CN111996155B

CN111996155B - Method for improving production capacity of L-histidine producing strain

Info

Publication number: CN111996155B
Application number: CN202010933228.1A
Authority: CN
Inventors: 范文超; 刘映淼; 王金刚; 高书良; 梁岩; 任亮
Original assignee: Zhejiang Huarui Biotechnology Co ltd
Current assignee: Huarui Biotechnology Chuzhou Co ltd
Priority date: 2020-09-08
Filing date: 2020-09-08
Publication date: 2022-02-11
Anticipated expiration: 2040-09-08
Also published as: CN111996155A

Abstract

The invention discloses a method for improving the production capacity of L-histidine producing bacteria, which comprises the following steps: mutating ATP phosphoribosyltransferase from escherichia coli to obtain a mutant with improved enzyme activity; designing a gene encoding the mutant; integrating the coding gene into the genome of the escherichia coli to obtain the genetically engineered bacterium. The method of the invention can improve the L-histidine production capacity of the L-histidine producing strain by more than 3.5 times.

Description

Method for improving production capacity of L-histidine producing strain

Technical Field

The invention belongs to the field of genetic engineering, and relates to a method for improving the production capacity of an L-histidine producing strain, an L-histidine genetic engineering producing strain and application thereof.

Background

L-histidine (L-histidine) is a semi-essential amino acid, which is particularly important for the growth of infants and animals, and which, as the human body begins to synthesize itself with age, is also used as a biochemical or pharmaceutical intermediate for the production of pharmaceuticals. According to the research report of the industry, in 2017, the global consumption of histidine is approximately 559.5 tons, 1516.2 tons of medicine and 393.1 tons of feed, and the consumption of L-histidine is estimated to reach 3000-4000 tons in 2023, so that the market demand is more and more large.

Currently, there are two main methods for preparing L-histidine: the protein hydrolysis method is mainly used for preparation in China (see China journal of biochemical medicine, 1982(02): 12-17; CN 101125831B); foreign high-yielding L-histidine strains obtained mainly by mutagenesis of Corynebacterium glutamicum or Escherichia coli were prepared by microbial fermentation (US 8071339; CA 02319283; CN 1749390A; CN 102286562A). Wherein the yield of cooperative and ajinomotoxin of Japan fermentation company accounts for more than 90%. At present, domestic medicinal L-histidine is mainly imported and is self-supplied by a microbial fermentation method.

In the E.coli L-histidine biosynthetic pathway, ATP phosphoribosyltransferase (HisG), a key enzyme, is inhibited by various intermediate metabolites and end products, especially by the end product L-histidine. Eliminating feedback inhibition by HisG is an important step in histidine breeding strains. Previously reported anti-feedback inhibition mutant enzyme HisG^E271KIs inhibited by purine nucleotides, particularly ADP and AMP, by competitive inhibition with ATP substrates, and 5-aminoimidazole-4-carboxamide ribonucleotides (AICAR) have also been found to be very potent inhibitors of ATP phosphoribosyltransferase (Malykh et al, micro Cell Fact (2018),17: 42). Through carrying out multiple amino acid site mutations on HisG in Corynebacterium glutamicum, an N215K/L231F/T235A mutant is finally obtained, the Ki value of the mutant is improved by 37 times, the concentration of a product in a fermentation broth is improved from 0.11mM to 4.15mM, and the high yield of histidine is further realized (Biochimie,94(2012) and p 829-838).

These studies demonstrated that obtaining higher yields of L-histidine producing strains by genetic engineering means is of great significance for increasing the benefits of L-histidine production.

Disclosure of Invention

In order to construct a genetic engineering strain with higher L-histidine yield, the invention utilizes a genetic engineering technology to transform Escherichia coli producing L-histidine. The histidine operon (his operon) is modified to improve the enzyme activity of feedback inhibition ATP phosphoribosyltransferase resistance and obtain a strain with obviously improved L-histidine yield, so that the production capacity of L-histidine is improved, and the strain has wide industrial development and application prospects. Specifically, the invention comprises the following technical scheme:

a method for improving the productivity of L-histidine producing bacteria comprises the following steps:

A. mutating ATP phosphoribosyltransferase from escherichia coli to obtain a mutant with improved enzyme activity;

B. designing a gene encoding the ATP phosphoribosyltransferase mutant obtained in the step A;

C. and B, integrating the gene in the step B into the genome of the escherichia coli, or replacing the original ATP phosphoribosyltransferase encoding gene in the genome of the escherichia coli to obtain the genetic engineering bacteria.

In one embodiment, the Escherichia coli described in step A is Escherichia coli W3110.

The amino acid sequence of ATP phosphoribosyltransferase from Escherichia coli W3110 is SEQ ID NO 18.

Preferably, the mutant can be K136E or S226Ter mutant (wherein Ter is a terminator) of SEQ ID NO. 18, and the amino acid sequence of the mutant is SEQ ID NO. 20.

The coding gene of the ATP phosphoribosyltransferase mutant can be SEQ ID NO: 21.

The gene encoding ATP phosphoribosyltransferase derived from Escherichia coli W3110 is SEQ ID NO 19.

Substitutions for the original ATP phosphoribosyltransferase encoding gene, such as SEQ ID NO:19, can be: the original gene in Escherichia coli is knocked out or inactivated, and then a coding gene of an ATP phosphoribosyl transferase mutant, such as SEQ ID NO:21, is integrated.

In one embodiment, step C is achieved by gene editing techniques.

Optionally, in another embodiment, step C may also be implemented by the following steps: constructing a plasmid for expressing the ATP phosphoribosyltransferase mutant, such as a vector pTrc99a, transferring the plasmid into an Escherichia coli competent cell, and screening to obtain a positive clone with correct gene sequencing verification.

The above gene editing techniques may employ a CRISPR-Cas system such as CRISPR/Cas9 system, CRISPR-Cas related transposition system INTEGRATE system, or CAST system.

According to a second aspect of the present invention, there is provided a genetically engineered bacterium constructed according to the above-described method.

According to a third aspect of the present invention, there is provided the use of the above genetically engineered bacterium in the production of L-histidine.

L-histidine is preferably produced by fermentation of the above-mentioned genetically engineered bacterium.

When the genetically engineered bacteria are fermented, the seed liquid culture medium can be composed of: 20g/L glucose, 4g/L yeast extract, 3g/L peptone, 1.2g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate 7H₂O, 10mg/L ferrous sulfate 7H₂O, 10mg/L manganese sulfate monohydrate, 1mg/L VB1, 1mg/L VB3, 1mg/L VB5, 1mg/L VB12, 1mg/L VH, sodium hydroxide adjusted to pH 7.0-7.2.

The shake flask fermentation medium may consist of: 20g/L glucose, 4g/L yeast extract, 3g/L peptone, 2g/L sodium citrate monohydrate, 2g/L potassium dihydrogen phosphate, 2g/L magnesium sulfate 7H₂O, 20mg/L ferrous sulfate, 20mg/L manganese sulfate, 2mg/L VB1, 2mg/L VB3, 2mg/L VB5, 2mg/L VB12 and 2mg/L VH, adjusting the pH value to 7.0-7.2 by sodium hydroxide, and supplementing ammonia water to the pH value of 7.2-7.4 every 4 hours during fermentation.

The method can improve the L-histidine production capacity of L-histidine producing bacteria, particularly Escherichia coli by at least 3.6 times, and has industrial development value.

Drawings

FIG. 1 is a schematic diagram of the structure of plasmid pTrc99a, which is presented by the molecular excellence center of the plant of Chinese academy of sciences, Sancheng researcher.

FIG. 2 is a schematic structural diagram of plasmid pTrc99a-EchisG constructed according to the present invention.

Detailed Description

The invention carries out directional mutagenesis on the endogenous wild ATP phosphoribosyltransferase of the escherichia coli by a high-throughput screening method, obtains a high-activity ATP phosphoribosyltransferase mutant resisting feedback inhibition by enzyme activity determination, then supplements the mutant gene to the wild ATP phosphoribosyltransferase mutant gene to knock out an escherichia coli strain, and then carries out fermentation test to obtain the genetic engineering production strain with improved L-histidine yield.

The terms "L-histidine-producing bacterium", "genetically engineered bacterium", "L-histidine-producing bacterium" herein mean the same.

The starting strain or original strain of the L-histidine-producing strain of the present invention, the wild-type (WT) strain, is Escherichia coli W3110, which may be abbreviated as W3110. Its endogenous ATP phosphoribosyltransferase (hisG), SEQ ID NO:18, is abbreviated as wild-type hisG, and its expression gene is SEQ ID NO: 19.

Herein, for the sake of simplicity of description, a certain protein such as ATP phosphoribosyltransferase (hisG) is sometimes mixed with the name of its encoding gene (DNA), and those skilled in the art will understand that they represent different substances at different description occasions. Their meaning will be readily understood by those skilled in the art based on the context and context. For example, for hisG, when used to describe ATP phosphoribosyltransferase function or class, it refers to proteins; when described as a gene, refers to the gene encoding the ATP phosphoribosyltransferase.

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

The addition amount, content and concentration of various substances are referred to herein, wherein the percentage refers to the mass percentage unless otherwise specified.

Examples

Materials and methods

The whole gene synthesis, primer synthesis and sequencing in the examples were performed by Nanjing Jinzhi Biotechnology Ltd.

The molecular biological experimental procedures in the examples include plasmid construction, enzyme digestion, competent cell preparation, transformation, and the like, which are mainly performed with reference to molecular cloning, a guide to experiments (third edition), J. SammBruk, D.W. Lassel (America), Huangpetang, et al, science publishers, Beijing, 2002). For example, the methods for competent cell transformation and competent cell preparation are described in Chapter 1, 96 of molecular cloning, A laboratory Manual (third edition). The specific experimental conditions can be determined by simple experiments if necessary.

Main culture medium:

LB culture medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride. (20 g/L agar powder was additionally added to the solid medium.)

Shake flask fermentation seed medium (1L): 20g glucose, 4g yeast extract, 3g peptone, 1.2g potassium dihydrogen phosphate, 0.5g magnesium sulfate 7H₂O, 10mg Festus 7H₂O, 10mg of manganese sulfate monohydrate, 1mg of VB1, 1mg of VB3, 1mg of VB5, 1mg of VB12, 1mg of VH, and sodium hydroxide to regulate the pH value to 7.0-7.2.

Shake flask fermentation medium (1L): 20g glucose, 4g yeast extract, 3g peptone, 2g sodium citrate monohydrate, 2g potassium dihydrogen phosphate, 2g magnesium sulfate 7H₂O, 20mg Festus 7H₂O, 20mg of manganese sulfate monohydrate, 2mg of VB1, 2mg of VB3, 2mg of VB5, 2mg of VB12, and 2mg of VH and sodium hydroxide are adjusted to a pH value of between 7.0 and 7.2, and ammonia is added to the mixture every 4 hours for fermentation until the pH value is between 7.2 and 7.4.

In the following examples, when a medium containing ampicillin resistance was used, the antibiotic concentration was 100. mu.g/ml; when kanamycin and spectinomycin medium were used, the final concentration of the antibiotic in the medium was 50. mu.g/ml.

PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the supplier of the plasmid or DNA template. If necessary, it can be adjusted by simple experiments.

The sequence information of the primers used in the examples is shown in Table 1.

TABLE 1 primer sequences

In Table 1, "-F" in the name represents the forward direction; "-R" represents reverse.

Example 1: construction of wild type hisG gene engineering bacteria

Primer pairs 99A-HisG-F and 99A-HisG-R sequences SEQ ID NO:1 and SEQ ID NO:2 were synthesized and PCR amplification was performed using the genome of the E.coli W3110 strain as a template (the following PCR reagents were purchased from KOD FX series of TOYOBO, Toyo Bombycis).

The 50 μ L PCR amplification system was: KOD FX 1. mu.L, KOD FX buffer 25. mu.L, dNTP 3. mu.L, genome template 0.5. mu.L, upstream and downstream primers 2. mu.L each, ddH₂O make up to 50. mu.L.

The PCR procedure was: heating the cover at 105 ℃ and pre-denaturing at 95 ℃ for 5 min; denaturation at 98 ℃ for 30s, annealing at 57 ℃ for 30s, and extension at 68 ℃ for 60s for 30 cycles; finally, extending for 10min at 68 ℃; cooling at 16 deg.C for 10 min.

The gene SEQ ID NO of the wild-type hisG is obtained by PCR amplification 19. The pTrc99a plasmid (see FIG. 1, which is given by the Cheng researchers in the excellence center of the plant molecule of Chinese academy of sciences) was double-digested with restriction enzymes NcoI and BamHI to obtain a linearized vector. Then obtaining pTrc99a-hisG plasmid by homologous recombination method, referring to figure 2, then transforming into Escherichia coli expression host W3110 competent cells by calcium chloride method, coating LB culture medium plate with benzyl resistance, culturing overnight at 37 ℃, selecting single colony, inoculating into LB culture medium test tube containing ampicillin, culturing overnight, centrifugally collecting thallus, extracting plasmid, determining correct gene sequencing, obtaining recombinant gene engineering strain expressing wild type ATP phosphoribosyltransferase SEQ ID NO. 18.

Example 2: construction of hisG random mutation point library by error-prone PCR method

The wild ATP phosphoribosyltransferase is mutated by an error-prone PCR method.

The 50 μ L error-prone PCR reaction system included: 50ng of pTrc99a-hisG plasmid template constructed in example 1, 30pmol of the HISG-EP-F and HISG-EP-R primer pairs SEQ ID NO:14 and SEQ ID NO:15, 1 XTaq buffer, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 7mM MgCl2, (0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM) MnCl₂2.5 units of Taq enzyme (fermentas).

The PCR reaction conditions are as follows: 5min at 95 ℃; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 2 min/kbp; 10min at 72 ℃.

The restriction enzyme DpnI was digested at 50 ℃ for 1 hour, and the 0.9kb mutant fragment was gel-recovered and subjected to homologous recombination with the pTrc99a linearized vector constructed in example 1, which was double digested with NcoI and BamHI, and then transformed into W3110 competent cells, an E.coli expression host, by the calcium chloride method, to obtain more than 10⁴Random mutant pools of individual clones.

Example 3: high throughput screening of hisG mutant pools

3.1 selecting transformants in the mutant library, inoculating the transformants into a 96-well deep-well culture plate containing 700 mu L of LB culture medium containing 100 mu g/mL of ampicillin, culturing at 37 ℃ for 6h, adding IPTG (0.1 mM final concentration), cooling to 25 ℃, and culturing overnight. Centrifuging at 5000rpm for 10min, discarding supernatant, freezing at-70 deg.C for 1h, and thawing at room temperature for 30 min. 200. mu.L of a buffer solution (pH8.0) containing 0.1M potassium phosphate salt was added thereto, and the cells were resuspended for measurement of hisG enzyme activity.

3.2 assay of HisG enzyme Activity

HisG enzyme activity assay reference (Biochimie,94, 2012, P829-838). Enzyme activity determination system: the reaction mixture contained 100mM Tris-HCl (pH 8.5), 150mM KCl, 10mM MgCl₂5mM ATP, 0.5mM PRPP, 1U yeast pyrophosphatase, in a volume of 0.1 mL. In 96-well microplate. The increase in absorbance was monitored with an ultraviolet spectrophotometer at 290 nm. The reaction was carried out at room temperature for 4min, and the reaction mixture without enzyme was a blank. One activity unit is defined as the amount of enzyme capable of increasing the absorbance by 0.02/min, corresponding to a conversion of 1.67nmol per minuteMinimum value of substance.

Screening about 1000 mutant clones in a random mutation library to find that the mutant enzyme activity of 4 amino acid site substitutions is improved. The hisG enzyme activities of 3 mutants were significantly improved, and the results are shown in Table 1.

TABLE 2 comparison of enzyme activities of partial hisG mutants

Mutants	Amino acid mutations	Relative enzyme activity (%)
			HisG	-	100
HisG-36	A175D	185
			HisG-117	K136E、S226Ter	387
HisG-833	K136E、A175S、S226Ter	316

In the table, the mutant amino acid a175D represents the substitution of the 175 th amino acid a (alanine, Ala) with D (aspartic acid, Asp), S226Ter represents the substitution of the 226 th amino acid S (serine, Ser) with Ter (stop codon), K136E represents the substitution of the 136 th amino acid K (lysine, Lys) with E (glutamic acid, Glu), and a175S represents the substitution of the 175 th amino acid a (alanine, Ala) with S (serine, Ser) on wild-type HisG. The mutant with the number of HisG-117 has the enzyme activity improved by nearly 3 times compared with the wild type. Hereinafter, the mutant HisG-117 was intensively studied, and its amino acid sequence was SEQ ID NO. 20 and the coding gene was SEQ ID NO. 21.

Example 4: construction of W3110 delta hisLG Gene engineering Strain

4.1 preparation of pTarget-hisL (together with pTarget-intron) plasmid

Primer pairs HisL-N20-F/HisL-N20-R sequences SEQ ID NO:3 and SEQ ID NO:4 were synthesized, and PCR-amplified using pTarget plasmid (Addgene ID 62226, a gift from Cheng researchers in the molecular excellence center of plants, academy of sciences, China) as a template.

The 50 μ L PCR amplification system was: KOD FX 1. mu.L, KOD FX buffer 25. mu.L, dNTP 3. mu.L, pTarget plasmid 0.5. mu.L, upstream and downstream primers 2. mu.L each, ddH₂O make up to 50. mu.L.

The PCR procedure was: heating the cover at 105 ℃ and pre-denaturing at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 68 ℃ for 2min, and 30 cycles; finally, extending for 10min at 68 ℃; cooling at 16 deg.C for 10 min. Amplification yielded an approximately 2.2Kb fragment.

The cells were digested with DpnI at 50 ℃ for 1 hour, 3. mu.L of the cells were transformed into DH 5. alpha. competent cells to obtain pTarget-hisL plasmid, and positive transformants were identified by sequencing.

The pTarget-intron plasmid is constructed by the same method, a primer pair HisL-N20-R/His-intron-N20-F sequence SEQ ID NO. 4 and SEQ ID NO. 5 are synthesized, and a PCR amplification system, a program and subsequent test steps are constructed with the pTarget-hisL plasmid by taking the pTarget plasmid as a template.

4.2 preparation of CRISPR-mediated hisLG knockout homology arms

A primer pair His-intron-N20-F/HisG-KO-UP-F sequences SEQ ID NO:5 and SEQ ID NO:6 was synthesized, and a hisLG knockout upstream homology arm (0.5kb) was prepared using the E.coli W3110 genome as a template. A primer pair HisG-KO-UP-R/HisG-KO-DN-F sequences SEQ ID NO:7 and SEQ ID NO:8 was synthesized, and a hisLG knockout downstream homology arm (0.5kb) was prepared using the E.coli W3110 genome as a template. Then synthesizing a primer pair of His-intron-N20-F/HisG-KO-DN-F sequences SEQ ID NO:5 and SEQ ID NO:8, taking the hisLG knockout upstream homology arm and the hisLG knockout downstream homology arm as templates, performing overlap PCR, and preparing the hisLG knockout homology arm by using a PCR amplification system and a program as above.

4.3 preparation of E.coli W3110. DELTA. hisLG/pCasSac Strain

Escherichia coli W3110 strain was prepared into calcification chloride-competent cells, which were transformed into pCas plasmid (Addgene ID 62225, presented by Yangshen researchers, the center of excellence in plant molecular, academy of China), 50. mu.l of which was plated with kanamycin antibiotic, and cultured overnight at 30 ℃ to obtain W3110/pCas monoclonal strain.

The W3110/pCasSac monoclonal was picked up and placed in LB + Kan test tube medium and cultured overnight on a shaker at 30 ℃ and 220 rpm. Inoculating 500 μ L of the strain to 50ml of LB + Kan +100mM arabinose liquid medium shake flask, culturing at 30 deg.C and 220rpm for 2-2.5h to OD₆₀₀The value reaches about 1.0. The whole amount of the suspension was transferred to a 50ml centrifuge tube on a clean bench, centrifuged at 4500 Xg at 4 ℃ and the supernatant was discarded, the cells were washed with 10% glycerol and resuspended, centrifuged at 4500 Xg at 4 ℃ and the supernatant was discarded after 1-time washing. And finally, adding 300 mu L of 10% glycerol to suspend the thalli, subpackaging the thalli into 1.5ml centrifuge tubes, preparing a competent cell every 90 mu L, adding 500ng of pTarget-hisL plasmid and 1 mu g of hisLG knockout homology arms (the total amount is not more than 10 mu L), uniformly mixing, transferring the mixture into an electric rotating cup, carrying out electric shock under the conditions of 2.5kV and 200 omega, wherein the electric shock time is 5.5ms, immediately transferring the mixture into 900 mu L of LB liquid culture medium after the electric shock, and then placing the mixture into a constant temperature shaking table to culture for 1h at 30 ℃ and 220rpm so as to recover the thalli. After recovery, 100. mu.l of the cells were spread on LB plates containing kanamycin and spectinomycin, and the plates were inverted and incubated overnight in a 30 ℃ incubator to obtain W3110. delta. hisLG/pCas + pTarget-hisL monoclonal strains. And (3) carrying out colony PCR identification on the transformation plate monoclonal by using primer pairs HISG-EP-F/HISG-EP-R sequences SEQ ID NO:14 and SEQ ID NO:15 to obtain the hisLG knockout positive strain.

Selecting positive clones to LB liquid (kan resistance), adding 10mM rhamnose, inducing sgRNA transcription of the targeting plasmid pTarget-hisL, shake culturing at 30 ℃ overnight, taking a little bacterial liquid, streaking on an LB (kan resistance) plate to separate a single colony, standing and culturing at 37 ℃ for about 12h, selecting a single clone to an LB (Spec resistance) plate, and obtaining a strain which can not grow on the LB (Spec resistance) plate, namely the pTarget-hisL eliminating strain W3110 delta hisLG/pCas. The strain does not express the original ATP phosphoribosyl transferase coding gene SEQ ID NO. 19 in the genome of Escherichia coli W3110 any more, and can be used for evaluating the enzyme activity of HisG mutant.

Example 5: construction of histidine-producing genetic engineering strain

Respectively synthesizing a primer pair HisG-KO-UP-F/HisG-AS-UP-R sequence SEQ ID NO:6/SEQ ID NO:10 and a primer pair HisG-KO-DN-R/HisG-AS-DN-F sequence SEQ ID NO:9/SEQ ID NO:13, respectively using the W3110 genome AS a template, and carrying out PCR amplification on Ptrchi G integrated upstream and downstream homology arm sequences. The PtrchisG fragment was PCR-amplified using the primer pair HisG-AS-F/HisG-AS-R sequence SEQ ID NO:11/SEQ ID NO:12, using the pTrc99a-hisG plasmid constructed in example 1 AS a template. Then using primers to perform overlap PCR on the sequence SEQ ID NO. 6/SEQ ID NO. 9 with the upstream and downstream homology arms of hisG and the three segments of PtrchisG fragment to obtain Uparm-PtrchisG-Dnarm fragment.

Likewise, plasmid pTrc99a-hisG obtained by site-directed mutagenesis in example 3 using the primer pair HisG-AS-F/HisG-AS-R sequences SEQ ID NO:11/SEQ ID NO:12^{K136E,S226Ter}Plasmid as template, PCR amplification of PtrchisG^{K136E,S226Ter}A fragment plasmid. Synthesizing a primer pair HisG-KO-UP-F/HisG-KO-DN-R sequence SEQ ID NO:6/SEQ ID NO:9, using the upstream and downstream homology arms of the hisG and PtrchisG^{K136E,S226Ter}The fragment was subjected to overlap PCR to obtain Uparm-PtrchisG^{K136E,S226Ter}A Dnarm fragment.

The W3110. DELTA.hisLG strain was used to prepare electroporation competent cells in the same manner as in example 4, and pTarget-intron plasmid and Upram-Ptrchis G-Dnarm fragment, pTarget-intron plasmid and Upram-Ptrchis G-into each of the two electroporation competent cells^{K136E,S226Ter}The Dnarm fragment was electroporated and threated, and then 100. mu.l of each cell was spread on LB plates containing kanamycin and spectinomycin, and the plates were inverted and incubated overnight in a 30 ℃ incubator to obtain W3110. delta. hisLG/pCas + pTarget-intron monoclonal strains. The primers HisLG-KO-V-F/HisLG-KO-V-R sequences SEQ ID NO:16 and SEQ ID NO:17 were used to perform colony PCR amplification on the single clones of the transformation plate, and the amplified fragments were sequenced to obtain PtrchisG and PtrchisG^{K136E,S226Ter}Integrating the strains.

Selecting positive clones to LB liquid (Kan resistance), adding 10mM rhamnose, inducing sgRNA transcription of targeting plasmid pTarget-intron, shake culturing at 37 deg.C overnight, taking a little bacterial liquid to streak out single colony on LB plate, standing culturing at 37 deg.C for about 12h, selecting single clone to LB (Kan resistance) plate, LB (Spec resistance) plate and LB plate, the strain which can grow on LB and can not grow on Kan and Spec resistance plate is W3110 without plasmid, PtrcHisG and W3110^{K136E,S226Ter}And (3) strain. Strain W3110 PtrcHisG^{K136E,S226Ter}The genes of the ATP phosphoribosyltransferase K136E and S226Ter mutant coding SEQ ID NO 21 are integrated in the genome of the transgenic microorganism.

Example 6: fermentation of histidine producing strain and determination of product content

6.1 seed Shake flask culture

Freshly cultured colonies were picked from LB plates into seed shake flasks (30ml/500ml) and incubated overnight at 37 ℃ with shaking at 220 rpm.

6.2 Shake flask fermentation

5ml of seed medium was taken from the seed shake flask into a fermentation shake flask (30ml/500ml single baffle shake flask) and incubated at 37 ℃ for about 24h with shaking at 220rpm (if the fermentation broth no longer changed color within 2h, the fermentation could be terminated earlier). And (3) adjusting the pH value by using 20% (v/v) ammonia water in the fermentation process (the ammonia water is stopped when the fermentation liquor turns red by shaking while adding). And (4) centrifuging the fermentation liquor at a high speed, taking the supernatant, diluting by 5 times, and detecting the yield of the L-histidine by HPLC.

6.3 high Performance liquid chromatography HPLC determination of histidine content

The L-histidine content of the fermentation broth was determined using OPA pre-column derivatized amino acid analysis. The first-order amino acid reacts with o-phthalaldehyde (OPA) in the presence of a sulfhydryl reagent to generate OPA-amino acid, the generated amino acid derivative is separated by reversed-phase high performance liquid chromatography and then is detected by ultraviolet or fluorescence, and the light absorption value of the amino acid derivative is in direct proportion to the concentration of the amino acid in a certain range.

Derivatizing agent and mobile phase formulation:

boric acid buffer: 6.183g of boric acid is accurately weighed by 0.4M boric acid buffer solution, dissolved in ultrapure water, adjusted to pH 10.2 by 10N NaOH solution and added into a volumetric flask with the volume of 250 ml.

A derivatizing agent: 500mg of an o-phthalaldehyde (OPA) solid was weighed out accurately, 5ml of absolute ethanol was added, 500. mu.l of mercaptopropionic acid was added, and a volume of 50ml was made up with 0.4M boric acid buffer solution of pH 10.2.

Mobile phase A: 40mM NaH₂PO₄Solution, accurately weighing 5.5g NaH₂PO₄·H₂Dissolving O in ultrapure water, adjusting the pH value to 7.8 by using 10NNaOH solution, metering to 1L, and filtering by using a 0.22 mu m filter membrane for later use.

Mobile phase B: methanol, reagent purity HPLC grade.

High performance liquid chromatography measurement conditions:

a chromatographic column: illite Hypersil BDS c184.6x 250mm, 5 μm; flow rate: 1.0 ml/min; stopping time: 20 min; column temperature: 40 degrees; setting the DAD: UV 334nm, 10nm (bandwidth), referenced 390nm, 20nm (bandwidth).

The elution procedure is shown in table 3.

TABLE 3 HPLC elution procedure

The sample was taken by an autosampler, and the sample was taken after mixing and derivatization, in each case, 0.5. mu.l of the sample, 2.5. mu.l of the boric acid buffer, 0.5. mu.l of the derivatization agent, and 32. mu.l of ultrapure water.

4.3L-histidine yield by fermentation of the Strain:

the L-histidine fermentation yields of the respective strains were compared according to the fermentation protocol determination method described above, and 3 parallel experiments were performed for each strain, and the results are shown in Table 4.

TABLE 4 comparison of L-histidine levels in fermentation of strains

As can be seen from Table 4, the genetically engineered strain W3110 (PtrcHisG)^{K136E,S226Ter}The yield of L-histidine was improved by about 3.75 times compared with W3110:PtrcHisG. Thus, hisG obtained by directed evolution in combination with high throughput screening was validated^K136E ^,S226TerThe mutant can effectively relieve the inhibition of histidine or an intermediate metabolite thereof on ATP phosphoribosyltransferase, improves the yield of L-histidine, lays a foundation for the subsequent genetic engineering modification of strains, and is worthy of further development and utilization.

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ccccgaggat caattagcag ctaaaccact ttcacgttag aaagc 45

<210> 8

<211> 45

<212> DNA

<213> Artificial sequence ()

<400> 8

gctgctaatt gatcctcggg gtcgccatga gctttaacac aatca 45

<210> 9

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 9

gatctcatcg gcaatcggcg gcg 23

<210> 10

<211> 45

<212> DNA

<213> Artificial sequence ()

<400> 10

gagccggatg attaattgtc aataaaccac tttcacgtta gaaag 45

<210> 11

<211> 45

<212> DNA

<213> Artificial sequence ()

<400> 11

ctttctaacg tgaaagtggt ttattgacaa ttaatcatcc ggctc 45

<210> 12

<211> 41

<212> DNA

<213> Artificial sequence ()

<400> 12

gtgttaaagc tcatggcgat cactccatca tcttctcaat c 41

<210> 13

<211> 41

<212> DNA

<213> Artificial sequence ()

<400> 13

gattgagaag atgatggagt gatcgccatg agctttaaca c 41

<210> 14

<211> 50

<212> DNA

<213> Artificial sequence ()

<400> 14

gataacaatt tcacacagga aacagaccat gacagacaac actcgtttac 50

<210> 15

<211> 48

<212> DNA

<213> Artificial sequence ()

<400> 15

gcctgcaggt cgactctaga ggatcctcac tccatcatct tctcaatc 48

<210> 16

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 16

cgctattttt ggtgccatca g 21

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 17

gctgtattcc cgcagggcct c 21

<210> 18

<211> 299

<212> PRT

<213> Escherichia coli W3110

<400> 18

Met Thr Asp Asn Thr Arg Leu Arg Ile Ala Met Gln Lys Ser Gly Arg

1 5 10 15

Leu Ser Asp Asp Ser Arg Glu Leu Leu Ala Arg Cys Gly Ile Lys Ile

20 25 30

Asn Leu His Thr Gln Arg Leu Ile Ala Met Ala Glu Asn Met Pro Ile

35 40 45

Asp Ile Leu Arg Val Arg Asp Asp Asp Ile Pro Gly Leu Val Met Asp

50 55 60

Gly Val Val Asp Leu Gly Ile Ile Gly Glu Asn Val Leu Glu Glu Glu

65 70 75 80

Leu Leu Asn Arg Arg Ala Gln Gly Glu Asp Pro Arg Tyr Phe Thr Leu

85 90 95

Arg Arg Leu Asp Phe Gly Gly Cys Arg Leu Ser Leu Ala Thr Pro Val

100 105 110

Asp Glu Ala Trp Asp Gly Pro Leu Ser Leu Asn Gly Lys Arg Ile Ala

115 120 125

Thr Ser Tyr Pro His Leu Leu Lys Arg Tyr Leu Asp Gln Lys Gly Ile

130 135 140

Ser Phe Lys Ser Cys Leu Leu Asn Gly Ser Val Glu Val Ala Pro Arg

145 150 155 160

Ala Gly Leu Ala Asp Ala Ile Cys Asp Leu Val Ser Thr Gly Ala Thr

165 170 175

Leu Glu Ala Asn Gly Leu Arg Glu Val Glu Val Ile Tyr Arg Ser Lys

180 185 190

Ala Cys Leu Ile Gln Arg Asp Gly Glu Met Glu Glu Ser Lys Gln Gln

195 200 205

Leu Ile Asp Lys Leu Leu Thr Arg Ile Gln Gly Val Ile Gln Ala Arg

210 215 220

Glu Ser Lys Tyr Ile Met Met His Ala Pro Thr Glu Arg Leu Asp Glu

225 230 235 240

Val Ile Ala Leu Leu Pro Gly Ala Glu Arg Pro Thr Ile Leu Pro Leu

245 250 255

Ala Gly Asp Gln Gln Arg Val Ala Met His Met Val Ser Ser Glu Thr

260 265 270

Leu Phe Trp Glu Thr Met Glu Lys Leu Lys Ala Leu Gly Ala Ser Ser

275 280 285

Ile Leu Val Leu Pro Ile Glu Lys Met Met Glu

290 295

<210> 19

<211> 900

<212> DNA

<213> Escherichia coli W3110

<400> 19

atgacagaca acactcgttt acgcatagct atgcagaaat ccggccgttt aagtgatgac 60

tcacgcgaat tgctggcgcg ctgtggcatt aaaattaatc ttcacaccca gcgcctgatc 120

gcgatggcag aaaacatgcc gattgatatt ctgcgcgtgc gtgacgacga cattcccggt 180

ctggtaatgg atggcgtggt agaccttggg attatcggcg aaaacgtgct ggaagaagag 240

ctgcttaacc gccgcgccca gggtgaagat ccacgctact ttaccctgcg tcgtctggat 300

ttcggcggct gtcgtctttc gctggcaacg ccggttgatg aagcctggga cggtccgctc 360

tccttaaacg gtaaacgtat cgccacctct tatcctcacc tgctcaagcg ttatctcgac 420

cagaaaggca tctcttttaa atcctgctta ctgaacggtt ctgttgaagt cgccccgcgt 480

gccggactgg cggatgcgat ttgcgatctg gtttccaccg gtgccacgct ggaagctaac 540

ggcctgcgcg aagtcgaagt tatctatcgc tcgaaagcct gcctgattca acgcgatggc 600

gaaatggaag aatccaaaca gcaactgatc gacaaactgc tgacccgtat tcagggtgtg 660

atccaggcgc gcgaatcaaa atacatcatg atgcacgcac cgaccgaacg tctggatgaa 720

gtcatcgccc tgctgccagg tgccgaacgc ccaactattc tgccgctggc gggtgaccaa 780

cagcgcgtag cgatgcacat ggtcagcagc gaaaccctgt tctgggaaac catggaaaaa 840

ctgaaagcgc tgggtgccag ttcaattctg gtcctgccga ttgagaagat gatggagtga 900

<210> 20

<211> 225

<212> PRT

<213> Artificial sequence ()

<400> 20

Met Thr Asp Asn Thr Arg Leu Arg Ile Ala Met Gln Lys Ser Gly Arg

1 5 10 15

Leu Ser Asp Asp Ser Arg Glu Leu Leu Ala Arg Cys Gly Ile Lys Ile

20 25 30

Asn Leu His Thr Gln Arg Leu Ile Ala Met Ala Glu Asn Met Pro Ile

35 40 45

Asp Ile Leu Arg Val Arg Asp Asp Asp Ile Pro Gly Leu Val Met Asp

50 55 60

Gly Val Val Asp Leu Gly Ile Ile Gly Glu Asn Val Leu Glu Glu Glu

65 70 75 80

Leu Leu Asn Arg Arg Ala Gln Gly Glu Asp Pro Arg Tyr Phe Thr Leu

85 90 95

Arg Arg Leu Asp Phe Gly Gly Cys Arg Leu Ser Leu Ala Thr Pro Val

100 105 110

Asp Glu Ala Trp Asp Gly Pro Leu Ser Leu Asn Gly Lys Arg Ile Ala

115 120 125

Thr Ser Tyr Pro His Leu Leu Glu Arg Tyr Leu Asp Gln Lys Gly Ile

130 135 140

Ser Phe Lys Ser Cys Leu Leu Asn Gly Ser Val Glu Val Ala Pro Arg

145 150 155 160

Ala Gly Leu Ala Asp Ala Ile Cys Asp Leu Val Ser Thr Gly Ala Thr

165 170 175

Leu Glu Ala Asn Gly Leu Arg Glu Val Glu Val Ile Tyr Arg Ser Lys

180 185 190

Ala Cys Leu Ile Gln Arg Asp Gly Glu Met Glu Glu Ser Lys Gln Gln

195 200 205

Leu Ile Asp Lys Leu Leu Thr Arg Ile Gln Gly Val Ile Gln Ala Arg

210 215 220

Glu

225

<210> 21

<211> 900

<212> DNA

<213> Artificial sequence ()

<400> 21

atgacagaca acactcgttt acgcatagct atgcagaaat ccggccgttt aagtgatgac 60

tcacgcgaat tgctggcgcg ctgtggcatt aaaattaatc ttcacaccca gcgcctgatc 120

gcgatggcag aaaacatgcc gattgatatt ctgcgcgtgc gtgacgacga cattcccggt 180

ctggtaatgg atggcgtggt agaccttggg attatcggcg aaaacgtgct ggaagaagag 240

ctgcttaacc gccgcgccca gggtgaagat ccacgctact ttaccctgcg tcgtctggat 300

ttcggcggct gtcgtctttc gctggcaacg ccggttgatg aagcctggga cggtccgctc 360

tccttaaacg gtaaacgtat cgccacctct tatcctcacc tgctcgagcg ttatctcgac 420

cagaaaggca tctcttttaa atcctgctta ctgaacggtt ctgttgaagt cgccccgcgt 480

gccggactgg cggatgcgat ttgcgatctg gtttccaccg gtgccacgct ggaagctaac 540

ggcctgcgcg aagtcgaagt tatctatcgc tcgaaagcct gcctgattca acgcgatggc 600

gaaatggaag aatccaaaca gcaactgatc gacaaactgc tgacccgtat tcagggtgtg 660

atccaggcgc gcgaatgaaa atacatcatg atgcacgcac cgaccgaacg tctggatgaa 720

gtcatcgccc tgctgccagg tgccgaacgc ccaactattc tgccgctggc gggtgaccaa 780

cagcgcgtag cgatgcacat ggtcagcagc gaaaccctgt tctgggaaac catggaaaaa 840

ctgaaagcgc tgggtgccag ttcaattctg gtcctgccga ttgagaagat gatggagtga 900

Claims

1. A method for improving the productivity of L-histidine producing bacteria comprises the following steps:

A. mutating ATP phosphoribosyltransferase from Escherichia coli W3110 to obtain mutant with improved enzyme activity, wherein the amino acid sequence of the ATP phosphoribosyltransferase is SEQ ID NO. 18, the mutant is K136E and S226Ter mutant of SEQ ID NO. 18, the amino acid sequence of the mutant is SEQ ID NO. 20, and Ter is terminator;

2. The method of claim 1, wherein the gene encoding the mutant is SEQ ID NO 21.

3. The method of claim 1, wherein step C is accomplished by gene editing techniques.

4. A genetically engineered bacterium constructed according to the method of any one of claims 1 to 3.

5. The use of the genetically engineered bacterium of claim 4 for the production of L-histidine.

6. The use of claim 5, wherein L-histidine is produced by fermentation of said genetically engineered bacteria.

7. The use of claim 6, wherein the seed medium consists of: 20g/L glucose, 4g/L yeast extract, 3g/L peptone, 1.2g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate 7H₂O, 10mg/L Festus 7H₂O, 10mg/L manganese sulfate monohydrate, 1mg/L VB1, 1mg/L VB3, 1mg/L VB5, 1mg/L VB12, 1mg/L VH, sodium hydroxide adjusted to pH 7.0-7.2.