Genetically engineered bacterium with high yield of L-histidine as well as construction method and application of genetically engineered bacterium

Abstract

The invention relates to the technical field of microbial metabolism engineering, and discloses a genetically engineered bacterium for high-yield L-histidine as well as a construction method and application of the genetically engineered bacterium. A nucleoside triphosphate pyrophosphate hydrolase coding gene mazG gene and a flagellin synthesis gene flhC gene are knocked out at the same time, an o-aminobenzoic acid phosphoribosyltransferase coding gene trpD gene and a ribulose phosphate 3-isomerase coding gene rpe gene are knocked down, and the recombinant protein is obtained. A ribose-5-phosphate isomerase gene coding rpiA gene and a lysine exponential coding gene lysE gene are overexpressed, and a promoter of 6-phosphoglucose isomerase pgi is replaced by a PfliC promoter, so that key substances for synthesis of L-histidine can be effectively accumulated, the fermentation yield of L-histidine is greatly increased, and compared with an initial strain, the yield of L-histidine is increased by 33.3%.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial metabolic engineering, and particularly relates to a genetically engineered bacterium with high yield of L-histidine, a construction method thereof, and an application thereof. Background Art

[0002] L-Histidine (L-His), also known as α-amino-β-imidazolepropionic acid, has a molecular formula of C6H9N3O2 and is a basic amino acid containing an imidazole nucleus. L-Histidine has various physiological functions and plays an important role in growth, tissue repair, treatment of ulcers and hyperacidity, etc. It can also be used as an additive for treating diseases such as allergies, rheumatoid arthritis, and anemia. Therefore, it is widely used in industries such as medicine and food.

[0003] The production methods of L-histidine mainly include protein hydrolysis method, chemical synthesis method, and microbial fermentation method. Among them, the protein hydrolysis method has a large demand for natural protein resources and a small output, resulting in a shortage of supply of L-histidine. The chemical synthesis method is prone to produce racemic compounds and is difficult to purify. The microbial fermentation method can obtain high-yield microorganisms through traditional mutation, screening, or metabolic engineering, and has the advantages of simple process, low raw material cost, mild reaction conditions, and easy separation and purification. The microbial fermentation method for producing L-histidine has broad prospects.

[0004] At present, the synthesis pathway of histidine has been widely studied in representative bacteria such as Escherichia coli, Corynebacterium glutamicum, and Salmonella. The transformation of strains for fermentative production of L-histidine mainly focuses on the following aspects: 1) Modifying the leader region of the histidine operon, for example, replacing the promoter of hisL with a strong promoter to relieve transcriptional attenuation; 2) Relieving the feedback inhibition of hisG and integrating multiple copies of the feedback-inhibition-resistant gene hisG (hisG fbr ); 3) Overexpressing the gene encoding phosphoribosyl pyrophosphate (PRPP) synthetase encoded by prs to enhance the supply of the precursor substance PRPP; 4) Enhancing the metabolic flux of the pentose phosphate pathway and overexpressing glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase.

[0005] However, the above-mentioned transformation only focuses on local metabolism, and there is easily instability during the fermentation production of L-histidine. The biosynthesis of histidine in Escherichia coli requires 10 biochemical reactions and 9 enzymes with 5-phosphoribosyl 1-pyrophosphate (PRPP) and adenosine triphosphate (ATP) as precursors. The synthesis route is long and involves numerous biochemical reactions. Therefore, the biosynthesis of L-histidine in Escherichia coli is strictly controlled by regulatory mechanisms, including transcriptional repression and enzyme inhibition. When local metabolic transformation ignores the global nature of metabolic engineering, the phenomenon that the biosynthesis yield of L-histidine is inhibited easily occurs. SUMMARY OF THE INVENTION

[0006] In order to solve the problem of improving the yield of biosynthesized L-histidine, the present invention provides a genetically engineered bacterium with high yield of L-histidine, a construction method thereof and an application thereof.

[0007] The specific technical solution of the present invention is as follows: On the one hand, the present invention provides a genetically engineered bacterium with high yield of L-histidine. The mazG gene and the flhC gene are knocked out on the chassis bacterium genome, the trpD gene and the rpe gene are knocked down, the rpiA gene and the lysE gene are overexpressed, and the promoter of the pgi gene is replaced with the P fliC promoter.

[0008] Preferably, the nucleotide sequence of the P fliC promoter is as shown in SEQ ID No.7.

[0009] The glycolysis pathway initiated by phosphoglucose isomerase (encoded by the pgi gene) is the main competing pathway of the pentose phosphate pathway, and the single knockout of the pgi gene will cause the problem of cell growth arrest. The P fliC promoter is a growth-coupled promoter. In the present invention, the phosphoglucose isomerase-encoding gene pgi gene is driven by the P fliC promoter, achieving the balance of cell growth and the metabolic flux of L-histidine. It can not only keep the strain cells in a good growth condition, but also make L-histidine have a high yield. The growth-related genes of Escherichia coli include the flgC, fliA, and fliC genes, which are involved in the formation of flagella. In the present invention, only the P fliC promoter can play a better role in balancing cell growth and the metabolic flux of L-histidine.

[0010] On the other hand, the present invention provides a construction method of a genetically engineered bacterium with high yield of L-histidine, and the following steps of transformation are carried out in the chassis bacterium: Knock out the nucleoside triphosphate pyrophosphohydrolase-encoding gene mazG gene; Knock out the flagellin synthesis gene flhC gene; Knock down the anthranilate phosphoribosyltransferase-encoding gene trpD; Knock down the ribulose phosphate 3-epimerase-encoding gene rpe; Overexpress the ribose-5-phosphate isomerase-encoding gene rpiA; Replace the promoter of the glucose-6-phosphate isomerase-encoding gene pgi with P fliC Promoter; Overexpress the lysine exporter-encoding gene lysE.

[0011] As an optimization of the above construction method, the method for knocking down the anthranilate phosphoribosyltransferase-encoding gene trpD is: mutate the start codon ATG of the trpD gene to GTG.

[0012] As an optimization of the above construction method, the method for knocking down the ribulose phosphate 3-epimerase-encoding gene rpe is: mutate the start codon ATG of the rpe gene to TTG.

[0013] As an optimization of the above construction method, the method for overexpressing the ribose-5-phosphate isomerase-encoding gene rpiA is: integrate the endogenous rpiA gene at the gene locus ppnN and replace P trc Promoter to control the rpiA gene.

[0014] As an optimization of the above construction method, the method for overexpressing the lysine exporter-encoding gene lysE is: integrate the lysE gene of exogenous Corynebacterium glutamicum at the gene locus yjiT and replace P trc Promoter to control the lysE gene.

[0015] As an optimization of the above construction method, the nucleotide sequence of the lysE gene is as shown in SEQ ID No.6.

[0016] As an optimization of the above construction method, the chassis bacterium is Escherichia coli.

[0017] The present invention also provides the application of the above genetic engineering bacterium in the preparation of L-histidine. After the transformation of "including knocking out the mazG gene and the flhC gene, knocking down the trpD gene and the rpe gene, overexpressing the rpiA gene and the lysE gene, and replacing the promoter of the pgi gene with P fliC Promoter", the yield of L-histidine can be increased from 2.04 g / L to 2.72 g / L, and the yield is increased by 33.3% compared with the starting strain chassis bacterium.

[0018] Compared with the prior art, the present invention has the following technical effects: In the present invention, by simultaneously knocking out the gene mazG encoding nucleoside triphosphate pyrophosphohydrolase, the gene flhC encoding flagellin, knocking down the gene trpD encoding anthranilate phosphoribosyltransferase, the gene rpe encoding phosphopentose 3-isomerase, overexpressing the gene rpiA encoding ribose-5-phosphate isomerase, the gene lysE encoding lysine exporter, and replacing the promoter of the gene pgi encoding glucose-6-phosphate isomerase with P fliC promoter, key substances for L-histidine synthesis can be effectively accumulated, greatly improving the fermentation yield of L-histidine. Compared with the starting strain, the L-histidine yield is increased by 33.3%. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shows the L-histidine yields and cell growth conditions OD of the starting strain LH0, recombinant strains LH1 and LH2 600 Figure; Figure 2 Shows the L-histidine yields and cell growth conditions OD of the starting strain LH0 and recombinant strains LH2, LH3, LH4, LH5 600 Figure.

[0020] Figure 3 Shows the L-histidine yields and cell growth conditions OD of the starting strain LH0 and recombinant strains LH5 and LH6-Δpgi, LH6-pgi GTG , LH6-pgi TTG , LH6-P flgC -pgi, LH6-P fliA -pgi, LH6-P fliC -pgi, LH6-P 600 Figure.

[0021] Figure 4 Shows the L-histidine yields and cell growth conditions OD of the starting strain LH0 and recombinant strain LH6-P fliC -pgi and LH7 600 Figure.

[0022] Figure 5 Shows the L-histidine yields and cell growth conditions OD of the starting strain LH0 and recombinant strains LH6-Δpgi-E, LH6-pgi GTG -E, LH6-pgi TTG -E, LH6-P flgC -pgi-E, LH6-P fliA -pgi-E and LH5-E 600 Figure. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention constructs a genetically engineered bacterium with high-yield L-histidine. The genetically engineered bacterium has the nucleoside triphosphate pyrophosphohydrolase-encoding gene mazG, the flagellin synthesis gene flhC, the downregulated anthranilate phosphoribosyltransferase-encoding gene trpD, and the ribulose 5-phosphate 3-epimerase-encoding gene rpe knocked down in the chassis bacterium genome, overexpresses the ribose-5-phosphate isomerase-encoding gene rpiA and the lysine exporter-encoding gene lysE, and replaces the promoter of glucose-6-phosphate isomerase pgi with P fliC promoter. Among them: The nucleotide sequence of the nucleoside triphosphate pyrophosphohydrolase-encoding gene mazG is shown in SEQ ID No.1; The nucleotide sequence of the flagellin synthesis gene flhC is shown in SEQ ID No.2; The method for downregulating the anthranilate phosphoribosyltransferase-encoding gene trpD is: mutating the start codon ATG of the trpD gene to GTG, and the nucleotide sequence of the trpD GTG gene obtained after mutation is shown in SEQ ID No.3; The method for downregulating the ribulose 5-phosphate 3-epimerase-encoding gene rpe is: mutating the start codon ATG of the rpe gene to TTG; the nucleotide sequence of the rpe TTG gene obtained after mutation is shown in SEQ ID No.4; The nucleotide sequence of the rpiA gene is shown in SEQ ID No.5; The nucleotide sequence of the lysE gene is shown in SEQ ID No.6; P fliC The nucleotide sequence of the promoter is shown in SEQ ID No.7.

[0024] The principle of constructing the genetically engineered bacterium with high-yield L-histidine in the present invention is as follows: First, block the branched metabolism of purine nucleotides to increase the intracellular ATP level; Second, indirectly promote the synthesis of PRPP by regulating the generation of 5-phosphoribose in the pentose phosphate pathway, and better accumulate the precursor substance PRPP required for the metabolism of L-histidine; Third, utilize P fliC promoter to better achieve the balance of cell growth and the metabolic flux of L-histidine.

[0025] The present invention will be further described below in conjunction with embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only a part of the embodiments of the present invention, rather than all of the embodiments. Therefore, all other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0026] In the following embodiments, the starting strain is E. coli W3110 LH0, and its genotype is E. coli W3110ΔlacIhisL::TrcΔhisG tdcD::Trc-hisG*ylbE::hisG gapC::hisG*ΔpurR ilvG::Trc-prsyghX::Trc-hisDCBHAFI yeeP::Trc-zwf yjiV::Trc-gndΔedd rpnD::Trc-purH ycjV::Trc-purA yncI::Trc-purB. It is a publicly available strain, and its construction can refer to the patent with the application publication number CN119220474A. In the following embodiments, the starting strain E. coli W3110LH0 is denoted as strain LH0.

[0027] In the following embodiments, the gene sources, sequence information, and functional information involved are shown in Table 1.

[0028] Table 1 Gene Name Nucleotide Sequence Involved Function mazG SEQ ID No.1 Nucleoside triphosphate pyrophosphohydrolase flhC SEQ ID No.2 Involved in flagellin synthesis trpD SEQ ID No.3 Anthranilate phosphoribosyltransferase rpe SEQ ID No.4 Phosphoribulokinase 3-isomerase rpiA SEQ ID No.5 Ribose-5-phosphate isomerase lysE SEQ ID No.6 Enhance L-histidine efflux <![CDATA[P fliC > SEQ ID No.7 Growth-coupled promoter <![CDATA[P trc > SEQ ID No.8 Strong promoter for gene overexpression In the following embodiments, the primer information involved is shown in Table 2.

[0029] Table 2 In the following embodiments, the liquid phase elution program for detecting the L-histidine content is shown in Table 3.

[0030] In the following embodiments, the CRISPR-Cas9 gene editing technology is a publicly available technical means. References: Yu Jiang et al. 2015 Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Applied Environmental Microbiology. 81: 2506-2514.

[0031] Table 3 Example 1 Using E. coli W3110 LH0 as the starting strain, the mazG gene was knocked out on the genome by using the CRISPR-Cas9 gene editing technology, and the recombinant E. coli strain E. coli W3110 LH0ΔmazG was constructed and subjected to shake flask fermentation. The specific operations are as follows: 1-1: Construction of the plasmid backbone pTarget-mazG: Using the pTarget F plasmid (Addgene Plasmid#62226) as the template, and mazG-pTar-F and mazG-pTar-R in Table 2 as primers, the pTarget plasmid was mutated. After PCR amplification, electrophoresis detection was carried out, and the size of the PCR amplification product band was observed with a gel imager. After success, 1 μL of DpnⅠ endonuclease was added to the PCR amplification product and digested at 37 °C for 1 h to eliminate the pTarget template plasmid. Using the pTarget-mazG backbone purified by the Clean up purification kit as the template, it was linearized with the pTarget-LINE-F and pTarget-LINE-R primers in Table 2. The amplified linearized product was subjected to agarose gel electrophoresis detection and purification and recovery to obtain the linearized pTarget-mazG backbone.

[0032] 1-2: Obtaining the Donor DNA fragment: Using the genome of the E. coli W3110 strain as the template, and Donor-mazG-1 / Donor-mazG- and Donor-mazG-3 / Donor-mazG-4 in Table 2 as primers respectively, the fragments Donor-mazG-up and Donor-mazG-down were obtained by PCR amplification; and the PCR products were subjected to electrophoresis detection and purification and recovery.

[0033] 1-3: Construction of the plasmid pTarget-mazG used for knocking out the key gene mazG The linearized pTarget-mazG backbone in step 1-1, the Donor-mazG-up fragment and the Donor-mazG-down fragment in step 1-2 were obtained through Nanjing Novoprotein Recombinant seamless cloning was performed using the MultiS One Step Cloning Kit. The ligated product was transformed into chemically competent DH5α and incubated in a 37°C incubator for 14 h. Single colonies on the plate were picked as templates, and colony PCR verification was performed using pTarget-YZF and pTarget-YZR in Table 2 as primers. The PCR amplification products were detected by agarose gel electrophoresis to verify the band size, and the PCR products with the same size as the target band were sent to Tsingke Biotechnology (Hangzhou) Co., Ltd. for further sequencing verification. After correct sequencing verification, single colonies with correct sequencing were picked into an LB tube containing spectinomycin resistance and cultured in a 37°C shaker for 15 h, and the plasmid was extracted to obtain the plasmid pTarget-mazG for knocking out the mazG gene.

[0034] 1 - 4: Preparation of electrocompetent LH0 cells and transformation of pCas plasmid: A small amount of LH0 bacterial solution was picked up with an inoculation loop from the glycerol cryopreservation tube and streaked on a solid LB medium without antibiotics, and cultured overnight (15 h) in a 37°C constant temperature incubator; Single colonies on the plate were picked and inoculated into a test tube containing 5 mL of LB medium, and cultured overnight (15 h) in a 37°C, 200 rpm constant temperature shaker incubator; 200 μL of the bacterial solution was transferred to 10 mL of LB medium without antibiotics (the amount of the bacterial solution was 2%), and cultured at 37°C and 200 rpm in a constant temperature shaker until the OD 600 was between 0.4 - 0.6. The shake flask was taken out and placed on ice for 10 - 15 min; 2 mL of the pre-cooled bacterial solution was placed in a centrifuge tube and centrifuged at 4°C and 4000 rpm for 2 min; The supernatant was discarded, 1 mL of pre-cooled sterile ultrapure water was added, resuspended and centrifuged at 4°C and 4500 rpm for 2 min, and this step was repeated twice; 100 μL of pre-cooled 10% glycerol solution was added and blown to resuspend to obtain electrocompetent LH0; 3 μL of pCas plasmid (Addgene Plasmid#62225) was put into a centrifuge tube containing 100 μL of LH0 competent cells; The sterile electroporation cuvette was pre-cooled on ice for 2 min; The BIO-RAD electroporator was turned on and adjusted to the EC2 setting; The competent cells mixed with pCas were transferred to the electroporation cuvette, and the electroporation cuvette was gently tapped to make the mixture evenly enter the bottom of the electroporation cuvette, and the external ice water of the electroporation cuvette was wiped dry; The electroporation cuvette was pushed into the electroporator, the PLUS button was pressed, and after hearing a beep, the electroporation cuvette was quickly withdrawn, and 700 μL of pre-prepared LB culture medium was added; It was placed in a 30°C incubator for 3 h, centrifuged at 5000 rpm for 3 min, 200 μL of the supernatant was taken, resuspended and spread on a plate containing 50 mg / L kanamycin, and cultured at 30°C for 15 h to obtain the LH0 / pCas strain.

[0035] 1 - 5: Preparation of LH0 / pCas competent cells and transformation with pTarget - mazG plasmid to complete the knockout of mazG gene: Pick a single colony from the LH0 / pCas plate in Step 1 - 4 and inoculate it into a 10 - mL LB test tube containing 50 mg / L kanamycin and 100 μL of 1 M arabinose. After culturing at 30 °C for 15 h, take 200 μL and inoculate it into a small LB shaker flask, and also add kanamycin and arabinose at the same final concentration. Culture at 30 °C for 3 h until the OD 600 is between 0.4 - 0.6. The subsequent preparation of competent cells is the same as that in 1 - 4. Finally, culture on a plate containing spectinomycin and kanamycin at 30 °C for 24 h. Design the verification primers mazG - YZF and mazG - YZR in Table 2 about 100 bp outside the Donor DNA. Pick a single colony on the plate as a template for colony PCR verification. After detection by agarose gel electrophoresis, send the PCR product to Tsingke Biological (Hangzhou) Company for sequencing for further verification to complete the knockout of the mazG gene.

[0036] 1 - 6: Elimination of pTarget - mazG plasmid and pCas plasmid: Inoculate the positive clone into a 10 - mL LB test tube containing kanamycin, and add 10 μL of 1 M IPTG mother liquor. Place it on a shaker at 30 °C and 180 rpm for 12 h; streak on a solid LB medium containing kanamycin and culture overnight at 30 °C; number the single colonies, and use a small pipette tip to pick some single colonies and streak them on solid LB media containing kanamycin and spectinomycin respectively, and place them at 30 °C and 37 °C for overnight culture; The single colonies that cannot grow in the area corresponding to the solid LB medium containing spectinomycin are the clones with successful elimination of pTarget - mazG, that is, LH1 / pCas. Inoculate the clone LH1 / pCas with successful elimination of pTarget - mazG into a 10 - mL LB test tube without resistance and place it on a shaker at 37 °C and 180 rpm for 12 h; streak on a solid LB medium without resistance and culture overnight at 37 °C; number the single colonies, and use a small pipette tip to pick some single colonies and streak them on solid LB media containing kanamycin and without resistance respectively, and place them at 30 °C and 37 °C for overnight culture; The single colonies that cannot grow in the area corresponding to the solid LB medium without resistance are the clones with successful elimination of pCas, which is the strain E. coli W3110 LH0ΔmazG, and the strain code is LH1.

[0037] 1-7: Shake flask fermentation and yield detection of recombinant strains: To verify the ability of the strain E. coli W3110LH0ΔmazG obtained in step 1-6 to produce L-histidine, single colonies of E. coli W3110 LH0ΔmazG and E. coli W3110LH0 were picked and inoculated into 10 mL LB test tubes, cultured overnight at 37 °C on a shaker at 180 rpm, transferred to the sterilized fermentation medium at a transfer volume ratio of 10% (volume ratio), and three parallels were set for each strain, cultured at 37 °C on a shaker at 200 rpm for 48 h. After fermentation, 1 mL of the fermentation broth was taken, centrifuged at 12000 rpm for 2 min, and the supernatant was used for high-performance liquid chromatography to detect the yield of L-histidine, and the precipitate was used to detect the OD 600 value. The results showed that the L-histidine yield of LH1 was 2.11 g / L, and the L-histidine yield of LH0 was 2.04 g / L.

[0038] Example 2 Using the CRISPR-Cas9 gene editing technology, the flhC gene was knocked out on the genome of LH1 to construct the recombinant Escherichia coli strain E. coli W3110 LH0ΔmazGΔflhC and perform shake flask fermentation. The specific operations are as follows: 2-1: Construction of plasmid backbone pTarget-flhC: Using the pTarget F plasmid (Addgene Plasmid #62226) as a template, pTarget plasmid was mutated with flhC-pTar-F and flhC-pTar-R primers in Table 2. After PCR amplification, electrophoresis detection was carried out, and the size of the PCR amplification product band was observed with a gel imager. After success, 1 μL of DpnⅠ endonuclease was added to the PCR amplification product and digested at 37 °C for 1 h to eliminate the pTarget template plasmid. Using the purified pTarget-flhC backbone by the Clean up purification kit as a template, it was linearized with the pTarget-LINE-F and pTarget-LINE-R primers in Table 2. The amplified linearized product was subjected to agarose gel electrophoresis detection and purification and recovery to obtain the linearized pTarget-flhC backbone.

[0039] 2-2: Obtaining the Donor DNA fragment: Using the genome of the E. coli W3110 strain as a template, the fragments Donor-flhC-up and Donor-flhC-down were obtained by PCR amplification with Donor-flhC-1 / Donor-flhC-2 and Donor-flhC-3 / Donor-flhC-4 primers in Table 2 respectively; and the PCR products were subjected to electrophoresis detection and purification and recovery.

[0040] 2-3: Construction of plasmid pTarget-flhC for knocking out the key gene flhC Recombine and seamlessly clone the linearized pTarget-flhC backbone in Step 2-1, the Donor-flhC-up fragment and the Donor-flhC-down fragment in Step 2-2 using the Nanjing Novoprotein MultiS One Step Cloning Kit; transform the ligated product into chemically competent DH5α and incubate it in a 37°C incubator for 14 h; pick a single colony on the plate as a template and use pTarget-YZF and pTarget-YZR in Table 2 as primers for colony PCR verification. Detect the size of the verification band by agarose gel electrophoresis of the PCR amplification product, and send the PCR product with the same size as the target band to Tsingke Biotechnology (Hangzhou) Co., Ltd. for further sequencing verification. After correct sequencing verification, pick a single colony with correct sequencing into an LB tube containing spectinomycin resistance and culture it on a shaker at 37°C for 15 h, then extract the plasmid to obtain the plasmid pTarget-flhC for knocking out the flhC gene.

[0041] 2-4: Preparation of LH1 / pCas competent cells and transformation of plasmid pTarget-flhC to complete the knockout of the flhC gene: Pick a single colony from the LH1 / pCas plate in Step 1-6 and prepare electrocompetent cells and electrotransformation according to Steps 1-5 in Example 1. Design the verification primers flhC-YZF and flhC-YZR in Table 2 about 100 bp outside the Donor DNA, pick a single colony on the plate as a template for colony PCR verification, and after agarose gel electrophoresis detection, send the PCR product to Tsingke Biotechnology (Hangzhou) Co., Ltd. for further sequencing verification to complete the knockout of the flhC gene.

[0042] 2-5: Eliminate plasmids pTarget-flhC and pCas according to Steps 1-6 in the example. The single colonies that cannot grow in the area corresponding to the LB solid medium with spectinomycin are the clones with successful elimination of pTarget-flhC, namely LH2 / pCas. The single colonies that cannot grow in the area corresponding to the LB solid medium without resistance are the clones with successful elimination of pCas, which is the strain E. coli W3110 LH1ΔmazGΔflhC, and the strain code is LH2.

[0043] Perform shake flask fermentation and detection on LH1 constructed in Example 1 and LH2 constructed in Example 2 according to Steps 1-7 in Example 1, using LH0 as a control group, OD 600 and the L-histidine content in the supernatant of the fermentation broth are as Figure 1 shown. Figure 1 ​Among them, LH1 represents the recombinant strain E. coli W3110 LH0ΔmazG, and LH2 represents the recombinant strain E. coli W3110 LH0ΔmazGΔflhC.

[0044] The fermentation results showed that the yields of L-histidine in the shake-flask fermentations of LH1 and LH2 were 2.11 g / L and 2.23 g / L respectively. The yield of L-histidine of the LH2 strain obtained by further knocking out the flhC gene from LH1 was 2.23 g / L, which was 9.3% higher than that of the control group LH0. Figure 1 It can be seen. This indicates that the mazG gene encodes a nucleoside triphosphate pyrophosphohydrolase, which can regulate the hydrolysis of nucleoside triphosphate pyrophosphate. The flhC gene is involved in flagellin synthesis. By knocking out the mazG gene and the flhC gene on the genome, the yield of L-histidine can be greatly improved. This shows that the knockout of the mazG gene and the flhC gene can ensure that there is enough ATP in metabolism for the synthesis of L-histidine.

[0045] Example 3 Knockdown of the trpD gene was achieved on the LH2 genome, that is, the start codon ATG of the anthranilate phosphoribosyltransferase-encoding gene trpD was mutated to GTG to construct the recombinant Escherichia coli strain E. coli W3110 LH0ΔmazGΔflhC trpD GTG And shake-flask fermentation was carried out. The specific operations are as follows: 3-1: Construction of the plasmid backbone pTarget-trpD: Using the pTarget F plasmid (Addgene Plasmid #62226) as a template, the pTarget plasmid was mutated with the primers trpD-pTar-F and trpD-pTar-R in Table 2. After PCR amplification, electrophoresis detection was carried out, and the size of the PCR amplification product bands was observed with a gel imager. After success, 1 μL of DpnⅠ endonuclease was added to the PCR amplification product and digested at 37 °C for 1 h to eliminate the pTarget template plasmid. Using the purified pTarget-trpD backbone after purification with the Clean up purification kit as a template, it was linearized with the primers pTarget-LINE-F and pTarget-LINE-R in Table 2. The amplified linearized product was subjected to agarose gel electrophoresis detection and purification and recovery to obtain the linearized pTarget-trpD backbone.

[0046] 3-2: Obtaining the Donor DNA fragment: Using the genome of E. coli W3110 strain as a template, and respectively using Donor-trpD-1 / Donor-trpD- and Donor-trpD-3 / Donor-trpD-4 in Table 2 as primers, PCR amplification was performed to obtain the fragment Donor-trpD-up and the fragment Donor-trpD-down; and the PCR products were subjected to electrophoresis detection and purification and recovery.

[0047] 3-3: Constructing the plasmid pTarget-trpD used for knocking out the key gene trpD The linearized pTarget-trpD backbone in step 3-1, the Donor-trpD-up fragment and the Donor-trpD-down fragment in step 3-2 were subjected to recombinant seamless cloning through the Nanjing Novoprotein MultiS One Step Cloning Kit; the ligated product was transformed into the chemically competent DH5α and placed in a 37°C incubator for 12 - 16 h; single colonies on the plate were picked as templates, and using pTarget-YZF and pTarget-YZR in Table 2 as primers, colony PCR verification was performed. The PCR amplification products were detected by agarose gel electrophoresis to verify the band size, and the PCR products with the same size as the target band were sent to Tsingke Biotech (Hangzhou) Co., Ltd. for further sequencing verification. After correct sequencing verification, single colonies with correct sequencing were picked into an LB test tube containing spectinomycin resistance and cultured in a 37°C shaker for 15 h, and the plasmid was extracted to obtain the plasmid pTarget-trpD for knocking down the trpD gene.

[0048] 3-4: Preparing LH2 / pCas competent cells and transforming the pTarget-trpD plasmid to complete the knockdown of the trpD gene: Single colonies were picked from the LH2 / pCas plate in step 2-5 of Example 2, and electrocompetent cells and electrotransformation were prepared according to step 1-5 of Example 1. Verification primers trpD-YZF and trpD-YZR in Table 2 were designed at about 100 bp outside the Donor DNA. Single colonies on the plate were picked as templates, colony PCR verification was performed, and after agarose gel electrophoresis detection, the PCR products were sent to Tsingke Biotech (Hangzhou) Co., Ltd. for further sequencing verification to complete the knockdown of the trpD gene.

[0049] 3 - 5: Eliminate the pTarget - trpD plasmid and the pCas plasmid according to steps 1 - 6 of Example 1. The single colonies that cannot grow in the area corresponding to the LB solid medium with spectinomycin are the clones with successful elimination of pTarget - trpD, namely LH3 / pCas. The single colonies that cannot grow in the area corresponding to the LB solid medium without resistance are the clones with successful elimination of pCas, which is the strain E. coli W3110 LH1ΔmazGΔflhC trpD GTG , and the strain is encoded as LH3.

[0050] Taking LH0 as the control group, ferment the LH3 strain constructed in this example in a shake flask and detect it according to steps 1 - 7 of Example 1. OD 600 and the L - histidine content in the supernatant of the fermentation broth are as Figure 2 shown.

[0051] Example 4 Knock down the rpe gene on the LH3 genome, that is, mutate the start codon ATG of the ribulose phosphate 3 - isomerase - encoding gene rpe to TTG, and construct the recombinant Escherichia coli strain E. coli W3110 LH0ΔmazGΔflhC trpD GTG rpe TTG And carry out shake - flask fermentation. The specific operations are as follows: 4 - 1: Construct the plasmid backbone pTarget - rpe TTG : Using the pTarget F plasmid (Addgene Plasmid#62226) as a template, mutate the pTarget plasmid with rpe - pTar - F and rpe - pTar - R in Table 2 as primers. After PCR amplification, perform electrophoresis detection and observe the size of the PCR amplification product bands with a gel imager. After success, add 1 μL of DpnⅠ endonuclease to the PCR amplification product and digest it at 37 °C for 1 h to eliminate the pTarget template plasmid. Using the pTarget - rpe backbone purified by the Clean up purification kit as a template, linearize it with the pTarget - LINE - F and pTarget - LINE - R primers in Table 2. Perform agarose gel electrophoresis detection and purification and recovery on the amplified linearized product to obtain the linearized rpe backbone.

[0052] 4 - 2: Obtain the Donor DNA fragment: Using the genome of the E. coli W3110 strain as a template, and using Donor - rpe - 1 / Donor - rpe - 2 and Donor - rpe - 3 / Donor - rpe - 4 in Table 2 as primers respectively, PCR amplify to obtain the fragment Donor - rpe TTG -up and the fragment Donor - rpe TTG-down; and the PCR products were detected by electrophoresis and purified and recovered.

[0053] 4-3: Construction of knockdown key gene rpe TTG Plasmid used: pTarget-rpe TTG Linearize the pTarget-rpe from step 4-1 TTG Skeleton, Donor-rpe of step 4-2 TTG -up fragment and Donor-rpe TTG -down clip by Nanjing Novi Sad The MultiS One Step Cloning Kit was used for seamless recombination cloning; the connected product was transformed into the competent DH5α and placed in a 37°C incubator for 15 hours; a single colony on the plate was picked as a template, and pTarget-YZF and pTarget-YZR in Table 2 were used as primers for colony PCR verification. The PCR amplification product was verified by agarose gel electrophoresis to verify the band size, and the PCR product with the same size as the target band was sent to Qingke Biotechnology (Hangzhou) for sequencing and further verification. After the sequencing verification was correct, a single colony with correct sequencing was picked and cultured in an LB test tube containing spectinomycin resistance at 37°C for 15 hours, and the plasmid was extracted to obtain the plasmid for knocking out rpe TTG Plasmid pTarget-rpe TTG .

[0054] 4-4: Preparation of LH3 / pCas competent cells and transformation of pTarget-rpe TTG Plasmid to complete the knockdown of rpe gene: Pick a single colony from the LH3 / pCas plate in steps 3-5 of Example 3, and prepare electroporation competent cells and electroporation according to steps 1-5 in Example 1. Design the verification primers rpe in Table 2 about 100 bp outside the donor DNA TTG -YZF and RPE TTG -YZR, a single colony on the plate was picked as a template for colony PCR verification. After agarose gel electrophoresis detection, the PCR product was sent to Qingke Biotechnology (Hangzhou) Company for sequencing for further verification to complete the knockdown of the rpe gene.

[0055] 4-5: Elimination of pTarget-rpe according to Example 1-6 TTG Plasmid and pCas plasmid. The single colony that cannot grow in the area corresponding to the LB solid medium containing spectinomycin is pTarget-rpe TTGSuccessfully eliminated clone, namely LH4 / pCas. The single colonies that could not grow in the area corresponding to the LB solid medium without resistance were the clones with pCas successfully eliminated, which were the strain E. coli W3110 LH0ΔmazGΔflhC trpD GTG rpe TTG , and the strain was encoded as LH4.

[0056] Taking LH0 as the control group, the LH4 strain constructed in this example was subjected to shake flask fermentation and detection according to the steps 1-7 of Example 1. OD 600 and the L-histidine content in the supernatant of the fermentation broth were as Figure 2 shown.

[0057] Example 5 Overexpress rpiA in the LH4 genome, that is, integrate the endogenous ribose-5-phosphate isomerase-encoding gene rpiA at the gene locus ppnN and be controlled by the P trc promoter to construct the recombinant Escherichia coli strain E. coli W3110 LH0ΔmazGΔflhCtrpD GTG rpe TTG ppnN::P trc -rpiA and carry out shake flask fermentation. The specific operations are as follows: 5-1: Construct the plasmid backbone pTarget-rpiA: Using the pTarget F plasmid (Addgene Plasmid#62226) as the template, mutate the pTarget plasmid with rpiA-pTar-F and rpiA-pTar-R in Table 2 as primers. After PCR amplification, electrophoresis detection was carried out, and the size of the PCR amplification product bands was observed with a gel imager. After success, add 1 μL of DpnⅠ endonuclease to the PCR amplification product and digest it at 37 °C for 1 h to eliminate the pTarget template plasmid. Using the pTarget-rpiA backbone purified by the Clean up purification kit as the template, linearize it with the pTarget-LINE-F and pTarget-LINE-R primers in Table 2, and perform agarose gel electrophoresis detection and purification and recovery on the amplified linearized product to obtain the linearized rpiA backbone.

[0058] 5-2: Obtain the Donor DNA fragment: Using the genome of the E. coli W3110 strain as the template, respectively use Donor-rpiA-1 / Donor-rpiA-2, Donor-rpiA-3 / Donor-rpiA-4 and rpiA-5 / rpiA-6 in Table 2 as primers, and PCR amplification to obtain the fragment Donor-rpiA-up, the fragment Donor-rpiA-down and the fragment P trc-rpiA; and subject the PCR product to electrophoresis detection and purification and recovery.

[0059] 5-3: Construction of the plasmid pTarget-ppnN::P used for overexpressing rpiA trc -rpiA Linearize the pTarget-ppnN::P in step 5-1 trc -rpiA backbone, the Donor-rpiA-up fragment in step 5-2, Donor-rpiA-down and the P trc -rpiA fragments are subjected to recombinant seamless cloning using the Nanjing Novoprotein MultiS One StepCloning Kit; transform the ligated product into chemically competent DH5α and place it in a 37°C incubator for 15 h; pick a single colony on the plate as a template and use pTarget-YZF and pTarget-YZR in Table 2 as primers for colony PCR verification. Detect the size of the PCR amplification product by agarose gel electrophoresis, and send the PCR product with the same size as the target band to Tsingke Biotechnology (Hangzhou) Co., Ltd. for further sequencing verification. After correct sequencing verification, pick a single colony with correct sequencing into an LB test tube containing spectinomycin resistance and culture it on a shaker at 37°C for 15 h, extract the plasmid, and the plasmid pTarget-ppnN::P used for overexpressing rpiA can be obtained -rpiA. trc -rpiA.

[0060] 5-4: Preparation of LH4 / pCas competent cells and transformation of pTarget-ppnN::P trc -rpiA plasmid to complete the overexpression of rpiA on the genome: Pick a single colony from the LH4 / pCas plate in step 4-6 and prepare electrocompetent cells and electrotransformation according to step 1-5. Design the verification primers rpiA-YZF and rpiA-YZR in Table 2 about 100 bp outside the Donor DNA, pick a single colony on the plate as a template for colony PCR verification, after agarose gel electrophoresis detection, send the PCR product to Tsingke Biotechnology (Hangzhou) Co., Ltd. for further sequencing verification to complete the overexpression of the rpiA gene.

[0061] 5-5: Eliminate the pTarget-ppnN::P trc -rpiA plasmid and pCas plasmid according to steps 1-6 of Example 1. Single colonies that cannot grow in the area corresponding to the LB solid medium with spectinomycin are pTarget-P fliC- Clones with successful pgi elimination, i.e., LH5 / pCas. Single colonies that cannot grow in the area corresponding to the LB solid medium without resistance are clones with successful pCas elimination, which are the strain E. coli W3110 LH0ΔmazGΔflhC trpD GTG rpe TTG ppnN::P trc - rpiA, and the strain encodes LH5.

[0062] Using LH0 as the control group, the LH5 strain constructed in this example was subjected to shake-flask fermentation and detection according to steps 1-7 of Example 1, and OD 600 and the L-histidine content in the supernatant of the fermentation broth are as Figure 2 shown. Among them, LH3 represents the recombinant strain E. coli W3110 LH0ΔmazGΔflhC trpD GTG , LH4 represents the recombinant strain E. coli W3110 LH0ΔmazGΔflhC trpD GTG rpe TTG , LH5 represents the recombinant strain E. coli W3110 LH0ΔmazGΔflhC trpD GTG rpe TTG ppnN::P trc - rpiA.

[0063] From Figure 2 it can be seen that the yields of L-histidine in the shake-flask fermentations of LH3, LH4, and LH5 are 2.34 g / L, 2.45 g / L, and 2.51 g / L respectively. Thus, it can be seen that the histidine yield of LH5 is 23% higher than that of the control group LH0. The results of the yield determination show that by knocking down the trpD and rpe genes on the genome and overexpressing the rpiA gene, the yield of L-histidine can be effectively accumulated. The knockdown of the trpD and rpe genes and the overexpression of the rpiA gene can cause the accumulation of precursors, which is more conducive to the production of L-histidine.

[0064] Example 6 Knock out the gene pgi encoding glucose-6-phosphate isomerase on the LH5 genome to construct the E. coli strain E. coli W3110 LH0ΔmazGΔflhC trpD GTG rpe TTG ppnN::P trc - rpiAΔpgi; Replace the start codon ATG of pgi with GTG on the LH5 genome to construct the E. coli strain E. coli W3110 LH0ΔmazGΔflhC trpD GTG rpe TTGppnN::P trc -rpiA pgi GTG ; Replace the start codon ATG of pgi with TTG on the LH5 genome to construct the Escherichia coli strain E. coli W3110LH0ΔmazGΔflhC trpD GTG rpe TTG ppnN::P trc -rpiA pgi TTG ; Replace the promoter of pgi with P on the LH5 genome flgC , and construct the Escherichia coli strain E. coli W3110 LH0ΔmazGΔflhC trpD GTG rpe TTG ppnN::P trc -rpiA P flgC -pgi; Replace the promoter of pgi with P on the LH5 genome fliA , and construct the Escherichia coli strain E. coli W3110 LH0ΔmazGΔflhC trpD GTG rpe TTG ppnN::P trc -rpiA P fliA -pgi; Replace the promoter of pgi with P on the LH5 genome fliC Promoter, and construct the recombinant Escherichia coli strain E. coli W3110 LH0ΔmazGΔflhC trpD GTG rpe TTG ppnN::P trc -rpiA P fliC ::P pgi And carry out shake-flask fermentation, and the specific operations are as follows: 6-1: Construct the plasmid backbone pTarget-pgi: Using the pTarget F plasmid (Addgene Plasmid #62226) as a template, mutate the pTarget plasmid with pgi-pTar-F and pgi-pTar-R primers in Table 2, detect by electrophoresis after PCR amplification, and observe the size of the PCR amplification product bands with a gel imager. After success, add 1 μL of DpnⅠ endonuclease to the PCR amplification product and digest it at 37 °C for 1 h to eliminate the pTarget template plasmid. Using the pTarget-pgi backbone purified by the Clean up purification kit as a template, linearize it with the pTarget-LINE-F / pTarget-LINE-R primer pair in Table 2, detect the amplified linearized product by agarose gel electrophoresis and purify and recover it to obtain the linearized pTarget-pgi backbone.

[0065] 6-2: Obtain Donor DNA fragment: Using the genome of E. coli W3110 strain as a template and using Donor-pgi-1 / Donor-pgi-2 and Donor-pgi-3 / Donor-pgi-4 in Table 2 as primers, PCR amplification was performed to obtain fragments Donor-pgi-up and Donor-pgi-down; and the PCR products were detected by electrophoresis and purified and recovered.

[0066] 6-3: Construction of plasmid pTarget-pgi for knocking out pgi The pTarget-pGI backbone linearized in step 6-1, the Donor-pGI-up fragment and the Donor-pGI-down fragment in step 6-2 were purified by Nanjing Novozymes The MultiS One Step Cloning Kit was used for seamless recombination cloning; the connected product was transformed into the competent DH5α and placed in a 37°C incubator for 15 hours; a single colony on the plate was picked as a template, and pTarget-YZF and pTarget-YZR in Table 2 were used as primers for colony PCR verification. The PCR amplification product was verified by agarose gel electrophoresis to verify the band size, and the PCR product with the same size as the target band was sent to Qingke Bio (Hangzhou) Company for sequencing and further verification. After the sequencing verification was correct, a single colony with correct sequencing was picked and cultured in an LB test tube containing spectinomycin resistance at 37°C for 15 hours, and the plasmid was extracted to obtain the plasmid pTarget-pgi for knocking out pgi.

[0067] 6-4: Prepare LH5 / pCas competent cells and transform pTarget-pgi plasmid to complete the replacement of pgi promoter: Pick a single colony from the LH5 / pCas plate in step 5-5, and prepare electroporation competent cells and electroporation according to steps 1-5. Design the verification primers pgi-QC-YZF and pgi-QC-YZR in Table 2 about 100bp outside the Donor DNA, pick a single colony on the plate as a template, perform colony PCR verification, and after agarose gel electrophoresis detection, send the PCR product to Qingke Bio (Hangzhou) Company for sequencing and further verification to complete the knockout of the pgi gene.

[0068] 6-5: Eliminate the pTarget-pgi plasmid and the pCas plasmid according to steps 1-6. The single colony that cannot grow in the area corresponding to the LB solid medium with spectinomycin is the clone from which pTarget-pgi has been successfully eliminated. The single colony that cannot grow in the area corresponding to the LB solid medium without resistance is the clone from which pCas has been successfully eliminated, that is, the strain ΔmazGΔflhCtrpD GTG rpe TTGppnN::P trc -rpiAΔpgi, the strain is encoded as LH6-Δpgi.

[0069] 6-6: Construction of the plasmid backbone pTarget-pgi-QJ: Using the pTarget F plasmid (Addgene Plasmid #62226) as a template, pgi-QJ-pTar-F and pgi-QJ-pTar-R in Table 2 were used as primers to mutate the pTarget plasmid. After PCR amplification, electrophoresis was performed for detection, and the size of the PCR amplification product bands was observed using a gel imager. After success, 1 μL of DpnⅠ endonuclease was added to the PCR amplification product and digested at 37 °C for 1 h to eliminate the pTarget template plasmid. Using the purified pTarget-pgi-QJ backbone by the Clean up purification kit as a template, it was linearized using the pTarget-LINE-F / pTarget-LINE-R primers in Table 2. The amplified linearized product was subjected to agarose gel electrophoresis detection and purification and recovery to obtain the linearized pTarget-pgi-QJ backbone.

[0070] 6-7: Obtaining the Donor DNA fragment: Using the genome of E. coli W3110 strain as a template, and using Donor-pgi-GTG-1 / Donor-pgi-GTG-2, Donor-pgi-GTG-3 / Donor-pgi-GTG-4, Donor-pgi-TTG-1 / Donor-pgi-TTG-2, Donor-pgi-TTG-3 / Donor-pgi-TTG-4 in Table 2 as primers respectively, PCR amplification was carried out to obtain the fragment Donor-pgi-GTG-up, the fragment Donor-pgi-GTG-down, the fragment Donor-pgi-TTG-up and the fragment Donor-pgi-TTG-down; and the PCR products were subjected to electrophoresis detection and purification and recovery.

[0071] 6-8: Construction of the plasmids pTarget-pgi GTG and pTarget-pgi TTG used to replace the start codon of pgi respectively: The linearized pTarget-pgi-QJ backbone in step 6-6 and the Donor-pgi-GTG-up and Donor-pgi-GTG-down fragments in step 6-7 were used through Nanjing Novoprotein Use the MultiS One Step Cloning Kit for recombinant seamless cloning; transform the ligated products into chemically competent DH5α respectively, and place them in an incubator at 37°C for 15 h; pick single colonies on the plate as templates, and use pTarget-YZF and pTarget-YZR in Table 2 as primers for colony PCR verification. The PCR amplification products are detected by agarose gel electrophoresis to verify the band size, and the PCR products with the same size as the target band are sent to Tsingke Biotechnology (Hangzhou) Co., Ltd. for sequencing for further verification. After correct sequencing verification, pick the single colonies with correct sequencing into an LB test tube containing spectinomycin resistance and culture them on a shaker at 37°C for 15 h, then extract the plasmid to obtain the plasmid pTarget-pgi for knocking down pgi GTG The linearized pTarget-pgi-QJ backbone in Step 6-6, the Donor-pgi-TTG-up fragment and the Donor-pgi-TTG-down fragment in Step 6-7 are used to construct the plasmid pTarget-pgi for knocking down pgi TTG The construction method is the same as that of the plasmid pTarget-pgi GTG Same.

[0072] 6-9: Prepare LH5 / pCas competent cells and transform pTarget-pgi GTG 、pTarget-pgi TTG plasmids respectively, so as to replace the start codon of pgi: Pick single colonies from the LH5 / pCas plate in Step 5-5, and prepare electrocompetent cells and electrotransformation according to Step 1-5. Design the verification primers pgi-QJ-YZF and pgi-QJ-YZR in Table 2 about 100 bp outside the Donor DNA. Pick single colonies on the plate as templates for colony PCR verification. After agarose gel electrophoresis detection, send the PCR products to Tsingke Biotechnology (Hangzhou) Co., Ltd. for sequencing for further verification to complete the replacement of the start codon of the pgi gene.

[0073] 6-10: Eliminate pTarget-pgi GTG 、pTarget-pgi TTG plasmids and pCas plasmid according to Step 1-6. Single colonies that cannot grow in the area corresponding to the LB solid medium with spectinomycin are clones with successful elimination of pTarget-pgi. Single colonies that cannot grow in the area corresponding to the LB solid medium without resistance are clones with successful elimination of pCas, that is, the strains ΔmazGΔflhC trpD GTG rpe TTG ppnN::P trc -rpiA pgi GTG, ΔmazGΔflhC trpD GTG rpe TTG ppnN::P trc -rpiA pgi TTG , the strains are respectively encoded as LH6-pgi GTG 、LH6-pgi TTG 。

[0074] 6-11: Construction of plasmid backbone pTarget-P-pgi: Using the pTarget F plasmid (Addgene Plasmid#62226) as a template, P-pgi-pTar-F and P-pgi-pTar-R in Table 2 were used as primers to mutate the pTarget plasmid. After PCR amplification, electrophoresis detection was carried out, and the size of the PCR amplification product bands was observed with a gel imager. After success, 1 μL of DpnⅠ endonuclease was added to the PCR amplification product and digested at 37 °C for 1 h to eliminate the pTarget template plasmid. Using the purified pTarget-P-pgi backbone by the Clean up purification kit as a template, it was linearized with the pTarget-LINE-F / pTarget-LINE-R primer pair in Table 2. The amplified linearized product was subjected to agarose gel electrophoresis detection and purification and recovery to obtain the linearized pTarget-P-pgi backbone.

[0075] 6-12: Obtaining the Donor DNA fragment: Using the genome of E. coli W3110 strain as a template, respectively using Donor-P flgC -pgi-1 / Donor-P flgC -pgi-2 / and Donor-P flgC -pgi-3 / Donor-P flgC -pgi-4 as primers, Donor-P fliA -pgi-1 / Donor-P fliA -pgi-2 / and Donor-P fliA -pgi-3 / Donor-P fliA -pgi-4 as primers, Donor-P fliC -pgi-1 / Donor-P fliC -pgi-2, Donor-P fliC -pgi-3 / Donor-P fliC -pgi-4 and P fliC -pgi-5 / P fliC -pgi-6 as primers, PCR amplification was respectively used to obtain the fragment Donor-P flgC -pgi-up, the fragment Donor-P flgC-pgi-down, fragment Donor-P fliA -pgi-up, fragment Donor-P fliA -pgi-down, fragment Donor-P fliC -pgi-up, fragment Donor-P fliC -pgi-down and fragment P fliC -pgi; and subject the PCR product to electrophoresis detection and purification and recovery.

[0076] 6 - 13: Construct plasmids pTarget-P used for replacing the pgi promoter respectively flgC -pgi, pTarget-P fliA -pgi, pTarget-P fliC -pgi: The linearized pTarget-P-pgi backbone in steps 6 - 10, Donor-P in steps 6 - 12 flgC -pgi-up and Donor-P flgC -pgi-down fragment through Nanjing Novoprotein MultiS One StepCloning Kit Perform recombinant seamless cloning; transform the ligated product into chemically competent DH5α, place it in a 37°C incubator for 15 h; pick single colonies on the plate as templates, use pTarget-YZF and pTarget-YZR in Table 2 as primers for colony PCR verification. Detect the size of the PCR amplification product by agarose gel electrophoresis, and send the PCR product with the same size as the target band to Tsingke Biotechnology (Hangzhou) Co., Ltd. for further sequencing verification. After correct sequencing verification, pick the single colony with correct sequencing into an LB test tube containing spectinomycin resistance and culture it on a shaker at 37°C for 15 h, extract the plasmid, and the plasmid pTarget-P for replacing the pgi promoter can be obtained flgC -pgi. The linearized pTarget-P-pgi backbone in steps 6 - 10 and Donor-P in steps 6 - 12 fliA -pgi-up and Donor-P fliA -pgi-down fragment is used to construct the plasmid pTarget-P for replacing the pgi promoter fliA -pgi. The linearized pTarget-P-pgi backbone in steps 6 - 10 and Donor-P in steps 6 - 12 fliC -pgi-up, Donor-P fliC -pgi-down is used to construct the plasmid pTarget-P for replacing the pgi promoter fliC -pgi. The construction method is the same as that of the plasmid pTarget-P flgC -pgi.

[0077] 6 - 14: Preparation of LH5 / pCas competent cells and transformation of pTarget - P respectively flgC -pgi, pTarget - P fliA -pgi, pTarget - P fliC - Replacement of the pgi promoter in the pg plasmid: Pick a single colony from the LH5 / pCas plate in step 5 - 5, prepare electrocompetent cells and perform electrotransformation according to step 1 - 5. Design the verification primers P - pgi - YZF and P - pgi - YZR in Table 2 about 100 bp outside the Donor DNA. Pick a single colony on the plate as the template, perform colony PCR verification, and after detecting by agarose gel electrophoresis, send the PCR product to Tsingke Biotechnology (Hangzhou) Co., Ltd. for sequencing for further verification to complete the replacement of the pgi promoter.

[0078] 6 - 15: Eliminate pTarget - P according to step 1 - 6 flgC -pgi, pTarget - P fliA -pgi, pTarget - P fliC - Single colonies that cannot grow in the area corresponding to the LB solid medium with spectinomycin are pTarget - P flgC -pgi, pTarget - P fliA -pgi, pTarget - P fliC - Clones with successful elimination of pg. Single colonies that cannot grow in the area corresponding to the LB solid medium without resistance are clones with successful elimination of pCas, that is, strains ΔmazGΔflhC trpD GTG rpe TTG ppnN::P trc -rpiA P flgC ::P pgi 、ΔmazGΔflhC trpD GTG rpe TTG ppnN::P trc -rpiA P fliA ::P pgi 、ΔmazGΔflhC trpD GTG rpe TTG ppnN::P trc -rpiA P fliC ::P pgi ,with strain codes LH6 - P flgC -pgi, LH6 - P fliA -pgi, LH6 - P fliC -pgi.

[0079] The LH6-Δpgi and LH6-pgi constructed in this example GTG 、LH6-pgi TTG 、LH6-P flgC -pgi、LH6-P fliA -pgi、LH6-P fliC -pgi and LH5 were subjected to shake-flask fermentation and detection according to the steps 1-7 of Example 1, with LH0 as the control group. The OD 600 and the L-histidine content in the supernatant of the fermentation broth are as Figure 3 shown. Figure 3 shows the cell growth conditions OD GTG 、LH6-pgi TTG 、LH6-P flgC -pgi、LH6-P fliA -pgi、LH6-P fliC -pgi of the starting strain LH0, the recombinant strain LH5 and the LH6-Δpgi, LH6-pgi 600 constructed in this example, which are 8.74, 8.09, 5.91, 7.23, 3.66, 5.83, 2.38 and 8.71 respectively. The yields of L-histidine are 2.05 g / L, 2.51 g / L, 1.48 g / L, 2.08 g / L, 1.75 g / L, 1.41 g / L, 0.86 g / L and 2.58 g / L respectively. It can be seen that although the L-histidine yield of LH6-P fliC -pgi has not been significantly improved, its growth has recovered to the state of the starting strain LH0, indicating that the fliC promoter has the effect of balancing growth during the regulation of pgi; while other growth-coupled promoters, such as the PfliA promoter and the PflgC promoter, when used to regulate the driving of the pgi gene, the obtained strains LH6-P flgC -pgi、LH6-P fliA -pgi have greatly reduced yields, and at the same time, the growth state has also deteriorated significantly.

[0080] Example 7 Integrate and overexpress the lysine exporter-encoding gene lysE of Corynebacterium glutamicum from outside on the genome of LH6-P fliC -pgi, that is, integrate the gene lysE of Corynebacterium glutamicum from outside at the gene locus yjiT and be controlled by the P trc promoter to construct the recombinant Escherichia coli strain E.coli W3110 LH0ΔmazGΔflhC trpD GTG rpe TTG ppnN::P trc -rpiAP fliC::P pgi yjiT::P trc -lysE and perform shake flask fermentation. The specific operations are as follows: 7-1: Construct the plasmid backbone pTarget-lysE: Using the pTarget F plasmid (Addgene Plasmid#62226) as a template, mutate the pTarget plasmid with lysE-pTar-F and lysE-pTar-R in Table 2 as primers. After PCR amplification, perform electrophoresis detection and observe the size of the PCR amplification product bands with a gel imaging system. After success, add 1 μL of DpnⅠ endonuclease to the PCR amplification product and digest it at 37 °C for 1 h to eliminate the pTarget template plasmid. Using the purified pTarget-lysE backbone by the Clean up purification kit as a template, linearize it with the pTarget-LINE-F / pTarget-LINE-R primer pair in Table 2. Perform agarose gel electrophoresis detection and purification and recovery on the amplified linearized product to obtain the linearized lysE backbone.

[0081] 7-2: Obtain the Donor DNA fragment: Using the genome of E. coli W3110 strain as a template, use Donor-lysE-1 / Donor-lysE-2, Donor-lysE-3 / Donor-lysE-4, and lysE-5 / lysE-6 in Table 2 as primers respectively, and PCR amplify to obtain the fragment Donor-lysE-up, the fragment Donor-lysE-down, and the fragment P trc -lysE; and perform electrophoresis detection and purification and recovery on the PCR product.

[0082] 7-3: Construct the plasmid pTarget-yjiT::P used for overexpressing lysE trc -lysE The linearized pTarget-yjiT::P trc -lysE backbone in step 7-1, the Donor-lysE-up fragment, Donor-lysE-down, and the P trc -lysE fragment by Nanjing Novoprotein Use the MultiS One Step Cloning Kit for recombinant seamless cloning; transform the ligated product into chemically competent DH5α and place it in an incubator at 37°C for 12 - 16 h; pick single colonies on the plate as templates and use pTarget-YZF and pTarget-YZR in Table 2 as primers for colony PCR verification. The PCR amplification products are detected by agarose gel electrophoresis to verify the band size, and the PCR products with the same size as the target band are sent to Tsingke Biotechnology (Hangzhou) Co., Ltd. for sequencing for further verification. After correct sequencing verification, pick the single colonies with correct sequencing into an LB test tube containing spectinomycin resistance and culture them on a shaker at 37°C for 14 - 16 h, then extract the plasmid to obtain the plasmid pTarget-yjiT::P used for overexpressing rpiA trc -lysE.

[0083] 7-4: Prepare LH6 / pCas competent cells and transform the pTarget-yjiT::P trc -lysE plasmid to complete the overexpression of lysE on the genome: Pick single colonies from the LH6 / pCas plate in Step 6-6, prepare electrocompetent cells and perform electrotransformation according to Step 1-5. Design the verification primers lysE-YZF and lysE-YZR in Table 2 about 100 bp outside the Donor DNA, pick single colonies on the plate as templates for colony PCR verification, and after agarose gel electrophoresis detection, send the PCR products to Tsingke Biotechnology (Hangzhou) Co., Ltd. for sequencing for further verification to complete the overexpression of the lysE gene.

[0084] 7-5: Eliminate the pTarget-yjiT::P trc -lysE plasmid and pCas plasmid according to Step 1-6. The single colonies that cannot grow in the area corresponding to the LB solid medium with spectinomycin are the clones with successful elimination of lysE, namely LH7 / pCas. The single colonies that cannot grow in the area corresponding to the LB solid medium without resistance are the clones with successful elimination of pCas, which is the strain E. coli W3110LH0ΔmazGΔflhC trpD GTG rpe TTG ppnN::P trc -rpiA P fliC ::P pgi yjiT::P trc -lysE, and the strain code is LH7.

[0085] Perform shake flask fermentation and detection on LH7 and LH6-P fliC -pgi constructed in Example 7 according to Steps 1-7 of Example 1, using LH0 as the control group, OD 600and the L-histidine content in the supernatant of the fermentation broth is as Figure 4 shown.

[0086] As Figure 4 can be seen, the yields of L-histidine in the shake-flask fermentation of LH6 and LH7 are 2.54 g / L and 2.72 g / L respectively. The histidine yield of LH7 is increased by 33.3% compared with the control group LH0.

[0087] The pTarget-yjiT::P trc -lysE plasmid constructed in Example 7 was integrated and overexpressed with the gene lysE on the genomes of strains LH6-Δpgi, LH6-pgi GTG , LH6-pgi TTG , LH6-P flgC -pgi, LH6-P fliA -pgi, and LH5 genome according to the methods of steps 7-1 to 7-5, and the obtained strains were respectively encoded as LH6-Δpgi-E, LH6-pgi GTG -E, LH6-pgi TTG -E, LH6-P flgC -pgi-E, LH6-P fliA -pgi-E and LH5-E. The obtained strains were subjected to shake-flask fermentation and detection. Using LH0 as the control group, OD 600 and the L-histidine content in the supernatant of the fermentation broth is as Figure 5 shown.

[0088] As Figure 5 can be seen, the OD GTG values of LH6-Δpgi-E, LH6-pgi TTG -E, LH6-pgi flgC -E, LH6-P fliA -pgi-E, LH6-P 600 -pgi-E and LH5-E are 5.83, 7.1, 3.53, 5.7, 2.33, 7.9 respectively, and the yields of L-histidine are 1.61 g / L, 2.22 g / L, 1.89 g / L, 1.57 g / L, 0.99 g / L, 2.62 g / L respectively. The growth-related genes of Escherichia coli include flgC, fliA, and fliC genes, which are involved in the formation of flagella. As Figure 4 and Figure 5 can be seen from the detection results, in the present invention, only the P fliC promoter can play a better role in balancing cell growth and the metabolic flux of L-histidine, resulting in a significant increase in the yield of L-histidine.

[0089] Unless otherwise specified, the raw materials and equipment used in the present invention are common raw materials and equipment in the art; unless otherwise specified, the methods used in the present invention are conventional methods in the art.

[0090] The above are only preferred embodiments of the present invention and do not impose any limitation on the present invention. Any simple modification, change, and equivalent transformation made to the above embodiments according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A genetically engineered bacterium for high production of L-histidine, characterized in that: In the genome of the chassis bacteria, the mazG gene and flhC gene were knocked out, the trpD gene and rpe gene were knocked down, the rpiA gene and lysE gene were overexpressed, and the promoter of the pgi gene was replaced by P fliC Promoter.

2. The genetically engineered bacteria for high L-histidine production according to claim 1, characterized in that: P fliC The nucleotide sequence of the promoter is shown in SEQ ID No.

7.

3. A method for constructing a genetically engineered bacterium with high L-histidine production, characterized in that: The transformation in the chassis bacteria includes the following steps: Knockout of the mazG gene encoding nucleoside triphosphate pyrophosphohydrolase; Knock out the flagellin synthesis gene flhC gene; Knock down the trpD gene encoding anthranilate phosphoribosyltransferase; Knock down the rpe gene encoding ribulose 3-phosphate isomerase; Overexpression of the ribose-5-phosphate isomerase gene encoding rpiA gene; Replace the promoter of 6-phosphoglucose isomerase pgi with P fliC Promoter; Overexpression of the lysine exporter encoding gene lysE.

4. The construction method according to claim 3, characterized in that: The method for knocking down the anthranilate phosphoribosyltransferase encoding gene trpD is: mutating the start codon ATG of the trpD gene to GTG.

5. The construction method according to claim 3, characterized in that: The method for knocking down the ribulose 3-isomerase encoding gene rpe is: mutating the start codon ATG of the rpe gene into TTG.

6. The construction method according to claim 3, characterized in that: The method for overexpressing the rpiA gene encoded by the ribose-5-phosphate isomerase gene is as follows: integrating the endogenous rpiA gene at the gene site ppnN and replacing P trc The promoter controls the rpiA gene.

7. The construction method according to claim 3, characterized in that: The method for overexpressing the lysine exporter encoding gene lysE is as follows: integrating the lysE gene of exogenous Corynebacterium glutamicum at the gene site yjiT and replacing P trc The promoter controls the lysE gene.

8. The construction method according to claim 3, characterized in that: The nucleotide sequence of the lysE gene is shown in SEQ ID No.

6.

9. The construction method according to claim 3, characterized in that: The bottom bacteria is Escherichia coli.

10. Use of the genetically engineered bacterium according to claim 1 or 2, or the genetically engineered bacterium constructed by the construction method according to any one of claims 3 to 9 in the preparation of L-histidine.

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