CN110184230A - The genetic engineering bacterium and its construction method of one plant height production L-Histidine and application - Google Patents
The genetic engineering bacterium and its construction method of one plant height production L-Histidine and application Download PDFInfo
- Publication number
- CN110184230A CN110184230A CN201910461591.5A CN201910461591A CN110184230A CN 110184230 A CN110184230 A CN 110184230A CN 201910461591 A CN201910461591 A CN 201910461591A CN 110184230 A CN110184230 A CN 110184230A
- Authority
- CN
- China
- Prior art keywords
- histidine
- gene
- hisg
- genome
- genetic engineering
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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)
Abstract
The present invention provides the genetic engineering bacterium and its construction method that a plant height produces L-Histidine, the bacterium is that nucleotide sequence Corynebacterium glutamicum ATP phosphoribosyl transferase HisG mutant code gene hisG* as shown in SEQ ID NO:1 is incorporated on the genome of Escherichia coli, to enhance the activity of histidine synthesis key enzyme HisG；The copy number of histidine operon gene is also increased in the genome, to enhance the terminal route of synthesis of histidine；Also incorporate the arginine from Corynebacterium glutamicum/lysine transport protein encoding gene lysE in the genome, with promote histidine intracellular to exocytosis.The genetic engineering bacterium is used for fermentation method of producing L-histidine, 5L ferment tank 40-50h can steady production histidine 40-55g/L, for production intensity up to 1.0-1.5g/ (L × h), conversion ratio is 0.18-0.22g histidine/g glucose.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of genetic engineering bacterium and its structure of high yield L-Histidine
Construction method and application.
Background technique
L-Histidine is protein structure element, is the 9th kind of necessary amino acid of humans and animals, participates in body development, resist
A variety of physiological and biochemical procedures such as oxidation and immunological regulation.In addition to as nutrition fortifier and feed addictive, L-Histidine is also got over
To be applied to pharmaceuticals industry more, is especially used to produce amino acid transfusion and comprehensive amino acid preparation, is used for heart disease, it is poor
The treatment of the diseases such as blood and gastroenteritic ulcer.Therefore, histidine is a kind of important products of amino acid of human production life, is had
Very high economy and society value.
The production method of histidine has chemical synthesis, Hydrolyze method and microbe fermentation method at present.Wherein, chemical synthesis
Mainly utilize dihydroxyacetone (DHA), formaldehyde and ammonia synthesis hydroxy methylimidazole, further synthesize histidine, but this method yield compared with
It is low, and the obtained histidine of this method is D, and L-Histidine, to prepare L-Histidine also needs to be torn open with crystallisation or enzyme process
Point, thus chemical synthesis prepare L-Histidine cost it is very high, practical application is few.Native protein Hydrolyze method produces amino acid
It is the common method of amino acids production, and histidine content highest in blood meal, pig or ox blood powder hydrolysis extraction method produce L- group ammonia
Acid is the main method for producing L-Histidine, but the loss late of this method L-Histidine is high, and equipment seriously corroded, separation costs are high,
It is not optimal histidine production method.Production by Microorganism Fermentation histidine, low raw-material cost is environmental-friendly, operation
Simply, the period is short, is suitble to industrialized production, is ideal L-Histidine production method.But it is limited to the fermentation level of strain, is sent out
Large-scale industrialization application is not yet received in ferment method production histidine.
Organism can synthesize L-Histidine by precursor of PRPP and ATP, but the biosynthesis pathway of L-Histidine is very long,
The metabolism network being related to is complicated, and is catalyzed the key enzyme HisG for the first step that histidine synthesizes also by stringent feedback regulation
Effect.Since 1970s, the researcher of the country such as Japan, the U.S., Russia from the bacterial strain of different genera,
The breeding work of L-Histidine strain is started, the method for strain improvement is substantially using more wheel mutagenesis screenings or in mutagenesis
Further genetic engineering transformation is carried out on the basis of bacterial strain.The starting strain of the bacterium of histidine production at present includes glutamic acid rod
Bacterium, thermophilic acetic acid bar bacterium, brevibacterium flavum, brevibacterium lactofermentum, Escherichia coli, Arthrobacter, Nocardia, withered grass bud
Spore bacillus, thermophilic ammonia microbacterium and serratia marcescens (e.g., United States Patent (USP): 7785860,3791925,4388405,7399618,
7871808,7067289,3716453,5294547；European patent: 1085086；Russ P: 2119536,2003677；
Japan Patent: JP19850116075).Emphasis of these researchs concentrate in vivo histidine terminal metabolic pathway repair
Decorations, especially solve the screening of the HisG mutant of regulation.Wherein, one plant of serratia marcescens of Japanese researcher's breeding
(Japan Patent, JP19850116075) fermentation 120h can accumulate 45g/L L-Histidine, be the fermentation method production L- of existing report
The highest level of histidine, the histidine operon gene that the plasmid pair which carries a low copy number has been mutated have carried out more
Copy.
Escherichia coli are easily cultivated, fermentation period is short, genetic properties are clear, genetic manipulation is simple, are commonly used for starting strain
Carry out breeding histidine Producing Strain.(Russ Ps, 2119536) such as Russian researchers Kljachko E V passes through traditional
Breeding method is selected with sulphaguanidine, D, L-1,2, the Escherichia coli of 4- triazole -3- alanine and streptomycin resistance, then into
One step carries out molecular modification to it, improves the transcription amount of its histidine operon gene, and obtaining yield is 16~17g/L, saccharic acid
The production bacterial strain VKPM B-7270 that conversion ratio is 24~26%；Sano of Japanese Ajincomoto Co., Inc etc. (United States Patent (USP),
4388405) strengthen L-Histidine in such a way that plasmid is overexpressed in production bacterium E.coli R-344 and synthesize relevant gene
Expression, so that L-Histidine output increased has been arrived 25g/L；Klyachko etc. is successively overexpressed in Escherichia coli production bacterium
PurH, talB and the prs gene of saltant type improve the yield of L-Histidine.
Mutagenic treatment is all passed through in existing L-histidine-producing bacteria strain substantially, and carries plasmid more.Due to negative sense mutation
Accumulation, plasmid, which exists, bears thalli growth bring, and the presence of the factors such as use of antibiotic leads to existing histidine strain
The problems such as slow growth, low to environment-stress adaptibility to response, nutritional need increases, and produces easily fluctuation, limits the industry of bacterial strain
Change application.From the clear wild-type strain of genetic background, histidine engineering bacteria is constructed by the method for metabolic engineering breeding,
It can largely solve the problems, such as that above-mentioned classic mutagenesis breeding exists.V.G.Doroshenko etc. is with wild Escherichia coli
MG1655+ is starting strain, by knocking out the leading peptide gene hisL of histidine operon, transcription regulatory factor purR, Yi Ji
Histidine, which synthesizes, introduces point mutation E271K in key enzyme HisG, so that bacterial strain be made to have histidine accumulation ability, shake flask fermentation can
Accumulate histidine 4.9g/L, but the too low requirement for not being able to satisfy industrialized production of its fermenting property.
Summary of the invention
In view of the above problems, it is an object of the present invention to provide a kind of bacillus coli gene engineerings of high yield L-Histidine
Bacterium and its construction method, and corresponding fermentation processes scheme has been formulated, which can be applied to the efficient industry of L-Histidine
Metaplasia produces.
Technical solution of the present invention is summarized as follows:
The present invention provides the genetically engineered E.coli WHY3 that a plant height produces L-Histidine, which is in Escherichia coli
Nucleotide sequence Corynebacterium glutamicum ATP phosphoribosyl transferase HisG as shown in SEQ ID NO:1 is incorporated on genome
Mutant code gene hisG* is acted on to release feedback regulation suffered by HisG and is made its strongly expressed；Also increase in the genome
The copy number of Escherichia coli itself histidine operon gene hisDBCHAFI, to enhance the terminal synthesis way of histidine
Diameter；The arginine from Corynebacterium glutamicum/lysine transport protein encoding gene lysE is also incorporated in the genome
And make its strongly expressed, with promote histidine intracellular to exocytosis.
Further, the Escherichia coli are E.coli W3110.
Further, the Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032.
Further, the histidine operon gene hisDBCHAFI, comprising hisD, hisB, hisC, hisH, hisA,
Seven genes of hisF and hisI.
Further, the Corynebacterium glutamicum ATP phosphoribosyl transferase HisG mutant code gene hisG* integration
On genome of E.coli at least two gene locis.
It is possible to further realize the strongly expressed of foreign gene by building strong promoter.
As a kind of better embodiment of the present invention, the Corynebacterium glutamicum ATP phosphoribosyl transferase HisG mutation
Body encoding gene hisG* is respectively integrated at genome of E.coli tdcD and ylbE gene loci, and by promoter PtrcStarting.
The present invention also provides the construction method of the genetically engineered E.coli WHY3 is as follows:
As a kind of better embodiment of the present invention, the genetically engineered E.coli WHY3 is using CRISPR/Cas
The 9 gene editing technologies mediated are oriented house of correction to E.coli W3110 and obtain, and specifically comprise the following steps:
(1) promoter P is constructedtrcWith the junction fragment of nucleotide sequence gene hisG* as shown in SEQ ID NO:1
Ptrc- hisG*, and it is respectively integrated at tdcD and ylbE gene loci on genome；
(2) promoter P is constructedtrcWith the junction fragment P of E. coli histidine operon genetrc-hisD-hisC-
HisB-hisH-hisA-hisF-hisI, and yghX gene loci on genome is integrated into the method for segmentation integration；
(3) promoter P is constructedtrcWith the junction fragment P of the lysE gene from Corynebacterium glutamicumtrc- lysE, and will
It integrates yjiT gene loci in the genome.
The present invention also provides the purposes using said gene engineering bacteria E.coli WHY3 fermenting and producing L-Histidine, packets
It includes:
(1) shake flask fermentation:
Seed liquor will be prepared after actication of culture, is inoculated into the triangular flask equipped with fermentation medium by 10-15% inoculum concentration,
The sealing of nine layers of gauze, 37 DEG C, 200r/min shaken cultivation maintains pH in 7.0-7.2 in fermentation process by adding ammonium hydroxide；It adds
60% (m/v) glucose solution maintains fermentation to carry out；
Preferred fermentation medium composition are as follows: glucose 20-40g/L, xylose 5-15g/L, yeast extract 2-5g/L, egg
White peptone 2-5g/L, KH2PO41-3g/L, MgSO4·7H2O 1-2g/L, FeSO4·7H2O 5-20mg/L, MnSO4·7H2O 5-
20mg/L, VB1、VB3、VB5、VB12、VHEach 1-3mg/L, remaining is water, pH 7.0-7.2.
Using the yield of histidine after 500mL shake flask fermentation 24-30h up to 6-10g/L.
(2) ferment tank:
Seed liquor will be prepared after actication of culture, fresh fermentation medium is accessed according to 15-20% inoculum concentration, starts to send out
Ferment, control pH stablizes 7.0 or so in fermentation process, and temperature maintains 37 DEG C, and dissolved oxygen is between 25-35%；When in culture medium
Glucose consumption it is complete after, stream plus 80% (m/v) glucose solution, maintain fermentation medium in concentration of glucose exist
0.1-5g/L；Preferred fermentation medium composition are as follows: glucose 10-30g/L, yeast extract 1-5g/L, peptone 1-5g/L,
K2HPO41-5g/L, MgSO4·7H2O 1-3g/L, FeSO4·7H2O 10-30mg/L, MnSO4·H2O 10-30mg/L, VB1、
VB3、VB5、VB12、VHEach 1-3mg/L, remaining is water, pH 7.0-7.2.
Histidine 40-55g/L can be produced using 5L ferment tank 40-50h, averagely production intensity is 1.0-1.5g/ (L
× h), conversion ratio is 0.18-0.22g histidine/g glucose.
The utility model has the advantages that
So far, histidine strain mostly uses the method for more wheel mutagenesis screenings or carries out on the basis of mutagenic strain
Further genetic engineering transformation obtains.On the one hand, there are many negative senses to be mutated for obtained strains, causes strains expressed to go out certain
Growth defect causes the problems such as increasing to the reduction of environment-stress adaptibility to response and nutritional need.On the other hand, existing histidine
The mode that production bacterial strain mostly uses plasmid to be overexpressed strengthens the expression of histidine synthesis related gene, and this reforming mode aggravates
The growth of thallus is born, at the same antibiotic use and plasmid it is easy to be lost caused by increased costs, antibiotic is residual in fermentation liquid
It stays and the problems such as production stability is poor limits its application in large-scale industrial production.
One plant of genetic background of present invention offer is clear, is free of plasmid, is capable of the genetic engineering bacterium of steady production L-Histidine,
The bacterium is used for fermentation method of producing L-histidine, can produce histidine 40-55g/L using 5L ferment tank 40-50h, production is strong
Degree is 1.0-1.5g/ (L × h), and it is current fermentation method of producing L-histidine that conversion ratio, which is 0.18-0.22g histidine/g glucose,
Highest level.
In addition, not relating to the transformation of histidine movement system in the research in relation to histidine high-yield strains breeding at present also
Report, the secretory protein of especially histidine is unknown, the arginine that confirmation is introduced into Corynebacterium glutamicum for the first time in the present invention/rely
Propylhomoserin transport protein LysE facilitates the promotion of histidine yield, is the important target spot of histidine strain transformation.
Detailed description of the invention
Fig. 1: (a) pREDCas9 plasmid map, (b) pGRB plasmid map.
Fig. 2: segment building and verifying electrophoretogram are integrated when tdcD gene loci integrates hisG*.Wherein: M:1kb DNA
marker；1: upstream homology arm；3:hisG* genetic fragment；3: downstream homology arm；4: overlapping fragments；5: opportunistic pathogen control；6: positive
The identification segment of bacterium.
Fig. 3: segment building and verifying electrophoretogram are integrated when ylbE gene loci integrates hisG*.Wherein: M:1kb DNA
marker；1: upstream homology arm；3:hisG* genetic fragment；3: downstream homology arm；4: overlapping fragments；5: opportunistic pathogen control；6: positive
The identification segment of bacterium.
Fig. 4: hisD integrates segment building and verifying electrophoretogram.Wherein: M:1kb DNA marker；1: upstream homology arm；
3:hisD genetic fragment；3: downstream homology arm；4: overlapping fragments；5: opportunistic pathogen control；6: the identification segment of positive bacteria.
Fig. 5: hisC-hisB integrates segment building and verifying electrophoretogram.Wherein: M:1kb DNA marker；On 1:hisC
Swim sequence-hisC-hisB segment；2: downstream homology arm；3: overlapping fragments；4: opportunistic pathogen control；5: the identification segment of positive bacteria.
Fig. 6: hisH-hisA-hisF-hisI integrates segment building and verifying electrophoretogram.Wherein: M:1kb DNA
marker；1:hisH upstream sequence-hisH-hisA-hisF-hisI segment；2: downstream homology arm；3: overlapping fragments；4: opportunistic pathogen
Control；5: the identification segment of positive bacteria.
Fig. 7: Ptrc- lysE integrates building and the verifying electrophoretogram of segment.Wherein: M:1kb DNA marker；1: upstream is same
Source arm；2:lysE genetic fragment；3: downstream homology arm；4: overlapping fragments；5: opportunistic pathogen control；6: positive bacteria identifies segment.
The shake flask fermentation experimental result of Fig. 8: bacterial strain E.coli WHY2-3 and bacterial strain E.coli WHY3.
Fermentation process curve of Fig. 9: the E.coli WHY3 on 5L fermentor.
Specific embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Range, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
Under the premise of invention spirit and scope, to the various changes or change of material component and dosage progress in these embodiments
It belongs to the scope of protection of the present invention.
The percentage sign " % " being related in following embodiment, if also not specified without definition well known in the art,
Refer to percent by volume；The percentage " % (m/v) " of solution refers to the grams in 100mL solution containing solute.
Embodiment 1:
The building of bacterial strain E.coli WHY3:
The method of 1 gene editing
Gene editing method reference literature (Li Y, Lin Z, Huang C, the et al.Metabolic used in the present invention
engineering of Escherichia coli using CRISPR–Cas9meditated genome
Editing.Metabolic engineering, 2015,31:13-21.) it carries out, two plasmid maps used in this method are shown in
Attached drawing 1.Wherein pREDCas9 carries the elimination system of gRNA expression plasmid pGRB, the Red recombination system and Cas9 egg of λ bacteriophage
White expression system, miramycin resistance (working concentration: 100mg/L), 32 DEG C of cultures；PGRB is using pUC18 as skeleton, including promoter
The bond area J23100, gRNA-Cas9 sequence and terminator sequence, amicillin resistance (working concentration: 100mg/L), 37
DEG C culture.
Specific step is as follows for this method:
1.1 pGRB plasmid constructions
Building plasmid pGRB purpose be in order to transcribe corresponding gRNA, thus with Cas9 albumen formed complex, and
By base pairing and PAM identifying purpose gene target site, target DNA double-strand break is realized.Using the DNA piece comprising target sequence
Section and the method for the carrier segments recombination of linearisation construct pGRB plasmid.
1.1.1 target sequence designs
Target sequence (PAM:5 '-NGG-3 ') is designed using CRISPR RGEN Tools
1.1.2 the preparation of the DNA fragmentation comprising target sequence
Design primer: 5 '-linearized vector end sequences (15bp)-restriction enzyme site-target sequence (not including PAM sequence)-
The primer of linearized vector end sequence (15bp) -3 ' and its reverse complemental includes target sequence by the annealing preparation of single stranded DNA
DNA fragmentation.Reaction condition: 95 DEG C of initial denaturation, 5min；30-50 DEG C of annealing, 1min.Annealing system is as follows:
Annealing system
1.1.3 the preparation of linear carrier
The method that the linearisation of carrier uses Inverse PCR amplification.
1.1.4 recombining reaction
The following table of recombination system.Recombinase used isII One Step Cloning Kit system
The enzyme of column, recombination condition: 37 DEG C, 30min.
Recombination system
1.1.5 the conversion of plasmid
10 μ L reaction solutions are taken, 100 μ L DH5 αization is added to and turns in competent cell, mix gently rear ice bath 20min, 42
DEG C heat shock 45-90s, ice bath 2-3min, is added 900 μ L SOC, in 37 DEG C of recovery 1h immediately.8000rpm is centrifuged 2min, abandons part
Supernatant is applied to the plate containing 100mg/L ampicillin after staying 200 μ L or so that thallus is resuspended, plate is inverted, in 37
It DEG C is incubated overnight.It is identified after plate grows single colonie by bacterium colony PCR, selects positive recombinant.
1.1.6 clone identification
PCR positive bacterium colony is seeded in the LB culture medium containing 100mg/L ampicillin after being incubated overnight and protects bacterium, mentioned
Take plasmid, digestion identification.
The preparation of 1.2 recombinant dna fragments
Recombinant fragment for knockout is made of that (upstream homology arm-downstream is homologous the upstream and downstream homology arm that need to knock out gene
Arm)；Recombinant fragment for integration forms that (upstream is homologous with the upstream and downstream homology arm and genetic fragment to be integrated of integration site
Arm-target gene-downstream homology arm).Using primer-design software primer5, with gene to be knocked out or to the upper of integration site
Downstream sequence is template, is designed upstream and downstream homology arm primer (amplification length about 400-500bp)；Using to integrator gene as template,
The amplimer of design integration gene.After expanding upstream and downstream homology arm and target gene fragment respectively by the method for PCR, then pass through
Lap over PCR preparation and reorganization segment.The system and method for PCR is as follows:
PCR amplification system
The system of over-lap PCR is as follows:
Over-lap PCR amplification system
Note: template is made of the amplified fragments and target gene equimolar of upstream and downstream homology arm, and total amount is no more than 10ng.
PCR reaction condition (treasured biology PrimeSTAR HS enzyme): initial denaturation (95 DEG C) 5min；Then 30 wheel circulations are carried out:
It is denaturalized (98 DEG C) 10s, anneal ((Tm-3/5) DEG C) 15s, and 72 DEG C extend (this enzyme activity 1min extends about 1kb)；72 DEG C after reneing
Stretch 10min；It maintains (4 DEG C).
The conversion of 1.3 plasmids and recombinant dna fragment
1.3.1 the conversion of pREDCas9
The electricity that pREDCas9 plasmid electricity goes to W3110 is turned in competence using the method that electricity turns, thallus is recovered and is cultivated
It is coated on the LB plate containing miramycin afterwards, 32 DEG C are incubated overnight.Single colonie is grown in resistant panel carries out bacterium with identification primer
PCR is fallen, positive recombinant is screened.
1.3.2 the purpose bacterial strain electrotransformation competence preparation containing pREDCas9
32 DEG C are cultivated to OD600When=0.1~0.2, the IPTG (making its final concentration of 0.1mM) of 0.1M is added, continues to cultivate
To OD600Competence preparation is carried out when=0.6~0.7.The purpose for adding IPTG is the recombination enzyme induction made on pREDCas9 plasmid
Expression.Culture medium needed for prepared by competence and preparation process are operated referring to conventional criteria.
1.3.3 the conversion of pGRB and recombinant dna fragment
By pGRB and donor DNA segment, electrotransformation to the electricity containing pREDCas9 turns in competent cell simultaneously.Electricity is turned
The thallus of culture of recovering after change is coated on the LB plate containing ampicillin and miramycin, and 32 DEG C are incubated overnight.It is same with upstream
The downstream primer of source arm upstream primer and downstream homology arm, or the special identification primer of design, carry out bacterium colony PCR verifying, screening
Positive recombinant simultaneously protects bacterium.
The elimination of 1.4 plasmids
1.4.1 the elimination of pGRB
Positive recombinant is placed in the LB culture medium containing 0.2% arabinose and is incubated overnight, is coated with after appropriate dilution
In on the LB plate containing miramycin resistance, 32 DEG C are incubated overnight.It is flat to LB of the point containing ampicillin and miramycin resistance
Plate is selected ampicillin plate and is not grown, and the single colonie of miramycin resistant panel growth protects bacterium.
1.4.2 the elimination of pREDCas9 plasmid
Positive recombinant is transferred in the LB liquid medium of non-resistant, 42 DEG C are incubated overnight, and are coated with after appropriate dilution
In on the LB plate of non-resistant, 37 DEG C are incubated overnight.To LB plate of the point containing miramycin resistance and non-resistant, miramycin is selected
Resistant panel is not grown, and the single colonie of non-resistant plated growth protects bacterium.
2. all primers involved in during strain construction see the table below:
The detailed process of 3 strain constructions
Feedback inhibition suffered by 3.1 releasing HisG simultaneously makes its strongly expressed
3.1.1 by Ptrc- hisG* is incorporated into tdcD gene loci
Using E.coli W3110 (ATCC 27325) genome as template, set according to the upstream and downstream sequence of its tdcD gene
Upstream homology arm primer (UP-tdcD-S, UP-tdcD-A) and downstream homology arm primer (DN-tdcD-S, DN-tdcD-A) are counted, and
Its upstream and downstream homology arm segment of PCR amplification；According to hisG* gene (nucleotide sequence is as shown in SEQ ID No.2) design primer
(hisG*-S, hisG*-A) then expands hisG* genetic fragment again.Promoter PtrcThen design is drawn in the downstream of upstream homology arm
In the upstream primer of object and hisG* gene.Above-mentioned segment obtained by the method for over-lap PCR hisG* gene integration segment (on
Swim homology arm-PtrcThe downstream-hisG*- homology arm), the DNA fragmentation containing target sequence that building pGRB-tdcD is used passes through primer
The annealing of gRNA-tdcD-S and gRNA-tdcD-A is made.The competent cell for preparing E.coli W3110, according to 1.3 and 1.4
Shown in method operation, it is final to obtain bacterial strain E.coli WHY1-1.Ptrc- hisG* integrates building and the positive strain of segment
The electrophoretogram of PCR verifying is shown in attached drawing 2.Wherein, the length of upstream homology arm should be 496bp, and the hisG* genetic fragment expanded is long
Degree should be 627bp, the length of downstream homology arm should be 1902bp, and the overall length for integrating segment should be 3024bp, with identification primer into
When row PCR is verified, positive bacteria pcr amplified fragment length should be 627bp, and opportunistic pathogen is without band.
3.1.2 by Ptrc- hisG* is incorporated into ylbE gene loci
Using E.coli W3110 (ATCC 27325) genome as template, according to the upstream and downstream sequence design of its ylbE gene
Upstream homology arm primer (UP-ylbE-S, UP-ylbE-A) and downstream homology arm primer (DN-ylbE-S, DN-ylbE-A), and
Its upstream and downstream homology arm segment of PCR amplification；With primer (hisG*-S, hisG*-A), hisG* genetic fragment is expanded.Above-mentioned segment
Integration segment (upstream homology arm-the P of hisG* gene is obtained by the method for over-lap PCRtrcThe downstream-hisG*- homology arm), structure
The DNA fragmentation containing target sequence that pGRB-ylbE is used is built to be made by the annealing of primer gRNA-ylbE-S and gRNA-ylbE-A.
The competent cell for preparing E.coli WHY1-1 is operated according to method shown in 1.3 and 1.4, final to obtain bacterial strain E.coli
WHY1-2。Ptrc- hisG* integrates the building of segment and the electrophoretogram of the PCR verifying of positive strain is shown in attached drawing 3.Wherein, upstream is same
The length of source arm should be 601bp, and the hisG* genetic fragment length expanded should be 627bp, and the length of downstream homology arm should be
547bp, the overall length for integrating segment should be 1815bp, when carrying out PCR verifying with identification primer, positive bacteria pcr amplified fragment length
It should be 903bp, opportunistic pathogen is without band.
3.2 by the histidine operon gene integration of E.coli W3110 in yghX gene loci
In the present invention by E.coli W3110 his operator (hisDBCHAFI, comprising hisD, hisB,
Seven genes of hisC, hisH, hisA, hisF and hisI) it is successively incorporated into the false base on E.coli WHY1-2 genome in order
At the site yghX, and by promoter PtrcThe transcriptional expression for starting the operon constructs bacterial strain E.coli HIS3-3.
The integration of histidine operon gene has been divided into three sections.
3.2.1 PtrcThe integration of-hisD
Using E.coli W3110 (ATCC27325) genome as template, according to the upstream and downstream sequence design of its yghX gene
Upstream homology arm primer (UP-yghX-S, UP-yghX-A) and downstream homology arm primer (DN-yghX-S1, DN-yghX-A), and
Its upstream and downstream homology arm segment of PCR amplification；According to hisD gene order design primer (hisD-S, hisD-A), and PCR amplification
HisD segment；Promoter PtrcThen design is in the downstream primer of upstream homology arm and the upstream primer of hisD gene.Above-mentioned segment
It is merged by the method for over-lap PCR, obtains PtrcIntegration segment (upstream homology arm-the P of-hisD genetrcThe downstream-hisD- is homologous
Arm), the moving back by primer gRNA-yghX-S and gRNA-yghX-A of the DNA fragmentation containing target sequence that building pGRB-yghX is used
Fire is made.The competent cell for preparing E.coli WHY1-2 operates according to method shown in 1.3 and 1.4, finally obtains bacterial strain
E.coli WHY2-1。PtrcIn-hisD segment integration process, the electrophoresis of the building of segment and the PCR verifying of positive strain is integrated
Figure is shown in attached drawing 4.Its middle and upper reaches homology arm length is 602bp, and hisD genetic fragment length is 1305bp, and the homologous arm lengths in downstream are
561bp, the length of overlapping fragments are 2542bp, and design identification primer simultaneously carries out PCR verifying, the segment that positive recombinant is expanded
Length should be 1208bp, and opportunistic pathogen is without band.
3.2.2 the integration of hisB-hisC
Using E.coli W3110 (ATCC27325) genome as template, according on hisB-hisC and its upstream sequence design
It swims homology arm primer (UP-hisBC-S, UP-hisBC-A), and PCR amplification its upstream homology arm segment；With E.coli HIS3-1
Genome is template, designs downstream homology arm primer (DN-yghX-S2, DN-yghX- according to the downstream sequence of its yghX gene
), and PCR amplification homology arm segment downstream A.Above-mentioned segment is merged by the method for over-lap PCR, obtains the whole of hisB-hisC
It closes segment (downstream the fragment upstream-hisB-hisC- homology arm of hisB).The DNA containing target sequence that building pGRB-his1 is used
Segment is made by the annealing of primer gRNA-his1-S and gRNA-his1-A.The competent cell of E.coli WHY2-1 is prepared,
It is operated according to method shown in 1.3 and 1.4, it is final to obtain bacterial strain E.coli WHY2-2.In hisB-hisC segment integration process,
The electrophoretogram for integrating the building of segment and the PCR verifying of positive strain is shown in attached drawing 4.Wherein fragment upstream-the hisB-hisC of hisB
Total length be 2696bp, the homologous arm lengths in downstream are 561bp, and the length of overlapping fragments is 3317bp, and design identification primer is simultaneously
PCR verifying is carried out, the length of positive recombinant amplified fragments is 1118bp, and opportunistic pathogen is without band.
3.2.3 the integration of hisH-hisA-hisF-hisI
Using E.coli W3110 (ATCC27325) genome as template, according to hisH-hisA-hisF-hisI and its upstream
Sequence design upstream homology arm primer (UP-hisHAFI-S, UP-hisHAFI-A), and PCR amplification its upstream homology arm segment；
Using E.coli HIS3-2 genome as template, downstream homology arm primer (DN- is designed according to the downstream sequence of its yghX gene
YghX-S3, DN-yghX-A), and PCR amplification homology arm segment downstream.Above-mentioned segment is merged by the method for over-lap PCR, is obtained
The integration segment of hisH-hisA-hisF-hisI (downstream fragment upstream-hisH-hisA-hisF-hisI- of hisH is homologous
Arm).The moving back by primer gRNA-his2-S and gRNA-his2-A of the DNA fragmentation containing target sequence that building pGRB-his2 is used
Fire is made.The competent cell for preparing E.coli WHY2-2 operates according to method shown in 1.3 and 1.4, finally obtains bacterial strain
E.coli WHY2-3.In the integration process of hisH-hisA-hisF-hisI, the PCR of the building and positive strain of integrating segment is tested
The electrophoretogram of card is shown in attached drawing 6.Wherein the total length of the fragment upstream-hisH-hisA-hisF-hisI of hisH is 3265bp, downstream
Homologous arm lengths are 561bp, and overlapping fragments total length is 3317bp, and design identification primer simultaneously carries out PCR verifying, positive recombinant
The length of amplified fragments is 1136bp, and opportunistic pathogen is without band.
3.3 PtrcThe integration of-lysE
Using E.coli W3110 (ATCC27325) genome as template, according to the upstream and downstream sequence design of its yjiT gene
Upstream homology arm primer (UP-yjiT-S, UP-yjiT-A) and downstream homology arm primer (DN-yjiT-S1, DN-yjiT-A), and
Its upstream and downstream homology arm segment of PCR amplification；Using the genome of Corynebacterium glutamicum (ATCC 13032) as template, according to lysE
(NCBI-GeneID:1019244) gene order design primer (lysE-S, lysE-A), and PCR amplification lysE segment；Promoter
PtrcThen design is in the downstream primer of upstream homology arm and the upstream primer of lysE gene.The side that above-mentioned segment passes through over-lap PCR
Method fusion, obtains PtrcIntegration segment (upstream homology arm-the P of-lysEtrcThe downstream-lysE- homology arm), building pGRB-yjiT makes
DNA fragmentation containing target sequence is made by the annealing of primer gRNA-yjiT-S and gRNA-yjiT-A.Prepare E.coli
The competent cell of WHY2-3 is operated according to method shown in 1.3 and 1.4, final to obtain bacterial strain E.coli WHY3.Ptrc-
In lysE segment integration process, the electrophoretogram for integrating the building of segment and the PCR verifying of positive strain is shown in attached drawing 7.Its middle and upper reaches
Homologous arm lengths are 372bp, and lysE genetic fragment length is 834bp, and the homologous arm lengths in downstream are 530bp, the length of overlapping fragments
Degree is 1655bp, and design identification primer simultaneously carries out PCR verifying, and the fragment length that positive recombinant is expanded should be 1429bp, former
Bacterium is then without band.
Embodiment 2:
Method using genetically engineered E.coli WHY3 fermenting and producing histidine is as follows:
Inclined-plane culture: taking -80 DEG C of preservation of bacteria strain streak inoculations in activated inclined plane, 37 DEG C of culture 12h, and passes on primary；
Shake-flask seed culture: a ring inclined-plane seed is scraped with oese and is inoculated in the 500mL equipped with 30mL seed culture medium
In triangular flask, nine layers of gauze sealing, 37 DEG C, 200rpm cultivates 6-8h；
Shake flask fermentation culture: (whole body in the 500mL triangular flask equipped with fermentation medium is inoculated by 10-15% inoculum concentration
Product is 30mL), the sealing of nine layers of gauze, 37 DEG C, 200r/min shaken cultivation maintains pH to exist in fermentation process by adding ammonium hydroxide
7.0-7.2；Adding 60% (m/v) glucose solution maintains fermentation to carry out；Fermentation period 24-30h；
Slant medium composition are as follows: 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, remaining is water, pH 7.0-7.2；
Seed culture medium composition are as follows: glucose 15-30g/L, yeast extract 5-10g/L, peptone 5-10g/L, KH2PO4
5-15g/L, MgSO4·7H2O 2-5g/L, FeSO4·7H2O 5-20mg/L, MnSO4·H2O 5-20mg/L, VB11-3mg/L,
VH0.1-1mg/L, defoaming agent 2 drip, remaining is water, pH 7.0-7.2；
Fermentation medium composition are as follows: glucose 20-30g/L, xylose 5-15g/L, yeast extract 2-5g/L, peptone 2-
4g/L, KH2PO41-3g/L, MgSO4·7H2O 1-2g/L, FeSO4·7H2O 5-20mg/L, MnSO4·7H2O 5-20mg/L,
VB1、VB3、VB5、VB12、VHEach 1-3mg/L, remaining is water, pH 7.0-7.2.
The yield of L-Histidine is up to 6-10g/L after shake flask fermentation 24-30h.
(2) ferment tank:
Slant activation culture: protecting in tube from -80 DEG C of refrigerators and scrape a ring strain, is spread evenly across activated inclined plane, 37 DEG C of trainings
12-16h is supported, switching eggplant-shape bottle continues to cultivate 12-16h；
Seed culture: taking appropriate amounts of sterilized water in eggplant-shape bottle, and bacteria suspension is accessed in seed culture medium, and pH stablizes 7.0
Left and right, temperature is constant at 37 DEG C, and dissolved oxygen is between 25-35%, culture to fermentation liquid OD600Value reaches 10-15；
Fresh fermentation medium is accessed according to 15-20% inoculum concentration, starts to ferment, and pH is controlled in fermentation process and is stablized
7.0 or so, temperature maintains 37 DEG C, and dissolved oxygen is between 25-35%；After the glucose consumption in culture medium is complete, stream adds
The glucose solution of 80% (m/v) maintains the concentration of glucose in fermentation medium in 0.1-5g/L.
Slant medium composition are as follows: 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, remaining is water, pH 7.0-7.2；
Seed culture medium composition are as follows: glucose 15-30g/L, yeast extract 5-10g/L, peptone 5-10g/L, KH2PO4
5-15g/L, MgSO4·7H2O 2-5g/L, FeSO4·7H2O 5-15mg/L, MnSO4·H2O 5-15mg/L, VB11-3mg/L,
VH0.1-1mg/L, defoaming agent 2 drip, remaining is water, pH 7.0-7.2；
Fermentation medium composition are as follows: glucose 10-25g/L, yeast extract 1-5g/L, peptone 1-5g/L, K2HPO4
1-5g/L, MgSO4·7H2O 1-3g/L, FeSO4·7H2O 10-30mg/L, MnSO4·H2O 10-30mg/L, VB1、VB3、VB5、
VB12、VHEach 1-3mg/L, remaining is water, pH 7.0-7.2.
Histidine 40-55g/L can be produced using 5L ferment tank 40-50h, averagely production intensity is 1.0-1.5g/ (L
× h), conversion ratio is 0.18-0.22g histidine/g glucose.
Embodiment 3:
With the shake flask fermentation experiment of the bacterial strain E.coli WHY2-3 and bacterial strain E.coli WHY3 that are constructed in embodiment 1:
The method that above-mentioned two plants of bacterium use shake flask fermentation under the same conditions produces L-Histidine, specific as follows:
Inclined-plane culture: taking -80 DEG C of preservation of bacteria strain streak inoculations in activated inclined plane, 37 DEG C of culture 12h, and passes on primary；
Shake-flask seed culture: a ring inclined-plane seed is scraped with oese and is inoculated in the 500mL equipped with 30mL seed culture medium
In triangular flask, nine layers of gauze sealing, 37 DEG C, 200rpm cultivates 8h；
Shake flask fermentation culture: it is inoculated into the 500mL triangular flask equipped with fermentation medium that (final volume is by 15% inoculum concentration
30mL), nine layers of gauze sealing, 37 DEG C, 200r/min shaken cultivation maintains pH in 7.0- in fermentation process by adding ammonium hydroxide
7.2；Adding 60% (m/v) glucose solution maintains fermentation to carry out；Fermentation period 30h.
Slant medium composition are as follows: glucose 1g/L, peptone 10g/L, beef extract 10g/L, yeast powder 5g/L, NaCl
2.5g/L, agar 20g/L, remaining is water, pH 7.0-7.2.
Seed culture medium composition are as follows: glucose 30g/L, yeast extract 5g/L, peptone 5g/L, KH2PO46g/L,
MgSO4·7H2O 2.5g/L, FeSO4·7H2O 10mg/L, MnSO4·H2O 10mg/L, VB12mg/L, VH1mg/L, defoaming
Agent 2 is dripped, remaining is water, pH 7.0-7.2.
Fermentation medium composition are as follows: glucose 20g/L, xylose 10g/L, yeast extract 5g/L, peptone 4g/L,
KH2PO43g/L, MgSO4·7H2O 2g/L, FeSO4·7H2O 10mg/L, MnSO4·7H2O 10mg/L, VB1、VB3、VB5、
VB12、VHEach 2mg/L, remaining is water, pH 7.0-7.2.
Experimental result such as Fig. 8.In terms of fermentation results, after incorporating the lysE gene from Corynebacterium glutamicum, bacterium
The growth of body and the sugared situation of consumption are uninfluenced, and the yield of histidine is then increased to 9.3g/L from 8g/L, improves 16.25%.
Embodiment 4:
Fermenting experiment of the E.coli WHY3 on 5L fermentor:
Histidine is produced using the bacterial strain E.coli WHY3 that embodiment 1 constructs as production bacterial strain:
Slant activation: glycerol is taken to save strain streak inoculation in test tube slant culture medium, 37 DEG C of cultures 12；Inclined-plane is protected again
Strain streak inoculation is deposited in eggplant-shape bottle slant medium, 37 DEG C of culture 14h.
Seed culture: taking 1 one, the fresh eggplant-shape bottle inclined-plane of activation, under the sterile washing of 150mL, connects under the protection of flames
Kind is into fermentor, and temperature controls 37 DEG C, and auto-feeding ammonium hydroxide controls pH 7.0, and initial Ventilation Rate is 2L/min, is initially stirred
Mixing revolving speed is 200rpm, maintains DO value between 20-30% in incubation, seed culture to OD600It is 15 or so.
Fermentation tank culture: fermentor seed (is discharged to 450mL, under the protection of flames with 15% inoculum concentration access seed liquor
Pour into the fermentation medium of sterilizing), temperature controls 35 DEG C, and auto-feeding ammonium hydroxide (or 20% sulfuric acid) controls pH 7.0, initially leads to
Gas velocity rate is 2L/min, and ventilation ratio 0.667vvm, initial speed of agitator is 400rpm, molten by adjusting revolving speed and Boiler pressure control
Oxygen is defoamed in 20-30% by manual drop addition GPE, and stream adds 80% glucose solution in fermentation process, guarantees sufficient sugar
It supplies and sugared concentration is not higher than 5g/L.
Slant medium composition are as follows: glucose 1g/L, peptone 10g/L, beef extract 10g/L, yeast powder 5g/L, NaCl
2.5g/L, agar 25g/L, remaining is water, pH 7.0-7.2；
Seed culture medium composition are as follows: glucose 10g/L, yeast extract 5g/L, peptone 5g/L, KH2PO45g/L,
MgSO4·7H2O 2g/L, FeSO4·7H2O 10mg/L, MnSO4·H2O 10mg/L, VB12mg/L, VH1mg/L, defoaming agent 2
Drop, remaining is water, pH 7.0-7.2.
Fermentation medium composition are as follows: glucose 10g/L, yeast extract 5g/L, tryptone 4g/L, K2HPO43g/L,
MgSO4·7H2O 1.5g/L, FeSO4·7H2O 20mg/L, MnSO4·H2O 20mg/L, VB1、VB3、VB5、VB12、VHEach 2mg/
L, remaining is water, pH 7.0-7.2.
Fermentation diagram of the E.coli WHY3 on 5L fermentor such as Fig. 9.
From fermentation diagram as can be seen that entering the Rapid Accumulation stage of histidine after fermentation 8 hours, maximum at this time is raw
Intensity is produced up to 2g/ (L × h)；Thalli growth enters stationary phase, the OD of fermentation liquid after 16 hours600It is 89；Fermentation is to 44 small
Constantly, histidine concentrations reach maximum value 55g/L, and the concentration of histidine and cell concentration begin with downward trend later；48 is small
When terminate to ferment, saccharic acid conversion ratio at this time is 0.2g histidine/g glucose.
Sequence table
<110>University Of Science and Technology Of Tianjin
The genetic engineering bacterium and its construction method of<120>one plant heights production L-Histidine and application
<141> 2019-05-30
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 627
<212> DNA
<213>artificial synthesized ()
<400> 1
atgttgaaaa tcgctgtccc aaacaaaggc tcgctgtccg agcgcgccat ggaaatcctc 60
gccgaagcag gctacgcagg ccgtggagat tccaaatccc tcaacgtttt tgatgaagca 120
aacaacgttg aattcttctt ccttcgccct aaagatatcg ccatctacgt tgctggtggc 180
cagctcgatt tgggtatcac cggccgcgac cttgctcgcg attcccaggc tgatgtccac 240
gaagttcttt ccctcggctt cggttcctcc actttccgtt acgcagcacc agctgatgaa 300
gagtggagca tcgaaaagct cgacggcaag cgcatcgcta cctcttaccc caaccttgtt 360
cgcgatgacc tcgcagcacg tgggctttcc gctgaggtgc tccgcctcga cggtgcagta 420
gaggtattca tcaagcttgg tgtcgcagat gccatcgccg atgttgtatc caccggccgc 480
acgctgcgtc agcaaggtct tgcacctttc ggcgaggttc tgtgcacctc tgaggctgtc 540
Claims (9)
1. the genetic engineering bacterium of plant height production L-Histidine, which is characterized in that the genetic engineering bacterium is the base in Escherichia coli
It is prominent because incorporating nucleotide sequence Corynebacterium glutamicum ATP phosphoribosyl transferase HisG as shown in SEQ ID NO:1 in group
Variant encoding gene hisG* simultaneously makes its strongly expressed；Also enhance in the genome E. coli histidine operon hisD,
The expression of seven genes of hisB, hisC, hisH, hisA, hisF and hisI；Also incorporated in the genome from glutamic acid rod
The arginine of bacillus/lysine transport protein encoding gene lysE simultaneously makes its strongly expressed.
2. genetic engineering bacterium as described in claim 1, which is characterized in that the Escherichia coli are E.coli W3110.
3. genetic engineering bacterium as described in claim 1, which is characterized in that the Corynebacterium glutamicum is Corynebacterium glutamicum
ATCC 13032。
4. genetic engineering bacterium as described in claim 1, which is characterized in that the Corynebacterium glutamicum ATP turns Phosphoribosyl
Enzyme HisG mutant code gene hisG* is incorporated on genome at least two gene locis, and is started by strong promoter.
5. genetic engineering bacterium as claimed in claim 1 or 2, which is characterized in that the Corynebacterium glutamicum ATP turns ribose phosphate
Base enzyme HisG mutant code gene hisG* is respectively integrated at tdcD and ylbE gene loci on genome, and by promoter Ptrc
Starting；The E. coli histidine operon gene fragment hisD-hisC-hisB-hisH-hisA-hisF-hisI is incorporated into
YghX gene loci on genome, and by promoter PtrcStarting；The arginine/lysine transport protein encoding gene
LysE integrates yjiT gene loci in the genome, and by promoter PtrcStarting.
6. the purposes that any one of the claim 1-5 genetic engineering bacterium is used for fermenting and producing L-Histidine.
7. purposes as claimed in claim 6, which is characterized in that carry out shake flask fermentation using the genetic engineering bacterium:
Seed liquor will be prepared after actication of culture, is inoculated into the triangular flask equipped with fermentation medium by 10-15% inoculum concentration, nine layers
Gauze sealing, 37 DEG C, 200r/min shaken cultivation maintains pH in 7.0-7.2 in fermentation process by adding ammonium hydroxide；Add 60%
(m/v) glucose solution maintains fermentation to carry out；Fermentation period 24-30h；
The fermentation medium composition are as follows: glucose 20-40g/L, yeast extract 2-5g/L, peptone 2-5g/L, KH2PO41-
3g/L, MgSO4·7H2O 1-2g/L, FeSO4·7H2O 5-20mg/L, MnSO4·7H2O 5-20mg/L, VB1、VB3、VB5、
VB12、VHEach 1-3mg/L, remaining is water, pH 7.0-7.2.
8. purposes as claimed in claim 6, which is characterized in that carry out ferment tank using the genetic engineering bacterium:
Seed liquor will be prepared after actication of culture, fresh fermentation medium is accessed according to 15-20% inoculum concentration, start to ferment, sent out
It controls pH during ferment to stablize 7.0 or so, temperature maintains 37 DEG C, and dissolved oxygen is between 25-35%；When the grape in culture medium
After sugar consumption is complete, the glucose solution of stream plus 80% (m/v) maintains the concentration of glucose in fermentation medium in 0.1-5g/
L；Fermentation period 40-50h；
The fermentation medium composition are as follows: glucose 10-30g/L, yeast extract 1-5g/L, peptone 1-5g/L, K2HPO41-
5g/L, MgSO4·7H2O 1-3g/L, FeSO4·7H2O 10-30mg/L, MnSO4·H2O 10-30mg/L, VB1、VB3、VB5、
VB12、VHEach 1-3mg/L, remaining is water, pH 7.0-7.2.
9. a kind of construction method of the genetic engineering bacterium of high yield histidine, which is characterized in that the genetic engineering bacterium is to use
The gene editing technology that CRISPR/Cas9 is mediated is oriented house of correction to E.coli W3110 and obtains, and includes the following steps:
(1) promoter P is constructedtrcWith the junction fragment P of nucleotide sequence gene hisG* as shown in SEQ ID NO:1trc-
HisG*, and it is respectively integrated at tdcD and ylbE gene loci on genome；
(2) promoter P is constructedtrcWith the junction fragment P of E. coli histidine operon genetrc-hisD-hisC-hisB-
HisH-hisA-hisF-hisI, and yghX gene loci on genome is integrated into the method for segmentation integration；
(3) promoter P is constructedtrcWith the junction fragment P of the lysE gene from Corynebacterium glutamicumtrc- lysE, and its is whole
Close yjiT gene loci in the genome.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910461591.5A CN110184230A (en) | 2019-05-30 | 2019-05-30 | The genetic engineering bacterium and its construction method of one plant height production L-Histidine and application |
| PCT/CN2019/089938 WO2020237701A1 (en) | 2019-05-30 | 2019-06-04 | High-yield l-histidine genetically engineered bacterium strain, and construction method therefor and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910461591.5A CN110184230A (en) | 2019-05-30 | 2019-05-30 | The genetic engineering bacterium and its construction method of one plant height production L-Histidine and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110184230A true CN110184230A (en) | 2019-08-30 |
Family
ID=67718785
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910461591.5A Pending CN110184230A (en) | 2019-05-30 | 2019-05-30 | The genetic engineering bacterium and its construction method of one plant height production L-Histidine and application |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN110184230A (en) |
| WO (1) | WO2020237701A1 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110964683A (en) * | 2019-12-02 | 2020-04-07 | 天津科技大学 | Genetically engineered bacterium for producing L-arginine and construction method and application thereof |
| CN111321102A (en) * | 2020-03-06 | 2020-06-23 | 浙江震元制药有限公司 | Genetically engineered bacterium for producing L-histidine and application thereof |
| CN111996155A (en) * | 2020-09-08 | 2020-11-27 | 浙江华睿生物技术有限公司 | Method for improving production capacity of L-histidine producing strain |
| CN113881726A (en) * | 2021-10-20 | 2022-01-04 | 广东肇庆星湖生物科技股份有限公司 | Method for improving histidine fermentation purity |
| CN113980882A (en) * | 2021-11-30 | 2022-01-28 | 天津科技大学 | Genetic engineering strain for dynamically regulating and controlling phosphoglucose isomerase to produce histidine, construction method and application thereof |
| CN115175994A (en) * | 2019-12-16 | 2022-10-11 | 银杏生物制品公司 | Enhanced production of histidine, purine pathway metabolites and plasmid DNA |
| US20220403348A1 (en) * | 2021-06-18 | 2022-12-22 | Zhejiang Zhenyuan Biotech Co., Ltd. | Genetically engineered bacterium for producing l-histidine and use thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101457244A (en) * | 2009-01-06 | 2009-06-17 | 天津科技大学 | Method for improving L-histidine fermentation process production rate |
| CN108130306A (en) * | 2018-01-10 | 2018-06-08 | 天津科技大学 | The genetic engineering bacterium and its construction method of high yield uridine and application |
| CN108913642A (en) * | 2018-07-27 | 2018-11-30 | 天津科技大学 | The purposes of Recombinant organism and its ferment synchronous production L-Trp and Valine |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BRPI0506369B1 (en) * | 2004-01-30 | 2020-11-03 | Ajinomoto Co., Inc | transgenic microorganism, and, method to produce a l-amino acid |
| DE102004005836A1 (en) * | 2004-02-06 | 2005-09-15 | Degussa Ag | Process for the preparation of L-amino acids using strains of the family Enterobacteriaceae |
| US20060008546A1 (en) * | 2004-05-28 | 2006-01-12 | Cargill, Incorporated | Organisms with enhanced histidine biosynthesis and their use in animal feeds |
| CN102453691B (en) * | 2011-12-02 | 2013-09-18 | 山东鲁抗生物制造有限公司 | Escherichia coli engineering bacteria capable of realizing high yield of L-tryptophan |
| CN104845923B (en) * | 2014-02-14 | 2018-03-23 | 中国科学院微生物研究所 | Produce the method and its special recombinant bacterium of L histidines |
| KR101904666B1 (en) * | 2017-08-02 | 2018-11-29 | 씨제이제일제당 (주) | An ATP phoshpolybosyltransferase mutation and a method of producing histidine using thereof |
-
2019
- 2019-05-30 CN CN201910461591.5A patent/CN110184230A/en active Pending
- 2019-06-04 WO PCT/CN2019/089938 patent/WO2020237701A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101457244A (en) * | 2009-01-06 | 2009-06-17 | 天津科技大学 | Method for improving L-histidine fermentation process production rate |
| CN108130306A (en) * | 2018-01-10 | 2018-06-08 | 天津科技大学 | The genetic engineering bacterium and its construction method of high yield uridine and application |
| CN108913642A (en) * | 2018-07-27 | 2018-11-30 | 天津科技大学 | The purposes of Recombinant organism and its ferment synchronous production L-Trp and Valine |
Non-Patent Citations (3)
| Title |
|---|
| A. BELLMANN 等: "Expression control and specificity of the basic amino acid exporter LysE of Corynebacterium glutamicum", 《MICROBIOLOGY》 * |
| MEIJUAN XU 等: "The Effect of a LYSE Exporter Overexpression on L-Arginine Production in Corynebacterium crenatum", 《CURR MICROBIOL》 * |
| 魏伟 等: "大肠杆菌L-组氨酸生物合成途径的改造及其对工程菌L-组氨酸产量的影响", 《天津科技大学学报》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110964683A (en) * | 2019-12-02 | 2020-04-07 | 天津科技大学 | Genetically engineered bacterium for producing L-arginine and construction method and application thereof |
| CN115175994A (en) * | 2019-12-16 | 2022-10-11 | 银杏生物制品公司 | Enhanced production of histidine, purine pathway metabolites and plasmid DNA |
| CN111321102A (en) * | 2020-03-06 | 2020-06-23 | 浙江震元制药有限公司 | Genetically engineered bacterium for producing L-histidine and application thereof |
| CN111321102B (en) * | 2020-03-06 | 2021-07-06 | 浙江震元制药有限公司 | Genetically engineered bacterium for producing L-histidine and application thereof |
| CN111996155A (en) * | 2020-09-08 | 2020-11-27 | 浙江华睿生物技术有限公司 | Method for improving production capacity of L-histidine producing strain |
| CN111996155B (en) * | 2020-09-08 | 2022-02-11 | 浙江华睿生物技术有限公司 | Method for improving production capacity of L-histidine producing strain |
| US20220403348A1 (en) * | 2021-06-18 | 2022-12-22 | Zhejiang Zhenyuan Biotech Co., Ltd. | Genetically engineered bacterium for producing l-histidine and use thereof |
| US11692178B2 (en) * | 2021-06-18 | 2023-07-04 | Zhejiang Zhenyuan Biotech Co., Ltd. | Genetically engineered bacterium for producing L-histidine and use thereof |
| CN113881726A (en) * | 2021-10-20 | 2022-01-04 | 广东肇庆星湖生物科技股份有限公司 | Method for improving histidine fermentation purity |
| CN113980882A (en) * | 2021-11-30 | 2022-01-28 | 天津科技大学 | Genetic engineering strain for dynamically regulating and controlling phosphoglucose isomerase to produce histidine, construction method and application thereof |
| CN113980882B (en) * | 2021-11-30 | 2023-09-26 | 天津科技大学 | Genetic engineering strain for dynamically regulating and controlling production of histidine by phosphoglucose isomerase as well as construction method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2020237701A1 (en) | 2020-12-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110184230A (en) | The genetic engineering bacterium and its construction method of one plant height production L-Histidine and application | |
| CN110607268B (en) | Genetically engineered bacterium for high yield of L-valine and method for producing L-valine by fermentation | |
| CN108130306B (en) | The genetic engineering bacterium and its construction method of high yield uridine and application | |
| CN110468092B (en) | Genetically engineered bacterium capable of producing L-valine at high yield, and construction method and application thereof | |
| CN108441460A (en) | The genetic engineering bacterium and its construction method of a kind of high yield hydroxy tetrahydro pyrimidine and application | |
| CN110964683A (en) | Genetically engineered bacterium for producing L-arginine and construction method and application thereof | |
| CN111321102B (en) | Genetically engineered bacterium for producing L-histidine and application thereof | |
| CN113667682B (en) | YH66-RS11190 gene mutant and application thereof in preparation of L-valine | |
| CN108949706B (en) | L-proline-4-hydroxylase, gene engineering bacterium thereof, construction method and application | |
| CN105420154A (en) | Double knockout recombinant rhodococcus as well as construction method and application thereof | |
| KR20220061146A (en) | Application of a transporter gene to improve L-tryptophan production efficiency in E. coli | |
| WO2022174597A1 (en) | Genetically engineered bacterium for producing l-sarcosine, construction method therefor and use thereof | |
| WO2022143763A1 (en) | Strain having enhanced l-glutamic acid productivity, construction method therefor and application thereof | |
| CN106591209A (en) | Recombinant strain and preparation method thereof and method for producing L-threonine | |
| CN113073074B (en) | Genetically engineered bacterium for efficiently synthesizing riboflavin and application thereof | |
| CN112280728B (en) | Genetic engineering strain for producing L-citrulline and application thereof | |
| CN109456987A (en) | The related gene and engineering bacteria construction method of high yield L-Leu and application | |
| WO2019136618A1 (en) | Gene engineering bacterium for producing uridine at high yield, construction method therefor and application thereof | |
| CN116854790A (en) | Mutant protein and application thereof in preparation of valine | |
| CN111471693B (en) | Corynebacterium glutamicum for producing lysine and construction method and application thereof | |
| WO2023236634A1 (en) | Ep6 promoter, related biomaterial, and use thereof | |
| CN106635945A (en) | Recombinant strain and preparation method thereof and method for producing L-threonine | |
| CN114181288B (en) | Process for producing L-valine, gene used therefor and protein encoded by the gene | |
| CN110106191A (en) | Artificial synthesized Vitreoscilla hemoglobin gene and corresponding engineered strain and application | |
| CN105018515A (en) | Method for improving yield of arginine by utilizing corynebacterium glutamicum and corynebacterium crenatum |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190830 |
|
| WD01 | Invention patent application deemed withdrawn after publication |