CN116121160A - Genetically engineered bacterium for over-expressing pyrB gene and method for producing L-arginine by using genetically engineered bacterium - Google Patents
Genetically engineered bacterium for over-expressing pyrB gene and method for producing L-arginine by using genetically engineered bacterium Download PDFInfo
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- CN116121160A CN116121160A CN202211353781.3A CN202211353781A CN116121160A CN 116121160 A CN116121160 A CN 116121160A CN 202211353781 A CN202211353781 A CN 202211353781A CN 116121160 A CN116121160 A CN 116121160A
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- engineered bacterium
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Abstract
The invention belongs to the technical field of genetic engineering, and in particular relates to a genetic engineering bacterium for over-expressing pyrB gene and a method for producing L-arginine by using the same. More specifically, the genetically engineered bacterium is modified in a mode of over-expressing pyrB gene for the strain E.coli W3110ARG10, and the modified genetically engineered bacterium is utilized for producing L-arginine by fermentation, so that the production performance of the L-arginine is remarkably improved.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and in particular relates to a genetic engineering bacterium for over-expressing pyrB gene and a method for producing L-arginine by using the same.
Background
L-arginine has wide application in the fields of medicine, industry, food, cosmetics, animal husbandry and the like, and has important economic and social values.
At present, three main methods for producing L-arginine are: microbial fermentation, chemical synthesis, and proteolytic processes. The microbial fermentation method has the advantages of wide sources of production raw materials, relatively simple production process, relatively small influence on environment, high product purity and the like, and is a main stream method for producing L-arginine.
The breeding of high-efficiency bacterial strain with L-arginine producing capacity is key to industrial application of microbial fermentation method. At present, two main methods for breeding L-arginine producing strains are available: 1) Irrational mutagenesis screening: the method mainly carries out mutagenesis treatment on wild chassis microorganisms by a physical or chemical method, and combines an L-arginine structural analogue resistance screening method to select and breed mutagenized strains with antagonism on L-arginine. Through multiple rounds of mutagenesis, a superior production strain with L-arginine synthesis ability is finally selected. For example, CN03112896.3 uses corynebacterium crenatum (Corynebacterium crenatum) SYA5 (histidine-deficient, sulfaguanidine-resistant) as a parent strain, performs physicochemical mutagenesis treatment according to a conventional method, and performs multiple structural analog resistance screening to obtain mutant strain SDNN403 (histidine-deficient, sulfaguanidine-resistant, D-arginine-resistant, homoarginine-resistant, methyl cysteine-resistant), and cultures under optimized conditions to produce L-arginine at an acid production level of 30-35g/L. 2) Rational metabolic engineering: the method mainly utilizes a high-efficiency gene editing technology to carry out systematic metabolic engineering transformation on an L-arginine synthesis network in chassis microorganisms so as to maximally redirect carbon metabolic flux to an L-arginine synthesis pathway, and mainly comprises the steps of blocking the L-arginine degradation pathway, relieving a key enzyme feedback inhibition regulation mechanism caused by L-arginine synthesis, strengthening the metabolic flux of the synthesis pathway, optimizing the coenzyme supply balance of chassis cells, modifying a transmembrane transport system of a target product and the like. For example, CN110964683a, in escherichia coli, analyzes and reconstructs arginine synthesis pathway in escherichia coli and metabolic flux related to arginine in whole amino acid metabolic network by integrating gene encoding carbamyl phosphate synthetase and gene encoding L-arginine biosynthesis pathway enzyme, so as to obtain genetically engineered bacterium which has clear genetic background, does not carry plasmid, does not undergo mutagenesis and can stably and efficiently produce L-arginine. With the rapid development of genetic engineering technology, the method for constructing the efficient L-arginine producing strain by rational metabolic engineering gradually replaces a mutation screening breeding method, and becomes a mainstream method for breeding the efficient and stable L-arginine producing strain.
Improving the L-arginine production performance of chassis microorganisms is a goal of the sustainability of rational metabolic engineering breeding. In the process of systematically reconstructing the chassis microbial metabolic network, the enhancement or weakening of the expression intensity of a target gene is a common strategy for improving the L-arginine production performance. For example, L-arginine synthesis may be significantly promoted by attenuating expression of one or more genes involved in L-arginine degradation, one or more genes involved in competing pathways for L-arginine synthesis, and genes involved in redistribution of carbon and nitrogen flux; in addition, the improvement of the expression of the key enzyme in the L-arginine synthesis pathway, the improvement of the expression of the L-arginine exomembrane protein and the improvement of the expression of the key enzyme in the L-arginine precursor synthesis pathway also has obvious beneficial effects on improving the L-arginine production of chassis microorganisms.
The pyrB gene encodes an aspartate carbamoyltransferase which catalyzes the binding of carbamoylphosphate to aspartate to aminomethylaspartate and phosphate, the two-way reaction being the first step in pyrimidine biosynthesis and having an important role in balancing the metabolic fluxes of the pyrimidine and purine synthetic pathways. However, the effect of modification of the pyrB gene on L-arginine production has not been reported.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide a method for producing L-arginine by genetically modified escherichia coli.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the invention provides a genetically engineered bacterium for producing L-arginine, in particular belonging to E.coli, more particularly the E.coli strain E.coli W3110ARG10 modified in such a way that the pyrB gene is overexpressed.
In a second aspect, the present invention provides a method for producing L-arginine using the genetically engineered bacterium as described above, comprising: culturing the genetically engineered bacterium in a medium to produce L-arginine; and collecting the L-arginine from the genetically engineered bacterium and/or the culture medium.
The method according to the second aspect of the present invention, wherein the genetically engineered bacterium has been modified in such a way that the pyrB gene is overexpressed, such that the pyrB gene has an increased expression strength or an increased expression level compared to an unmodified E.coli.
The beneficial effects of the invention are as follows:
the pyrB gene encodes aspartate carbamoyltransferase, and the relationship and function of this gene to the L-arginine synthesis pathway is not clear. The present invention demonstrates that the level of L-arginine production by E.coli modified in such a way that the pyrB gene is overexpressed is significantly increased. Specifically, the L-arginine accumulation concentration in the genetically engineered strain modified in a manner of over-expressing pyrB gene is increased from 31.2g/L to 36.5g/L by taking an L-arginine engineering strain E.coli W3110ARG10 as an original strain, and the L-arginine yield is increased by 17%.
Drawings
Fig. 1: ygaY:: P trc -pyrB gene editing electropherograms. Wherein: m:1kb DNAmarker;1: an upstream homology arm; 2: a downstream homology arm; 3: overlapping the segments; 4: a primordium control; 5: positive bacteria identify fragments.
Detailed Description
The invention is described below by means of specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention.
In a first aspect, the invention provides a genetically engineered bacterium for producing L-arginine, in particular belonging to E.coli, more particularly the E.coli strain E.coli W3110ARG10 modified in such a way that the pyrB gene is overexpressed.
According to a first aspect of the invention, the E.coli W3110ARG10 strain is modified in such a way that the pyrB gene is overexpressed, such that the expression level of the pyrB gene is increased compared to an unmodified E.coli strain, or the total enzyme activity of the pyrB gene expression product, e.g.aspartate carbamoyltransferase, is increased, e.g.by 150% or more, 200% or more, 300% or more.
According to a first aspect of the invention, the strain E.coli W3110ARG10 (described in detail in China patent CN 110964683B) is based on E.coli W3110 and contains genes pyrA and pyrAB encoding carbamoyl phosphate synthetase from B.subtilisA260; comprises the following arginine biosynthetic pathway enzymes: argC, argJ, argB, argD, argF, argG and argH, the gene encoding an arginine biosynthetic pathway enzyme consisting of P trc Starting a promoter; knocking out coding genes speA, adiA, astA and argE for decomposing L-arginine; contains a gene lysE encoding an arginine transporter derived from Corynebacterium validum.
According to the first aspect of the invention, the pyrB gene may be overexpressed by introducing and/or increasing the copy number of the pyrB gene in the bacterial genome (e.g., increasing the copy number of the pyrB gene by autonomously replicating plasmids such as pET28a, pTrc99a, pSTV28, etc., or increasing the copy number of the pyrB gene in the bacterial chromosome), or modifying the expression control sequences of the pyrB gene (e.g., promoters, ribosome binding sites, etc.), or a combination of the above.
According to a preferred embodiment of the invention, the pyrB gene is overexpressed by ligating the gene with a strong promoter and integrating it into the E.coli genome, wherein the integration site of the gene is selectable according to routine knowledge by those skilled in the art at pseudogene sites which do not significantly affect bacterial growth and basal metabolism, such as for example, the yeeP, ygiP, yghX, ygaY, yjiT, ycjV, ycgH, ygaY, yeeL, ilvG isogenic site; the strong promoter can be any one of Ptrc, BBa-J23100 and T7.
According to the first aspect of the present invention, the pyrB gene is not limited to the nucleotide sequence shown in NCBI GeneID 948767, but may also include a mutant nucleotide sequence which is a sequence shown in NCBI GeneID 948767 or a gene which is homologous to a sequence shown in NCBI GeneID 948767 and which encodes a mutant of pyrB protein. The pyrB gene may be a variant nucleotide sequence due to the degeneracy of the genetic code.
In a second aspect, the present invention provides a method for producing L-arginine using the genetically engineered bacterium as described above, comprising: culturing the genetically engineered bacterium in a medium to produce L-arginine; and collecting the L-arginine from the genetically engineered bacterium and/or the culture medium.
According to the second aspect of the present invention, the L-arginine includes not only L-arginine in a free form but also salts or hydrates of L-arginine.
According to the second aspect of the present invention, the culturing of the genetically engineered bacterium may be performed by a method conventional in the art. The medium used for producing L-arginine may be a synthetic or natural medium, such as a typical medium containing a carbon source, a nitrogen source, a sulfur source, inorganic ions and other organic and inorganic components as desired.
The genetically engineered bacterium may be cultured under aerobic conditions for 16 to 72 hours, or 20 to 48 hours, or 26 to 30 hours; the culture temperature may be controlled within 30 to 45℃or 30 to 37 ℃; and the pH may be adjusted between 5.0 and 8.0, or between 6.0 and 7.5, or between 6.8 and 7.2. The pH can be adjusted by using inorganic or organic acidic or basic substances, as well as ammonia gas.
After culturing, solids such as cells and cell debris can be removed from the liquid medium by conventional techniques (e.g., centrifugation, membrane filtration), and L-arginine can then be recovered from the fermentation broth by any combination of conventional techniques (e.g., concentration, ion exchange chromatography, crystallization).
The bacteria may be subjected to strain activation, seed expansion and the like before inoculation and fermentation according to the preservation state thereof, and suitable conditions and culture media may be selected according to conventional techniques in the art, for example, the seed culture media may be the same composition as the fermentation culture media, or may be appropriately adjusted on the basis of the same.
Other specific procedures for molecular biology, genetic engineering, etc. may be implemented according to technical manuals, textbooks, or literature reports readily available to those skilled in the art, and the detailed description of the procedures is not necessary here. In the following examples, specific strains are selected as hosts, and thus specific gene integration sites, target genes, primers therefor, and the like are selected according to hosts, but it is not intended that the objects of the present invention can be achieved only by these specific selections, and the scope of the present invention is not limited thereto, but only by the spirit and scope of the present invention.
Example 1: construction of genetically engineered Strain ARG-pyrB modified in such a way that the pyrB Gene is overexpressed
1 method of Gene editing
The gene editing methods employed in the examples of the present invention were performed, unless specifically stated, with reference to the literature (Li Y, lin Z, huang C, et al, metabolic engineering of Escherichia coli using CRISPR-Cas9 mediated genome editing, metabolic engineering,2015, 31:13-21.). Wherein pREDCas9 carries the elimination system of gRNA expression plasmid pGRB, the Red recombination system of lambda phage and Cas9 protein expression system, and the resistance of the Qimamycin (working concentration: 100 mg/L) is cultivated at 32 ℃; pGRB was grown at 37℃with pUC18 as the backbone, comprising the promoter J23100, the gRNA-Cas9 binding region sequence and the terminator sequence, ampicillin resistance (working concentration: 100 mg/L).
The method comprises the following specific steps:
1.1 construction of pGRB plasmid
The purpose of constructing plasmid pGRB is to transcribe the corresponding gRNA, thereby forming a complex with Cas9 protein, and to recognize the target site of the target gene by base pairing and PAM, achieving the target DNA double strand break. pGRB plasmids were constructed by recombination of DNA fragments containing the target sequence with linearized vector fragments.
1.1.1 target sequence design
Target sequence design Using CRISPR RGEN Tools (PAM: 5 '-NGG-3')
1.1.2 preparation of DNA fragments comprising the target sequence
Designing a primer: 5 '-linearization vector terminal sequence (15 bp) -cleavage site-target sequence (excluding PAM sequence) -linearization vector terminal sequence (15 bp) -3' and reverse complement primer thereof, DNA fragment containing target sequence was prepared by annealing single-stranded DNA. Reaction conditions: pre-denaturation at 95℃for 5min; annealing at 30-50deg.C for 1min. The annealing system is as follows:
1.1.3 preparation of Linear Carriers
Linearization of the vector uses inverse PCR amplification.
1.1.4 recombination reactions
The recombinant enzymes used were ClonExpress
II One Step Cloning Kit series of enzymes, recombination conditions: 37℃for 30min. The recombination system is as follows: />
1.1.5 transformation of plasmids
10 mu L of the reaction solution is taken and added into 100mL of DH5 alphabetized competent cells, the mixture is lightly mixed, then the ice bath is carried out for 20min, the heat shock is carried out at 42 ℃ for 45-90s, the ice bath is immediately carried out for 2-3min, 900 mu L of SOC is added, and the mixture is resuscitated for 1h at 37 ℃. Centrifugation at 8000rpm for 2min, discarding part of the supernatant, leaving about 200. Mu.L of the cell mass to be resuspended and spread on a plate containing 100mg/L ampicillin, inverting the plate and culturing overnight at 37 ℃. After single colony is formed on the plate, the positive recombinants are selected through colony PCR identification.
1.1.6 clone identification
The PCR positive colony is inoculated into LB culture medium containing 100mg/L ampicillin for overnight culture and then is preserved, plasmid is extracted and enzyme-cut for identification.
1.2 preparation of recombinant DNA fragments
The recombinant fragment for promoter replacement consists of the upstream and downstream homology arms of the pyrB gene (upstream homology arm-downstream homology arm). The primer design software primer5 is utilized, the upstream and downstream homology arm primers (the amplification length is about 400-500 bp) are designed by taking the upstream and downstream sequences of pyrB genes as templates, the upstream and downstream homology arms and target gene fragments are respectively amplified by a PCR method, and then the recombinant fragments are prepared by overlapping PCR. The PCR amplification system is shown in the following table:
the system of overlap PCR is as follows:
note that: the template consists of amplified fragments of upstream and downstream homology arms and target genes in equimolar mode, and the total amount is not more than 10ng.
PCR reaction conditions (Bao biological PrimeSTAR HS enzyme): pre-denaturation (95 ℃) for 5min; then 30 cycles were performed: denaturation (98 ℃) for 10s, annealing ((Tm-3/5) ℃for 15s, extension at 72℃for 1min for about 1 kb; continuing to extend for 10min at 72 ℃; maintained (4 ℃ C.).
1.3 transformation of plasmids and recombinant DNA fragments
1.3.1 Transformation of pREDCas9
The pREDCas9 plasmid is electrotransferred into electrotransfer competence of an original strain by using an electrotransfer method, and the thallus is coated on an LB plate containing the azithromycin for culture after resuscitating and culturing, and is cultured overnight at 32 ℃. Single colonies were grown on the resistance plates and subjected to colony PCR with the identification primers to screen positive recombinants.
1.3.2 preparation of pREDCas 9-containing target Strain electrotransformation competence
Culturing at 32deg.C to OD 600 When the concentration is=0.1 to 0.2, 0.1M IPTG (to a final concentration of 0.1 mM) is added, and the culture is continued until the OD is reached 600 Competent preparation was performed at=0.6 to 0.7. The purpose of IPTG addition is to induce expression of the recombinase on the pREDcas9 plasmid. The culture medium and the preparation process required for competent preparation are operated according to conventional standard.
1.3.3 transformation of pGRB and recombinant DNA fragments
pGRB and donor DNA fragments were simultaneously electrotransformed into electrocompetent cells containing pREDCas 9. Resultured cells after electrotransformation were plated on LB plates containing ampicillin and Qixamycin and incubated overnight at 32 ℃. And (3) performing colony PCR verification by using specially designed identification primers, screening positive recombinants and preserving bacteria.
1.4 elimination of plasmid
1.4.1 Elimination of pGRB
Positive recombinants were placed in LB medium containing 0.2% arabinose for overnight culture, diluted in appropriate amounts, plated on LB plates containing resistance to Qixime, and incubated overnight at 32 ℃. Ampicillin and Qamycin resistant LB plates were selected for individual colonies growing on ampicillin and Qamycin resistant plates without growing.
1.4.2 Elimination of pREDCas9 plasmid
The positive recombinants were transferred to a non-resistant LB liquid medium, cultured overnight at 42℃and then plated on a non-resistant LB plate after appropriate dilution, and cultured overnight at 37 ℃. And selecting a single colony for bacteria protection, wherein the single colony contains the resistance and no resistance of the Qamycin, and the resistance of the Qamycin.
2. The primers used in the strain construction are shown in the following table:
3.ygaY::P trc construction of the pyrB Gene
Using coligenome as template, designing upstream homology arm primer (UP-ygaY-S, UP-ygaY-A) and downstream homology arm primer (DN-ygaY-S, DN-ygaY-A) according to upstream and downstream sequence of ygaY gene (NCBI GeneID: 2847696); based on the sequence upstream and downstream of the pyrB gene (NCBI GeneID: 948767) and P trc The promoter sequences were designed to amplify the primers pyrB-S and pyrB-A required for the pyrB gene. The fragment is fused by an overlapping PCR method to obtain ygaY:: P trc pyrB (upstream homology arm-P) trc Downstream homology arms). Construction of pGRB-ygaY: the DNA fragment containing the target sequence used was prepared by annealing the primers gRNA-ygaY-S and gRNA-ygaY-A. Competent cells of E.coli W3110ARG10 were prepared, and the procedure described above under 1.3 and 1.4 was followed to construct a genetically engineered strain ARG-pyrB modified in such a way that the pyrB gene was overexpressed. ygaY:: P trc Construction of the pyrB fragment and PCR verification of the positive strain the electrophoretogram is shown in FIG. 1. Wherein the length of the upstream homology arm is 616bp, P trc The length of the promoter is 74bp, the length of the pyrB fragment is 660bp, the length of the downstream homology arm is 617bp, the total length of the overlapped fragments is 1967bp, the length of the positive bacteria PCR amplified fragment is 1967bp when the PCR is verified, and the length of the primordial bacteria PCR amplified fragment is 1424bp.
Example 2: production of L-arginine using E.coli ARG-pyrB with over-expressed pyrB gene
Coli ARG-pyrB is modified based on E.coli ARG10 in such a way that the pyrB gene is overexpressed. ARG10 strain (described in detail in Chinese patent CN 110964683B) is based on E.coli W3110 and contains genes pyrA and pyrAB encoding carbamoyl phosphate synthetase from B.subtilisA260; comprises the following arginine biosynthetic pathway enzymes: argC, argJ, argB, argD, argF, argG and argH, which encode an arginine biosynthetic pathway enzymeIs composed of gene P trc Starting a promoter; knocking out coding genes speA, adiA, astA and argE for decomposing L-arginine; contains a gene lysE encoding an arginine transporter derived from Corynebacterium validum.
The same culture medium and culture method are adopted to shake flask culture and ferment the escherichia coli ARG-pyrB and the control escherichia coli ARG10 to produce the L-arginine.
Composition of the medium: glucose 20g/L, yeast extract 3g/L, peptone 2g/L, K 2 HPO 4 6g/L，MgSO 4 ·7H 2 O 1g/L，FeSO 4 ·7H 2 O 20mg/L，MnSO 4 ·7H 2 O 20mg/L，V B1 、V B3 、V B5 、V B12 、V H 1mg/L of each and the balance of water, and the pH value is 7.0-7.2.
The shake flask culture method comprises the following steps: inoculating into 500mL triangular flask (final volume of 30 mL) containing fermentation medium according to inoculum size of 10% of seed culture solution volume, sealing nine layers of gauze, shake culturing at 37deg.C at 200r/min, and maintaining pH at 7.0-7.2 by adding ammonia water during fermentation; the fermentation was maintained by adding 60% (m/v) glucose solution.
After 28h fermentation culture in shake flask, 1mL of each fermentation broth was taken for 13000 rpm to remove the thalli at high speed, and fermentation supernatant was obtained. The fermentation supernatant was assayed for L-arginine concentration by high performance liquid chromatography (as shown in Table 1).
TABLE 1 results of shaking flask fermentation of ARG-pyrB Strain
The results in Table 1 show that overexpression of the pyrB gene does not have a significant effect on the growth of the engineered strain; at the same time, the concentration of L-arginine in ARG-pyrB is promoted to be increased from 31.2g/L to 36.5g/L, and the yield of L-arginine is improved by 17 percent. The experimental result shows that the L-arginine is produced by fermenting genetically engineered bacteria modified in a mode of over-expressing pyrB gene, the L-arginine production performance is obviously improved, and the modified strain has stronger L-arginine production capacity.
Although the present invention has been described with reference to preferred embodiments, it is not intended to be limited to the embodiments shown, but rather, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations in form and details can be made therein without departing from the spirit and principles of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims (4)
1. A genetically engineered bacterium for producing L-arginine, wherein the genetically engineered bacterium has been modified to over-express the pyrB gene from strain e.coli W3110ARG 10.
2. The genetically engineered bacterium of claim 1, wherein said strain e.coli W3110ARG10 is based on e.coli W3110 and comprises genes pyrAA and pyrAB encoding carbamoyl phosphate synthetase from b.subtilisa260; comprises the following arginine biosynthetic pathway enzymes: argC, argJ, argB, argD, argF, argG and argH, the gene encoding an arginine biosynthetic pathway enzyme consisting of P trc Starting a promoter; knocking out coding genes speA, adiA, astA and argE for decomposing L-arginine; contains a gene lysE encoding an arginine transporter derived from Corynebacterium validum.
3. The genetically engineered bacterium of claim 1, wherein said pyrB gene is integrated at the ygaY gene locus of the strain e.coli W3110ARG10 genome and is defined by P trc The promoter is started.
4. A method for producing L-arginine using the genetically engineered bacterium of any one of claims 1 to 3, comprising: culturing the genetically engineered bacterium in a medium to produce L-arginine; and collecting the L-arginine from the genetically engineered bacterium and/or the culture medium.
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