WO2020237701A1 - 一株高产l-组氨酸的基因工程菌及其构建方法与应用 - Google Patents

一株高产l-组氨酸的基因工程菌及其构建方法与应用 Download PDF

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WO2020237701A1
WO2020237701A1 PCT/CN2019/089938 CN2019089938W WO2020237701A1 WO 2020237701 A1 WO2020237701 A1 WO 2020237701A1 CN 2019089938 W CN2019089938 W CN 2019089938W WO 2020237701 A1 WO2020237701 A1 WO 2020237701A1
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histidine
gene
fermentation
genome
genetically engineered
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French (fr)
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谢希贤
张悦
樊伟明
蒋卫
吴鹤云
陈燕娜
田道光
屠建情
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天津科技大学
浙江震元制药有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/24—Proline; Hydroxyproline; Histidine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/02017—ATP phosphoribosyltransferase (2.4.2.17)

Definitions

  • the invention belongs to the technical field of genetic engineering, and specifically relates to a genetically engineered bacterium with high L-histidine production and a construction method and application thereof.
  • L-histidine is a structural element of protein and the ninth essential amino acid for humans and animals. It participates in various physiological and biochemical processes such as body development, anti-oxidation and immune regulation. In addition to being used as a nutritional supplement and feed additive, L-histidine is also increasingly used in the pharmaceutical industry, especially for the production of amino acid infusion and comprehensive amino acid preparations, for heart disease, anemia and gastrointestinal ulcers. the treatment. Therefore, histidine is an important amino acid product for human production and life, and has high economic and social value.
  • the current production methods of histidine include chemical synthesis, hydrolysis and microbial fermentation.
  • the chemical synthesis method mainly uses dihydroxyacetone, formaldehyde and ammonia to synthesize hydroxymethyl imidazole, and then further synthesize histidine, but the yield of this method is low, and the histidine obtained by this method is D, L-group To prepare L-histidine, it needs to be resolved by crystallization or enzymatic method. Therefore, the cost of preparing L-histidine by chemical synthesis is very high, and there are not many practical applications.
  • the production of amino acids by natural proteolysis is a common method for the production of amino acids, and the histidine content in blood meal is the highest.
  • L-histidine by hydrolysis of pig or cattle blood meal is the main method for producing L-histidine.
  • the loss rate of histidine is high, the equipment is corroded seriously, and the separation cost is high, which is not the best histidine production method.
  • the production of histidine by microbial fermentation has low raw material cost, environmental friendliness, simple operation, short cycle, suitable for industrial production, and is an ideal L-histidine production method.
  • the production of histidine by fermentation has not yet been applied in large-scale industrial applications.
  • Organisms can synthesize L-histidine with PRPP and ATP as precursors, but the biosynthetic pathway of L-histidine is very long, the metabolic network involved is complex, and the key enzyme HisG, which catalyzes the first step of histidine synthesis, is also Subject to strict feedback regulation.
  • researchers in Japan, the United States, Russia and other countries began to select strains of L-histidine strains from strains of different species. The methods of strain selection basically adopted multiple strains. Round of mutagenesis screening or further genetic engineering on the basis of mutagenic strains.
  • the starting strains of histidine producing bacteria include Corynebacterium glutamicum, Corynebacterium acetate, Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Arthrobacter, Nocardia, Bacillus subtilis, Ammophilus Bacillus and Serratia marcescens (for example, U.S. Patent: 7785860, 3791925, 4388405, 7398618, 7871808, 7067289, 3716453, 5294547; European Patent: 1085086; Russian Patent: 2119536, 2003677; Japanese Patent: JP19850116075). These studies focused on the modification of histidine terminal metabolic pathways in organisms, especially the screening of unregulated HisG mutants.
  • a strain of Serratia marcescens selected by Japanese researchers can accumulate 45g/L L-histidine after 120 hours of fermentation, which is the most effective method for producing L-histidine by fermentation.
  • the bacterium carries a low copy number plasmid that has multiple copies of the mutated histidine operon gene.
  • Escherichia coli is easy to cultivate, has a short fermentation cycle, clear genetics, and simple genetic manipulation. It is often used to make a hair strain to breed histidine high-producing bacteria.
  • Russian researchers Kljachko E V and others (Russian patent, 2119536) used traditional breeding methods to select sulfaguanidine, D,L-1,2,4-triazole-3-alanine and streptomycin resistance Escherichia coli, and then further molecularly modified it to increase the transcription of its histidine operon gene, resulting in a production strain VKPM B-7270 with a yield of 16-17 g/L and a sugar-acid conversion rate of 24-26%; Japan Sano et al.
  • the existing L-histidine producing strains have basically undergone mutagenesis treatment, and most of them carry plasmids. Due to the accumulation of negative mutations, the burden of plasmids on the growth of bacteria, the use of antibiotics and other factors have led to the slow growth of existing histidine strains, low response to environmental stress, increased nutritional requirements, and easy production. Problems such as fluctuations have restricted the industrial application of strains. Starting from wild-type strains with clear genetic background, constructing histidine engineering bacteria through metabolic engineering breeding methods can largely solve the above-mentioned problems of traditional mutation breeding. VGDoroshenko et al. used wild E.
  • the purpose of the present invention is to provide a genetically engineered Escherichia coli strain with high L-histidine production and its construction method, and to formulate a corresponding fermentation process control plan, which can be applied to L-histidine Efficient industrial production.
  • the present invention provides a genetically engineered strain E. coli WHY3 with high L-histidine production, which is a Corynebacterium glutamicum ATP whose nucleotide sequence is integrated into the genome of E. coli as shown in SEQ ID NO:1 Transphosphoribosylase HisG mutant encoding gene hisG*, to relieve the feedback regulation of HisG and make it strongly expressed; also increase the copy number of E.
  • hisDBCHAFI histidine operon gene hisDBCHAFI in the genome, thereby enhancing The terminal synthesis pathway of histidine; the arginine/lysine transporter encoding gene lysE derived from Corynebacterium glutamicum is also integrated in the genome and strongly expressed to promote intracellular histidine Extracellular secretion.
  • Escherichia coli is E. coli W3110.
  • Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032.
  • histidine operon gene hisDBCHAFI includes seven genes hisD, hisB, hisC, hisH, hisA, hisF and hisI.
  • the coding gene hisG* of the ATP-transphosphoribosylase HisG mutant of Corynebacterium glutamicum is integrated at at least two gene sites on the E. coli genome.
  • strong expression of foreign genes can be achieved by constructing strong promoters.
  • the coding gene hisG* of the Corynebacterium glutamicum ATP-transphosphoribosylase HisG mutant is integrated into the tdcD and ylbE gene sites of the E. coli genome, and is activated by the promoter P trc .
  • the present invention also provides the method for constructing the genetically engineered strain E. coli WHY3 as follows:
  • the genetically engineered bacterium E. coli WHY3 is obtained by directional transformation of E. coli W3110 using CRISPR/Cas 9-mediated gene editing technology, which specifically includes the following steps:
  • the present invention also provides the use of the above-mentioned genetically engineered bacteria E.coli WHY3 to ferment to produce L-histidine, including:
  • the seed solution is prepared, and 10-15% of the inoculum is inoculated into an Erlenmeyer flask containing fermentation medium, sealed with nine layers of gauze, cultivated at 37°C, 200r/min shaking, and maintained by adding ammonia water during the fermentation process
  • the pH is 7.0-7.2; 60% (m/v) glucose solution is added to maintain the fermentation;
  • the preferred fermentation medium composition is: glucose 20-40g/L, xylose 5-15g/L, yeast extract 2-5g/L, peptone 2-5g/L, KH 2 PO 4 1-3g/L, MgSO 4 ⁇ 7H 2 O 1-2g/L, FeSO 4 ⁇ 7H 2 O 5-20mg/L, MnSO 4 ⁇ 7H 2 O 5-20mg/L, V B1 , V B3 , V B5 , V B12 , V H each 1-3mg/L, the rest is water, pH 7.0-7.2.
  • histidine can reach 6-10g/L after 24-30h fermentation in a 500mL shake flask.
  • the seed solution is prepared, and 15-20% of the inoculum is connected to the fresh fermentation medium to start the fermentation.
  • the pH is stabilized at about 7.0, the temperature is maintained at 37°C, and the dissolved oxygen is at 25-35%.
  • the preferred composition of the fermentation medium is glucose 10-30g/L, yeast extract 1-5g/L, peptone 1-5g/L, K 2 HPO 4 1-5g/L, MgSO 4 ⁇ 7H 2 O 1-3g/L, FeSO 4 ⁇ 7H 2 O 10-30mg/L, MnSO 4 ⁇ H 2 O 10-30mg/L, V B1 , V B3 , V B5 , V B12 , V H each 1-3 mg/L, the rest is water, pH 7.0-7.2.
  • a 5L fermenter can produce 40-55g/L of histidine for 40-50h, with an average production intensity of 1.0-1.5g/(L ⁇ h), and a conversion rate of 0.18-0.22g histidine/g glucose.
  • histidine strains are mostly obtained by multiple rounds of mutagenesis screening methods or by further genetic engineering on the basis of mutagenic strains.
  • the obtained strain has many negative mutations, which causes the strain to show certain growth defects, resulting in problems such as reduced ability to respond to environmental stress and increased nutritional requirements.
  • the existing histidine production strains mostly use plasmid overexpression to enhance the expression of histidine synthesis-related genes. This modification method increases the growth burden of the bacteria, and at the same time the use of antibiotics and the easy loss of plasmids Increased costs, antibiotic residues in the fermentation broth, and poor production stability limit its application in large-scale industrial production.
  • the present invention provides a genetically engineered bacteria with a clear genetic background, no plasmid, and stable production of L-histidine.
  • the bacteria is used to produce L-histidine by a fermentation method, and can be fermented for 40-50h in a 5L fermentor to produce
  • the acid is 40-55g/L
  • the production intensity is 1.0-1.5g/(L ⁇ h)
  • the conversion rate is 0.18-0.22g histidine/g glucose, which is the highest level of L-histidine produced by fermentation at present.
  • Figure 1 (a) pREDCas9 plasmid map, (b) pGRB plasmid map.
  • Figure 2 The construction and verification electrophoresis diagram of the integrated fragment when hisG* is integrated at the tdcD gene locus. Among them: M: 1kb DNA marker; 1: upstream homology arm; 3: hisG* gene fragment; 3: downstream homology arm; 4: overlapping fragment; 5: original bacteria control; 6: identification fragment of positive bacteria.
  • Figure 3 The electrophoresis diagram of the integration fragment construction and verification when integrating hisG* at the ylbE gene locus. Among them: M: 1kb DNA marker; 1: upstream homology arm; 3: hisG* gene fragment; 3: downstream homology arm; 4: overlapping fragment; 5: original bacteria control; 6: identification fragment of positive bacteria.
  • Figure 4 The construction and verification electropherogram of hisD integration fragment. Among them: M: 1kb DNA marker; 1: upstream homology arm; 3: hisD gene fragment; 3: downstream homology arm; 4: overlapping fragment; 5: original bacteria control; 6: identification fragment of positive bacteria.
  • Figure 5 The construction and verification electropherogram of hisC-hisB integration fragment. Among them: M: 1kb DNA marker; 1: hisC upstream sequence-hisC-hisB fragment; 2: downstream homology arm; 3: overlapping fragment; 4: original bacteria control; 5: identification fragment of positive bacteria.
  • Figure 6 The construction and verification electropherogram of hisH-hisA-hisF-hisI integration fragment.
  • M 1kb DNA marker
  • 1 hisH upstream sequence-hisH-hisA-hisF-hisI fragment
  • 2 downstream homology arm
  • 3 overlapping fragment
  • 4 original bacteria control
  • 5 identification fragment of positive bacteria.
  • Figure 7 Construction and verification electropherogram of P trc- lysE integration fragment. Among them: M: 1kb DNA marker; 1: upstream homology arm; 2: lysE gene fragment; 3: downstream homology arm; 4: overlapping fragment; 5: original bacteria control; 6: positive bacteria identification fragment.
  • Figure 8 Shake flask fermentation experiment results of strain E.coli WHY2-3 and strain E.coli WHY3.
  • Figure 9 Fermentation process curve of E.coli WHY3 on a 5L fermentor.
  • the gene editing method used in the present invention is carried out with reference to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated gene editing. Metabolic engineering, 2015, 31: 13-21.), The two plasmid maps used in this method are shown in Figure 1.
  • pREDCas9 carries gRNA expression plasmid pGRB elimination system, lambda phage Red recombination system and Cas9 protein expression system, spectinomycin resistance (working concentration: 100mg/L), cultured at 32°C;
  • pGRB uses pUC18 as backbone, including promoter J23100, gRNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance (working concentration: 100mg/L), cultured at 37°C.
  • the purpose of constructing the plasmid pGRB is to transcribe the corresponding gRNA to form a complex with the Cas9 protein, and to recognize the target gene target site through base pairing and PAM to achieve the target DNA double-strand break.
  • the pGRB plasmid was constructed by recombining the DNA fragment containing the target sequence with the linearized vector fragment.
  • Design primers 5'-linearized vector end sequence (15bp)-restriction site-target sequence (excluding PAM sequence)-linearized vector end sequence (15bp)-3' and its reverse complementary primer, through a single
  • the annealing of stranded DNA produces a DNA fragment containing the target sequence.
  • Reaction conditions pre-denaturation at 95°C, 5min; annealing at 30-50°C, 1min.
  • the annealing system is as follows:
  • the linearization of the vector adopts the method of inverse PCR amplification.
  • the reorganization system is as follows. All recombinases used are II One Step Cloning Kit series of enzymes, recombination conditions: 37°C, 30min.
  • PCR-positive colonies were inoculated into LB medium containing 100mg/L ampicillin and cultured overnight to preserve the bacteria, and the plasmids were extracted and identified by restriction enzyme digestion.
  • the recombination fragment used for knockout consists of the upstream and downstream homology arms of the gene to be knocked out (upstream homology arm-downstream homology arm); the recombination fragment used for integration is the upstream and downstream homology arms of the integration site and to be integrated Composition of gene fragments (upstream homology arm-target gene-downstream homology arm).
  • primer design software primer5 use the upstream and downstream sequences of the gene to be knocked out or the site to be integrated as a template to design the upstream and downstream homology arm primers (amplification length is about 400-500bp); use the gene to be integrated as a template to design the integrated gene
  • the amplification primers After amplifying the upstream and downstream homology arms and the target gene fragments by PCR, the recombinant fragments are prepared by overlapping PCR.
  • the PCR system and method are as follows:
  • the template is composed of the amplified fragments of the upstream and downstream homology arms and the target gene equimolar, and the total amount does not exceed 10ng.
  • PCR reaction conditions (PrimeSTAR HS enzyme): pre-denaturation (95°C) for 5min; then 30 cycles of denaturation (98°C) for 10s, annealing ((Tm-3/5)°C) for 15s, 72°C extension (this Enzyme activity is extended for about 1 kb in 1 minute); extended at 72°C for 10 minutes; maintained (4°C).
  • the pREDCas9 plasmid was electrotransformed into the electrotransformation competence of W3110 by electrotransformation, the bacteria were resuscitated and cultured and spread on an LB plate containing spectinomycin, and cultured overnight at 32°C. A single colony grown on the resistant plate was subjected to colony PCR with identification primers to screen positive recombinants.
  • IPTG to make the final concentration 0.1 mM
  • Competent preparation required medium and preparation process refer to routine standard operation.
  • the pGRB and donor DNA fragments were simultaneously electrotransformed into electrocompetent cells containing pREDCas9. After electrotransformation, the resuscitated cells were spread on LB plates containing ampicillin and spectinomycin, and cultured overnight at 32°C.
  • the positive recombinants were cultured overnight in an LB medium containing 0.2% arabinose, diluted appropriately and spread on an LB plate containing spectinomycin resistance, and cultured overnight at 32°C. To spot LB plates containing ampicillin and spectinomycin resistance, select a single colony that does not grow on the ampicillin plate and the spectinomycin resistant plate grows to protect the bacteria.
  • the positive recombinants were transferred to non-resistant LB liquid medium and cultured at 42°C overnight, diluted appropriately and spread on a non-resistant LB plate, and cultured overnight at 37°C. Spot the LB plates containing spectinomycin resistance and non-resistant, select the spectinomycin-resistant plates that do not grow, and the single colonies that grow on the non-resistant plates are protected.
  • upstream homology arm primers UP-tdcD-S, UP-tdcD-A
  • downstream homology arm primers DN-
  • hisG* gene nucleotide sequence shown in SEQ ID No. 2
  • the promoter P trc is designed in the downstream primer of the upstream homology arm and the upstream primer of hisG* gene.
  • the above fragments were used to obtain the integrated fragment of hisG* gene (upstream homology arm-P trc- hisG*-downstream homology arm) by overlapping PCR method, and the DNA fragment containing the target sequence used in the construction of pGRB-tdcD was passed through the primer gRNA-tdcD- S and gRNA-tdcD-A annealing.
  • Prepare competent cells of E.coli W3110 follow the methods shown in 1.3 and 1.4, and finally obtain the strain E.coli WHY1-1.
  • the construction of the P trc -hisG* integration fragment and the electrophoresis diagram of the PCR verification of the positive strains are shown in Figure 2.
  • the length of the upstream homology arm should be 496bp
  • the length of the amplified hisG* gene fragment should be 627bp
  • the length of the downstream homology arm should be 1902bp
  • the total length of the integrated fragment should be 3024bp.
  • the length of the PCR amplified fragment of the positive bacteria should be 627bp, and the original bacteria have no bands.
  • upstream homology arm primers UP-ylbE-S, UP-ylbE-A
  • downstream homology arm primers DN-ylbE
  • primers hisG*-S, hisG*-A
  • the length of the upstream homology arm should be 601bp
  • the length of the amplified hisG* gene fragment should be 627bp
  • the length of the downstream homology arm should be 547bp
  • the total length of the integrated fragment should be 1815bp.
  • the length of the PCR amplified fragment of the positive bacteria should be 903bp, and the original bacteria have no bands.
  • the histidine operator in E. coli W3110 (hisDBCHAFI, including hisD, hisB, hisC, hisH, hisA, hisF and hisI seven genes) is sequentially integrated into the E. coli WHY1-2 genome.
  • HisDBCHAFI including hisD, hisB, hisC, hisH, hisA, hisF and hisI seven genes
  • the strain E.coli HIS3-3 was constructed.
  • the integration of the histidine operon gene is divided into three segments.
  • the upstream homology arm primers (UP-yghX-S, UP-yghX-A) and downstream homology arm primers (DN-yghX-A) were designed according to the upstream and downstream sequences of its yghX gene. S1, DN-yghX-A), and PCR amplify its upstream and downstream homology arm fragments; design primers (hisD-S, hisD-A) according to the hisD gene sequence, and PCR amplify hisD fragments; the promoter P trc is designed In the downstream primer of the upstream homology arm and the upstream primer of the hisD gene.
  • the construction of the integrated fragment and the electrophoresis diagram of the PCR verification of the positive strains are shown in Figure 4.
  • the length of the upstream homology arm is 602bp
  • the length of the hisD gene fragment is 1305bp
  • the length of the downstream homology arm is 561bp
  • the length of the overlapping fragment is 2542bp.
  • the identification primers are designed and verified by PCR.
  • the length of the fragment amplified by the positive recombinant should be 1208bp, the original bacteria has no bands.
  • design upstream homology arm primers (UP-hisBC-S, UP-hisBC-A) according to hisB-hisC and its upstream sequence, and amplify the upstream homology arm fragments by PCR ;
  • design downstream homology arm primers (DN-yghX-S2, DN-yghX-A) according to the downstream sequence of its yghX gene, and PCR amplify its downstream homology arm fragments. The above fragments are fused by overlapping PCR to obtain an integrated fragment of hisB-hisC (upstream fragment of hisB-hisB-hisC-downstream homology arm).
  • the DNA fragment containing the target sequence used to construct pGRB-his1 is prepared by annealing the primers gRNA-his1-S and gRNA-his1-A. Prepare competent cells of E. coli WHY2-1, follow the methods shown in 1.3 and 1.4, and finally obtain the strain E. coli WHY2-2. During the integration of hisB-hisC fragments, the construction of the integrated fragments and the electrophoresis diagram of the PCR verification of the positive strains are shown in Figure 4. The total length of the upstream fragment of hisB-hisB-hisC is 2696bp, the length of the downstream homology arm is 561bp, and the length of the overlapping fragment is 3317bp. The identification primers are designed and verified by PCR. The length of the amplified fragment of the positive recombinant is 1118bp. The bacteria have no bands.
  • design upstream homology arm primers (UP-hisHAFI-S, UP-hisHAFI-A) according to hisH-hisA-hisF-hisI and its upstream sequence, and amplify its upstream by PCR Homologous arm fragments; using E.coli HIS3-2 genome as a template, design downstream homologous arm primers (DN-yghX-S3, DN-yghX-A) according to the downstream sequence of its yghX gene, and PCR amplify the downstream homology Source arm fragment.
  • the above fragments were fused by overlapping PCR to obtain an integrated fragment of hisH-hisA-hisF-hisI (upstream fragment of hisH-hisH-hisA-hisF-hisI-downstream homology arm).
  • the DNA fragment containing the target sequence used in the construction of pGRB-his2 was prepared by annealing the primers gRNA-his2-S and gRNA-his2-A.
  • the construction of the integrated fragment and the electrophoresis diagram of the PCR verification of the positive strains are shown in Figure 6.
  • the total length of the upstream fragment of hisH-hisH-hisA-hisF-hisI is 3265bp, the length of the downstream homology arm is 561bp, and the total length of overlapping fragments is 3317bp.
  • the identification primers are designed and verified by PCR, the length of the amplified fragments of the positive recombinants It is 1136bp, and the original bacteria has no bands.
  • upstream homology arm primers (UP-yjiT-S, UP-yjiT-A) and downstream homology arm primers (DN-yjiT-A) were designed according to the upstream and downstream sequences of its yjiT gene.
  • the length of the upstream homology arm is 372bp
  • the length of the lysE gene fragment is 834bp
  • the length of the downstream homology arm is 530bp
  • the length of the overlapping fragment is 1655bp.
  • the identification primers are designed and verified by PCR.
  • the length of the fragment amplified by the positive recombinant should be 1429bp, the original bacteria has no bands.
  • the method of using genetically engineered bacteria E.coli WHY3 to ferment to produce histidine is as follows:
  • Slope culture streak and inoculate the preserved strains on the activated slant at -80°C, cultivate for 12h at 37°C, and pass them once;
  • Shake flask seed culture use an inoculating loop to scrape a loop of slant seeds and inoculate them in a 500mL Erlenmeyer flask containing 30mL seed culture medium, seal with nine layers of gauze, cultivate for 6-8h at 37°C, 200rpm;
  • Shake flask fermentation culture inoculate 10-15% of the inoculum into a 500mL Erlenmeyer flask containing fermentation medium (final volume is 30mL), seal with nine layers of gauze, culture at 37°C, 200r/min shaking, and supplement during fermentation. Add ammonia water to maintain pH at 7.0-7.2; add 60% (m/v) glucose solution to maintain fermentation; fermentation cycle 24-30h;
  • the slant medium composition is: glucose 1-5g/L, peptone 5-10g/L, beef extract 5-10g/L, yeast powder 1-5g/L, NaCl 1-2.5g/L, agar 20-25g/L , The rest is water, pH 7.0-7.2;
  • composition of seed medium is: glucose 15-30g/L, yeast extract 5-10g/L, peptone 5-10g/L, KH 2 PO 4 5-15g/L, MgSO 4 ⁇ 7H 2 O 2-5g/L , FeSO 4 ⁇ 7H 2 O 5-20mg/L, MnSO 4 ⁇ H 2 O 5-20mg/L, V B1 1-3mg/L, V H 0.1-1mg/L, 2 drops of defoamer, the rest is water , PH 7.0-7.2;
  • the composition of the fermentation medium is: glucose 20-30g/L, xylose 5-15g/L, yeast extract 2-5g/L, peptone 2-4g/L, KH 2 PO 4 1-3g/L, MgSO 4 ⁇ 7H 2 O 1-2g/L, FeSO 4 ⁇ 7H 2 O 5-20mg/L, MnSO 4 ⁇ 7H 2 O 5-20mg/L, V B1 , V B3 , V B5 , V B12 , V H each 1- 3mg/L, the rest is water, pH 7.0-7.2.
  • the yield of L-histidine can reach 6-10g/L after 24-30h fermentation in shake flasks.
  • Slope activation culture scrape a ring of bacteria from the -80°C refrigerator preservation tube, spread it evenly on the activation slope, incubate at 37°C for 12-16 h, transfer to eggplant-shaped bottle and continue culturing for 12-16 h;
  • Seed culture Take a suitable amount of sterile water in an eggplant-shaped bottle, connect the bacterial suspension to the seed culture medium, the pH is stable at about 7.0, the temperature is constant at 37°C, and the dissolved oxygen is between 25-35%, cultivate until fermentation The liquid OD 600 value reaches 10-15;
  • the slant medium composition is: glucose 1-5g/L, peptone 5-10g/L, beef extract 5-10g/L, yeast powder 1-5g/L, NaCl 1-2.5g/L, agar 20-25g/L , The rest is water, pH 7.0-7.2;
  • composition of seed medium is: glucose 15-30g/L, yeast extract 5-10g/L, peptone 5-10g/L, KH 2 PO 4 5-15g/L, MgSO 4 ⁇ 7H 2 O 2-5g/L , FeSO 4 ⁇ 7H 2 O 5-15mg/L, MnSO 4 ⁇ H 2 O 5-15mg/L, V B1 1-3mg/L, V H 0.1-1mg/L, 2 drops of defoamer, the rest is water , PH 7.0-7.2;
  • the composition of the fermentation medium is: glucose 10-25g/L, yeast extract 1-5g/L, peptone 1-5g/L, K 2 HPO 4 1-5g/L, MgSO 4 ⁇ 7H 2 O 1-3g/L , FeSO 4 ⁇ 7H 2 O 10-30mg/L, MnSO 4 ⁇ H 2 O 10-30mg/L, V B1 , V B3 , V B5 , V B12 , V H each 1-3mg/L, the rest is water, pH 7.0-7.2.
  • a 5L fermenter can produce 40-55g/L of histidine for 40-50h, with an average production intensity of 1.0-1.5g/(L ⁇ h), and a conversion rate of 0.18-0.22g histidine/g glucose.
  • Slope culture streak and inoculate the preserved strains on the activated slant at -80°C, cultivate for 12h at 37°C, and pass them once;
  • Shake flask seed culture Scrape a ring of slant seeds with an inoculating loop and inoculate them in a 500 mL Erlenmeyer flask containing 30 mL of seed culture medium, seal with nine layers of gauze, and cultivate for 8 hours at 37°C at 200 rpm;
  • Shake flask fermentation culture inoculate 15% inoculum into a 500mL Erlenmeyer flask containing fermentation medium (final volume is 30mL), seal with nine layers of gauze, culture at 37°C, 200r/min shaking, add ammonia water during the fermentation process Maintain the pH at 7.0-7.2; add 60% (m/v) glucose solution to maintain the fermentation; the fermentation cycle is 30h.
  • the slant medium composition is: glucose 1g/L, peptone 10g/L, beef extract 10g/L, yeast powder 5g/L, NaCl 2.5g/L, agar 20g/L, the rest is water, pH 7.0-7.2.
  • composition of the seed medium is: glucose 30g/L, yeast extract 5g/L, peptone 5g/L, KH 2 PO 4 6g/L, MgSO 4 ⁇ 7H 2 O 2.5g/L, FeSO 4 ⁇ 7H 2 O 10mg/ L, MnSO 4 ⁇ H 2 O 10mg/L, V B1 2mg/L, V H 1mg/L, 2 drops of defoamer, the rest is water, pH 7.0-7.2.
  • the composition of the fermentation medium is: glucose 20g/L, xylose 10g/L, yeast extract 5g/L, peptone 4g/L, KH 2 PO 4 3g/L, MgSO 4 ⁇ 7H 2 O 2g/L, FeSO 4 ⁇ 7H 2 O 10mg/L, MnSO 4 ⁇ 7H 2 O 10mg/L, V B1 , V B3 , V B5 , V B12 , V H each 2 mg/L, the rest is water, pH 7.0-7.2.
  • Slope activation streak the glycerin-preserved strain and inoculate it on the slant medium of the test tube and incubate at 37°C for 12; then streak the preserved slant strain into the eggplant-shaped flask slant medium and cultivate for 14h at 37°C.
  • Seed culture Take a freshly activated eggplant-shaped bottle slant, wash it with 150mL sterile water, inoculate it into the fermenter under flame protection, temperature control 37°C, automatic flow of ammonia water to control the pH at 7.0, the initial aeration rate is 2L/min, the initial stirring speed is 200rpm, and the DO value is maintained between 20-30% during the cultivation process, and the seeds are cultivated until the OD 600 is about 15.
  • Fermentation tank culture Fermentation tank seeds are inserted into the seed solution with 15% inoculum amount (discharge to 450mL, pour sterilized fermentation medium under flame protection), temperature control 35°C, automatic flow of ammonia (or 20% sulfuric acid) ) Control the pH at 7.0, the initial aeration rate is 2L/min, the aeration ratio is 0.667vvm, the initial stirring speed is 400rpm, the dissolved oxygen is controlled at 20-30% by adjusting the speed and air volume, and the foam is added manually to defoam and ferment During the process, add 80% glucose solution to ensure sufficient sugar supply and sugar concentration not higher than 5g/L.
  • composition of the slant medium is: glucose 1g/L, peptone 10g/L, beef extract 10g/L, yeast powder 5g/L, NaCl 2.5g/L, agar 25g/L, the rest is water, pH 7.0-7.2;
  • composition of seed culture medium is: glucose 10g/L, yeast extract 5g/L, peptone 5g/L, KH 2 PO 4 5g/L, MgSO 4 ⁇ 7H 2 O 2g/L, FeSO 4 ⁇ 7H 2 O 10mg/L , MnSO 4 ⁇ H 2 O 10mg/L, V B1 2mg/L, V H 1mg/L, 2 drops of defoamer, the rest is water, pH 7.0-7.2.
  • the composition of the fermentation medium is: glucose 10g/L, yeast extract 5g/L, tryptone 4g/L, K 2 HPO 4 3g/L, MgSO 4 ⁇ 7H 2 O 1.5g/L, FeSO 4 ⁇ 7H 2 O 20mg /L, MnSO 4 ⁇ H 2 O 20mg/L, V B1 , V B3 , V B5 , V B12 , V H each 2 mg/L, the rest is water, pH 7.0-7.2.
  • the fermentation curve that after 8 hours of fermentation, it enters the rapid accumulation stage of histidine, and the maximum production intensity at this time can reach 2g/(L ⁇ h); after 16 hours, the bacterial growth enters a stable period, and the OD of the fermentation broth 600 is 89; when the fermentation reaches 44 hours, the histidine concentration reaches the maximum value of 55g/L, and then the histidine concentration and the cell concentration begin to decrease; after 48 hours, the fermentation is over, the sugar-acid conversion rate at this time is 0.2 g histidine/g glucose.

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Abstract

提供了一株高产L-组氨酸的基因工程菌及其构建方法，是在大肠杆菌的基因组上整合了谷氨酸棒杆菌ATP转磷酸核糖基酶（HisG）突变体编码基因以增强HisG的活性，还在基因组上增加了组氨酸操纵子基因的拷贝数以增强组氨酸的合成，并在基因组上整合了来源于谷氨酸棒杆菌的精氨酸/赖氨酸转运蛋白的编码基因lysE，以促进胞内组氨酸向胞外分泌。该基因工程菌用于发酵法生产L-组氨酸，在5L发酵罐发酵40-50h可稳定生产组氨酸40-55g/L，生产强度可达1.0-1.5g/(L×h)，转化率为0.18-0.22g组氨酸/g葡萄糖。

Description

一株高产L-组氨酸的基因工程菌及其构建方法与应用 技术领域
本发明属于基因工程技术领域，具体涉及一种高产L-组氨酸的基因工程菌及其构建方法与应用。
背景技术
L-组氨酸是蛋白质结构元件，是人和动物的第九种必须氨基酸，参与机体发育，抗氧化和免疫调节等多种生理生化过程。除作为营养强化剂和饲料添加剂外，L-组氨酸还越来越多地应用于医药行业，特别是用来生产氨基酸输液及综合氨基酸制剂，用于心脏病，贫血和胃肠溃疡等疾病的治疗。因此，组氨酸是人类生产生活的一种重要的氨基酸制品，具有很高的经济和社会价值。
目前组氨酸的生产方法有化学合成法、水解法和微生物发酵法。其中，化学合成法主要利用二羟基丙酮、甲醛和氨合成羟甲基咪唑，再进一步合成组氨酸，但是此法产率较低，并且此法所得到的组氨酸为D，L-组氨酸，要制备L-组氨酸还需用结晶法或酶法进行拆分，因而化学合成法制备L-组氨酸的成本很高，实际应用不多。天然蛋白水解法生产氨基酸是氨基酸生产的常用方法，而血粉中组氨酸含量最高，猪或牛血粉水解提取法生产L-组氨酸是生产L-组氨酸的主要方法，但此法L-组氨酸的损失率高，设备腐蚀严重，分离成本高，并不是最佳的组氨酸生产方法。微生物发酵法生产组氨酸，原料成本低廉，环境友好，操作简单，周期短，适合工业化生产，是理想的L-组氨酸生产方法。但受限于菌种的发酵水平，发酵法生产组氨酸尚未得到大规模的工业化应用。
生物体可以PRPP和ATP为前体合成L-组氨酸，但是L-组氨酸的生物合成途径很长，涉及的代谢网络复杂，并且催化组氨酸合成的第一步的关键酶HisG还受到严格的反馈调节作用。从上世纪70年代开始，日本、美国、俄罗斯等国家的研究人员从不同种属的菌株出发，开始了L-组氨酸菌种的选育工作，菌种选育的方法基本都是采用多轮诱变筛选或者在诱变菌株的基础上进行进一步的基因工程改造。目前组氨酸生产菌的出发菌株包括谷氨酸棒杆菌、嗜醋酸棒杆菌、黄色短杆菌、乳糖发酵短杆菌、大肠杆菌、节杆菌属、诺卡氏菌属、枯草芽孢杆菌、嗜氨微杆菌和粘质沙雷氏菌(如，美国专利：7785860，3791925，4388405，7399618，7871808，7067289，，3716453，5294547；欧洲专利：1085086；俄罗斯专利：2119536，2003677； 日本专利：JP19850116075)。这些研究的侧重点集中在生物体内组氨酸终端代谢途径的修饰，特别是解调控的HisG突变体的筛选。其中，日本的研究人员选育的一株粘质沙雷氏菌(日本专利，JP19850116075)发酵120h可积累45g/L L-组氨酸，是现有报道的发酵法生产L-组氨酸的最高水平，该菌携带一个低拷贝数的质粒对已突变的组氨酸操纵子基因进行了多拷贝。
大肠杆菌易培养、发酵周期短、遗传学性质清晰、基因操作简单，常用作出发菌株来选育组氨酸高产菌。俄罗斯研究人员Kljachko E V等(俄罗斯专利，2119536)通过传统的育种方法选育出具有磺胺胍、D,L-1,2,4-三唑-3-丙氨酸和链霉素抗性的大肠杆菌，然后进一步对其进行分子改造，提高其组氨酸操纵子基因的转录量，得到产量为16～17g/L，糖酸转化率为24～26％的生产菌株VKPM B-7270；日本味之素株式会社的Sano等(美国专利，4388405)在生产菌E.coli R-344中通过质粒过表达的方式强化L-组氨酸合成相关的基因的表达，从而将L-组氨酸产量提高到了25g/L；Klyachko等先后在大肠杆菌生产菌中过表达了purH、talB和突变型的prs基因提高了L-组氨酸的产量。
现有的L-组氨酸生产菌株基本都经过诱变处理，且多携带质粒。由于负向突变的累积，质粒存在对菌体生长带来的负担，抗生素的使用等因素的存在，导致现有组氨酸菌种生长缓慢，对环境胁迫应对能力低，营养需求增高，生产易波动等问题，限制了菌株的工业化应用。从遗传背景清楚的野生型菌株出发，通过代谢工程育种的方法构建组氨酸工程菌，可以很大程度上解决上述传统诱变育种存在的问题。V.G.Doroshenko等以野生大肠杆菌MG1655+为出发菌株，通过敲除组氨酸操纵子前导肽基因hisL，转录调控因子purR，以及在组氨酸合成关键酶HisG中引入点突变E271K，从而使菌株具备组氨酸积累能力，摇瓶发酵可积累组氨酸4.9g/L，但其发酵性能太低不能满足工业化生产的要求。
发明内容
针对上述存在的问题，本发明目的是提供一种高产L-组氨酸的大肠杆菌基因工程菌及其构建方法，并制定了相应的发酵过程控制方案，该菌可应用于L-组氨酸的高效工业化生产。
本发明技术方案概述如下：
本发明提供一株高产L-组氨酸的基因工程菌E.coli WHY3，该菌是在大肠杆菌的基因组上整合了核苷酸序列如SEQ ID NO:1所示的谷氨酸棒杆菌ATP转磷酸核糖基酶HisG突变体编码基因hisG*，以解除HisG所受的反馈调节作用并使其强表达；还在基因组上增加了大肠杆菌自身组氨酸操纵子基因hisDBCHAFI的拷贝数，从而增强组氨酸的终端合成途径；还在基因组上整合了来源于谷氨酸棒杆菌的精氨酸/赖氨酸转运蛋白的编码基因lysE并使其强表达，以促进胞内组氨酸的向胞外分泌。
进一步地，所述大肠杆菌为E.coli W3110。
进一步地，所述谷氨酸棒杆菌为谷氨酸棒杆菌ATCC 13032。
进一步地，所述组氨酸操纵子基因hisDBCHAFI，包含hisD、hisB、hisC、hisH、hisA、hisF和hisI七个基因。
进一步地，所述谷氨酸棒杆菌ATP转磷酸核糖基酶HisG突变体编码基因hisG*整合在大肠杆菌基因组上至少两个基因位点处。
进一步地，可以通过构建强启动子实现外源基因的强表达。
作为本发明一种较佳实施方式，所述谷氨酸棒杆菌ATP转磷酸核糖基酶HisG突变体编码基因hisG*分别整合在大肠杆菌基因组tdcD和ylbE基因位点，并由启动子P trc启动。
本发明还提供所述基因工程菌E.coli WHY3的构建方法如下：
作为本发明一种较佳实施方式，所述基因工程菌E.coli WHY3是采用CRISPR/Cas 9介导的基因编辑技术对E.coli W3110进行定向改造所得，具体包括如下步骤：
(1)构建启动子P trc与核苷酸序列如SEQ ID NO:1所示的基因hisG*的连接片段P trc-hisG*，并将其分别整合在基因组上tdcD和ylbE基因位点；
(2)构建启动子P trc与大肠杆菌组氨酸操纵子基因的连接片段P trc-hisD-hisC-hisB-hisH-hisA-hisF-hisI，并用分段整合的方法将其整合在基因组上yghX基因位点；
(3)构建启动子P trc与来源于谷氨酸棒杆菌的lysE基因的连接片段P trc-lysE，并将其整合在基因组上yjiT基因位点。
本发明还提供了利用上述基因工程菌E.coli WHY3发酵生产L-组氨酸的用途，包括：
(1)摇瓶发酵：
将菌种活化后制备种子液，按10-15％接种量接种到装有发酵培养基的三角瓶中，九层纱布封口，37℃，200r/min振荡培养，发酵过程中通过补加氨水维持pH在7.0-7.2；补加60％(m/v)葡萄糖溶液维持发酵进行；
优选的发酵培养基组成为：葡萄糖20-40g/L，木糖5-15g/L，酵母提取物2-5g/L，蛋白胨2-5g/L，KH 2PO 4 1-3g/L，MgSO 4·7H 2O 1-2g/L，FeSO 4·7H 2O 5-20mg/L，MnSO 4·7H 2O 5-20mg/L，V B1、V B3、V B5、V B12、V H各1-3mg/L，其余为水，pH 7.0-7.2。
利用500mL摇瓶发酵24-30h后组氨酸的产量可达6-10g/L。
(2)发酵罐发酵：
将菌种活化后制备种子液，按照15-20％接种量接入新鲜的发酵培养基，开始发酵，发酵过程中控制pH稳定在7.0左右，温度维持在37℃，溶氧在25-35％之间；当培养基中的葡萄 糖消耗完之后，流加80％(m/v)的葡萄糖溶液，维持发酵培养基中的葡萄糖浓度在0.1-5g/L；优选的发酵培养基组成为：葡萄糖10-30g/L，酵母提取物1-5g/L，蛋白胨1-5g/L，K 2HPO 4 1-5g/L，MgSO 4·7H 2O 1-3g/L，FeSO 4·7H 2O 10-30mg/L，MnSO 4·H 2O 10-30mg/L，V B1、V B3、V B5、V B12、V H各1-3mg/L，其余为水，pH 7.0-7.2。
利用5L发酵罐发酵40-50h可生产组氨酸40-55g/L，平均生产强度为1.0-1.5g/(L×h)，转化率为0.18-0.22g组氨酸/g葡萄糖。
有益效果：
目前为止，组氨酸菌种多采用多轮诱变筛选的方法或者在诱变菌株的基础上进行进一步的基因工程改造获得。一方面，所得菌株存在许多负向突变，导致菌株表现出一定的生长缺陷，造成对环境胁迫应对能力降低以及营养需求增高等问题。另一方面，现有组氨酸生产菌株多采用质粒过表达的方式强化组氨酸合成相关基因的表达，这种改造方式加重了菌体的生长负担，同时抗生素的使用以及质粒易丢失造成的成本增加，发酵液中抗生素残留以及生产稳定性差等问题限制了其在大规模工业化生产中的应用。
本发明提供一株遗传背景清晰，不含质粒，能够稳定生产L-组氨酸的基因工程菌，该菌用于发酵法生产L-组氨酸，利用5L发酵罐发酵40-50h可生产组氨酸40-55g/L，生产强度为1.0-1.5g/(L×h)，转化率为0.18-0.22g组氨酸/g葡萄糖，是目前发酵法生产L-组氨酸的最高水平。
另外，目前有关组氨酸高产菌种选育的研究中还未有涉及组氨酸转运系统改造的报道，特别是组氨酸的分泌蛋白未知，本发明中首次证实引入谷氨酸棒杆菌中的精氨酸/赖氨酸转运蛋白LysE有助于组氨酸产量的提升，是组氨酸菌种改造的重要靶点。
附图说明
图1：(a)pREDCas9质粒图谱，(b)pGRB质粒图谱。
图2：在tdcD基因位点整合hisG*时整合片段构建及验证电泳图。其中：M：1kb DNA marker；1：上游同源臂；3：hisG*基因片段；3：下游同源臂；4：重叠片段；5：原菌对照；6：阳性菌的鉴定片段。
图3：在ylbE基因位点整合hisG*时整合片段构建及验证电泳图。其中：M：1kb DNA marker；1：上游同源臂；3：hisG*基因片段；3：下游同源臂；4：重叠片段；5：原菌对照；6：阳性菌的鉴定片段。
图4：hisD整合片段构建及验证电泳图。其中：M：1kb DNA marker；1：上游同源臂；3：hisD基因片段；3：下游同源臂；4：重叠片段；5：原菌对照；6：阳性菌的鉴定片段。
图5：hisC-hisB整合片段构建及验证电泳图。其中：M：1kb DNA marker；1：hisC上游序列-hisC-hisB片段；2：下游同源臂；3：重叠片段；4：原菌对照；5：阳性菌的鉴定片段。
图6：hisH-hisA-hisF-hisI整合片段构建及验证电泳图。其中：M：1kb DNA marker；1：hisH上游序列-hisH-hisA-hisF-hisI片段；2：下游同源臂；3：重叠片段；4：原菌对照；5：阳性菌的鉴定片段。
图7：P trc-lysE整合片段的构建及验证电泳图。其中：M：1kb DNA marker；1：上游同源臂；2：lysE基因片段；3：下游同源臂；4：重叠片段；5：原菌对照；6：阳性菌鉴定片段。
图8：菌株E.coli WHY2-3和菌株E.coli WHY3的摇瓶发酵实验结果。
图9：E.coli WHY3在5L发酵罐上的发酵过程曲线。
具体实施方式
下面通过具体的实施方案叙述本发明。除非特别说明，本发明中所用的技术手段均为本领域技术人员所公知的方法。另外，实施方案应理解为说明性的，而非限制本发明的范围，本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言，在不背离本发明实质和范围的前提下，对这些实施方案中的物料成分和用量进行的各种改变或改动也属于本发明的保护范围。
以下实施例中涉及到的百分号“％”，如果没有本领域公知的定义也未特别说明，指的是体积百分比；溶液的百分比“％(m/v)”指的是100mL溶液中含有溶质的克数。
实施例1：
菌株E.coli WHY3的构建：
1 基因编辑的方法
本发明中采用的基因编辑方法参照文献(Li Y,Lin Z,Huang C,et al.Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing.Metabolic engineering,2015,31:13-21.)进行，该方法所用的两个质粒图谱见附图1。其中pREDCas9携带gRNA表达质粒pGRB的消除系统，λ噬菌体的Red重组系统及Cas9蛋白表达系统，奇霉素抗性(工作浓度：100mg/L)，32℃培养；pGRB以pUC18为骨架，包括启动子J23100，gRNA-Cas9结合区域序列和终止子序列，氨苄青霉素抗性(工作浓度：100mg/L)，37℃培养。
该方法的具体步骤如下：
1.1 pGRB质粒构建
构建质粒pGRB的目的是为了转录相应的gRNA，从而与Cas9蛋白形成的复合体，并通过碱基配对和PAM识别目的基因靶位点，实现目的DNA双链断裂。采用包含靶序列的DNA 片段与线性化的载体片段重组的方法构建pGRB质粒。
1.1.1 靶序列设计
使用CRISPR RGEN Tools设计靶序列(PAM:5’-NGG-3’)
1.1.2 包含靶序列的DNA片段的制备
设计引物：5’-线性化载体末端序列(15bp)-酶切位点-靶序列(不包括PAM序列)-线性化载体末端序列(15bp)-3’及其反向互补的引物，通过单链DNA的退火制备包含靶序列的DNA片段。反应条件：预变性95℃，5min；退火30-50℃，1min。退火体系如下：
退火体系
Figure PCTCN2019089938-appb-000001
1.1.3 线性载体的制备
载体的线性化采用反向PCR扩增的方法。
1.1.4 重组反应
重组体系如下表。所用重组酶均为
Figure PCTCN2019089938-appb-000002
II One Step Cloning Kit系列的酶，重组条件：37℃，30min。
重组体系
Figure PCTCN2019089938-appb-000003
1.1.5 质粒的转化
取10μL反应液，加入到100μL DH5α化转感受态细胞中，轻轻混匀后冰浴20min，42℃热激45-90s，立即冰浴2-3min，加入900μL SOC，于37℃复苏1h。8000rpm离心2min，弃部分上清，留200μL左右将菌体重悬后涂布到含有100mg/L氨苄青霉素的平板，将平板倒置，于37℃过夜培养。待平板长出单菌落后通过菌落PCR鉴定，挑选阳性重组子。
1.1.6 克隆鉴定
将PCR阳性菌落接种至含有100mg/L氨苄青霉素的LB培养基中过夜培养后保菌，提取质粒，酶切鉴定。
1.2 重组DNA片段的制备
用于敲除的重组片段由需敲除基因的上下游同源臂组成(上游同源臂-下游同源臂)；用于整合的重组片段以整合位点的上下游同源臂及待整合的基因片段组成(上游同源臂-目的基因-下游同源臂)。利用引物设计软件primer5，以待敲除基因或待整合位点的上下游序列为模板，设计上下游同源臂引物(扩增长度约400-500bp)；以待整合基因为模板，设计整合基因的扩增引物。通过PCR的方法分别扩增上下游同源臂和目的基因片段后，再经过重叠PCR制备重组片段。PCR的体系和方法如下表：
PCR扩增体系
Figure PCTCN2019089938-appb-000004
重叠PCR的体系如下表：
重叠PCR扩增体系
Figure PCTCN2019089938-appb-000005
注：模板由上下游同源臂的扩增片段和目的基因等摩尔组成，且总量不超过10ng。
PCR反应条件(宝生物PrimeSTAR HS酶)：预变性(95℃)5min；然后进行30轮循环：变性(98℃)10s，退火((Tm-3/5)℃)15s，72℃延伸(此酶活力1min延伸约1kb)；72℃继续延伸10min；维持(4℃)。
1.3 质粒和重组DNA片段的转化
1.3.1 pREDCas9的转化
利用电转的方法将pREDCas9质粒电转至W3110的电转感受态中，将菌体复苏培养后涂布于含奇霉素的LB平板上，32℃过夜培养。抗性平板上生长单菌落用鉴定引物进行菌落PCR, 筛选阳性重组子。
1.3.2 含pREDCas9的目的菌株电转化感受态制备
32℃培养至OD 600＝0.1～0.2时，添加0.1M的IPTG(使其终浓度为0.1mM)，继续培养至OD 600＝0.6～0.7时进行感受态制备。添加IPTG的目的是使pREDCas9质粒上的重组酶诱导表达。感受态制备所需培养基及制备过程参照常规标准操作。
1.3.3 pGRB和重组DNA片段的转化
将pGRB和供体DNA片段同时电转化至含有pREDCas9的电转感受态细胞中。将电转化后复苏培养的菌体涂布于含氨苄青霉素和奇霉素的LB平板上，32℃过夜培养。用上游同源臂上游引物和下游同源臂的下游引物，或设计专门的鉴定引物，进行菌落PCR验证，筛选阳性重组子并保菌。
1.4 质粒的消除
1.4.1 pGRB的消除
将阳性重组子置于含有0.2％阿拉伯糖的LB培养基中过夜培养，适量稀释后涂布于含有奇霉素抗性的LB平板上，32℃过夜培养。对点含有氨苄青霉素和奇霉素抗性的LB平板，挑选氨苄青霉素平板不生长，奇霉素抗性平板生长的单菌落保菌。
1.4.2 pREDCas9质粒的消除
将阳性重组子转接到无抗性的LB液体培养基中，42℃过夜培养，适量稀释后涂布于无抗性的LB平板上，37℃过夜培养。对点含有奇霉素抗性和无抗性的LB平板，挑选奇霉素抗性平板不生长，无抗性平板生长的单菌落保菌。
2.菌株构建过程中所涉及的所有引物见下表：
Figure PCTCN2019089938-appb-000006
Figure PCTCN2019089938-appb-000007
3 菌株构建的具体过程
3.1 解除HisG所受的反馈抑制并使其强表达
3.1.1 将P trc-hisG*整合在tdcD基因位点
以E.coli W3110(ATCC 27325)基因组为模板，根据其tdcD基因的上、下游序列设计上游 同源臂引物(UP-tdcD-S、UP-tdcD-A)和下游同源臂引物(DN-tdcD-S、DN-tdcD-A)，并PCR扩增其上下游同源臂片段；根据hisG*基因(核苷酸序列如SEQ ID No.2所示)设计引物(hisG*-S、hisG*-A)，然后再扩增hisG*基因片段。启动子P trc则设计在上游同源臂的下游引物和hisG*基因的上游引物中。上述片段通过重叠PCR的方法获得hisG*基因的整合片段(上游同源臂-P trc-hisG*-下游同源臂)，构建pGRB-tdcD使用的含靶序列的DNA片段通过引物gRNA-tdcD-S和gRNA-tdcD-A的退火制得。制备E.coli W3110的感受态细胞，按照1.3和1.4所示的方法操作，最终获得菌株E.coli WHY1-1。P trc-hisG*整合片段的构建和阳性菌株的PCR验证的电泳图见附图2。其中，上游同源臂的长度应为496bp，所扩增的hisG*基因片段长度应为627bp，下游同源臂的长度应为1902bp，整合片段的总长应为3024bp，用鉴定引物进行PCR验证时，阳性菌PCR扩增片段长度应为627bp，原菌无条带。
3.1.2 将P trc-hisG*整合在ylbE基因位点
以E.coli W3110(ATCC 27325)基因组为模板，根据其ylbE基因的上下游序列设计上游同源臂引物(UP-ylbE-S、UP-ylbE-A)和下游同源臂引物(DN-ylbE-S、DN-ylbE-A)，并PCR扩增其上下游同源臂片段；用引物(hisG*-S、hisG*-A)，扩增hisG*基因片段。上述片段通过重叠PCR的方法获得hisG*基因的整合片段(上游同源臂-P trc-hisG*-下游同源臂)，构建pGRB-ylbE使用的含靶序列的DNA片段通过引物gRNA-ylbE-S和gRNA-ylbE-A的退火制得。制备E.coli WHY1-1的感受态细胞，按照1.3和1.4所示的方法操作，最终获得菌株E.coli WHY1-2。P trc-hisG*整合片段的构建和阳性菌株的PCR验证的电泳图见附图3。其中，上游同源臂的长度应为601bp，所扩增的hisG*基因片段长度应为627bp，下游同源臂的长度应为547bp，整合片段的总长应为1815bp，用鉴定引物进行PCR验证时，阳性菌PCR扩增片段长度应为903bp，原菌无条带。
3.2 将E.coli W3110的组氨酸操纵子基因整合在yghX基因位点
本发明中将E.coli W3110中的组氨酸操纵基因(hisDBCHAFI，包含hisD、hisB、hisC、hisH、hisA、hisF和hisI七个基因)按顺序依次整合在E.coli WHY1-2基因组上的假基因yghX位点处，并由启动子P trc启动该操纵子的转录表达，构建了菌株E.coli HIS3-3。
组氨酸操纵子基因的整合共分了三段。
3.2.1 P trc-hisD的整合
以E.coli W3110(ATCC27325)基因组为模板，根据其yghX基因的上下游序列设计上游同源臂引物(UP-yghX-S、UP-yghX-A)和下游同源臂引物(DN-yghX-S1、DN-yghX-A)，并PCR扩增其上下游同源臂片段；根据hisD基因序列设计引物(hisD-S、hisD-A)，并PCR扩增hisD片段；启动子P trc则设计在上游同源臂的下游引物和hisD基因的上游引物中。上述片段通过 重叠PCR的方法融合，获得P trc-hisD基因的整合片段(上游同源臂-P trc-hisD-下游同源臂)，构建pGRB-yghX使用的含靶序列的DNA片段通过引物gRNA-yghX-S和gRNA-yghX-A的退火制得。制备E.coli WHY1-2的感受态细胞，按照1.3和1.4所示的方法操作，最终获得菌株E.coli WHY2-1。P trc-hisD片段整合过程中，整合片段的构建和阳性菌株的PCR验证的电泳图见附图4。其中上游同源臂长度为602bp，hisD基因片段长度为1305bp，下游同源臂长度为561bp，重叠片段的长度为2542bp，设计鉴定引物并进行PCR验证，阳性重组子所扩增的片段长度应为1208bp，原菌无条带。
3.2.2 hisB-hisC的整合
以E.coli W3110(ATCC27325)基因组为模板，根据hisB-hisC及其上游序列设计上游同源臂引物(UP-hisBC-S、UP-hisBC-A)，并PCR扩增其上游同源臂片段；以E.coli HIS3-1基因组为模板，根据其yghX基因的下游序列设计下游同源臂引物(DN-yghX-S2、DN-yghX-A)，并PCR扩增其下游同源臂片段。上述片段通过重叠PCR的方法融合，获得hisB-hisC的整合片段(hisB的上游片段-hisB-hisC-下游同源臂)。构建pGRB-his1使用的含靶序列的DNA片段通过引物gRNA-his1-S和gRNA-his1-A的退火制得。制备E.coli WHY2-1的感受态细胞，按照1.3和1.4所示的方法操作，最终获得菌株E.coli WHY2-2。hisB-hisC片段整合过程中，整合片段的构建和阳性菌株的PCR验证的电泳图见附图4。其中hisB的上游片段-hisB-hisC的总长度为2696bp，下游同源臂长度为561bp，重叠片段的长度为3317bp，设计鉴定引物并进行PCR验证，阳性重组子扩增片段的长度为1118bp，原菌无条带。
3.2.3 hisH-hisA-hisF-hisI的整合
以E.coli W3110(ATCC27325)基因组为模板，根据hisH-hisA-hisF-hisI及其上游序列设计上游同源臂引物(UP-hisHAFI-S、UP-hisHAFI-A)，并PCR扩增其上游同源臂片段；以E.coli HIS3-2基因组为模板，根据其yghX基因的下游序列设计下游同源臂引物(DN-yghX-S3、DN-yghX-A)，并PCR扩增其下游同源臂片段。上述片段通过重叠PCR的方法融合，获得hisH-hisA-hisF-hisI的整合片段(hisH的上游片段-hisH-hisA-hisF-hisI-下游同源臂)。构建pGRB-his2使用的含靶序列的DNA片段通过引物gRNA-his2-S和gRNA-his2-A的退火制得。制备E.coli WHY2-2的感受态细胞，按照1.3和1.4所示的方法操作，最终获得菌株E.coli WHY2-3。hisH-hisA-hisF-hisI的整合过程中，整合片段的构建和阳性菌株的PCR验证的电泳图见附图6。其中hisH的上游片段-hisH-hisA-hisF-hisI的总长度为3265bp，下游同源臂长度为561bp，重叠片段总长度为3317bp，设计鉴定引物并进行PCR验证，阳性重组子扩增片段的长度为1136bp，原菌无条带。
3.3 P trc-lysE的整合
以E.coli W3110(ATCC27325)基因组为模板，根据其yjiT基因的上下游序列设计上游同源臂引物(UP-yjiT-S、UP-yjiT-A)和下游同源臂引物(DN-yjiT-S1、DN-yjiT-A)，并PCR扩增其上下游同源臂片段；以谷氨酸棒杆菌(ATCC 13032)的基因组为模板，根据lysE(NCBI-GeneID:1019244)基因序列设计引物(lysE-S、lysE-A)，并PCR扩增lysE片段；启动子P trc则设计在上游同源臂的下游引物和lysE基因的上游引物中。上述片段通过重叠PCR的方法融合，获得P trc-lysE的整合片段(上游同源臂-P trc-lysE-下游同源臂)，构建pGRB-yjiT使用的含靶序列的DNA片段通过引物gRNA-yjiT-S和gRNA-yjiT-A的退火制得。制备E.coli WHY2-3的感受态细胞，按照1.3和1.4所示的方法操作，最终获得菌株E.coli WHY3。P trc-lysE片段整合过程中，整合片段的构建和阳性菌株的PCR验证的电泳图见附图7。其中上游同源臂长度为372bp，lysE基因片段长度为834bp，下游同源臂长度为530bp，重叠片段的长度为1655bp，设计鉴定引物并进行PCR验证，阳性重组子所扩增的片段长度应为1429bp，原菌则无条带。
实施例2：
利用基因工程菌E.coli WHY3发酵生产组氨酸的方法如下：
斜面培养：取-80℃保藏菌种划线接种于活化斜面，37℃培养12h，并传代一次；
摇瓶种子培养：用接种环刮取一环斜面种子接种于装有30mL种子培养基的500mL三角瓶中，九层纱布封口，37℃，200rpm培养6-8h；
摇瓶发酵培养：按10-15％接种量接种到装有发酵培养基的500mL三角瓶中(终体积为30mL)，九层纱布封口，37℃，200r/min振荡培养，发酵过程中通过补加氨水维持pH在7.0-7.2；补加60％(m/v)葡萄糖溶液维持发酵进行；发酵周期24-30h；
斜面培养基组成为：葡萄糖1-5g/L，蛋白胨5-10g/L，牛肉膏5-10g/L，酵母粉1-5g/L，NaCl 1-2.5g/L，琼脂20-25g/L，其余为水，pH 7.0-7.2；
种子培养基组成为：葡萄糖15-30g/L，酵母提取物5-10g/L，蛋白胨5-10g/L，KH 2PO 4 5-15g/L，MgSO 4·7H 2O 2-5g/L，FeSO 4·7H 2O 5-20mg/L，MnSO 4·H 2O 5-20mg/L，V B1 1-3mg/L，V H 0.1-1mg/L，消泡剂2滴，其余为水，pH 7.0-7.2；
发酵培养基组成为：葡萄糖20-30g/L，木糖5-15g/L，酵母提取物2-5g/L，蛋白胨2-4g/L，KH 2PO 4 1-3g/L，MgSO 4·7H 2O 1-2g/L，FeSO 4·7H 2O 5-20mg/L，MnSO 4·7H 2O 5-20mg/L，V B1、V B3、V B5、V B12、V H各1-3mg/L，其余为水，pH 7.0-7.2。
摇瓶发酵24-30h后L-组氨酸的产量可达6-10g/L。
(2)发酵罐发酵：
斜面活化培养：从-80℃冰箱保菌管中刮一环菌种，均匀涂布于活化斜面，37℃培养12-16 h，转接茄形瓶继续培养12-16h；
种子培养：取适量无菌水于茄形瓶中，将菌悬液接入种子培养基中，pH稳定在7.0左右，温度恒定在37℃，溶氧在25-35％之间，培养至发酵液OD 600值达10-15；
按照15-20％接种量接入新鲜的发酵培养基，开始发酵，发酵过程中控制pH稳定在7.0左右，温度维持在37℃，溶氧在25-35％之间；当培养基中的葡萄糖消耗完之后，流加80％(m/v)的葡萄糖溶液，维持发酵培养基中的葡萄糖浓度在0.1-5g/L。
斜面培养基组成为：葡萄糖1-5g/L，蛋白胨5-10g/L，牛肉膏5-10g/L，酵母粉1-5g/L，NaCl 1-2.5g/L，琼脂20-25g/L，其余为水，pH 7.0-7.2；
种子培养基组成为：葡萄糖15-30g/L，酵母提取物5-10g/L，蛋白胨5-10g/L，KH 2PO 4 5-15g/L，MgSO 4·7H 2O 2-5g/L，FeSO 4·7H 2O 5-15mg/L，MnSO 4·H 2O 5-15mg/L，V B1 1-3mg/L，V H 0.1-1mg/L，消泡剂2滴，其余为水，pH 7.0-7.2；
发酵培养基组成为：葡萄糖10-25g/L，酵母提取物1-5g/L，蛋白胨1-5g/L，K 2HPO 4 1-5g/L，MgSO 4·7H 2O 1-3g/L，FeSO 4·7H 2O 10-30mg/L，MnSO 4·H 2O 10-30mg/L，V B1、V B3、V B5、V B12、V H各1-3mg/L，其余为水，pH 7.0-7.2。
利用5L发酵罐发酵40-50h可生产组氨酸40-55g/L，平均生产强度为1.0-1.5g/(L×h)，转化率为0.18-0.22g组氨酸/g葡萄糖。
实施例3：
以实施例1中构建的菌株E.coli WHY2-3和菌株E.coli WHY3的摇瓶发酵实验：
上述两株菌在相同条件下采用摇瓶发酵的方法生产L-组氨酸，具体如下：
斜面培养：取-80℃保藏菌种划线接种于活化斜面，37℃培养12h，并传代一次；
摇瓶种子培养：用接种环刮取一环斜面种子接种于装有30mL种子培养基的500mL三角瓶中，九层纱布封口，37℃，200rpm培养8h；
摇瓶发酵培养：按15％接种量接种到装有发酵培养基的500mL三角瓶中(终体积为30mL)，九层纱布封口，37℃，200r/min振荡培养，发酵过程中通过补加氨水维持pH在7.0-7.2；补加60％(m/v)葡萄糖溶液维持发酵进行；发酵周期30h。
斜面培养基组成为：葡萄糖1g/L，蛋白胨10g/L，牛肉膏10g/L，酵母粉5g/L，NaCl2.5g/L，琼脂20g/L，其余为水，pH 7.0-7.2。
种子培养基组成为：葡萄糖30g/L，酵母提取物5g/L，蛋白胨5g/L，KH 2PO 4 6g/L，MgSO 4·7H 2O 2.5g/L，FeSO 4·7H 2O 10mg/L，MnSO 4·H 2O 10mg/L，V B1 2mg/L，V H 1mg/L，消泡剂2滴，其余为水，pH 7.0-7.2。
发酵培养基组成为：葡萄糖20g/L，木糖10g/L，酵母提取物5g/L，蛋白胨4g/L，KH 2PO 4  3g/L，MgSO 4·7H 2O 2g/L，FeSO 4·7H 2O 10mg/L，MnSO 4·7H 2O 10mg/L，V B1、V B3、V B5、V B12、V H各2mg/L，其余为水，pH 7.0-7.2。
实验结果如图8。从发酵结果看，在整合了来源于谷氨酸棒杆菌的lysE基因后，菌体的生长和耗糖状况未受影响，而组氨酸的产量则从8g/L提高到9.3g/L，提高了16.25％。
实施例4：
E.coli WHY3在5L发酵罐上的发酵实验：
以实施例1构建的菌株E.coli WHY3作为生产菌株生产组氨酸：
斜面活化：取甘油保存菌种划线接种于试管斜面培养基，37℃培养12；再将斜面保存菌种划线接种于茄形瓶斜面培养基，37℃培养14h。
种子培养：取活化的新鲜茄形瓶斜面1一支，用150mL无菌水洗下，在火焰保护下接种到发酵罐中，温度控制37℃，自动流加氨水控制pH在7.0，初始通气速率为2L/min，初始搅拌转速为200rpm，培养过程中维持DO值在20-30％之间，种子培养至OD 600为15左右。
发酵罐培养：发酵罐种子以15％接种量接入种子液(放料至450mL，在火焰保护下倒入灭菌的发酵培养基)，温度控制35℃，自动流加氨水(或20％硫酸)控制pH在7.0，初始通气速率为2L/min，通气比为0.667vvm，初始搅拌转速为400rpm，通过调整转速和风量控制溶氧在20-30％，通过手动滴加泡敌消泡，发酵过程中流加80％的葡萄糖溶液，保证充足的糖供应并且糖浓度不高于5g/L。
斜面培养基组成为：葡萄糖1g/L，蛋白胨10g/L，牛肉膏10g/L，酵母粉5g/L，NaCl2.5g/L，琼脂25g/L，其余为水，pH 7.0-7.2；
种子培养基组成为：葡萄糖10g/L，酵母提取物5g/L，蛋白胨5g/L，KH 2PO 4 5g/L，MgSO 4·7H 2O 2g/L，FeSO 4·7H 2O 10mg/L，MnSO 4·H 2O 10mg/L，V B1 2mg/L，V H 1mg/L，消泡剂2滴，其余为水，pH 7.0-7.2。
发酵培养基组成为：葡萄糖10g/L，酵母提取物5g/L，胰蛋白胨4g/L，K 2HPO 4 3g/L，MgSO 4·7H 2O 1.5g/L，FeSO 4·7H 2O 20mg/L，MnSO 4·H 2O 20mg/L，V B1、V B3、V B5、V B12、V H各2mg/L，其余为水，pH 7.0-7.2。
E.coli WHY3在5L发酵罐上的发酵曲线如图9。
从发酵曲线可以看出，发酵8小时以后进入组氨酸的快速积累阶段，此时的最大生产强度可达2g/(L×h)；16小时以后菌体生长进入稳定期，发酵液的OD 600为89；发酵至44小时时，组氨酸浓度达到最大值55g/L，之后组氨酸的浓度和菌体浓度开始有下降趋势；48小时结束发酵，此时的糖酸转化率为0.2g组氨酸/g葡萄糖。

Claims (9)

  1. 一株高产L-组氨酸的基因工程菌，其特征在于，所述基因工程菌是在大肠杆菌的基因组上整合了核苷酸序列如SEQ ID NO:1所示的谷氨酸棒杆菌ATP转磷酸核糖基酶HisG突变体编码基因hisG*并使其强表达；还在基因组上增强了大肠杆菌组氨酸操纵子hisD、hisB、hisC、hisH、hisA、hisF和hisI七个基因的表达；还在基因组上整合了来源于谷氨酸棒杆菌的精氨酸/赖氨酸转运蛋白的编码基因lysE并使其强表达。
  2. 如权利要求1所述的基因工程菌，其特征在于，所述大肠杆菌为E.coli W3110。
  3. 如权利要求1所述的基因工程菌，其特征在于，所述谷氨酸棒杆菌为谷氨酸棒杆菌ATCC 13032。
  4. 如权利要求1所述的基因工程菌，其特征在于，所述谷氨酸棒杆菌ATP转磷酸核糖基酶HisG突变体编码基因hisG*整合在基因组上至少两个基因位点处，并由强启动子启动。
  5. 如权利要求1或2所述的基因工程菌，其特征在于，所述谷氨酸棒杆菌ATP转磷酸核糖基酶HisG突变体编码基因hisG*分别整合在基因组上tdcD和ylbE基因位点，并由启动子P trc启动；所述大肠杆菌组氨酸操纵子基因片段hisD-hisC-hisB-hisH-hisA-hisF-hisI整合在基因组上yghX基因位点，并由启动子P trc启动；所述精氨酸/赖氨酸转运蛋白的编码基因lysE整合在基因组上yjiT基因位点，并由启动子P trc启动。
  6. 权利要求1-5任一项所述基因工程菌用于发酵生产L-组氨酸的用途。
  7. 如权利要求6所述的用途，其特征在于，利用所述基因工程菌进行摇瓶发酵：
    将菌种活化后制备种子液，按10-15％接种量接种到装有发酵培养基的三角瓶中，九层纱布封口，37℃，200r/min振荡培养，发酵过程中通过补加氨水维持pH在7.0-7.2；补加60％(m/v)葡萄糖溶液维持发酵进行；发酵周期24-30h；
    所述发酵培养基组成为：葡萄糖20-40g/L，酵母提取物2-5g/L，蛋白胨2-5g/L，KH 2PO 4 1-3g/L，MgSO 4·7H 2O 1-2g/L，FeSO 4·7H 2O 5-20mg/L，MnSO 4·7H 2O 5-20mg/L，V B1、V B3、V B5、V B12、V H各1-3mg/L，其余为水，pH 7.0-7.2。
  8. 如权利要求6所述的用途，其特征在于，利用所述基因工程菌进行发酵罐发酵：
    将菌种活化后制备种子液，按照15-20％接种量接入新鲜的发酵培养基，开始发酵，发酵过程中控制pH稳定在7.0左右，温度维持在37℃，溶氧在25-35％之间；当培养基中的葡萄糖消耗完之后，流加80％(m/v)的葡萄糖溶液，维持发酵培养基中的葡萄糖浓度在0.1-5g/L；发酵周期40-50h；
    所述发酵培养基组成为：葡萄糖10-30g/L，酵母提取物1-5g/L，蛋白胨1-5g/L，K 2HPO 4  1-5g/L，MgSO 4·7H 2O 1-3g/L，FeSO 4·7H 2O 10-30mg/L，MnSO 4·H 2O 10-30mg/L，V B1、V B3、V B5、V B12、V H各1-3mg/L，其余为水，pH 7.0-7.2。
  9. 一种高产组氨酸的基因工程菌的构建方法，其特征在于，所述基因工程菌是采用CRISPR/Cas 9介导的基因编辑技术对E.coli W3110进行定向改造所得，包括如下步骤：
    (1)构建启动子P trc与核苷酸序列如SEQ ID NO:1所示的基因hisG*的连接片段P trc-hisG*，并将其分别整合在基因组上tdcD和ylbE基因位点；
    (2)构建启动子P trc与大肠杆菌组氨酸操纵子基因的连接片段P trc-hisD-hisC-hisB-hisH-hisA-hisF-hisI，并用分段整合的方法将其整合在基因组上yghX基因位点；
    (3)构建启动子P trc与来源于谷氨酸棒杆菌的lysE基因的连接片段P trc-lysE，并将其整合在基因组上yjiT基因位点。
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