CN117701480A - High-throughput screening system and method for high-yield bacteria of 5-aminolevulinic acid and application of high-throughput screening system and method - Google Patents
High-throughput screening system and method for high-yield bacteria of 5-aminolevulinic acid and application of high-throughput screening system and method Download PDFInfo
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- CN117701480A CN117701480A CN202211730317.1A CN202211730317A CN117701480A CN 117701480 A CN117701480 A CN 117701480A CN 202211730317 A CN202211730317 A CN 202211730317A CN 117701480 A CN117701480 A CN 117701480A
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Abstract
The invention belongs to the technical fields of synthetic biology and microbial metabolism engineering, and particularly relates to a high-throughput screening system and method for high-yield bacteria of 5-aminolevulinic acid and application of the high-throughput screening system and method. The present invention has found through research that 5-aminolevulinic acid (ALA) causes higher levels of oxidative stress in a dose-dependent manner, whereas ALA-induced oxidative stress reduces intracellular cAMP levels in E.coli. Based on the above, the invention constructs cAMP response promoters with different dynamic ranges, and establishes a high-throughput screening system of ALA high-yield strains on the basis. The invention uses the system to screen the enzyme high-yield mutants in two ALA production paths (C4 path and C5 path) by Fluorescence Activated Cell Sorting (FACS), and successfully screens out the excellent mutant strains of ALA production path enzyme, thereby having good practical application value.
Description
Technical Field
The invention belongs to the technical fields of synthetic biology and microbial metabolism engineering, and particularly relates to a high-throughput screening system and method for high-yield bacteria of 5-aminolevulinic acid and application of the high-throughput screening system and method.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
5-aminolevulinic acid (ALA), a non-protein amino acid, is 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. ALA has wide application in agriculture, animal husbandry, and medicine fields due to its safety, environmental compatibility, and biodegradability. Today, the production of ALA by microbial fermentation is an environmentally friendly, simple, inexpensive and sustainable process which avoids the disadvantages of complex reaction steps, high costs and environmental pollution in chemical synthesis.
The biosynthetic pathway of ALA is widely found in plants, animals and microorganisms and is divided into the C4 (Shemin) pathway and the C5 pathway. In the C4 pathway, succinyl-coa and glycine are condensed in one step by ALA synthase to produce ALA. In the C5 pathway, glutamate undergoes three sequential enzymatic reactions of glutamyl tRNA synthetase, glutamyl tRNA reductase and glutamate-1-semialdehyde transaminase, which produce ALA, which is more complex than in the C4 pathway. So far, enzyme screening, pathway engineering, fermentation process optimization and tolerance engineering have been studied, and the microbial yield of ALA has been significantly improved. In terms of protein engineering, kang et al significantly improved ALA production in the E.coli C5 pathway by modifying the N-terminal amino acid sequence of the glutamyl tRNA reductase (hemA) of Salmonella arizona. Tan et al reduced the heme inhibition of rhodopseudomonas palustris ALA synthetase by computer aided protein rational design, and the ALA yield in microbial fermentation was higher than that of wild type. This suggests that protein engineering of ALA production pathway enzymes is an effective strategy for increasing ALA microbial production. However, the inventors found that there are still few reports on high throughput screening methods for ALA production pathway enzymes. Thus, the lack of a method for high throughput screening of good mutants of ALA production pathway enzymes is thus a limiting factor for high ALA production in microorganisms.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a high-throughput screening system and method for high-yield bacteria of 5-aminolevulinic acid and application thereof. The invention successfully constructs cAMP response promoters with different dynamic ranges, establishes a high-throughput screening system of ALA high-yield strains on the basis, screens enzyme high-yield mutants in two ALA production paths (C4 path and C5 path) by Fluorescence Activated Cell Sorting (FACS), and screens out excellent mutant strains of ALA production path enzymes. Based on the above results, the present invention has been completed.
Specifically, the invention relates to the following technical scheme:
in a first aspect of the invention, there is provided a high throughput screening system for high yield 5-aminolevulinic acid comprising at least cAMP responsive to 5-aminolevulinic acid, a CAMP Receptor Protein (CRP), and a CRP regulated promoter and a reporter gene, wherein the reporter gene is located downstream of and under the control of the promoter.
Such promoters include, but are not limited to, P cspE 、P epd 、P glpD 、P gntK 、P cspP 、P cdd 、P mglB 、P gapA 、P galS And P galP The method comprises the steps of carrying out a first treatment on the surface of the Experiments prove that the promoter has good dose-response relationship with cAMP, ALA and the like.
In a second aspect of the invention, there is provided a recombinant expression vector carrying the high throughput screening system described above.
In a third aspect of the invention, there is provided a cell comprising the recombinant expression vector described above or carrying the high throughput screening system described above.
The cells may be prokaryotic or eukaryotic.
In a fourth aspect of the invention there is provided the use of a high throughput screening system, recombinant expression vector or cell as described above for high throughput screening of strains metabolically producing 5-aminolevulinic acid or an upstream or downstream product thereof.
In a fifth aspect of the invention, there is provided a method for high throughput screening of high yield 5-aminolevulinic acid strains, the method comprising screening high yield 5-aminolevulinic acid strains using Fluorescence Activated Cell Sorting (FACS) based on the high throughput screening system, recombinant expression vector or cell described above.
Wherein the high-yield 5-aminolevulinic acid strain can be escherichia coli and derivative bacteria thereof, and has 5-aminolevulinic acid C4 production pathway related enzyme or 5-aminolevulinic acid C5 production pathway related enzyme;
when the strain has a 5-aminolevulinic acid C4 production pathway-related enzyme, it has a mutant of glutamyl tRNA reductase (hemA) obtained by mutating wild-type hemA at one or more of the following positions: v69 5292 91 8238 391A, R97H, T317 5652 228I, G152R, A290G, V I, R292H, A223T, V322L, D379G, said wild hemA being derived from rhodopseudomonas palustris (Rhodopseudomonas palustris KUGB 306).
When the strain has a 5-aminolevulinic acid C5 production pathway-related enzyme, it has a hemA mutant and/or a hemL mutant;
the hemA mutant is obtained from a wild-type hemA mutated at one or more sites in the group: E265V, L112M, D381G, F127Y, A164T, I233M, Q R, R272H, K125Q, D226V, V I, said wild-type hemA being derived from salmonella typhimurium (Salmonella typhimurium);
the hemL mutants were obtained from wild-type hemL mutated at one or more sites in the following group: V203A, V206L, M62I, N67H, A338S, V47I, A157T, T S, M385L, A R, E389K, K425G, A70P, P395S, A G, said wild type hemL being derived from escherichia coli (Escherichia coli MG 1655).
The beneficial technical effects of one or more of the technical schemes are as follows:
the above protocol was studied to find that ALA leads to higher levels of oxidative stress in a dose dependent manner, whereas ALA-induced oxidative stress reduces intracellular cAMP levels in E.coli. Based on the above technical scheme, cAMP response promoters with different dynamic ranges are constructed, and a high-throughput screening system of ALA high-yield strains is established on the basis. The invention uses the system to screen the high-yield mutants of the enzymes in two ALA production paths (C4 path and C5 path) by Fluorescence Activated Cell Sorting (FACS), and successfully screens out the excellent mutant strains of the ALA production path enzymes, thereby having considerable application value and prospect.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a graph showing the relationship between ALA and the level of oxidative stress and the intracellular cAMP of E.coli in the present invention, wherein A is ALA and H 2 O 2 Concentration relationship diagram, B is the relationship diagram of ALA and intracellular oxidative stress level of colibacillus, C is the relationship diagram of ALA and intracellular cAMP level of colibacillus.
FIG. 2 shows the expression levels of GFP under the control of a responsive cAMP promoter at different intracellular cAMP levels in examples of the invention.
FIG. 3 shows the GFP expression levels of the artificial promoter and its original promoter in response to cAMP levels in the examples of the present invention.
FIG. 4 is a graph showing the dose response curves of two artificial promoters responding to cAMP levels and their original promoters to cAMP and ALA in the examples of the present invention.
FIG. 5 shows the production of ALA by the C4 and C5 pathway enzyme mutants and wild type in the examples of the present invention.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof. Experimental methods in the following embodiments, unless specific conditions are noted, are generally in accordance with conventional methods and conditions of molecular biology within the skill of the art, and are fully explained in the literature. See, e.g., sambrook et al, molecular cloning: the techniques and conditions described in the handbook, or as recommended by the manufacturer.
In one exemplary embodiment of the invention, a high throughput screening system for high yield 5-aminolevulinic acid is provided comprising at least cAMP responsive to 5-aminolevulinic acid, CAMP Receptor Protein (CRP), and a promoter and a reporter gene under the control of the CRP, wherein the reporter gene is located downstream of and under the control of the promoter.
The promoters are typically constitutive expression promoters and regulated promoters, and the invention selects a regulated promoter regulated by CRP which, when cAMP binds to CRP protein, causes a conformational change in the CRP protein which in turn binds to a promoter upstream of the promoter with the CRP binding motif to regulate the strength of the promoter. Changes in intracellular cAMP concentration result in corresponding changes in the strength of the CRP regulated promoter. By adding ALA to affect intracellular cAMP level and thus affect expression intensity of a promoter regulated by CRP, by using the promoter regulated by CRP to express a reporter gene, the intensity change level of the promoter can be converted into the level change of the reporter gene, so that the change level of the promoter regulated by CRP can be reflected by detecting the change level of the reporter gene, and thus the level of ALA can be laterally reflected.
The promoters used are promoters (inhibitory and activating) which contain CRP binding sequences and are regulated by CRP, and verification of several promoters regulated by CRP shows that the promoters have better dose-response relation with cAMP, ALA and the like.
In one embodiment of the invention, 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 The method comprises the steps of carrying out a first treatment on the surface of the Experiments prove that the promoter has good dose-response relationship with cAMP, ALA and the like.
In one embodiment of the present invention, the reporter gene may be at least any one of genes encoding a fluorescent protein, and more particularly, the fluorescent protein may be GFP.
In one embodiment of the present invention, due to P mglB And P galP Has a larger dynamic range and higher fluorescence intensity after cAMP and ALA induction, so the promoter is preferably P mglB And P galP To further increase the dynamic range of the promoters, both promoters were engineered by altering the CRP binding site and tandem CRP binding site. The original CRP binding site of these two promoters was replaced with a strong binding site (5'-AAATGTGATCTAGATCATATTT-3', SEQ ID NO. 1) to obtain P respectively mglB-ST And P galP-ST . Addition of CRP strong binding sites at three positions-61.5, -72.5 and-83.5 upstream of these two promoters was also adopted 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 promoters PgalP-83.5 and PmiglB-83.5, with the addition of the CRP binding site at the-83.5 position, showed the highest fold activation. And P mglB (49.76X) compared with P mglB-83.5 The dynamic range of (62.64 ×) improves significantly. Based on the above results, the present invention considers that the addition of a strong CRP binding site at the-83.5 position can significantly improve its dynamic range.
In yet another embodiment of the present invention, a recombinant expression vector carrying the high throughput screening system described above is provided.
Specifically, the recombinant expression vector is obtained by operably linking the polynucleotide molecule encoding the high-throughput screening system described above to an expression vector, which is any one or more of a viral vector, a plasmid, a phage, a cosmid, or an artificial chromosome; viral vectors may include adenovirus vectors, retrovirus vectors, or adeno-associated virus vectors, and artificial chromosomes include Bacterial Artificial Chromosomes (BACs), phage P1-derived vectors (PACs), yeast Artificial Chromosomes (YACs), or Mammalian Artificial Chromosomes (MACs).
More specifically, the recombinant expression vector may be a plasmid carrying the high throughput screening system described above. In one embodiment of the invention, the plasmid may be pCDFDuet-1.
In yet another embodiment of the invention, a cell is provided comprising the recombinant expression vector described above or carrying the high throughput screening system described above.
The cell may be a prokaryotic cell or a eukaryotic cell, and in one embodiment of the invention, the prokaryotic cell may be a 5-aminolevulinic acid-producing bacterium, which may be E.coli or a derivative thereof.
In yet another embodiment of the present invention, there is provided the use of the high throughput screening system, recombinant expression vector or cell described above for high throughput screening of strains metabolically producing 5-aminolevulinic acid or an upstream or downstream product thereof.
In yet another embodiment of the present invention, a method for high throughput screening of high yield 5-aminolevulinic acid strains is provided, the method comprising screening high yield 5-aminolevulinic acid strains using Fluorescence Activated Cell Sorting (FACS) based on the high throughput screening system, recombinant expression vector or cell described above.
More specifically, the specific method for sorting fluorescence activated cells comprises sorting strains to be screened according to GFP fluorescence intensity by using a flow cytometer.
Wherein the strain to be screened may be a strain having a C4 production pathway (related enzyme) or a C5 production pathway (related enzyme) of 5-aminolevulinic acid prepared by error-prone PCR.
More specifically, the high-yield 5-aminolevulinic acid strain may be Escherichia coli and a derivative thereof.
In yet another embodiment of the present invention, a high yield 5-aminolevulinic acid strain is provided, which may be E.coli and derivatives thereof having a 5-aminolevulinic acid C4 production pathway-related enzyme or a 5-aminolevulinic acid C5 production pathway-related enzyme;
when the strain has a 5-aminolevulinic acid C4 production pathway-related enzyme, it has a mutant of glutamyl tRNA reductase (hemA) obtained by mutating wild-type hemA at one or more of the following positions: V69A, P91T, V391A, R97H, T317I, V173M, V228I, G152R, A G, V266I, R292H, A223T, V322L, D379G, the wild type hemA is derived from rhodopseudomonas palustris (Rhodopseudomonas palustris KUGB) and has the amino acid sequence shown in SEQ ID NO. 2.
When the strain has a 5-aminolevulinic acid C5 production pathway-related enzyme, it has a hemA mutant and/or a hemL mutant;
the hemA mutant is obtained from a wild-type hemA mutated at one or more sites in the group: E265V, L112M, D381G, F127Y, A164T, I233M, Q R, R272H, K125Q, D226V, V I, said wild hemA is derived from Salmonella typhimurium (Salmonella typhimurium) and has the amino acid sequence shown in SEQ ID NO. 3;
the hemL mutants were obtained from wild-type hemL mutated at one or more sites in the following group: V203A, V206L, M62I, N67H, A338S, V47I, A157T, T S, M385L, A241R, E549K, K425G, A70P, P S, A406G, said wild type hemL being derived from E.coli (Escherichia coli MG 1655) having the amino acid sequence shown in SEQ ID NO. 4.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Plasmids and primers used in the examples are shown in tables 1 and 2 below.
TABLE 1 plasmid
TABLE 2 primers
Examples
1. Experimental method
1.1 Strain, medium and culture conditions
Coli DH 5. Alpha. Cells were used for plasmid construction, characterization of green fluorescent protein and production of 5-ALA. LB medium containing 10g/L peptone, 5g/L yeast extract and 5g/L NaCl was used for cell culture and characterization. Contains 20g/L glucose, 2g/L yeast extract, 16g/L (NH) 4 ) 2 SO 4 ,3g/LKH 2 PO 4 ，16g/LNa 2 HPO 4 ·12H 2 O,1g/L MgSO 4 ·7H 2 O and 0.01g/LMnSO 4 ·7H 2 The fermentation medium of O was used for the fermentation of the 5-ALAC5 pathway, supplemented with 10g/L succinic acid and 4g/L glycine for the fermentation of the C4 pathway, and 4g/L glycine was supplemented every 12h during the C4 fermentation. Culturing and fermenting at 37deg.C and 220 rpm. Antibiotics were added to the medium at appropriate concentrations, if necessary, including streptomycin (100. Mu.g/mL), ampicillin (100. Mu.g/mL), chloramphenicol (34. Mu.g/mL) and kanamycin (50. Mu.g/mL). 0 was added.1mM isopropyl-beta-d-thiogalactoside (IPTG) induces the expression of genes.
1.2 construction of plasmid
The fragment of interest was amplified from E.coli MG1655 genomic DNA by using Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech, nanjing, china) and the corresponding primers. The desired fragment was purified and recovered, and then the construction of the corresponding plasmid was carried out using ClonExpress II One Step Cloning Kit (Vazyme Biotech, nanjing, china), and restriction enzymes were purchased from Thermo Fisher Scientific (Waltham, mass.). Primer synthesis and Sanger sequencing were performed by Tsingke (Beijin, china).
1.3 measurement of intracellular ROS levels
To determine ALA-treated intracellular ROS levels, the previously described 2',7' -dichlorofluorescein-acetoacetate (DCFH-DA) based assay was used. Non-fluorescent DCFH-DA can permeate into cells, once entering cells, is hydrolyzed by cellular esterases to generate 2',7' -dichlorofluorescein (2 ',7' -dichlorofluorescein, DCFH), and then is rapidly oxidized by ROS to generate strong fluorescent product 2',7' -dichlorofluorescein (2 ',7' -dichlorofluorescein, DCF), which can be detected by fluorescence spectroscopy (Ex/em=504/529 nm). Coli cells were incubated in 10. Mu.M DCFH-DA and incubated in the dark at 37℃for 30 minutes.
1.4H 2 O 2 Measurement
H purchased from Sangon Biotech (Shanghai, china) was used 2 O 2 Quantitative determination kit (Water compatible) for determination of H 2 O 2 Horizontal. The kit utilizes dye molecules with absorption peaks at 595nm after reaction with Fe3+, fe 3+ Is made of Fe 2+ Quilt H 2 O 2 Oxidation.
1.5cAMP analysis
Inoculating the strain into LB culture medium, culturing, and harvesting culture in exponential phase to obtain OD of sample 600 And adjusting to the same value. cAMP concentration in the bacterial lysates was measured using cAMP enzyme-linked immunosorbent assay (ELISA) kit (NewEast Biosciences, malvern, pa., USA) according to the manufacturer's instructions.
1.6 mutant library construction and screening
We constructed a library of mutants using error-prone PCR, and PCR amplified on the gene of interest using GeneMorph II kit (Agilent Technologies, USA) to construct a library of random mutations of the gene of interest. To increase the mutation frequency of the library, we performed a second round of error-prone PCR amplification using the product of the first round of error-prone PCR as a template, for a total of three rounds of error-prone PCR amplification. The error-prone PCR product was then transformed into DH 5. Alpha. Strain with the selected plasmid for overnight culture, the overnight cultured strain was inoculated into another bottle of LB liquid medium at an inoculation ratio of 2% on the next day, and the strain was inoculated at OD 600 When the expression reaches 0.4 to 0.6, IPTG is added to induce the expression of genes. And then a certain amount of bacterial liquid is sucked and diluted to a proper concentration for subsequent flow cytometry sorting, a sorting area is selected according to the fluorescence intensity of bacteria in the sorting process, a sorting gate is defined to ensure that the bacterial strain ratio of low green fluorescence falling in the gate is about 3%, the sorted bacterial liquid is inoculated into LB culture medium with corresponding resistance for culture for the next continuous sorting, and after the total sorting is carried out for 2-3 times, the final library is streaked on an agar plate to pick single bacterial colony for fermentation verification.
1.7 analysis method of ALA
The broth supernatant was transferred to a new centrifuge tube. Diluting according to a certain proportion. 400. Mu.L of the diluted solution was taken, 200. Mu.L of sodium acetate buffer and 100. Mu.L of acetylacetone were added respectively, and the mixture was boiled for 15min. Cooled to room temperature, added with Modified Ehrlich's reagent for reaction for 20min, then detected by a spectrophotometer at a wavelength of 554nm using a 1cM cuvette, and measured according to ALA/OD 554 The concentration of ALA was calculated from the standard curve of (C).
2. Test results
ALA has high reactivity and instability as an amino ketone compound. This property of ALA enables it to generate Reactive Oxygen Species (ROS), which are produced in three main ways. First, ALA undergoes enolization and aerobic oxidation to produce ROS; second, the spontaneous dimerization of ALA is irreversibly broken down into 2,5- (β -carboxyethyl) pyrazine, with the production of ROS; third, the downstream product of ALA protoporphyrin IX, upon irradiation, produces ROS. The large amount of ROS induces oxidative stress and affects the physiological state of cells. Oxidative stress is reported to result in a decrease in intracellular ATP levels, which act as precursors for cAMP synthesis and thus affect changes in intracellular cAMP levels and affect CAMP Receptor Protein (CRP) binding to its regulatory genes, giving the cells different physiological properties. At the same time, low levels of intracellular cAMP can make E.coli more resistant 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 would render E.coli resistant to the oxidative stress induced by ALA.
To demonstrate our hypothesis, we measured the levels of ROS produced by ALA and intracellular cAMP levels in escherichia coli at different concentrations of ALA. Since ALA-induced ROS are mainly hydrogen peroxide and superoxide anion radicals, under physiological conditions superoxide anion radicals spontaneously convert to hydrogen peroxide. We use the level of hydrogen peroxide to represent ALA-induced ROS. First, different concentrations of ALA were added to the liquid medium to determine ROS levels resulting from ALA addition. Since the addition of ALA will change the pH of the medium, we adjusted the pH of the medium to 6.5-7.0 after the addition of ALA. By measuring H in the medium 2 O 2 Level, we found H 2 O 2 The level increased with the addition of ALA (fig. 1A). Next, intracellular ROS levels of e.coli were detected at different ALA concentrations. The results indicate that with ALA addition, intracellular ROS levels in E.coli also increased (FIG. 1B). Based on the above results, we demonstrate that ALA-induced ROS levels are positively correlated with the amount of ALA added. Then, we measured intracellular cAMP levels in e.coli for different concentrations of ALA. As expected, with increasing ALA addition, intracellular cAMP levels in E.coli decreased. ALA addition correlated inversely with intracellular cAMP levels in E.coli (FIG. 1C). These results indicate that ALA-induced ROS can reduce intracellular cAMP levels in e.coli and correlate with ALA concentration.
We have demonstrated that ALA-induced ROS can reduce intracellular cAMP levels in e.coli in a dose-dependent manner, and we therefore intend to further characterize the relationship of ALA to intracellular cAMP by using CRP-specific regulated promoters. Schulte previously used endogenous CRP regulatory promoters to detect intracellular cAMP levels in corynebacterium glutamicum. This also demonstrates that changes in intracellular cAMP levels can be well reflected in this way.
We selected 10 promoters (P) exclusively under CRP based on information on CRP-regulated promoters on the EcoCyc website (https:// ecc. Org) 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 GFP expressed from these promoters was constructed into pCDFDuet-1 plasmid vectors to characterize these promoters. Since glucose can significantly reduce the level of cAMP in E.coli cells, we established two conditions for measuring the range of variation in fluorescence intensity, both in the presence and absence of glucose. The sensitivity of each promoter to cAMP was determined by characterization of the fluorescence intensity as described above. The measured fluorescence intensities showed that 6 out of 10 promoters had significant fluorescence intensity changes. Wherein the two promoters with the largest dynamic range are P mglB (49.76X) and P galP (45.26×). Although the fluorescence intensity of these two promoters was not as good as that of P in the absence of glucose cspE But in the presence of glucose, the background leakage of both promoters was small, that is to say the response to ALA was more stringent (fig. 2). Thus, we optimized two promoters with large dynamic range, P mglB And P galP To further increase the dynamic range of both promoters.
We intend to engineer both promoters by altering the CRP binding site and 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), obtaining P separately mglB-ST And P galP-ST . Also take the steps of starting at the two positionsThree positions-61.5, -72.5 and-83.5 upstream of the child add a strong CRP binding site 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 indicate that CRP binding site, P, is added at the-83.5 position galP-83.5 And P mglB-83.5 Shows the highest fluorescence intensity. And (3) with PmglB (49.76X) compared with P mglB-83.5 The dynamic range of (62.64 ×) was significantly improved (fig. 3). Based on the above results, the present invention considers that the addition of a strong CRP binding site at the-83.5 position can significantly improve its dynamic range. The dynamic range of the screened promoter is obviously improved after the screened promoter is artificially modified. We next characterized the response relationship of cAMP and ALA for these two artificial promoters. The results indicate that these promoters all had the highest response intensity when 10mM cAMP was exogenously added (FIG. 4). Meanwhile, the artificial promoter has a good response relationship to ALA, and the maximum response concentration is reached when the ALA is 40 g/L. And the dynamic range of the artificial promoter is obviously better than that of a wild type promoter. Wherein P is mglB-83.5 With the best cAMP dose-response and ALA dose-response. Therefore we have chosen P mglB-83.5 For subsequent construction of the screening system.
Pmiglbe-83.5 has been shown to have a good dose-response relationship to ALA. We used E.coli DH 5. Alpha. With the PmiglB-83.5 plasmid as host strain for Fluorescence Activated Cell Sorting (FACS) high throughput screening of ALA producing strains to obtain high yielding mutants of ALA producing pathway enzymes. We used the ALA gene from R.palustris KUGB306 hemA for the C4 pathway; ALA production from the C5 pathway was performed using genes from S.tyrphinium hemA and E.coli MG1655 hemL. We amplified the genes of the two production pathways by error-prone PCR, and generated random mutations on the enzyme genes of the two pathways during the amplification process, so as to prepare random mutation libraries of the enzyme genes of the two pathways. The prepared mutant libraries (C4 and C5) of ALA production pathway enzymes were used for subsequent flow screening based on fluorescence levels, the number of mutant libraries per production pathway was approximately-3X 10 6 . We use flow cytometry according to GThe library strains were sorted for FP fluorescence intensity. ALA yield verification is carried out on the separated monoclonal strains, and high-yield mutants of the enzymes of the C4 and C5 production paths are obtained. We used E.coli DH 5. Alpha. As the production strain, which showed almost no detectable ALA production in the absence of the C4 and C5 production pathway enzymes, and which showed ALA production of 2.65.+ -. 0.09g/L and 1.38.+ -. 0.09g/L, respectively, after the production of the enzymes by the pathway in E.coli DH 5. Alpha. Over-expressing C4 and C5. ALA yields when we overexpress the mutant pathway enzymes we screened based on FACS were as follows: the C4 pathway: the yield of the strain 1 (V69A, P91T, V391A) is 4.27+/-0.25 g/L, which is improved by 61.15 percent compared with the wild type; the yield of the strain No.2 (R97H, T317I) is 4.18+/-0.20 g/L, which is 57.94 percent higher than that of the wild type strain; the yield of the strain 3 (V173M, V228I) is 3.68+/-0.13 g/L, which is 38.98 percent higher than that of the wild type strain; the yield of the strain No.4 (G152R, A and 290G) is 3.50+/-0.10G/L, which is improved by 25.68 percent compared with the wild type; the yield of the strain No. 5 (V266I, R292H) is 3.33+/-0.11 g/L, which is improved by 32.26 percent compared with the wild type; the yield of strain 6 (A223T, V322L, D379G) is 3.25+/-0.15G/L, which is improved by 22.78% compared with the wild type. The C5 pathway: the yield of the strain 1 (hemA: E265V hemL: V203A, V206L) is 2.46+/-0.20 g/L, which is 78.51 percent higher than that of the wild type; the yield of the strain No.2 (hemA: L112M, D381G hemL: M62I, N67H, A338S) is 2.36+/-0.16G/L, which is improved by 71.45 percent compared with the wild type; strain 3 (hemL: V47I, A157T, T241S, M385L) had a yield of 2.20+ -0.20 g/L, 59.69% higher than the wild type; the yield of the strain No.4 (hemA: F127Y, A164T, I M, Q371R hemL: A304R) is 2.18+/-0.19 g/L, which is 58.66% higher than that of the wild type strain; the yield of the strain No. 5 (hemA: R272HhemL: E389K, K425G) is 2.07+/-0.16G/L, which is 50.57 percent higher than that of the wild type; strain 6 (hemA: K125Q, D226V, V I hemL: A70P, P395S, A G) had a yield of 2.07.+ -. 0.11G/L, which was improved by 50.28% compared to the wild type (FIG. 5).
The amino acid sequences of proteins referred to in the above examples
The C4 pathway:
hemA:Rhodopseudomonas palustris KUGB306(SEQ ID NO.2)
mdytkffada ldrlhaerry rvfadlerva grfphatwhs psgerdvviw csndylgmgqhpkvvgamve tatrlgtgag gtrniagthh plvmlerela dlhgkeaall ftsgyvsnqtgistlaklip nclilsdaln hnsmiegirq sgcerivwrh ndtahleell ravepgrpvl
iafeslysmd gdvapmakic dlaekygamt ycdevhavgm ygargagvae rdgvmhridiieatlakafg clggyisgkk dvidavrsya pgfifttalp ppicaaataa irhlktstwe
rerhqdraar lkavlntagl pvmptdthiv pvfvgdaerc kkasdlllek hgiyiqpinyptvakgkerl ritpspyhdd dlmdrlaeal vdvwetlelp lgakplaae
the C5 pathway:
hemA:Salmonella typhimurium(SEQ ID NO.3)
mtkkllalgi nhktapvslr ervtfspdtl dqaldsllaq pmvqggvvls tcnrtelyls
veeqdnlqea lirwlcdyhn lneddlrnsl ywhqdndavs hlmrvasgld slvlgepqilgqvkkafads qkghlnasal ermfqksfsv akrvrtetdi gasavsvafa actlarqifeslstvtvllv gagetielva rhlrehkvqk miianrtrer aqaladevga evislsdida
rlqdadiiis stasplpiig kgmveralks rrnqpmllvd iavprdvepe vgklanaylysvddlqsiis hnlaqrqaaa veaetiveqe asefmawlra qgasetirey rsqseqirdelttkalsalq qggdaqailq dlawkltnrl ihaptkslqq aardgdderl nilrdslglehemL:Escherichia coli MG1655(SEQ ID NO.4)
msksenlysa arelipggvn spvraftgvg gtplfiekad gaylydvdgk ayidyvgswgpmvlghnhpa irnavieaae rglsfgapte mevkmaqlvt elvptmdmvr mvnsgteatmsairlargft grdkiikfeg cyhghadcll vkagsgaltl gqpnspgvpa dfakytltct
yndlasvraa feqypqeiac iivepvagnm ncvpplpefl pglralcdef galliidevmtgfrvalaga qdyygvvpdl tclgkiiggg mpvgafggrr dvmdalaptg pvyqagtlsgnpiamaagfa clnevaqpgv hetldelttr laeglleaae eagiplvvnh vggmfgifftdaesvtcyqd vmacdverfk rffhmmldeg vylapsafea gfmsvahsme dinntidaarrvfakl
finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A high throughput screening system for high yield bacteria of 5-aminolevulinic acid, comprising at least cAMP responsive to 5-aminolevulinic acid, cAMP receptor protein CRP, and a CRP regulated promoter and reporter gene, wherein the reporter gene is downstream of and under the control of the promoter.
2. The high throughput screening system of claim 1, wherein said reporter gene comprises a gene encoding a fluorescent protein; further, the fluorescent protein is GFP.
3. The high throughput screening system of claim 1, wherein said promoter comprises P cspE 、P epd 、P glpD 、P gntK 、P cspP 、P cdd 、P mglB 、P gapA 、P galS And P galP 。
4. A recombinant expression vector carrying the high throughput screening system of any one of claims 1-3.
5. A cell comprising the recombinant expression vector of claim 4 or carrying the high throughput screening system of any one of claims 1-3.
6. The cell of claim 5, wherein the cell is a prokaryotic cell or a eukaryotic cell, further wherein the prokaryotic cell is a 5-aminolevulinic acid-producing bacterium, and wherein the bacterium is escherichia coli or a derivative thereof.
7. Use of the high throughput screening system of any one of claims 1-3, the recombinant expression vector of claim 4, or the cell of claim 5 or 6 for high throughput screening of strains metabolically producing 5-aminolevulinic acid or an upstream or downstream product thereof.
8. A method of high throughput screening of a high yield 5-aminolevulinic acid strain, comprising screening the high yield 5-aminolevulinic acid strain using fluorescence activated cell sorting based on the high throughput screening system of any one of claims 1-3, the recombinant expression vector of claim 4, or the cells of claim 5 or 6.
9. The method of claim 8, wherein the specific method for fluorescence activated cell sorting comprises sorting strains to be screened according to GFP fluorescence intensity using a flow cytometer;
the strain to be screened is a strain prepared by error-prone PCR and having a C4 production pathway or a C5 production pathway of 5-aminolevulinic acid.
10. A strain of high-yield 5-aminolevulinic acid, characterized in that it is escherichia coli and its derivative, which has a 5-aminolevulinic acid C4 production pathway-related enzyme or a 5-aminolevulinic acid C5 production pathway-related enzyme;
when the strain has a 5-aminolevulinic acid C4 production pathway-related enzyme, it has a hemA mutant obtained from a wild-type hemA mutated at one or more sites in the group: v69 5292 91 8238 391A, R97H, T317 5652 228I, G152R, A290G, V I, R292H, A223T, V322L, D379G, said wild hemA being derived from rhodopseudomonas palustris (Rhodopseudomonas palustris KUGB) rhodopseudomonas palustris (62306);
when the strain has a 5-aminolevulinic acid C5 production pathway-related enzyme, it has a hemA mutant and/or a hemL mutant;
the hemA mutant is obtained from a wild-type hemA mutated at one or more sites in the group: E265V, L112M, D381G, F127Y, A164T, I233M, Q R, R272H, K125Q, D226V, V I, said wild-type hemA being derived from salmonella typhimurium (Salmonella typhimurium);
the hemL mutants were obtained from wild-type hemL mutated at one or more sites in the following group: V203A, V206L, M62I, N67H, A338S, V47I, A157T, T S, M385L, A R, E389K, K425G, A70P, P395S, A G, said wild type hemL being derived from escherichia coli (Escherichia coli MG 1655).
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| CN102206606A (en) * | 2011-03-31 | 2011-10-05 | 山东大学 | Recombinant escherichia coli and application thereof in production of 5-aminolevulinic acid (ALA) |
| CN112280820A (en) * | 2020-10-09 | 2021-01-29 | 吉林医药学院 | Application of FRT cell strain in preparation of preparation or kit for screening CFTR (circulating fluid transfer) regulator |
| CN114032225A (en) * | 2018-07-13 | 2022-02-11 | 中国科学院天津工业生物技术研究所 | 5-amino-acetopropionic acid synthetase mutant and host cell and application thereof |
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| CN102206606A (en) * | 2011-03-31 | 2011-10-05 | 山东大学 | Recombinant escherichia coli and application thereof in production of 5-aminolevulinic acid (ALA) |
| CN114032225A (en) * | 2018-07-13 | 2022-02-11 | 中国科学院天津工业生物技术研究所 | 5-amino-acetopropionic acid synthetase mutant and host cell and application thereof |
| CN112280820A (en) * | 2020-10-09 | 2021-01-29 | 吉林医药学院 | Application of FRT cell strain in preparation of preparation or kit for screening CFTR (circulating fluid transfer) regulator |
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| JULIA SCHULTE ET AL.: "Development of a single-cell GlxR-based cAMP biosensor for Corynebacterium glutamicum", JOURNAL OF BIOTECHNOLOGY, vol. 258, 8 July 2017 (2017-07-08), pages 1 - 7 * |
| QI WANG ET AL.: "Design of a Genetically Encoded Biosensor for High-Throughput Screening and Engineering 5‑Aminolevulinic Acid Hyper-Producing Escherichia coli", ACS SUSTAINABLE CHEM. ENG., vol. 12, 11 March 2024 (2024-03-11), pages 4846 - 4857 * |
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