Construction method of escherichia coli genetic engineering strain for improving yield of L-histidine

Abstract

The invention discloses a construction method of genetically engineered bacteria for improving the yield of L-histidine, and belongs to the technical field of bioengineering. Escherichia coli is used as an original strain, and vitreoscilla hemoglobin genes are integrated and overexpressed on a genome of the escherichia coli, so that effective supply of oxygen and ATP is enhanced, and the yield of L-histidine is increased. The genetically engineered bacterium is used for producing the L-histidine by a fermentation method, and the yield of the L-histidine reaches 2.76 g / L at most after fermentation in a shake flask for 48 hours, and is increased by 52.49% compared with an original strain.

Description

Technical Field

[0001] The invention relates to a method for constructing a genetic engineering strain of Escherichia coli for improving the production of L-histidine, and belongs to the technical field of bioengineering. Background Art

[0002] L-histidine, also known as L-α-amino-β-imidazole propionic acid, is a basic amino acid with an imidazole group in the side chain. Its molecular formula is C6H9N3O2 and its relative molecular mass is 155.16.

[0003] In recent years, L-histidine has been widely used in the pharmaceutical, food and feed industries. L-histidine plays an important role in growth and tissue repair. It can be used to prepare amino acid infusions and comprehensive amino acid preparations to treat ulcers, hyperacidity, rheumatoid arthritis and other diseases. In addition, L-histidine can also be used as a synthetic precursor for a variety of drugs. L-histidine can be combined with β-alanine to form carnosine, which can be used to synthesize drugs for the treatment of renal failure, atherosclerosis, Alzheimer's disease and other diseases.

[0004] The traditional method for synthesizing L-histidine is mainly protein hydrolysis, but its production volume depends on the availability of raw materials rich in natural protein such as soybeans and starch, and the production volume is relatively low. Microbial fermentation is currently the mainstream method for producing L-histidine. The microorganisms commonly used to produce L-histidine mainly include Corynebacterium glutamicum, Escherichia coli and Serratia marcescens. As a model strain, Escherichia coli has a clear genetic background, grows faster and has mature genetic manipulation methods. Therefore, Escherichia coli can be used as an ideal host for fermentation production of L-histidine.

[0005] In Escherichia coli, L-histidine is synthesized through a ten-step catalytic reaction using glucose as raw material and adenosine triphosphate (ATP) and 5-phosphoribosyl pyrophosphate (PRPP) as precursors. First, ATP phosphoribosyltransferase (encoded by the HisG gene) catalyzes the connection between the C1 of PRPP and the N1 of ATP, removes the pyrophosphate, opens the ring, decomposes, and releases 5-aminoimidazole-4-carbamoyl nucleotide (AICAR) for purine synthesis. The remaining imidazole glycerol phosphate is oxidized, transaminated, and dephosphorylated to form histidinol, which is oxidized to generate histidine. The reaction catalyzed by ATP phosphoribosyltransferase is the rate-limiting step in the L-histidine synthesis pathway.

[0006] The highest yield of L-histidine fermentation reported in the current literature is an article published by Professor Xie Xixian of Tianjin University of Science and Technology in 2020, which demonstrated the work of increasing histidine production by genetically modifying wild-type Escherichia coli. Ultimately, the modified strain HW6-3 was able to produce 11.8g / L of histidine. And 66.5g / L of histidine was produced under fed-batch fermentation in a 5L fermenter.

[0007] The histidine-producing strain B25 was based on BL21 (DE3) and was modified as follows: the leader region hisL of the L-histidine operon was replaced with an artificial open reading frame (hisL′-λattB′-trpE), which relieved the feedback inhibition of hisL; the key enzyme hisG (from S. marcescens) was overexpressed; Prs (from S. marcescens) was overexpressed to enhance the synthesis of the precursor substance PRPP. However, among the two precursor substances, ATP and PRPP, the ATP synthesis of the B25 strain was not enhanced. Moreover, in histidine fermentation, especially in large-tank production, the dissolved oxygen conditions are relatively poor, and the oxygen utilization rate is low, which further limits the synthesis of ATP.

[0008] Therefore, in the prior art, insufficient ATP supply and low oxygen utilization are still the key factors that limit the synthesis of L-histidine. Summary of the invention

[0009] In view of the above-mentioned deficiencies in the prior art, the present invention describes a method for constructing a genetically engineered Escherichia coli strain with increased L-histidine production, the purpose of which is to increase L-histidine production by overexpressing CgPPK2 and VHb.

[0010] The first technical solution provided by the present invention is a genetically engineered Escherichia coli bacterium, wherein the genetically engineered bacterium overexpresses polyphosphate kinase CgPPK2 from Corynebacterium glutamicum and Vitreoscilla hemoglobin VHb in Escherichia coli.

[0011] In certain embodiments, the nucleotide sequence of the CgPPK2 gene is shown as SEQ ID NO:1, and the nucleotide sequence of the VHb gene is shown as SEQ ID NO:2.

[0012] In certain embodiments, the CgPPK2 gene and the VHb gene are integrated into the ycgH and caiB gene sites of the Escherichia coli genome, respectively.

[0013] In certain embodiments, the Escherichia coli is Escherichia coli B25.

[0014] In certain embodiments, the Escherichia coli B25 is based on Escherichia coli BL21 (DE3) as the starting strain, and the leader region hisL of the L-histidine operon is replaced with an open reading frame (hisL′-λattB′-trpE) to release the feedback inhibition of hisL; the key enzyme hisG (from S. marcescens) is overexpressed; and Prs (from S. marcescens) is overexpressed to enhance the synthesis of the precursor substance PRPP.

[0015] The second technical solution provided by the present invention is a method for constructing a genetically engineered Escherichia coli bacterium, wherein the method uses Escherichia coli as a starting strain, and overexpresses polyphosphate kinase CgPPK2 from Corynebacterium glutamicum and Vitreoscilla hemoglobin VHb in the starting strain.

[0016] In certain embodiments, the nucleotide sequence of the CgPPK2 gene is shown as SEQ ID NO:1, and the nucleotide sequence of the VHb gene is shown as SEQ ID NO:2.

[0017] In certain embodiments, the CgPPK2 gene and the VHb gene are integrated into the ycgH and caiB gene sites of the Escherichia coli genome, respectively.

[0018] Preferably, the CgPPK2 gene and the VHb gene are respectively integrated into the genome of the Escherichia coli using CRISPR-mediated gene editing technology.

[0019] In certain embodiments, the CgPPK2 gene and the VHb are expressed by a strong constitutive promoter; optionally, the strong constitutive promoter is promoter trc, and the nucleotide sequence is as shown in SEQ ID NO:3.

[0020] The third technical solution provided by the present invention is a method for producing histidine, wherein the method comprises using the genetically engineered bacteria described in the first technical solution to perform fermentation to synthesize histidine.

[0021] In certain embodiments, the method is to introduce the activated liquid of the genetically engineered bacteria described in the first technical solution into a fermentation medium to perform fermentation to prepare histidine.

[0022] In certain embodiments, the fermentation medium components are glucose 30-40 g / L, yeast extract 1-5 g / L, (NH4)2SO4 10-20 g / L, K2HPO4·3H2O 1-5 g / L, MnSO4·5H2O 1-10 mg / L, FeSO4·7H2O 1-10 mg / L, CaCO3 20-30 g / L, sodium hexametaphosphate 1-10 g / L, and MgSO4·7H2O 1-5 g / L.

[0023] In certain embodiments, the fermentation parameters are: fermentation at a temperature of 35.0-40.0°C and a speed of 200-250 rpm for 36-40 hours.

[0024] The fourth technical solution provided by the present invention is the use of the genetically engineered bacteria described in the first technical solution, or the method described in the second technical solution, or the method described in the third technical solution in the preparation of histidine or a histidine-containing product.

[0025] The technical effects of the present invention are as follows:

[0026] The present invention overexpresses CgPPK2 and VHb genes in Escherichia coli, thereby ensuring sufficient ATP supply and improving the utilization rate of oxygen by VHb gene cloned bacteria at the molecular level, thereby increasing the production of L-histidine in genetically engineered bacteria. The production ultimately reaches 2.76 g / L, which is 52.49% higher than that of the starting strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the pEcCpf1 plasmid map.

[0028] Figure 2 This is the pcrEG plasmid map.

[0029] Figure 3 This is the HPLC chart of histidine standard. The retention time of histidine is 4.147min.

[0030] Figure 4 The HPLC chart of shake flask fermentation sample liquid. The retention time of histidine is 4.159min.

[0031] Figure 5 These are the results of shake flask fermentation experiments. DETAILED DESCRIPTION

[0032] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0033] The raw materials used in the examples are:

[0034] 1. Escherichia coli B25 uses BL21 (DE3) as the starting strain, removes the feedback inhibition of hisL, that is, replaces the leader region hisL of the L-histidine operon of BL21 (DE3) with an artificial open reading frame (hisL′-λattB′-trpE); overexpresses the key enzyme hisG (from S. marcescens); overexpresses Prs (from S. marcescens) to enhance the synthesis of the precursor substance 5-phosphoribosyl-1-pyrophosphate (PRPP). (Published in "Metabolic Engineering of Escherichia coli to Synthesize L-histidine" by Li Mengying, Lü Xueqin, Liu Yanfeng, Li Jianghua, Du Guocheng, Wu Jianrong, and Liu Long.)

[0035] 2. Source of plasmid used:

[0036] The gene editing method and tool plasmid pEcCpf1 / pcrEG used in the present invention refer to the literature (Xuewen Zhu, Yaokang Wu, et al. Combining CRISPR-Cpf1 and Recombineering Facilitates Fast and Efficient Genome Editing in Escherichia coli, ACS Synthetic Biology, Volume 11, Issue 5, 2022). Among them, pEcCpf1 (kanamycin resistance, such as Figure 1 As shown) carrying the λ-Red recombination system and Cpf1 protein expression system, pcrEG (spectinomycin resistance, as Figure 2 As shown) is constructed to carry the corresponding target sequence and target gene.

[0037] Primers involved in the embodiments:

[0038] Table 1 Primer sequences involved in the present invention

[0039]

[0040]

[0041] Example 1 Construction of a recombinant Escherichia coli strain overexpressing CgPPK2

[0042] CgPPK2 was connected to the trc promoter and integrated into the gene sites of ycgH, caiB, and xylG respectively. The construction steps are explained below using the ycgH site as an example, and the other sites are similar.

[0043] In the following steps, the concentration of kanamycin is 50 μg / mL, and the concentration of spectinomycin is 80 μg / mL. The LB medium formula is peptone 10.0 g / L, yeast extract 5.0 g / L, and sodium chloride 10.0 g / L. The culture temperature is 37°C.

[0044] 1.1 Construction of pcrEG plasmid

[0045] The purpose of constructing plasmid pcrEG is to transcribe the corresponding gRNA, so that the complex formed with Cpf1 protein can recognize the target site on the genome through base pairing, and achieve double-strand break of the target DNA. And through homologous arms at both ends of the target gene, homologous recombination is carried out to integrate the target gene into the genome. The construction method is as follows.

[0046] 1.1.1N23 target sequence design

[0047] The target sequence (PAM: TTV or TTTV) was designed using CRISPR RGEN Tools. Then, primers were synthesized (the primers described in this article were ordered from Shanghai Sangon Biotechnology Co., Ltd.), the sequence of which was: 5'-restriction site-N23 target sequence-3', and its reverse complementary primer, to obtain N23-ycgH-F and N23-ycgH-R (see Table 1), and the two primers were slowly annealed by single-stranded DNA to obtain the target sequence DNA fragment.

[0048] 1.1.2 Construction of pcrEG-N23(ycgH) plasmid

[0049] First, the plasmid pcrEG was digested with BsaI (purchased from Nanjing Novozyme Technology Co., Ltd.) and recovered by running on a gel to obtain a linearized vector.

[0050] Secondly, the linearized vector and the target sequence fragment obtained in 1.1.1 were connected by T4 ligase (purchased from Shanghai Sangon Biotechnology Co., Ltd.). Then, the connection system was chemically transformed into competent DH5α, and the transformation steps were as follows:

[0051] (1) Thaw the competent cells on ice.

[0052] (2) Add DNA to the competent medium, the volume of which should not exceed 10% of the competent medium volume, flick gently to mix, and place in an ice bath for 30 minutes. (3) Place the centrifuge tube in a 42°C water bath for 60-90 seconds, quickly transfer to an ice bath, and place to cool for 2-3 minutes.

[0053] (4) Add 900 μL of LB medium to the centrifuge tube and incubate at 37°C, 150 rpm for 45-60 min.

[0054] (5) Spread an appropriate amount of competent cells on LB plates with corresponding antibiotics and culture at 37°C overnight.

[0055] Finally, single clones were selected for colony PCR verification, positive clones were obtained and sequenced to confirm, and the pcrEG-N23 plasmid was obtained. 1.1.3 Construction of pcrEG-N23-CgPPK2 plasmid

[0056] First, the pcrEG-N23 plasmid obtained above was reversely amplified by primers pcrEG-F / pcrEG-R to obtain a linearized vector. The homology arms were amplified by primers ycgH-up-F / ycgH-up-R and ycgH-down-F / ycgH-down-R in Table 1 using the B25 genome as a template. The gene CgPPK2 was amplified by primers CgPPK2-F / CgPPK2-R. The promoter (nucleotide sequence is shown in SEQ ID NO.3) and the terminator (nucleotide sequence is shown in SEQ ID NO.4) were connected to CgPPK2 and the upper and lower homology arm fragments by primers.

[0057] Secondly, the vector and fragment obtained by the above PCR amplification were connected by a one-step cloning kit (purchased from Nanjing Novozyme Technology Co., Ltd.). Then, the connection system was chemically transformed into competent DH5α (the transformation method is the same as 1.1.2).

[0058] Finally, single clones were picked for colony PCR verification, positive clones were obtained and sequenced to confirm, and the pcrEG-N23-CgPPK2 plasmid was obtained.

[0059] 1.2 Gene Editing Steps

[0060] 1.2.1 Transformation of plasmid pEcCpf1

[0061] First, the B25 strain to be edited is made into a competent colon cell. The competent cell preparation method is as follows:

[0062] (1) Pick a single colony the day before (or directly use 100 μL of bacterial solution stored at -80°C), inoculate it into 5 mL of LB medium and culture it in a shaking incubator at 37°C overnight (about 12-16 hours).

[0063] (2) Take 2 mL of overnight culture and transfer it to 100 ml of LB medium. Incubate the culture on a shaker at 37°C at 220 rpm for about 2.5-3 hours. Detect the OD 600 At 0.5±0.1, the bacterial solution was dispensed into 50 mL centrifuge tubes, 25 mL in each tube, and precooled on ice for 10 min.

[0064] (3) Centrifuge at 4000 rpm for 10 min at 4°C.

[0065] (4) Discard the supernatant and add 10 mL of pre-cooled 0.1 M CaCl2 solution to each tube. Use a pipette to gently pipette up and down to mix the cells to resuspend them. Place on ice for 10-20 min.

[0066] (5) Centrifuge at 4000 rpm for 10 min at 4°C.

[0067] (6) Repeat (4)-(5).

[0068] (7) Discard the supernatant and add 1 mL of pre-cooled 0.1 M CaCl2 solution containing 15% glycerol to each tube. Mix gently up and down with a pipette to resuspend the cells. Aliquot 100 μL / tube into 1.5 mL centrifuge tubes on ice, seal and label, and store at -80°C until needed.

[0069] The plasmid pEcCpf1 (100-200 ng) was transferred into the prepared B25 competent cells (the transformation method is the same as 1.1.2) to obtain the B25 strain containing pEcCpf1.

[0070] 1.2.2 Transformation of plasmid pcrEG-N23-CgPPK2

[0071] Then, the E. coli B25 strain containing pEcCpf1 was prepared into competent cells (the preparation method is the same as 1.2.1, except that kanamycin is added when culturing in 100 mL LB to maintain the stability of pEcCpf1 and the OD 600 When the concentration reaches 0.3, 10 mM arabinose is added to induce the expression of λ-Red recombinase. The prepared competent cells can be used directly for transformation or stored in a -80℃ refrigerator for later use.

[0072] Then, 500-1000 ng of plasmid pcrEG-N23-CgPPK2 was transferred into the B25 strain containing pEcCpf1 constructed in 1.2.1 (the transformation method was the same as 1.1.2, except that the incubation time was 1-1.5 h).

[0073] 1.2.3 Colony Verification

[0074] Positive colonies were screened by colony PCR and sequencing, and the strain in which CgPPK2 was successfully integrated into the B25 genome was obtained.

[0075] 1.3 Plasmid loss

[0076] The next round of gene editing can only be performed after the plasmid is lost, and the steps are as follows.

[0077] 1.3.1 Loss of pcrEG-N23-CgPPK2 plasmid

[0078] The confirmed Ec01 colonies were picked and cultured overnight at 37°C in 2 mL of LB medium containing 50 μg / mL kanamycin and L-rhamnose (10 mM). The next day, the culture was streaked on a plate containing 50 μg / mL kanamycin and cultured overnight at 37°C. A single colony was picked and streaked on an LB plate containing 50 μg / mL kanamycin and 100 μg / mL spectinomycin. The single colony that was sensitive to spectinomycin but not to kanamycin was the colony that successfully lost the pcrEG-N23-CgPPK2 plasmid.

[0079] 1.3.2 Loss of pEcCpf1 plasmid

[0080] The colony that lost the pEcCpf1 plasmid was further cultured in 2 mL of LB medium containing 5 g / L glucose at 37°C for 12-16 h. The bacterial liquid was then streaked on a plate containing 5 g / L glucose and 10 g / L sucrose, cultured overnight at 37°C, and single colonies were selected for screening on LB plates with and without 50 μg / mL kanamycin. The colony that was sensitive to kanamycin was the colony that lost the pEcCpf1 plasmid, i.e., the Ec01 strain.

[0081] Following the same method, CgPPK2 was integrated into the caiB and xylG sites, and the obtained strains were named Ec02 and Ec03, respectively.

[0082] Example 2: Shake flask fermentation of strains overexpressing CgPPK2 to compare the yield of L-histidine

[0083] 2.1 Shake flask fermentation method

[0084] Seed culture: The medium used for seeds is LB medium, with a formula of 10.0 g / L peptone, 5.0 g / L yeast extract, and 10.0 g / L sodium chloride. The plate is streaked for 24 hours, and a single colony is picked into a 250 mL shake flask filled with 25 mL LB medium and cultured for 10-12 hours until the OD 600 =5.0±1.0.

[0085] Shake flask fermentation: The fermentation medium used was composed of 40 g / L glucose, 2 g / L yeast extract, 16 g / L (NH4)2SO4, 1 g / L K2HPO4·3H2O, 5 mg / L MnSO4·5H2O, 5 mg / L FeSO4·7H2O, 30 g / L CaCO3, 5 g / L sodium hexametaphosphate, and 1 g / L MgSO4·7H2O. Three parallels were made for each strain, and each 250 mL shake flask was filled with 15 mL of liquid. Fermentation was carried out at 37.0°C and 220 rpm for 48 h.

[0086] 2.2 Histidine production detection method

[0087] The histidine content was determined by HPLC.

[0088] Preparation of histidine standard: weigh histidine and place it in a 25mL volumetric flask, fill half of the volume with 0.1N HCl, and shake or sonicate until dissolved. Then dilute to the mark with water to make the concentration of histidine stock solution 10nmol / μL, store at -20℃, and dilute with water when using.

[0089] The instrument used was Agilent HPLC (model 1260); column oven temperature: 40°C; chromatographic column: Agilent AdvanceBio AAA chromatographic column (4.6×100 mm); derivatization reagents: borate buffer, OPA and FMOC (all purchased from Agilent), online derivatization by automatic liquid injector; mobile phase A: 10 mM Na2HPO4 and 10 mM Na2B4O7, pH 8.2, mobile phase B: acetonitrile: methanol: water (45:45:10, v:v:v); gradient elution program is shown in Table 2; flow rate: 1.5 mL / min; detection by diode array detector (DAD) at a wavelength of 262 nm. Figure 3 This is the HPLC chart of histidine standard. The retention time of histidine is 4.147min.

[0090] Table 2 HPLC mobile phase gradient elution program

[0091]

[0092]

[0093] 2.3 Shake flask fermentation results

[0094] After 48h of fermentation, samples were taken to measure the OD value of the fermentation liquid and the histidine production ( Figure 4 The HPLC chart of shake flask fermentation sample liquid. The retention time of histidine is 4.159min). The fermentation liquid was diluted or not diluted and centrifuged at 10000rpm for 5min. The supernatant was diluted and filtered with a 0.22μm filter membrane before loading. The yield results were calculated as shown in Table 3.

[0095] Table 3 Histidine production and corresponding bacterial solution OD of overexpressed CgPPK2 strains in shake flask fermentation 600

[0096]

[0097] like Figure 5As shown in the figure, compared with the starting strain B25, the L-histidine production of Ec01, Ec02 and Ec03 was increased, indicating that overexpression of CgPPK2 was helpful for the production of histidine, and finally reached 2.09 g / L, an increase of 15.47%. 600 It can be seen that overexpression of CgPPK2 has an effect on OD 600 The impact is not significant.

[0098] Example 3: Construction of a recombinant E. coli strain overexpressing VHb

[0099] The VHb was connected to the trc promoter and integrated into the caiB and xylG sites of Ec01. The method was basically the same as in Example 1. Taking the caiB site as an example, the specific steps were as follows:

[0100] The target sequence design method is the same as 1.1.1 in Example 1, except that the integration site is the caiB site, and the designed primers are N23-caiB-F / N23-caiB-R (see Table 1). The two primers obtain the target sequence DNA fragment by slow annealing of single-stranded DNA. The construction method of pcrEG-N23 (caiB) plasmid is the same as 1.1.2 in Example 1, except that the linearized pcrEG vector is connected with the target sequence fragment obtained in 2.1.1, and then transformed and verified to obtain the pcrEG-N23 (caiB) plasmid.

[0101] The construction method of pcrEG-N23-VHb plasmid is the same as 1.1.3 in Example 1. The homology arms are amplified by primers caiB-up-F / caiB-up-R and caiB-down-F / caiB-down-R using the B25 genome as a template. Gene VHb is obtained by gene synthesis and amplified with VHb-F / VHb-R primers for subsequent connection. The promoter and terminator are connected to VHb and the upper and lower homology arm fragments by primer PCR, and the fragments are connected.

[0102] The pcrEG-N23-VHb plasmid was obtained by transformation and verification.

[0103] According to step 1.2 of Example 1, pEcCpf1 and pcrEG-N23-VHb plasmids were successively transformed into the strain Ec01. After verification, a strain integrating both CgPPK2 and VHb was screened and named Ec04.

[0104] Following the same method, VHb was integrated into the xylG site of Ec01 and the resulting strain was named Ec05.

[0105] Example 4: Shake flask fermentation of strains overexpressing VHb to compare the yield of L-histidine

[0106] The strain constructed in Example 3 was fermented in a shake flask and the yield was detected. The fermentation and detection methods were consistent with those in Example 2. The results are shown in Table 4.

[0107] Table 4 Histidine production and corresponding bacterial liquid OD of strains overexpressing VHb in shake flask fermentation 600

[0108]

[0109] From the perspective of fermentation yield, the L-histidine yield of Ec04 and Ec05 was higher than that of Ec01, indicating that overexpression of VHb is helpful for the production of histidine. Finally, the L-histidine yield of Ec05 was the highest, reaching 2.76 g / L, which was 52.49% higher than that of B25. 600 It can be seen that overexpression of CgPPK2 has an effect on OD 600 The effect was not significant. After overexpression of VHb, the bacterial OD 600 There is a significant improvement, indicating that increasing the supply of oxygen is also helpful for the growth of bacteria.

[0110] Example 5: Effect of overexpression of VHb using different promoters on L-histidine production

[0111] The promoter of VHb itself (Pvgb, nucleotide sequence as shown in SEQ ID NO.5), tac promoter (nucleotide sequence as shown in SEQ ID NO.6), and trc promoter were used to express VHb, and integrated into the ycgH site of Ec01. The construction method was consistent with Example 3, and the shake flask fermentation method was consistent with Example 2. The fermentation yields obtained were shown in Table 5. It was found that the three promoters all increased the yield of histidine, among which the trc promoter was used to express VHb, and the L-histidine yield was the highest, reaching 2.76 g / L.

[0112] Table 5 Histidine yield and corresponding bacterial liquid OD in shake flask fermentation using different promoters to overexpress VHb 600

[0113]

[0114] Example 6: Effect of overexpression of VHb homologous protein on L-histidine production

[0115] The effects of three other different VHb homologous proteins on L-histidine production were compared, namely BacA (WP_027099064.1) from Clostridium paraputrificum, Hb (CDB15533.1) from Clostridium sp. CAG:221, and HP (ETI93048.1) from Intestinibacter bartlettii DORA_8_9. All used trc promoters, integrated into the ycgH site of Ec01, the construction method was consistent with Example 3, the shake flask fermentation method was consistent with Example 2, and the fermentation yields obtained were shown in Table 6. It was found that overexpression of BacA reduced the yield, overexpression of Hb and HP slightly increased the yield, and the strain overexpressing VHb had the highest L-histidine yield, reaching 2.76 g / L.

[0116] Table 6 Histidine production and corresponding bacterial liquid OD in shake flask fermentation overexpressing VHb homologous protein 600

[0117]

[0118] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A genetically engineered Escherichia coli, characterized in that: The genetically engineered bacteria overexpress polyphosphate kinase CgPPK2 from Corynebacterium glutamicum and Vitreoscilla hemoglobin VHb in Escherichia coli, the nucleotide sequence of the CgPPK2 gene is shown in SEQ ID NO:1, and the nucleotide sequence of the VHb gene is shown in SEQ ID NO:

2.

2. The genetically engineered bacterium according to claim 1, characterized in that: The Escherichia coli is Escherichia coli B25.

3. A method for constructing a genetically engineered Escherichia coli, characterized in that: The method uses Escherichia coli as a starting strain, overexpresses polyphosphate kinase CgPPK2 from Corynebacterium glutamicum and Vitreoscilla hemoglobin VHb in the starting strain, the nucleotide sequence of the CgPPK2 gene is shown in SEQ ID NO: 1, and the nucleotide sequence of the VHb gene is shown in SEQ ID NO:

2.

4. The method according to claim 3, characterized in that: The CgPPK2 gene and the VHb gene are respectively integrated into the ycgH and caiB gene sites of the Escherichia coli genome.

5. The method according to claim 3, characterized in that: The CgPPK2 gene and the VHb are driven to express by a strong constitutive promoter; optionally, the strong constitutive promoter is promoter trc.

6. A method for producing histidine, characterized in that The method comprises using the genetically engineered bacteria of claim 1 or 2 to ferment and synthesize histidine.

7. The method according to claim 6, characterized in that The method comprises introducing the activated liquid of the genetically engineered bacteria according to claim 1 or 2 into a fermentation medium to perform fermentation to prepare histidine.

8. The method according to claim 6, characterized in that The fermentation medium comprises 30-40 g / L of glucose, 1-5 g / L of yeast extract, 10-20 g / L of (NH4)2SO4, 1-5 g / L of K2HPO4·3H2O, 1-10 mg / L of MnSO4·5H2O, 1-10 mg / L of FeSO4·7H2O, 20-30 g / L of CaCO3, 1-10 g / L of sodium hexametaphosphate and 1-5 g / L of MgSO4·7H2O.

9. The method according to claim 6, characterized in that The fermentation parameters are: fermentation for 36-60 hours at a temperature of 35.0-40.0°C and a speed of 200-250 rpm.

10. Use of the genetically engineered bacteria according to claim 1 or 2, or the method according to any one of claims 3 to 5, or the method according to any one of claims 6 to 9 in the preparation of histidine or a product containing histidine.

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