Genetically engineered bacteria overexpressing pyrB gene and method for producing L-arginine

Abstract

The present invention belongs to the field of genetic engineering technology, and specifically relates to a genetically engineered bacterium that overexpresses the pyrB gene and a method for producing L-arginine using the same. More specifically, the genetically engineered bacterium is a strain of E. coli W3110ARG10 that is modified in a manner that overexpresses the pyrB gene, and the modified genetically engineered bacterium is used to ferment and produce L-arginine, which significantly improves the L-arginine production performance.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a genetic engineering bacterium over-expressing a pyrB gene and a method for producing L-arginine by using the same. Background Art

[0002] L-arginine is widely used in medicine, industry, food, cosmetics, animal husbandry and other fields, and has important economic and social value.

[0003] At present, there are three main methods for producing L-arginine: microbial fermentation, chemical synthesis, and protein hydrolysis. Among them, microbial fermentation has the advantages of a wide range of raw materials, relatively simple production process, relatively small impact on the environment, and high product purity. It has now become the mainstream method for L-arginine production.

[0004] Breeding efficient strains with L-arginine production capacity is the key to the industrial application of microbial fermentation. There are two main methods for breeding L-arginine production strains at present: 1) irrational mutagenesis screening: This method mainly uses physical or chemical methods to mutagenize wild-type chassis microorganisms, combined with L-arginine structural analog resistance screening methods, to breed mutagenized strains with antagonistic effects on L-arginine. After multiple rounds of mutagenesis, the excellent production strain with L-arginine synthesis ability is finally screened out. For example, CN03112896.3 uses Corynebacterium crenatum SYA5 (histidine deficiency, sulfaguanidine resistance) as the parent strain, and performs physical and chemical mutagenesis according to the conventional method, and obtains mutant SDNN403 (histidine deficiency, sulfaguanidine resistance, D-arginine resistance, homoarginine resistance, methylcysteine ​​resistance) through multiple structural analog resistance screening, and cultivates and produces L-arginine under optimized conditions, and the acid production level reaches 30-35g / L. 2) Rational metabolic engineering: This method mainly uses efficient gene editing technology to systematically metabolize the L-arginine synthesis network in chassis microorganisms to maximize the redirection of carbon metabolic flux to the L-arginine synthesis pathway, mainly including blocking the L-arginine degradation pathway, removing the key enzyme feedback inhibition regulation mechanism caused by L-arginine synthesis, strengthening the metabolic flux of the synthesis pathway, optimizing the balance of chassis cell coenzyme supply, modifying the transmembrane transport system of the target product, etc. For example, CN110964683A analyzes and reconstructs the metabolic flux related to arginine in the arginine synthesis pathway and the entire amino acid metabolic network in Escherichia coli by integrating genes encoding carbamoyl phosphate synthetase and genes encoding enzymes of the L-arginine biosynthesis pathway in Escherichia coli, and obtains genetically engineered bacteria with a clear genetic background, no plasmid, no mutagenesis, and stable and efficient production of L-arginine. With the rapid development of genetic engineering technology, the method of rational metabolic engineering to construct efficient L-arginine production strains has gradually replaced the mutagenesis screening breeding method and has become the mainstream method for breeding efficient and stable L-arginine production strains.

[0005] Improving the L-arginine production performance of chassis microorganisms is a continuous goal of rational metabolic engineering breeding. In the process of systematic reconstruction of the chassis microbial metabolic network, the enhancement or reduction of the expression intensity of the target gene is a common strategy to improve the L-arginine production performance. For example, weakening the expression of one or several genes for L-arginine degradation, one or several genes in the competitive pathway for L-arginine synthesis, and genes involved in the redistribution of carbon and nitrogen flux can significantly promote the synthesis of L-arginine; in addition, increasing the expression of key enzymes in the L-arginine synthesis pathway, increasing the expression of L-arginine efflux membrane proteins, and increasing the expression of key enzymes in the L-arginine precursor synthesis pathway also has a significant beneficial effect on improving the production of L-arginine in chassis microorganisms.

[0006] The pyrB gene encodes aspartate transcarbamylase, which catalyzes the fusion of carbamoyl phosphate and aspartate to form aminomethyl aspartate and phosphate. This bidirectional reaction is the first step in pyrimidine biosynthesis and plays an important role in the balance of metabolic flux in the pyrimidine and purine biosynthesis pathways. However, the effect of modification of the pyrB gene on L-arginine production has not been reported. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention aims to provide a method for producing L-arginine by genetically modified Escherichia coli.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a genetically engineered bacterium for producing L-arginine, specifically, it belongs to Escherichia coli, and more specifically, the Escherichia coli is a strain E. coli W3110 ARG10 modified in a manner of overexpressing the pyrB gene.

[0010] In a second aspect, the present invention provides a method for producing L-arginine using the genetically engineered bacteria as described above, comprising: culturing the genetically engineered bacteria in a culture medium to produce L-arginine; and collecting the L-arginine from the genetically engineered bacteria and / or the culture medium.

[0011] As in the method provided by the present invention in the second aspect, the genetically engineered bacteria have been modified in a manner of overexpressing the pyrB gene, so that the expression intensity or expression level of the pyrB gene is enhanced compared to unmodified Escherichia coli.

[0012] The beneficial effects of the present invention are as follows:

[0013] The pyrB gene encodes aspartate carbamoyltransferase, and the relationship and role of this gene with the L-arginine biosynthesis pathway are still unclear. The present invention confirms that the level of L-arginine production by Escherichia coli modified by overexpressing the pyrB gene is significantly improved. Specifically, using an L-arginine engineering strain E. coli W3110 ARG10 as the starting strain, the L-arginine accumulation concentration in the genetically engineered strain modified by overexpressing the pyrB gene was increased from 31.2 g / L to 36.5 g / L, and the L-arginine yield was increased by 17%. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 :ygaY::P trc-pyrB gene editing electrophoresis diagram. Where: M: 1kb DNA marker; 1: upstream homology arm; 2: downstream homology arm; 3: overlapping fragments; 4: original bacteria control; 5: positive bacteria identification fragment. DETAILED DESCRIPTION

[0015] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0016] In a first aspect, the present invention provides a genetically engineered bacterium for producing L-arginine, specifically, it belongs to Escherichia coli, and more specifically, the Escherichia coli is a strain E. coli W3110 ARG10 modified in a manner of overexpressing the pyrB gene.

[0017] According to the first aspect of the present invention, the Escherichia coli is a strain E. coli W3110 ARG10 modified by overexpressing the pyrB gene, so that the expression level of the pyrB gene is increased compared with the unmodified Escherichia coli, or the total enzyme activity of the pyrB gene expression product, such as aspartate carbamoyltransferase, is increased, for example, by 150% or more, 200% or more, 300% or more.

[0018] According to the first aspect of the present invention, the strain E. coli W3110 ARG10 (described in detail in Chinese patent CN110964683B) is based on E. coli W3110, contains genes pyrAA and pyrAB encoding carbamoyl phosphate synthetase from Bacillus subtilis B. subtilis A260; contains the following arginine biosynthetic pathway enzymes: argC, argJ, argB, argD, argF, argG and argH, and the genes encoding the arginine biosynthetic pathway enzymes are derived from P trc The promoter is started; the genes speA, adiA, astA and argE encoding the decomposition of L-arginine are knocked out; and the gene lysE encoding the arginine transporter derived from effective bacterium is contained.

[0019] According to the first aspect of the present invention, the pyrB gene can be overexpressed by introducing and / or increasing the copy number of the pyrB gene in the bacterial genome (for example, increasing the copy number of the pyrB gene by autonomously replicating plasmids such as pET28a, pTrc99a, pSTV28, or increasing the copy number of the pyrB gene in the bacterial chromosome), or modifying the expression regulatory sequence of the pyrB gene (for example, a promoter, a ribosome binding site, etc.), or a combination of the above methods.

[0020] According to a preferred embodiment of the present invention, the method of overexpressing the pyrB gene is to connect the gene to a strong promoter and integrate it into the Escherichia coli genome, wherein the integration site of the gene can be selected according to the conventional knowledge of those skilled in the art to select some pseudogene sites that will not have a significant effect on bacterial growth and basal metabolism, such as yeeP, ygiP, yghX, ygaY, yjiT, ycjV, ycgH, ygaY, yeeL, ilvG and other gene sites can be selected; the strong promoter can be selected from any one of Ptrc, BBa-J23100, and T7.

[0021] 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 of the sequence shown in NCBI GeneID: 948767 or a gene homologous to the sequence shown in NCBI GeneID: 948767 and encoding a mutant of the pyrB protein. The pyrB gene may be a variant nucleotide sequence due to the degeneracy of the genetic code.

[0022] In a second aspect, the present invention provides a method for producing L-arginine using the genetically engineered bacteria as described above, comprising: culturing the genetically engineered bacteria in a culture medium to produce L-arginine; and collecting the L-arginine from the genetically engineered bacteria and / or the culture medium.

[0023] According to the second aspect of the present invention, the L-arginine includes not only free form of L-arginine but also a salt or hydrate of L-arginine.

[0024] According to a second aspect of the present invention, the cultivation of the genetically engineered bacteria can be carried out by conventional methods in this area. The substratum used to produce the L-arginine can be a synthetic or natural substratum, for example a typical substratum containing a carbon source, a nitrogen source, a sulphur source, an inorganic ion and other organic and inorganic components required.

[0025] The genetically engineered bacteria can be cultured under aerobic conditions for 16 to 72 hours, or 20 to 48 hours, or 26 to 30 hours; the culture temperature can be controlled within 30 to 45°C, or 30 to 37°C; and the pH can 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 alkaline substances, and ammonia gas.

[0026] After cultivation, solids such as cells and cell debris can be removed from the liquid culture 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).

[0027] Before inoculation and fermentation, bacteria can be activated and seed cultured according to their storage status. Appropriate conditions and culture media can be selected according to conventional techniques in the art. For example, the seed culture medium can have the same composition as the fermentation medium, or it can be appropriately adjusted on this basis.

[0028] Other specific operation methods such as molecular biology and genetic engineering involved can be implemented according to technical manuals, textbooks or literature reports that are easily available to those skilled in the art, and it is not necessary to describe the operation process in detail here. In addition, specific strains are selected as hosts in the following embodiments, and thus specific gene integration sites, target genes and primers thereof are selected according to the hosts, but it does not mean that the purpose of the present invention can only be achieved through these specific selections, and the scope of the present invention cannot be limited to this. The essence and scope of the present invention are only limited by the claims.

[0029] Example 1: Construction of genetically engineered strain ARG-pyrB modified by overexpressing the pyrB gene

[0030] 1. Gene Editing Methods

[0031] Unless otherwise specified, the gene editing method used in the embodiment of the present invention is carried out with reference to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9meditated genome editing. Metabolic engineering, 2015, 31: 13-21.). Among them, pREDCas9 carries the elimination system of the gRNA expression plasmid pGRB, the Red recombination system of λ phage and the Cas9 protein expression system, spectinomycin resistance (working concentration: 100 mg / L), and is cultured at 32°C; pGRB uses pUC18 as the skeleton, including the promoter J23100, the gRNA-Cas9 binding region sequence and the terminator sequence, ampicillin resistance (working concentration: 100 mg / L), and is cultured at 37°C.

[0032] The specific steps of this method are as follows:

[0033] 1.1 Construction of pGRB plasmid

[0034] The purpose of constructing the plasmid pGRB is to transcribe the corresponding gRNA, thereby forming a complex with the Cas9 protein, and recognize the target gene target site through base pairing and PAM to achieve the target DNA double-strand break. The pGRB plasmid is constructed by recombination of a DNA fragment containing the target sequence with a linearized vector fragment.

[0035] 1.1.1 Target sequence design

[0036] Design target sequence (PAM: 5'-NGG-3') using CRISPR RGEN Tools

[0037] 1.1.2 Preparation of DNA fragments containing target sequences

[0038] Design primers: 5'-linearized vector end sequence (15bp)-restriction site-target sequence (excluding PAM sequence)-linearized vector end sequence (15bp)-3' and its reverse complementary primer, and prepare DNA fragments containing target sequences by annealing single-stranded DNA. Reaction conditions: pre-denaturation at 95℃, 5min; annealing at 30-50℃, 1min. The annealing system is as follows:

[0039]

[0040] 1.1.3 Preparation of linear vector

[0041] The vector was linearized by inverse PCR amplification.

[0042] 1.1.4 Recombination reaction

[0043] All recombinant enzymes used were ClonExpress II One Step Cloning Kit series enzymes, recombination conditions: 37°C, 30min. The recombination system is as follows:

[0044]

[0045] 1.1.5 Plasmid transformation

[0046] Take 10μL of the reaction solution and add it to 100mL of DH5α competent cells. Mix gently and place on ice for 20min. Heat shock at 42℃ for 45-90s. Place on ice for 2-3min. Add 900μL of SOC and resuscitate at 37℃ for 1h. Centrifuge at 8000rpm for 2min, discard part of the supernatant, and keep about 200μL to resuspend the cells and apply them to a plate containing 100mg / L ampicillin. Invert the plate and culture at 37℃ overnight. After a single colony grows on the plate, identify it by colony PCR and select the positive recombinants.

[0047] 1.1.6 Clone identification

[0048] The PCR-positive colonies were inoculated into LB medium containing 100 mg / L ampicillin and cultured overnight to maintain the bacteria. The plasmids were extracted and identified by enzyme digestion.

[0049] 1.2 Preparation of recombinant DNA fragments

[0050] The recombinant fragment used for promoter replacement consists of the upstream and downstream homology arms of the pyrB gene (upstream homology arm-downstream homology arm). Using the primer design software primer5, the upstream and downstream sequences of the pyrB gene were used as templates to design upstream and downstream homology arm primers (amplification length of about 400-500bp). After amplifying the upstream and downstream homology arms and the target gene fragments by PCR, the recombinant fragments were prepared by overlapping PCR. The PCR amplification system is as follows:

[0051]

[0052] The overlapping PCR system is as follows:

[0053]

[0054] Note: The template consists of equimolar amounts of the amplified fragments of the upstream and downstream homology arms and the target gene, and the total amount does not exceed 10 ng.

[0055] PCR reaction conditions (Bao Bio PrimeSTAR HS enzyme): pre-denaturation (95°C) for 5 min; then 30 cycles: denaturation (98°C) for 10 s, annealing ((Tm-3 / 5)°C) for 15 s, extension at 72°C (this enzyme activity extends about 1 kb per 1 min); continue extension at 72°C for 10 min; maintain (4°C).

[0056] 1.3 Transformation of plasmids and recombinant DNA fragments

[0057] 1.3.1 Transformation with pREDCas9

[0058] The pREDCas9 plasmid was electroporated into the electroporation competent state of the starting strain by electroporation. After the bacteria were revived and cultured, they were spread on LB plates containing spectinomycin and cultured overnight at 32°C. Single colonies grown on the resistant plates were subjected to colony PCR using identification primers to screen positive recombinants.

[0059] 1.3.2 Preparation of electroporation competent cells of target strains containing pREDCas9

[0060] Culture at 32°C until OD 600 =0.1-0.2, add 0.1M IPTG (to make the final concentration 0.1mM), and continue to culture until OD 600 =0.6-0.7 for competent cell preparation. The purpose of adding IPTG is to induce the expression of the recombinase on the pREDCas9 plasmid. The culture medium and preparation process required for competent cell preparation refer to conventional standard operations.

[0061] 1.3.3 Transformation of pGRB and recombinant DNA fragments

[0062] pGRB and donor DNA fragments were electro-transformed into electro-competent cells containing pREDCas9 at the same time. The revived bacteria after electro-transformation were spread on LB plates containing ampicillin and spectinomycin and cultured at 32°C overnight. Colony PCR verification was performed using specially designed identification primers to screen positive recombinants and preserve the bacteria.

[0063] 1.4 Elimination of plasmid

[0064] 1.4.1 Elimination of pGRB

[0065] The positive recombinants were cultured overnight in LB medium containing 0.2% arabinose, and then appropriately diluted and spread on LB plates containing spectinomycin resistance, and cultured overnight at 32°C. For LB plates containing ampicillin and spectinomycin resistance, single colonies that did not grow on the ampicillin plate but grew on the spectinomycin resistance plate were selected for preservation.

[0066] 1.4.2 Elimination of pREDCas9 plasmid

[0067] Transfer the positive recombinant to LB liquid medium without resistance, culture at 42℃ overnight, dilute appropriately and spread on LB plate without resistance, culture at 37℃ overnight. For LB plates containing spectinomycin resistance and LB plates without resistance, select single colonies that do not grow on spectinomycin resistance plates but grow on plates without resistance.

[0068] 2. The primers used in the strain construction process are shown in the following table:

[0069]

[0070]

[0071] 3.ygaY::P trc -pyrB gene editing strain construction

[0072] Using the Escherichia coli genome as a template, upstream homology arm primers (UP-ygaY-S, UP-ygaY-A) and downstream homology arm primers (DN-ygaY-S, DN-ygaY-A) were designed according to the upstream and downstream sequences of the ygaY gene (NCBI GeneID: 2847696); trc The promoter sequence was designed to amplify the primers pyrB-S and pyrB-A required for the pyrB gene. The above fragments were fused by overlapping PCR to obtain ygaY::P trc -pyrB (upstream homology arm-P trc - downstream homology arm). Construction of pGRB-ygaY: The DNA fragment containing the target sequence was prepared by annealing primers gRNA-ygaY-S and gRNA-ygaY-A. Prepare competent cells of E. coli W3110 ARG10, and construct the genetically engineered strain ARG-pyrB modified by overexpressing the pyrB gene according to the methods shown in 1.3 and 1.4 above. ygaY::P trc The electrophoresis diagram of the construction of the -pyrB fragment and the PCR verification of the positive strains is attached. Figure 1 Among them, the length of the upstream homology arm should be 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 should be 617bp, and the total length of the overlapping fragments should be 1967bp. During PCR verification, the length of the PCR amplification fragment of positive bacteria should be 1967bp, and the length of the PCR amplification fragment of the original bacteria should be 1424bp.

[0073] Example 2: Production of L-arginine using E. coli ARG-pyrB with overexpressed pyrB gene

[0074] E. coli ARG-pyrB is modified by overexpressing the pyrB gene based on E. coli ARG10. The ARG10 strain (described in detail in Chinese patent CN110964683B) is based on E. coli W3110 and contains the genes pyrAA and pyrAB encoding carbamoyl phosphate synthetase from Bacillus subtilis A260; contains the following arginine biosynthetic pathway enzymes: argC, argJ, argB, argD, argF, argG and argH, and the genes encoding the arginine biosynthetic pathway enzymes are derived from P trc The promoter is started; the genes speA, adiA, astA and argE encoding the decomposition of L-arginine are knocked out; and the gene lysE encoding the arginine transporter derived from effective bacterium is contained.

[0075] The same culture medium and culture method were used to culture Escherichia coli ARG-pyrB and control Escherichia coli ARG10 in shake flasks to produce L-arginine.

[0076] The composition of the culture medium: glucose 20 g / L, yeast extract 3 g / L, peptone 2 g / L, K2HPO4 6 g / L, MgSO4·7H2O 1 g / L, FeSO4·7H2O 20 mg / L, MnSO4·7H2O 20 mg / L, V B1 、V B3 、V B5 、V B12 、V H 1 mg / L of each, the rest is water, pH 7.0-7.2.

[0077] Shake flask culture method: inoculate 10% of the volume of the seed culture solution into a 500mL Erlenmeyer flask containing fermentation medium (final volume is 30mL), seal with nine layers of gauze, and culture at 37°C, 200r / min with shaking. During the fermentation process, the pH is maintained at 7.0-7.2 by adding ammonia water; and 60% (m / v) glucose solution is added to maintain the fermentation process.

[0078] After 28 hours of fermentation in a shake flask, 1 mL of the fermentation broth was taken from each sample and the cells were removed at 13,000 rpm to obtain the fermentation supernatant. The concentration of L-arginine in the fermentation supernatant was measured by high performance liquid chromatography (as shown in Table 1).

[0079] Table 1 Shake flask fermentation results of ARG-pyrB strain

[0080]

[0081] The results in Table 1 show that overexpression of the pyrB gene did not significantly affect the growth of the engineered strain; at the same time, the concentration of L-arginine in ARG-pyrB was increased from 31.2 g / L to 36.5 g / L, and the L-arginine yield was increased by 17%. The above experimental results show that the production of L-arginine by fermentation of genetically engineered bacteria modified by overexpressing the pyrB gene significantly improves the L-arginine production performance, indicating that the modified strain has a stronger L-arginine production capacity.

[0082] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A genetically engineered bacterium for producing L-arginine, characterized in that: The genetically engineered bacteria are strains E. coli W3110 ARG10 is overexpressed pyrB The strain has been modified in a genetic manner; E. coli W3110 ARG10 is based on Escherichia coli W3110 and contains B. subtilis A260 gene encoding carbamoyl phosphate synthetase pyrAA and pyrAB ; contains the following arginine biosynthetic pathway enzymes: argC, argJ, argB, argD, argF, argG and argH , the genes encoding enzymes of the arginine biosynthetic pathway are P trc Promoter start; Knocked out the gene encoding the breakdown of L-arginine speA, adiA, astA, and argE; Contains a gene encoding an arginine transporter from Corynebacterium efficacious lysE .

2. The genetically engineered bacterium according to claim 1, characterized in that: Said pyrB Gene integration in strains E. coli W3110ARG10 genome Y gene locus, and by P trc The promoter starts.

3. A method for producing L-arginine using the genetically engineered bacteria according to claim 1 or 2, comprising: Cultivating the genetically engineered bacteria in a culture medium to produce L-arginine; And, collecting the L-arginine from the genetically engineered bacteria and / or the culture medium.

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