid high-yield strain is provided, which comprises using fluorescence-activated cell sorting (FACS) to screen a 5-amino levulinic acid high-yield strain based on the above-mentioned high-throughput screening system, recombinant expression vector or cell.

[0014] The high 5-aminolevulinic acid-producing strain can be Escherichia coli and a derivative thereof, and has 5-aminolevulinic acid C4 production pathway-related enzymes or 5-aminolevulinic acid C5 production pathway-related enzymes.

[0015] When the strain has 5-aminolevulinic acid C4 production pathway-related enzymes, the strain has a glutamyl tRNA reductase (hemA) mutant, and the hemA mutant is obtained by mutating a wild-type hemA at one or more sites in the following group: V69A, P91T, V391A, R97H, T317I, V173M, V228I, G152R, A290G, V266I, R292H, A223T, V322L, D379G, and the wild-type hemA is derived from Rhodopseudomonas palustris KUGB306.

[0016] When the strain has 5-aminolevulinic acid C5 production pathway-related enzymes, the strain has a hemA mutant and / or a hemL mutant.

[0017] The hemA mutant is obtained by mutating a wild-type hemA at one or more sites in the following group: E265V, L112M, D381G, F127Y, A164T, I233M, Q371R, R272H, K125Q, D226V, V302I, and the wild-type hemA is derived from Salmonella typhimurium;

[0018] The hemL mutant is obtained by mutating a wild-type hemL at one or more sites in the following group: V203A, V206L, M62I, N67H, A338S, V47I, A157T, T241S, M385L, A304R, E389K, K425G, A70P, P395S, A406G, and the wild-type hemL is derived from Escherichia coli MG1655.

[0019] The beneficial technical effects of one or more of the above technical solutions are:

[0020] The above technical solution is found through research that ALA causes higher level of oxidative stress in a dose-dependent manner, and the oxidative stress caused by ALA reduces the intracellular cAMP level of E. coli. Based on this, the above technical solution constructs cAMP response promoters with different kinetic ranges, and establishes a high-throughput screening system for ALA high-yield strains on this basis. The system is used to screen enzyme high-yield mutants in two ALA production pathways (C4 pathway and C5 pathway) by fluorescence-activated cell sorting (FACS), and excellent mutant strains of ALA production pathway enzymes are successfully screened, so the system has considerable application value and prospect. BRIEF DESCRIPTION OF DRAWINGS

[0021] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The embodiments of these drawings are set to explain the application, and do not constitute an improper limitation on the application.

[0022] Figure 1 A is the relationship between ALA and H2O2 concentration, B is the relationship between ALA and intracellular oxidative stress level of E. coli, and C is the relationship between ALA and intracellular cAMP level of E. coli.

[0023] Figure 2 The expression level of GFP regulated by the cAMP response promoter under different intracellular cAMP levels in the embodiment of the application.

[0024] Figure 3 The expression level of GFP of the cAMP level response artificial promoter and its original promoter in the embodiment of the application.

[0025] Figure 4 The dose response curves of the two cAMP level response artificial promoters and their original promoters to cAMP and ALA in the embodiment of the application.

[0026] Figure 5 The ALA production of C4 and C5 pathway enzyme mutants and wild type in the embodiment of the application. DETAILED DESCRIPTION

[0027] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0028] It is to be understood that the terms used herein are for the purpose of describing specific embodiments and are not intended to limit exemplary embodiments according to the present application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. If not mentioned otherwise, the experimental methods in the following detailed description are generally carried out according to conventional methods and conditions in the art of molecular biology, which are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual, or according to the conditions suggested by the manufacturer.

[0029] In one exemplary embodiment of the present application, a high-throughput screening system for 5-aminolevulinic acid high-yield bacteria is provided, which comprises at least cAMP, cAMP receptor protein (CRP), and a CRP-regulated promoter and a reporter gene responsive to 5-aminolevulinic acid, wherein the reporter gene is located downstream of the promoter and is regulated by the promoter.

[0030] Generally, a promoter is used to regulate the expression of a downstream gene. Generally, a constitutive expression promoter and a regulatory promoter are used. In the present application, a CRP-regulated regulatory promoter is selected. When cAMP binds to CRP protein, the conformation of CRP protein changes, and then it binds to the upstream of the promoter with a CRP binding motif to regulate the strength of the promoter. Changes in the concentration of intracellular cAMP will cause corresponding changes in the strength of the CRP-regulated promoter. By adding ALA to affect the intracellular cAMP level, the expression strength of the CRP-regulated promoter is affected. By using the CRP-regulated promoter to express the reporter gene, the change level of the strength of the promoter can be converted into the change level of the reporter gene. Therefore, the change level of the CRP-regulated promoter can be detected by detecting the change level of the reporter gene, and then the high and low levels of ALA content can be indirectly reflected.

[0031] The promoter used is a CRP-regulated promoter (inhibitory and activating) containing a CRP binding sequence. Several CRP-regulated promoters are selected for verification, which shows that these promoters have a good dose-response relationship with cAMP and ALA, etc.

[0032] In one embodiment of the present application, the promoter includes but is not limited to P cspE , P epd , P glpD , P gntK , P cspP , P cdd , PmglB , P gapA , P galS and P galP ; and it has been proved by experiments that the above promoters have good dose-response relationship with cAMP and ALA, etc.

[0033] In one specific embodiment of the present application, the reporter gene can be at least any one of fluorescent protein coding genes, and more specifically, the fluorescent protein can be GFP.

[0034] In one specific embodiment of the present application, since P mglB and P galP have greater dynamic range and higher fluorescence intensity after induction by cAMP and ALA, the promoter is preferably P mglB and P galP , to further improve the dynamic range of the promoter, the two promoters are modified by changing the CRP binding site and the tandem CRP binding site. The original CRP binding site of the two promoters is replaced by a strong binding site (5'-AAATGTGATCTAGATCATATTT-3', SEQ ID NO. 1) to obtain P mglB-ST and P galP-ST . CRP strong binding sites are also added at three positions -61.5, -72.5 and -83.5 upstream of the two promoters to obtain P galP-61.5 , P galP-72.5 , P galP-83.5 , P mglB-61.5 , P mglB-72.5 and P mglB-83.5 . The results show that the promoters PgalP-83.5 and PmglB-83.5 with CRP binding sites added at the -83.5 position show the highest activation fold. Compared with P mglB (49.76x), the dynamic range of P mglB-83.5 (62.64x) is significantly improved. Based on the above results, the present application considers that the addition of CRP strong binding sites at the -83.5 position can significantly improve the dynamic range.

[0035] In another specific embodiment of the present application, a recombinant expression vector is provided, which carries the above high-throughput screening system.

[0036] Specifically, the recombinant expression vector is obtained by effectively connecting the polynucleotide encoding the high-throughput screening system to an expression vector, which is any one or more of a viral vector, a plasmid, a bacteriophage, a cosmid, or an artificial chromosome; the viral vector can include an adenovirus vector, a retrovirus vector, or an adeno-associated virus vector, and the artificial chromosome includes a bacterial artificial chromosome (BAC), a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).

[0037] More specifically, the recombinant expression vector can be a plasmid carrying the high-throughput screening system. In a specific embodiment of the present application, the plasmid can be pCDFDuet-1.

[0038] In another specific embodiment of the present application, a cell comprising the recombinant expression vector or carrying the high-throughput screening system is provided.

[0039] The cell can be a prokaryotic cell or a eukaryotic cell, and in a specific embodiment of the present application, the prokaryotic cell can be a 5-amino levulinic acid-producing bacterium, which can be Escherichia coli and its derivative bacteria.

[0040] In another specific embodiment of the present application, the high-throughput screening system, the recombinant expression vector, or the cell is used in high-throughput screening of a strain producing 5-amino levulinic acid or its upstream or downstream product.

[0041] In another specific embodiment of the present application, a method for high-throughput screening of a 5-amino levulinic acid-producing strain is provided, which comprises using fluorescence-activated cell sorting (FACS) to screen a 5-amino levulinic acid-producing strain based on the high-throughput screening system, the recombinant expression vector, or the cell.

[0042] More specifically, the fluorescence-activated cell sorting method comprises using a flow cytometer to sort the strain to be screened according to the GFP fluorescence intensity.

[0043] The strain to be screened can be a strain with a 5-amino levulinic acid C4 production pathway (related enzyme) or a C5 production pathway (related enzyme) prepared by error-prone PCR.

[0044] More specifically, the 5-amino levulinic acid-producing strain can be Escherichia coli and its derivative bacteria.

[0045] In another specific embodiment of the present application, a 5-amino levulinic acid-producing strain is provided, which can be Escherichia coli and its derivative bacteria, and has a 5-amino levulinic acid C4 production pathway-related enzyme or a 5-amino levulinic acid C5 production pathway-related enzyme.

[0046] When the strain has 5-amino levulinic acid C4 production pathway related enzymes, it has a glutamyl tRNA reductase (hemA) mutant, which is obtained by mutating wild-type hemA at one or more sites in the following group: V69A, P91T, V391A, R97H, T317I, V173M, V228I, G152R, A290G, V266I, R292H, A223T, V322L, D379G, the wild-type hemA being derived from Rhodopseudomonas palustris KUGB306, the amino acid sequence of which is shown as SEQ ID NO. 2.

[0047] When the strain has 5-amino levulinic acid C5 production pathway related enzymes, it has a hemA mutant and / or a hemL mutant;

[0048] The hemA mutant is obtained by mutating wild-type hemA at one or more sites in the following group: E265V, L112M, D381G, F127Y, A164T, I233M, Q371R, R272H, K125Q, D226V, V302I, the wild-type hemA being derived from Salmonella typhimurium, the amino acid sequence of which is shown as SEQ ID NO. 3;

[0049] The hemL mutant is obtained by mutating wild-type hemL at one or more sites in the following group: V203A, V206L, M62I, N67H, A338S, V47I, A157T, T241S, M385L, A304R, E389K, K425G, A70P, P395S, A406G, the wild-type hemL being derived from Escherichia coli MG1655, the amino acid sequence of which is shown as SEQ ID NO. 4.

[0050] The present application is further explained by the following examples, which do not limit the present application. It should be understood that these examples are merely for the purpose of illustration and are not to be taken as limiting the scope of the present application. The plasmids and primers used in the examples are shown in Table 1 and Table 2 below.

[0051] Table 1 Plasmids

[0052]

[0053] Table 2 Primers

[0054]

[0055]

[0056]

[0057] Embodiments

[0058] 1. Experimental methods

[0059] 1.1 Strains, media and culture conditions

[0060] E. coli DH5a cells were used for plasmid construction, GFP characterization and 5-ALA production. LB medium containing 10 g / L of peptone, 5 g / L of yeast extract and 5 g / L of NaCl was used for cell cultivation and characterization. Fermentation medium containing 20 g / L of glucose, 2 g / L of yeast extract, 16 g / L of (NH4)2SO4, 3 g / L of KH2PO4, 16 g / L of Na2HPO4-12H2O, 1 g / L of MgSO4-7H2O and 0.01 g / L of MnSO4-7H2O was used for 5-ALA C5 pathway fermentation, supplemented with 10 g / L of succinate and 4 g / L of glycine for C4 pathway fermentation, and 4 g / L of glycine was supplemented every 12 h for C4 fermentation. Cultivation and fermentation were performed at 37 °C, 220 rpm. Appropriate concentrations of antibiotics were added to the medium when needed, including streptomycin (100 pg / mL), ampicillin (100 pg / mL), chloramphenicol (34 pg / mL) and kanamycin (50 pg / mL). Expression of genes was induced by adding 0.1 mM of isopropyl-β-d-thiogalactopyranoside (IPTG).

[0061] 1.2 Plasmid construction

[0062] The target fragments were amplified from E. coli MG1655 genomic DNA by using Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China) and the corresponding primers. After purification and recovery of the target fragments, the corresponding plasmid construction was performed using ClonExpress II One Step Cloning Kit (Vazyme Biotech, Nanjing, China), and the restriction enzymes were purchased from Thermo Fisher Scientific (Waltham, MA). Primer synthesis and Sanger sequencing were performed by Tsingke (Beijing, China).

[0063] 1.3 Measurement of intracellular ROS levels

[0064] To determine the ROS level in ALA-treated cells, a previously described 2',7'-dichlorodihydrofluorescein-acetate (DCFH-DA)-based assay was used. Non-fluorescent DCFH-DA can penetrate into cells, once inside the cells, it is hydrolyzed by cellular esterases to generate 2',7'-dichlorodihydrofluorescein (DCFH), which is then rapidly oxidized by ROS to generate the strongly fluorescent product 2',7'-dichlorofluorescein (DCF), which can be detected by fluorescence spectroscopy (Ex / Em = 504 / 529 nm). E. coli cells were incubated in 10 mM DCFH-DA and incubated at 37 °C in the dark for 30 min.

[0065] 1.4 H202assay

[0066] H202level was determined using H202quantification assay kit (water compatible) purchased from Sangon Biotech (Shanghai, China). This kit utilizes dye molecules that have an absorption peak at 595 nm after reaction with Fe3+, Fe 3+ is generated by the oxidation of H202. 2+

[0067] 1.5 cAMP assay

[0068] Strains were inoculated in LB medium for culture and the culture was harvested at exponential phase, the OD 600 of the samples measured were adjusted to the same value. The cAMP enzyme-linked immunosorbent assay (ELISA) kit (NewEast Biosciences, Malvern, PA, USA) was used to measure the cAMP concentration in bacterial lysates according to the manufacturer's instructions.

[0069] 1.6 Mutant library construction and screening

[0070] We used error-prone PCR to construct the mutant library, the GeneMorph II kit (Agilent Technologies, USA) was used to perform PCR amplification of the target gene to construct a random mutation library of the target gene. To improve the mutation frequency of the library, we used the product of the first round of error-prone PCR as a template for the second round of error-prone PCR amplification, a total of three rounds of error-prone PCR amplification were performed. Subsequently, the product of error-prone PCR was subjected to plasmid construction and transformed into DH5a strain with a screening plasmid for overnight culture, the next day the overnight cultured strain was inoculated into another bottle of LB liquid medium at an inoculation ratio of 2%, and the OD​600 When the OD600 reaches 0.4-0.6, IPTG is added to induce the expression of the gene. Then a certain amount of bacteria solution is taken and diluted to an appropriate concentration for subsequent flow cytometry sorting. During the sorting process, the sorting area is selected according to the fluorescence intensity of the bacteria, and a sorting gate is circled to make the proportion of low green fluorescence bacteria in the gate about 3%. The sorted bacteria solution is inoculated into LB medium containing the corresponding resistance for culture for the next sorting. After a total of 2-3 times of sorting, the final library is streaked on agar plates for single colony fermentation verification.

[0071] 1.7 Analysis method of ALA

[0072] The fermentation supernatant is transferred to a new centrifuge tube. Dilute it according to a certain proportion. Take 400 μL of the diluent, add 200 μL of sodium acetate buffer and 100 μL of acetylacetone, respectively, and boil for 15 min. Cool to room temperature, add Modified Ehrlich’s reagent and react for 20 min, then use a 1 cM cuvette to detect the OD value at a wavelength of 554 nm using a spectrophotometer. According to the standard curve of ALA / OD 554 , the concentration of ALA is calculated.

[0073] 2. Test results

[0074] ALA, as an amino ketone compound, has high reactivity and instability. This property of ALA enables it to produce reactive oxygen species (ROS), which are produced in three main ways. First, ALA undergoes enolization and aerobic oxidation to produce ROS; second, ALA spontaneously dimerizes and is irreversibly decomposed into 2,5-(β-carboxyethyl)pyrazine, while generating ROS; third, the downstream product of ALA, protoporphyrin IX, generates ROS after irradiation. A large amount of ROS triggers oxidative stress and affects the physiological state of cells. It has been reported that oxidative stress can lead to a decrease in intracellular ATP levels, which serves as a precursor for cAMP synthesis, thereby affecting the change in intracellular cAMP levels and affecting the binding of cAMP receptor protein (CRP) to its regulatory genes, making cells have different physiological characteristics. At the same time, low levels of intracellular cAMP can make E. coli have stronger resistance to stress. Based on this, we speculate that when ALA induces oxidative stress in E. coli, it may reduce the level of intracellular cAMP in E. coli, which will make E. coli resistant to oxidative stress induced by ALA.

[0075] To prove our hypothesis, we measured the level of ROS and intracellular cAMP in E. coli under different concentrations of ALA. Since the ROS caused by ALA is mainly hydrogen peroxide and superoxide anion radical, under physiological conditions, superoxide anion radical will spontaneously convert to hydrogen peroxide. We used the level of hydrogen peroxide to represent the ROS caused by ALA. First, we added different concentrations of ALA to the liquid medium to determine the level of ROS caused by the addition of ALA. Since the addition of ALA will change the pH value of the medium, we adjusted the pH value of the medium to 6.5-7.0 after adding ALA. By measuring the level of H2O2 in the medium, we found that the level of H2O2 increased with the addition of ALA Figure 1 A). Next, we detected the intracellular ROS level of E. coli under different concentrations of ALA. The results showed that the level of intracellular ROS in E. coli also increased with the addition of ALA Figure 1 B). Based on the above results, we proved that the level of ROS caused by ALA was positively correlated with the amount of ALA added. Then, we measured the intracellular cAMP level of E. coli under different concentrations of ALA. As expected, the intracellular cAMP level in E. coli decreased with the increase of the amount of ALA added. The amount of ALA added was negatively correlated with the level of intracellular cAMP in E. coli Figure 1 C). These results showed that the ROS caused by ALA could reduce the intracellular cAMP level in E. coli and was related to the concentration of ALA.

[0076] We have confirmed that the ROS caused by ALA can reduce the intracellular cAMP level in E. coli in a dose-dependent manner, so we intend to further characterize the relationship between ALA and intracellular cAMP by using a CRP-specific regulated promoter. Schulte previously used an endogenous CRP-regulated promoter to detect the intracellular cAMP level of C. glutamicum. This also proved that the change of intracellular cAMP level can be well reflected in this way.

[0077] We selected 10 promoters specifically regulated by CRP (P cspE , P epd , P glpD , P gntK , P cspP , P cdd , P mglB , P gapA , P galS , P galP). Each of these 10 promoters was used to express GFP, and the GFP expressed by these promoters was constructed into pCDFDuet-1 plasmid vector to characterize these promoters. Since glucose can significantly reduce the level of cAMP in E. coli cells, we established two conditions, with and without glucose, to measure the range of fluorescence intensity. The sensitivity of each promoter to cAMP was determined by the above characterization of fluorescence intensity. The measured fluorescence intensity showed that 6 of the 10 promoters had a significant change in fluorescence intensity. Among them, the two promoters with the largest dynamic range were P mglB (49.76x) and P galP (45.26x). Although the fluorescence intensity of these two promoters was not as high as that of P cspE without glucose, in the presence of glucose, the background leakage of these two promoters was smaller, that is, the response to ALA was more stringent Figure 2 . Therefore, we optimized two promoters with large dynamic range, P mglB and P galP , to further improve the dynamic range of these two promoters.

[0078] We intended to modify these two promoters by changing the CRP binding site and the tandem CRP binding site. We first replaced the original CRP binding site of these two promoters with a strong binding site (5'-AAATGTGATCTAGATCATATTT-3', SEQ ID NO. 1) to obtain P mglB-ST and P galP-ST , respectively. We also added a CRP strong binding site at three positions upstream of these two promoters, -61.5, -72.5 and -83.5, to obtain P galP-61.5 , P galP-72.5 , P galP-83.5 , P mglB-61.5 , P mglB-72.5 and P mglB-83.5 . The results showed that the addition of a CRP binding site at position -83.5, P galP-83.5 and P mglB-83.5 showed the highest fluorescence intensity. Compared with P PmglB (49.76x), the dynamic range of P mglB-83.5 (62.64x) was significantly improved Figure 3 . Based on the above results, the present application believes that the addition of a CRP strong binding site at position -83.5 can significantly improve its dynamic range. By artificially modifying the screened promoters, the dynamic range of the promoters was significantly improved. We next characterized the response relationship of these two artificial promoters to cAMP and ALA. The results showed that when 10 mM cAMP was added exogenously, these promoters all had the highest response intensityFigure 4 ). Meanwhile, the artificial promoters also have a good response relationship with ALA, and reach the maximum response concentration when ALA is 40 g / L. And the dynamic range of the artificial promoters is significantly better than that of the wild-type promoter. Among them, P mglB-83.5 has the best cAMP dose-response and ALA dose-response. Therefore, we selected P mglB-83.5 for the subsequent construction of the screening system.

[0079] PmglB-83.5 has been proved to have a good dose-response relationship with ALA. We used E. coli DH5a with PmglB-83.5 plasmid as the host strain for fluorescence-activated cell sorting (FACS) high-throughput screening of ALA-producing strains to obtain high-yield mutants of ALA production pathway enzymes. We used the hemA gene from R. palustris KUGB306 for ALA production in the C4 pathway; and the hemA gene from S. typhimurium and the hemL gene from E. coli MG1655 for ALA production in the C5 pathway. We amplified the two production pathway genes by error-prone PCR, which produced random mutations in the two pathway enzyme genes during amplification, in order to prepare random mutation libraries of the two pathway enzyme genes. The prepared mutant libraries of ALA production pathway enzymes (C4 and C5) were used for subsequent fluorescence level-based flow screening, and the number of each production pathway mutant library was about ~3 x 10 6We used flow cytometry to sort these library strains according to GFP fluorescence intensity. We verified the ALA production of the sorted single clone strains, from which we obtained high-yield mutants of C4 and C5 pathway enzymes. We used E. coli DH5a as the production strain, which produced almost undetectable amounts of ALA without C4 and C5 pathway enzymes, and produced 2.65 ± 0.09 g / L and 1.38 ± 0.09 g / L of ALA when overexpressing the C4 and C5 pathway enzymes, respectively. When overexpressing the mutant pathway enzymes we selected based on FACS screening, the ALA production was as follows: C4 pathway: Strain No. 1 (V69A, P91T, V391A) produced 4.27 ± 0.25 g / L, which was 61.15% higher than the wild type; Strain No. 2 (R97H, T317I) produced 4.18 ± 0.20 g / L, which was 57.94% higher than the wild type; Strain No. 3 (V173M, V228I) produced 3.68 ± 0.13 g / L, which was 38.98% higher than the wild type; Strain No. 4 (G152R, A290G) produced 3.50 ± 0.10 g / L, which was 25.68% higher than the wild type; Strain No. 5 (V266I, R292H) produced 3.33 ± 0.11 g / L, which was 32.26% higher than the wild type; Strain No. 6 (A223T, V322L, D379G) produced 3.25 ± 0.15 g / L, which was 22.78% higher than the wild type. C5 pathway: Strain No. 1 (hemA: E265V hemL: V203A, V206L) produced 2.46 ± 0.20 g / L, which was 78.51% higher than the wild type; Strain No. 2 (hemA: L112M, D381G hemL: M62I, N67H, A338S) produced 2.36 ± 0.16 g / L, which was 71.45% higher than the wild type; Strain No. 3 (hemL: V47I, A157T, T241S, M385L) produced 2.20 ± 0.20 g / L, which was 59.69% higher than the wild type; Strain No. 4 (hemA: F127Y, A164T, I233M, Q371R hemL: A304R) produced 2.18 ± 0.19 g / L, which was 58.66% higher than the wild type; Strain No. 5 (hemA: R272H hemL: E389K, K425G) produced 2.07 ± 0.16 g / L, which was 50.57% higher than the wild type; Strain No. 6 (hemA: K125Q, D226V, V302I hemL: A70P, P395S, A406G) produced 2.07 ± 0.11 g / L, which was 50.28% higher than the wild type. Figure 5 ​

[0080] Protein amino acid sequences involved in the above examples

[0081] C4 pathway:

[0082] hemA: Rhodopseudomonas palustris KUGB306 (SEQ ID NO. 2)

[0083] mdytkffada ldrlhaerry rvfadlerva grfphatwhs psgerdvviw csndylgmgq

[0084] hpkvvgamve tatrlgtgag gtrniagthh plvmlerela dlhgkeaall ftsgyvsnqt

[0085] gistlaklip nclilsdaln hnsmiegirq sgcerivwrh ndtahleell ravepgrpvl

[0086] iafeslysmd gdvapmakic dlaekygamt ycdevhavgm ygargagvae rdgvmhridi

[0087] ieatlakafg clggyisgkk dvidavrsya pgfifttalp ppicaaataa irhlktstwe

[0088] rerhqdraar lkavlntagl pvmptdthiv pvfvgdaerc kkasdlllek hgiyiqpiny

[0089] ptvakgkerl ritpspyhdd dlmdrlaeal vdvwetlelp lgakplaae

[0090] C5 pathway:

[0091] hemA: Salmonella typhimurium (SEQ ID NO. 3)

[0092] mtkkllalgi nhktapvslr ervtfspdtl dqaldsllaq pmvqggvvls tcnrtelyls

[0093] veeqdnlqea lirwlcdyhn lneddlrnsl ywhqdndavs hlmrvasgld slvlgepqil

[0094] gqvkkafads qkghlnasal ermfqksfsv akrvrtetdi gasavsvafa actlarqife

[0095] slstvtvllv gagetielva rhlrehkvqk miianrtrer aqaladevga evislsdida

[0096] rlqdadiiis stasplpiig kgmveralks rrnqpmllvd iavprdvepe vgklanayly

[0097] svddlqsiis hnlaqrqaaa veaetiveqe asefmawlra qgasetirey rsqseqirde

[0098] lttkalsalq qggdaqailq dlawkltnrl ihaptkslqq aardgdderlnilrdslglehemL: Escherichia coli MG1655 (SEQ ID NO. 4)

[0099] msksenlysa arelipggvn spvraftgvg gtplfiekad gaylydvdgk ayidyvgswg

[0100] pmvlghnhpa irnavieaae rglsfgapte mevkmaqlvt elvptmdmvr mvnsgteatm

[0101] sairlargft grdkiikfeg cyhghadcll vkagsgaltl gqpnspgvpa dfakytltct

[0102] yndlasvraa feqypqeiac iivepvagnm ncvpplpefl pglralcdef galliidevm

[0103] Tgfr Val AgA QDYYGVVPDL TCLGKIIGGG MPVGAFGGRR DVMDALAPTG PVYQAGTISG

[0104] Npi Ama AgF A CLNEVAQPGV HETLDELTTR LAEGLLEAAE EAGIPLVVNH VGGMFGIFFT

[0105] DaesvtCyQD VMACDVERFK RFFHMMLDEG VYLAPSAFEA GFMSVAHSME DINNTIDAAR

[0106] RvfAkl

[0107] Finally, it should be noted that the above only for the preferred embodiments of the present application, and is not intended to limit the present application, although the foregoing detailed description of the present application has been made with reference to the foregoing embodiments, for those skilled in the art, it still can be modified, or part of the technical features of the equivalent replacement of the technical solutions described in the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A high-yield strain of 5-aminolevulinic acid, characterized in that, It is an *Escherichia coli* species containing enzymes related to the 5-aminolevulinic acid (5-levulinic acid) C4 production pathway, and it possesses an *hemA* mutant, which is obtained by mutations in the wild-type *hemA* at the V69A, P91T, and V391A sites; the wild-type *hemA* originates from *Rhodopseudomonas palustris*. Rhodopseudomonas palustris KUGB306, whose amino acid sequence is shown in SEQ ID NO.

2.

2. The screening method for high-yielding 5-aminolevulinic acid strains according to claim 1, the method comprising screening high-yielding 5-aminolevulinic acid strains using fluorescence-activated cell sorting based on a high-throughput screening system, recombinant expression vector or cells; in, The high-throughput screening system includes at least cAMP responsive to 5-aminolevulinic acid, cAMP receptor protein CRP, and a promoter and reporter gene regulated by CRP, wherein the reporter gene is located downstream of the promoter and is regulated by the promoter; The reporter gene includes a gene encoding a fluorescent protein; the fluorescent protein is GFP. The promoter includes P cspE P epd P glpD P gntK P mglB and P galP ; The recombinant expression vector carries a high-throughput screening system; The cells contain recombinant expression vectors or carry high-throughput screening systems; The cells were Escherichia coli; The specific method for fluorescently activated cell sorting includes using flow cytometry to sort the strains to be screened based on the GFP fluorescence intensity. The strains to be screened are strains with either the C4 or C5 production pathway of 5-aminolevulinic acid, prepared by error-prone PCR.

3. The application of the screening method of claim 2 in high-throughput screening of strains that metabolize and produce 5-aminolevulinic acid.

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