CN111235079A - Method for screening escherichia coli with high tryptophan yield - Google Patents

Method for screening escherichia coli with high tryptophan yield Download PDF

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CN111235079A
CN111235079A CN201910736581.8A CN201910736581A CN111235079A CN 111235079 A CN111235079 A CN 111235079A CN 201910736581 A CN201910736581 A CN 201910736581A CN 111235079 A CN111235079 A CN 111235079A
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screening
tryptophan
escherichia coli
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coli
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张沨
王新飞
王兴春
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Shandong Huiguan Kangbo Biotechnology Co Ltd
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • 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
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/227—Tryptophan

Abstract

The invention discloses a method for screening high-tryptophan-producing escherichia coli, which comprises the steps of cloning a tryptophan transport RNA gene trpT from escherichia coli MG1655 to change an anticodon loop sequence, cloning a constitutive promoter llp and a terminator rrnB from the escherichia coli MG1655, then constructing a plasmid containing tRNA and a screening gene, transferring the plasmid into escherichia coli C321. delta A.exp to obtain converted thalli, and carrying out enrichment culture on the converted thalli to select a strain with the fastest growth of the thalli as a high-yield strain of amino acid.

Description

Method for screening escherichia coli with high tryptophan yield
Technical Field
The invention relates to the technical field of biological engineering, in particular to a method for screening high-yield tryptophan escherichia coli.
Background
Tryptophan is one of 22 standard amino acids constituting protein, and is also one of eight essential amino acids for human body. Tryptophan is not synthesized in many animals and can only be obtained by exogenous ingestion. When livestock and poultry lack tryptophan, the conditions of growth retardation, weight loss, fat accumulation reduction and the like can occur. L-tryptophan can be used as an additive to be used in animal feed, and tryptophan ingested by animals can participate in the renewal of plasma protein in the bodies of the animals, promote the exertion of riboflavin, enhance the synthesis of nicotinic acid and heme, obviously increase the antibody concentration in the bodies of the newborn animals of the pregnant animals, has the function of promoting lactation of cows and sows in the lactation period, and is an amino acid with high application value and commercial prospect.
The early synthetic method of tryptophan mainly utilizes tryptophan synthetase to catalyze indole and L-serine in one step to obtain L-tryptophan, which is also the main reaction for synthesizing L-tryptophan in most animals and plants. Subsequently, a process for converting and synthesizing L-tryptophan by using anthranilic acid or DL-hydantoin as a substrate appears. Since the precursors and enzymes used in the above processes are expensive, the most economical method for producing tryptophan is currently a microbial fermentation using sugar as a substrate.
3-deoxy-D-arabinoheptonate-7-phosphate synthase (3-deoxy-D-arabinoheptonate-7-phosphate synthase) and anthranilate synthase (anthranilate synthase) are key nodes in the biosynthetic pathway of L-tryptophan. The conventional method constructs a strain capable of producing L-tryptophan by screening mutants having resistance to various tryptophan analogs. For example, mutants of Brevibacterium flavum (Brevibacterium flavum) are tyrosine auxotrophic and are resistant to both 5-fluorotryptophan (5-fluorotryptophan) and azaserine (azaserine). The method also screens out tyrosine and phenylalanine double auxotrophic Corynebacterium glutamicum (Corynebacterium glutamicum) mutants which are resistant to 6 different tryptophan analogs such as 5-methyltryptophan ((5-methyltryptophan), tryptophan hydroxamate (tryptophan hydroxamate) and tyrosine hydroxamate (tyrosine hydroxamate), mutants of Bacillus subtilis which are resistant to 5-fluorotryptophan (5-fluorotryptophan) and indomycin (indomycin) are highly toxic to cells in actual fermentation, however, tryptophan analogs used at present are extremely toxic to cells, and high concentrations of the analogs inhibit normal growth of most cells, so that mutants which are normally grown are enriched, mutant strains which escape screening pressure by means of discharging, degrading and the like also exist, so that false positives are eliminated by methods of multi-round re-screening, one-by-one identification and the like in the process of obtaining the true tryptophan high-yield strains.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for screening escherichia coli with high tryptophan yield so as to solve the problems that the screening method in the prior art is complex and needs to consume a large amount of human resources.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a method for screening Escherichia coli with high tryptophan production is characterized by comprising the following steps: the method comprises the following steps:
step 1, searching and annotating trpT fragment of tryptophan transport RNA gene in Escherichia coli MG1655, and replacing CCA with CTA;
step 2, replacing a tryptophan codon TGG in the screened gene with a termination codon TAG according to the content of tryptophan in different screened genes to obtain an artificially synthesized screened gene;
step 3, connecting the artificially synthesized screening gene sequence obtained in the step 2 with a plasmid vector to obtain a plasmid for screening, and converting the plasmid into a C321. delta A.exp mutant bacteria library to obtain a converted bacterial liquid;
step 4, enriching and culturing the bacterial liquid obtained in the step 3 in a culture medium, coating the bacterial liquid on a flat plate for 10-48 h, and selecting a strain with the fastest thallus growth as an amino acid high-yield strain;
and 5, inoculating the amino acid high-yield strain obtained in the step 4 into a fermentation medium, and determining the yield of the final amino acid.
2. The method for screening E.coli having a high tryptophan production according to claim 1, wherein: the protein expressed by the artificially synthesized screening gene comprises fluorescent protein, chromogenic protein or antibiotic resistance protein.
3. The method for screening E.coli having a high tryptophan production according to claim 2, wherein: the culture medium comprises: 5-10g/L of tryptone, 1-10g/L of yeast powder and 5-10g/L of sodium chloride.
4. The method for screening E.coli having a high tryptophan production according to claim 3, wherein: the fermentation medium comprises: 5-20g/L of phosphate, 1-10g/L of ammonium chloride, 10-40g/L of glucose, 0.1-5g/L of sodium chloride, 0.3g/L of magnesium sulfate, 0.015g/L of calcium chloride and 15-50 mu g/L of vitamin B.
By adopting the technical scheme, the tryptophan high-yield strain can be screened by applying the endogenous stress of bacteria under the condition of no need of amino acid analogues, no toxic reagent is generated in the operation process, the screening process is simplified, and the working strength is reduced.
Drawings
FIG. 1 is a graph showing the comparison between the growth of the E.coli strain with high tryptophan production screened by the present invention and the growth of the control strain.
Detailed Description
The following further describes embodiments of the present invention with reference to the drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
A method for screening high-tryptophan-producing Escherichia coli comprises the following steps:
step 1, construction of screening plasmids:
a trpT fragment annotated as a tryptophan transport RNA gene is searched in Escherichia coli MG1655, an anticodon loop sequence CCA is replaced by CTA, an lpp promoter and an rrnB terminator are respectively added at the upstream and the downstream, and the synthetic primers of the trpT are used for artificial synthesis by using a PCR method.
The gene total synthesis system is as follows: 2 XA 8 PCR MasterMix 25. mu.L, primer premix 5. mu.L, distilled water 20. mu.L, total volume 50. mu.L. The amplification conditions were denaturation at 95 ℃ for 20 seconds, annealing at 60 ℃ for 15 seconds, and extension at 72 ℃ for 10 seconds (30 cycles); extension at 72 ℃ for 5 min (1 cycle).
And 2, constructing a vector pSK-Trp:
amplifying a fragment from the vector pSK for connecting a transfer RNA gene fragment,
the amplification product contains the transport RNA gene and a segment overlapping with the vector segment by 20 bases, and is ligated to the pSK vector. A connection system: 1.5. mu.L of the LPCR amplification product, 1. mu.L of the amplified fragment of the pSK vector, 7.5. mu.L of Gibson
Figure BDA0002162360710000041
MasterMix (from NEW ENGLAND BioLabs) was gently mixed and reacted in a water bath at 50 ℃ for 60 minutes. Then 50 μm of lXL10-GOLD competent cells (from Beijing Bomaide Gene technology, Inc.) were added, and the mixture was ice-washed for 30 minutes, heat-shocked at 42 ℃ for 60 seconds, and immediately placed on ice for 2 minutes. Adding 1000. mu.L SOC culture medium, shake culturing for 1 hr in shaker at 37 deg.C and 200rpm, spreading 200. mu.L bacterial liquid on LB plate containing spectacular, overnight culturing, performing PCR sequencing verification, performing liquid culture on positive clone, and extracting plasmid for sequencing verification. Sequencing results show that a newly synthesized transfer RNA gene is inserted into the vector pSK, the plasmid construction is proved to be correct, and the obtained recombinant plasmid is named as pSK-Trp.
Step 3, artificially synthesizing a kanamycin resistance gene: the kanamycin resistance gene sequence is obtained by inquiring from a Genbank gene bank, 6 tryptophan codons TGG are replaced by a stop codon TAG, and a primer is chemically synthesized and then PCR is carried out to obtain a kanR-UAG gene sequence. The kanamycin resistance gene shown in the sequence 1 is obtained by a gene total synthesis method by using a synthetic primer of a kanR-UAG gene sequence.
The gene total synthesis system is as follows: 2 XA 8 PCR MasterMix 25. mu.L, primer premix 5. mu.L, distilled water 20. mu.L, total volume 50. mu.L. The amplification conditions were denaturation at 95 ℃ for 20 seconds, annealing at 60 ℃ for 15 seconds, and extension at 72 ℃ for 30 seconds (30 cycles); extension at 72 ℃ for 5 min (1 cycle). The amplification product contains kanamycin resistance gene and 1 of each upstream and downstream thereof00 bases and ligated into pSK-Trp vector. A connection system: 1.5. mu.L of PCR amplification product, 1. mu.L of pSK-Trp vector fragment, 7.5. mu.L of Gibson
Figure BDA0002162360710000051
MasterMix (from NEW ENGLAND BioLabs) was gently mixed and reacted in a water bath at 50 ℃ for 60 minutes. Subsequently 50. mu.L of XL10-GOLD competent cells (from Beijing Bomaide Gene technology, Inc.) were added, ice-washed for 30 minutes, heat-shocked at 42 ℃ for 60 seconds, and immediately placed on ice for 2 minutes. Add 250. mu.L SOC medium and shake culture in a shaker at 200rpm and 37 ℃ for 1 hour. And (3) coating 200 mu L of bacterial liquid on an LB plate containing spectinomycin, performing overnight culture, performing PCR sequencing verification, performing liquid culture on positive clones, and extracting plasmids for sequencing verification. Sequencing results show that newly synthesized kanamycin resistance gene and 100 bases on the upstream and the downstream of the newly synthesized kanamycin resistance gene are inserted into the vector pSK-Trp, the construction of the plasmid is proved to be correct, and the obtained recombinant plasmid is named as pSK-Trp-UAG.
Step 4, construction of the screened strains: single colonies of C321. DELTA.A.exp were picked from the plate, transferred to LB medium, and cultured at 37 ℃ and 200rpm for 3 to 5 hours until OD600To 0.4-0.6, the bacterial solution was placed on ice for 15-30 minutes, then centrifuged at 4000 Xg for 10 minutes at 4 ℃ and the supernatant was discarded, the cells were resuspended with 500. mu.L of sterile ice water, centrifuged again at 4000 Xg for 10 minutes at 4 ℃ and the supernatant was carefully discarded. Resuspend cells to 50 μ L with 20% glycerol solution. And (3) electrotransfer conditions: 50. mu.L of the prepared competent cells were placed on ice, and 1. mu.L of the plasmid vector pSK-Trp-UAG constructed in example 1 was added thereto, and after being placed on ice for 10 minutes, the cells were transferred to a 0.2em Bio-Red electric rotary cup. The electroporation system of Gene Pulser XcellTM (Bio-Red) was used, and the electric shock voltage was 2.5 kV. Immediately after electric shock, the electric rotor was rinsed with 500. mu.L of LB medium, gently aspirated for 5 times, transferred to a test tube, cultured at 37 ℃ for half an hour at 200rpm, spread on an LB plate containing spectinomycin, and cultured overnight at 37 ℃ to obtain a strain C321. delta. A.exp containing the plasmid pSK-Trp-UAG, which was designated as C321 (Trp-UAG).
Step 5, determination of screening conditions: the single colony of the strain C321(Trp-UAG) obtained in step 4 was transferred to 5mL of the selection medium A and the selection medium B, ampicillin, spectinomycin and kanamycin were added simultaneously, the mixture was cultured at 37 ℃ and 200rpm for 12 to 72 hours, 200. mu.L of the bacterial solution was taken every 12 hours to a 96-well plate, and the growth of the bacterial cells was measured at 600nm, as shown in FIG. 1.
Wherein, the screening medium A: peptone 5-10g/L, yeast extract 1-5g/L, sodium chloride 5-10g/L
Wherein, screening medium B: 10-15g/L of peptone, 20-30g/L of yeast extract, 1-5g/L of glycerol, 1-3g/L of sodium dihydrogen phosphate and 5-15g/L of disodium hydrogen phosphate.
Step 6, screening the high-yield L-tryptophan strains: escherichia coli C321. delta. A.exp was used as a starting strain, and the C321. delta. A.exp was mutagenized by ultraviolet light, nitrosoguanidine or an ordinary temperature and pressure plasma mutagenesis system (ARTP), and the treated bacterial solution was transferred to 2mL of LB medium. After culturing at 37 ℃ and 200rpm for 3-5 hours until the OD600 is 0.4-0.6, the bacterial solution is placed on ice for 15-30 minutes, then centrifuged at 4 ℃ and 4000 Xg for 10 minutes, the supernatant is discarded, the cells are resuspended in 500. mu.L of sterile ice water, and centrifuged again at 4 ℃ and 4000 Xg for 10 minutes, and the supernatant is carefully discarded. Resuspend cells to 50 μ L with 20% glycerol solution. And (3) electrotransfer conditions: 50 μ L of the prepared competent cells were placed on ice, 1 μ L of the vector pSK-Trp-UAG was added, and after 10 minutes on ice, they were transferred to a 0.2em Bio-Red electric rotor. The electroporation system of Gene Pulser XcellTM (Bio-Red) was used, and the electric shock voltage was 2.5 kV. Immediately after the electric shock, the electric beaker was rinsed with 500. mu.L of LB medium, gently aspirated 5 times, transferred to a test tube, supplemented with 4.5mL of LB medium containing ampicillin and kanamycin, and cultured at 37 ℃ and 200rpm for 12 hours. The bacterial liquid is coated on an LB plate containing ampicillin and kanamycin, and cultured for 12 hours at 37 ℃ to obtain the mutant flora containing high-yield tryptophan. Randomly pick 10 single colonies from the plate, pick the single colony to inoculate in seed medium.
The seed medium in a 50mL Erlenmeyer flask was 10mL and sterilized at 121 ℃ for 20 minutes. After cooling, 10 escherichia coli c321. delta. A.exp mutant strains and 1 escherichia coli c321. delta. A.exp strain were inoculated, the culture temperature was 37 ℃, the rotation speed of a shaker was 200rpm, and after 12 hours of culture, the absorbance values of all the strains were measured. The cells were collected by centrifugation at 8000 Xg, washed 3 times with sterile water, adjusted to the same absorbance of all seed solutions, and used for inoculation of fermentation medium.
20mL of fermentation medium in a 250mL triangular flask, and sterilizing at 115 ℃ for 20 min. The inoculum size was 0.1% (v/v), the fermentation temperature was 37 ℃, the shaker speed was 250rpm, and the fermentation time was 24 hours.
Wherein, the seed culture medium comprises: 10g/L of tryptone, 5g/L of yeast powder, 10g/L of sodium chloride, 50 mu g/mL of kanamycin and 7.0 of culture medium pH.
Wherein the fermentation medium comprises the following components: 5-20g/L of phosphate, 1-5g/L of ammonium chloride, 1-5g/L of glucose, 0.1-10g/L of sodium chloride, 0.3g/L of magnesium sulfate, 0.015g/L of calcium chloride, 15-50 mu g/L of vitamin B, and pH 6.0-8.0.
The analysis method comprises the following steps: l-tryptophan in the fermentation broth was measured using Agilent high performance liquid chromatograph 1290. L-tryptophan was quantified by treating the fermentation broth with phenyl isothiocyanate derivative method and separating and quantifying the L-tryptophan by means of a CelmerfeI C18 column.
The results of the invention are shown in FIG. 1, the L-tryptophan yield of all mutant strains is higher than that of the control strain C321. delta. A.exp, the L-tryptophan yield of WP-8 in the screened mutant strains can reach 1.942g/L, and is improved by 7.1 times compared with that of the control strain C321. delta. A.exp. The screening method has obvious effect, and provides an efficient and reliable method for screening the high-yield strains of the tryptophan of the escherichia coli.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Claims (4)

1. A method for screening Escherichia coli with high tryptophan production is characterized by comprising the following steps: the method comprises the following steps:
step 1, searching a trpT fragment annotated as a tryptophan transport RNA gene from Escherichia coli MG1655, and replacing CCA with CTA;
step 2, replacing a tryptophan codon TGG in the screened gene with a termination codon TAG according to the content of tryptophan in different screened genes to obtain an artificially synthesized screened gene;
step 3, connecting the artificially synthesized screening gene sequence obtained in the step 2 with a plasmid vector to obtain a plasmid for screening, and converting the plasmid into a C321. delta A.exp mutant bacteria library to obtain a converted bacterial liquid;
step 4, enriching and culturing the bacterial liquid obtained in the step 3 in a culture medium, coating the bacterial liquid on a flat plate, culturing for 10-48 h, and selecting a strain with the fastest thallus growth as an amino acid high-yield strain;
and 5, inoculating the amino acid high-yield strain obtained in the step 4 into a fermentation medium, and determining the yield of the final amino acid.
2. The method for screening E.coli having a high tryptophan production according to claim 1, wherein: the protein expressed by the artificially synthesized screening gene comprises fluorescent protein, chromogenic protein or antibiotic resistance protein.
3. The method for screening E.coli having a high tryptophan production according to claim 2, wherein: the culture medium comprises: 5-10g/L of tryptone, 1-10g/L of yeast powder and 5-10g/L of sodium chloride.
4. The method for screening E.coli having a high tryptophan production according to claim 3, wherein: the fermentation medium comprises: 5-20g/L of phosphate, 1-10g/L of ammonium chloride, 10-40g/L of glucose, 0.1-5g/L of sodium chloride, 0.3g/L of magnesium sulfate, 0.015g/L of calcium chloride and 15-50 mu g/L of vitamin B.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101107355A (en) * 2005-01-19 2008-01-16 味之素株式会社 A method for producing an l-amino acid using a bacterium of the enterobacteriaceae family having a pathway of glycogen biosynthesis disrupted
CN101812426A (en) * 2010-04-02 2010-08-25 安徽丰原发酵技术工程研究有限公司 Method for constructing colibacillus strain for producing tryptophan and constructed strain
CN102453691A (en) * 2011-12-02 2012-05-16 山东鲁抗生物制造有限公司 Escherichia coli engineering bacteria capable of realizing high yield of L-tryptophan
CN109486737A (en) * 2018-12-03 2019-03-19 江南大学 A kind of recombination bacillus coli and its construction method of high yield L-Trp

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101107355A (en) * 2005-01-19 2008-01-16 味之素株式会社 A method for producing an l-amino acid using a bacterium of the enterobacteriaceae family having a pathway of glycogen biosynthesis disrupted
CN101812426A (en) * 2010-04-02 2010-08-25 安徽丰原发酵技术工程研究有限公司 Method for constructing colibacillus strain for producing tryptophan and constructed strain
CN102453691A (en) * 2011-12-02 2012-05-16 山东鲁抗生物制造有限公司 Escherichia coli engineering bacteria capable of realizing high yield of L-tryptophan
CN109486737A (en) * 2018-12-03 2019-03-19 江南大学 A kind of recombination bacillus coli and its construction method of high yield L-Trp

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
唐任天: "大肠杆菌色氨酸操纵子的结构与克隆", 《微生物学通报》 *
郝大利等: "大肠杆菌aroG基因的定点突变及与trpBA基因的串联表达", 《应用与环境生物学报》 *

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Application publication date: 20200605